Section 1 Epidemiology

**3**

**Chapter 1**

**1. Introduction**

Introductory Chapter: Zika

2015-2020 - Knowledge and

Experience in the Americas

**2. Fundamental aspects of Zika virus and Zika disease**

*and Alfonso J. Rodriguez-Morales*

*Carlos Andrés Rosero-Oviedo, D. Katterine Bonilla-Aldana,* 

Zika virus is an arbovirus that was discovered many decades ago but remains fundamentally silent until a couple of decades ago. Nevertheless, only when arrived in the Americas, was able to cause significant epidemics and new clinical consequences, including microcephaly and the Guillain-Barré syndrome, among others. In this chapter, we introduce general concepts and our position regard the relevance of Zika and their knowledge and experience in the Americas over the last years, 2015–2020.

The Zika virus (ZIKV) was first isolated in Uganda in 1947 and was confined for almost 60 years in Africa and Asia. Later, in 2007, the Yap outbreak allowed its spread to French Polynesia and other Pacific islands in 2013–2015, finally reaching the Americas in 2015 and being declared a Public Health Emergency of International Importance in 2016. Currently, no area is reporting Zika outbreaks; however, its circulation through sporadic cases remains a global threat. The discovery of ZIKV and many other arboviruses was the result of research conducted within the Rockefeller Foundation-sponsored yellow fever research programs [1]. The current East African Virus Research Institute (Entebbe, Uganda), was a focal point for research on pathogenic viruses. In April 1947, in an attempt to map the spread of yellow fever, the temperature of Rhesus monkeys, of the Asian species (*Macaca mulatta*), used in six sentinel platforms in the Zika forest in Entebbe, Uganda, was measured periodically [2]. On April 18, 1947, the temperature of one of these monkeys, Rhesus 766, was reported to be 39.7°C and the next day 40°C, so it was taken to the Entebbe laboratory where it was kept under observation for 30 days, with no evidence of other symptoms. On the third day of fever, a blood sample was taken that would subsequently allow the isolation of what was called ZIKV (strain 766). In the same report, the first isolation of ZIKV is described in 86 Aedes africanus mosquitoes trapped on a tree platform in the Zika forest in January 1948 [2]. Interestingly, although there was no evidence that ZIKV caused disease among Ugandan residents, the prevalence of antibodies to ZIKV was 9.5–20%, suggesting that the virus was already circulating in the human population (or was a consequence of cross-reactivity with other flaviviruses) [3]. A more detailed

*Jaime A. Cardona-Ospina, Wilmer E. Villamil-Gómez* 

## **Chapter 1**

## Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas

*Carlos Andrés Rosero-Oviedo, D. Katterine Bonilla-Aldana, Jaime A. Cardona-Ospina, Wilmer E. Villamil-Gómez and Alfonso J. Rodriguez-Morales*

## **1. Introduction**

Zika virus is an arbovirus that was discovered many decades ago but remains fundamentally silent until a couple of decades ago. Nevertheless, only when arrived in the Americas, was able to cause significant epidemics and new clinical consequences, including microcephaly and the Guillain-Barré syndrome, among others. In this chapter, we introduce general concepts and our position regard the relevance of Zika and their knowledge and experience in the Americas over the last years, 2015–2020.

#### **2. Fundamental aspects of Zika virus and Zika disease**

The Zika virus (ZIKV) was first isolated in Uganda in 1947 and was confined for almost 60 years in Africa and Asia. Later, in 2007, the Yap outbreak allowed its spread to French Polynesia and other Pacific islands in 2013–2015, finally reaching the Americas in 2015 and being declared a Public Health Emergency of International Importance in 2016. Currently, no area is reporting Zika outbreaks; however, its circulation through sporadic cases remains a global threat. The discovery of ZIKV and many other arboviruses was the result of research conducted within the Rockefeller Foundation-sponsored yellow fever research programs [1]. The current East African Virus Research Institute (Entebbe, Uganda), was a focal point for research on pathogenic viruses. In April 1947, in an attempt to map the spread of yellow fever, the temperature of Rhesus monkeys, of the Asian species (*Macaca mulatta*), used in six sentinel platforms in the Zika forest in Entebbe, Uganda, was measured periodically [2]. On April 18, 1947, the temperature of one of these monkeys, Rhesus 766, was reported to be 39.7°C and the next day 40°C, so it was taken to the Entebbe laboratory where it was kept under observation for 30 days, with no evidence of other symptoms. On the third day of fever, a blood sample was taken that would subsequently allow the isolation of what was called ZIKV (strain 766). In the same report, the first isolation of ZIKV is described in 86 Aedes africanus mosquitoes trapped on a tree platform in the Zika forest in January 1948 [2]. Interestingly, although there was no evidence that ZIKV caused disease among Ugandan residents, the prevalence of antibodies to ZIKV was 9.5–20%, suggesting that the virus was already circulating in the human population (or was a consequence of cross-reactivity with other flaviviruses) [3]. A more detailed

description of the origins of ZIKV is presented elsewhere [4]. Although the initial isolation and characterisation of ZIKV in Uganda's Zika forest are unquestionable, there is some controversy as to which report described the first human ZIKV infection [5]. Several authors suggest that the first isolation in humans was in 1954, and it was a 10-year-old African girl with fever and headache associated with a malaria co-infection [6]. Cross-neutralisation tests with convalescent sera from monkeys infected with different viruses indicated that only ZIKV-infected serum neutralised the virus from the patient's serum, strongly suggesting ZIKV infection. However, a later published report indicated that the virus isolated in West Africa was more closely related to the Sponweni virus, and not to the Zika virus [7]. Ten years later, in 1964, a report is published of a worker at the East African Virus Research Institute in Entebbe who became infected with ZIKV while working on a series of new ZIKV strains, and his clinical presentation was characterised headache, diffuse pink maculopapular rash, myalgia, fever, and general malaise [8]. Interestingly, the author of the report is the same infected patient.

Outside of Africa, ZIKV was first isolated in 1969, in one of 58 groups of 1,277 *Aedes aegypti* mosquitoes collected from cities and towns on the Malay Peninsula (now Malaysia, Asia) [9]. Then, in 1977 in Indonesia (Asia), human infections of ZIKV infection were described, which were clinically characterised by high fever, malaise, stomach pain, dizziness and anorexia [10]. Notably, apart from direct evidence on ZIKV circulation from previously described human isolates and cases of infection, serological studies conducted in the 1950s suggest that ZIKV had a widespread distribution in both Africa (East, Central, West and South) as in several Asian countries [11]. However, the interpretation of serological results should be made with caution because the cross-reactions were not well characterised at that time [12]. From the first report of ZIKV infection in humans in 1954 (or 1964) through the early 2000s, only a few isolated cases of ZIKV infection disease have been documented. However, the outbreak in Yap State "initiates" the events that would make ZIKV a potential pandemic threat and are briefly described in **Figure 1**. Other publications describe these milestones in more detail [13–26].

The ZIKV is a member of the Flaviviridae family of viruses, which includes small viruses with a positive single-stranded RNA genome (9000–13000 bases) and which in turn is composed of 4 genera (Flavivirus, Hepacivirus [hepatitis C virus], Pegivirus and Pestivirus) [27]. Although transmission through mosquito bites is the

#### **Figure 1.**

*Chronology of ZIKV infection and reported cases. \*ZIKV = Zika virus; GBS = Guillain-Barré syndrome; M-F = Maternal-fetal; PHEIC = Public Health Emergency of International Concern.*

**5**

**4. Diagnosis**

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas*

primary mechanism for the spread of ZIKV, other routes of transmission have been

Vector transmission: Transmission through the bite of infected mosquitoes is the primary mechanism for the spread of ZIKV. The *Aedes aegypti* mosquito is the primary vector for urban transmission of ZIKV throughout the world. At the same time, other Aedes species can act as vectors for ZIKV in specific environments where its abundance is essential (e.g. *A. albopictus* that lives in temperate regions) [29]. Aedes mosquitoes can also transmit dengue and chikungunya viruses.

Non-vector transmission: Non-vector transmission events have been reported including maternal-fetal transmission, sexual transmission, transmission associated with transfusion of blood products or organ transplantation, and laboratory

Possibly the first clinical description of a patient with ZIKV was reported in 1956 [32]. It was a 34-year-old European volunteer who was inoculated subcutaneously with a strain of ZIKV from Nigeria. After an incubation period of 82 hours, he developed a frontal headache associated with mild and short-term fever. On the afternoon of the fifth day, the headache, fever, and malaise increased in severity. It was accompanied by nausea and vertigo (which was attributed to a histamine reaction that responded to a small dose of aspirin). By the seventh day, the patient had fully recovered [32]. The percentage of asymptomatic ZIKV infections is estimated to be 50–80% [16, 33]. Data obtained from the Yap Island seroprevalence study showed that only 19% of those infected had symptoms attributable to ZIKV [34]. However, the retrospective seroprevalence study in French Polynesia showed that, among ZIKV seropositive patients, the percentage of symptomatic infections was 47% in adults [35]. That suggests that the virus strain could influence the proportion of symptomatic ZIKV infections.

For symptomatic infections, the incubation period varies from 3 to 14 days [36], and in most cases, the disease is self-limited [37]. It generally manifests as an itchy rash, mild fever, fatigue, myalgia/arthralgia, conjunctivitis, and headache, with an average duration of 1 week [37, 38]. The clinical characteristics appear to be similar in all age groups, regardless of sex and gestational status. Serious illness requiring hospitalisation is rare [9–48]. As observed, especially in some countries of Latin

The frequency of complications related to ZIKV infection appears to be low, but when they do occur, they are severe and can be fatal [39]. The most commonly reported complications to include complications associated with ZIKV infection during pregnancy such as congenital ZIKV syndrome (CZS), microcephaly, congenital malformations or abnormalities, brain abnormalities (e.g., delayed brain growth), eye disorders, pregnancy, fetal or perinatal death, hearing disorders, cardiovascular damage, neurological complications, intrauterine growth restriction, amniotic fluid abnormalities and epilepsy; neurological complications in adults, such as Guillain-Barré syndrome (https://www.who.int/csr/disease/zika/case-definition/en/); and

Because the clinical manifestations of acute ZIKV infection are nonspecific, the definitive diagnosis is made by molecular and serological methods [43]. However,

America, comorbidities would complicated cases [21–48].

finally death associated with ZIKV infection [40–49].

clinical and epidemiological criteria justify screening tests.

*DOI: http://dx.doi.org/10.5772/intechopen.95537*

proposed that contribute to the epidemic.

exposure [30, 31].

**3. Clinical features**

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas DOI: http://dx.doi.org/10.5772/intechopen.95537*

primary mechanism for the spread of ZIKV, other routes of transmission have been proposed that contribute to the epidemic.

Vector transmission: Transmission through the bite of infected mosquitoes is the primary mechanism for the spread of ZIKV. The *Aedes aegypti* mosquito is the primary vector for urban transmission of ZIKV throughout the world. At the same time, other Aedes species can act as vectors for ZIKV in specific environments where its abundance is essential (e.g. *A. albopictus* that lives in temperate regions) [29]. Aedes mosquitoes can also transmit dengue and chikungunya viruses.

Non-vector transmission: Non-vector transmission events have been reported including maternal-fetal transmission, sexual transmission, transmission associated with transfusion of blood products or organ transplantation, and laboratory exposure [30, 31].

### **3. Clinical features**

*Current Concepts in Zika Research*

author of the report is the same infected patient.

description of the origins of ZIKV is presented elsewhere [4]. Although the initial isolation and characterisation of ZIKV in Uganda's Zika forest are unquestionable, there is some controversy as to which report described the first human ZIKV infection [5]. Several authors suggest that the first isolation in humans was in 1954, and it was a 10-year-old African girl with fever and headache associated with a malaria co-infection [6]. Cross-neutralisation tests with convalescent sera from monkeys infected with different viruses indicated that only ZIKV-infected serum neutralised the virus from the patient's serum, strongly suggesting ZIKV infection. However, a later published report indicated that the virus isolated in West Africa was more closely related to the Sponweni virus, and not to the Zika virus [7]. Ten years later, in 1964, a report is published of a worker at the East African Virus Research Institute in Entebbe who became infected with ZIKV while working on a series of new ZIKV strains, and his clinical presentation was characterised headache, diffuse pink maculopapular rash, myalgia, fever, and general malaise [8]. Interestingly, the

Outside of Africa, ZIKV was first isolated in 1969, in one of 58 groups of 1,277 *Aedes aegypti* mosquitoes collected from cities and towns on the Malay Peninsula (now Malaysia, Asia) [9]. Then, in 1977 in Indonesia (Asia), human infections of ZIKV infection were described, which were clinically characterised by high fever, malaise, stomach pain, dizziness and anorexia [10]. Notably, apart from direct evidence on ZIKV circulation from previously described human isolates and cases of infection, serological studies conducted in the 1950s suggest that ZIKV had a widespread distribution in both Africa (East, Central, West and South) as in several Asian countries [11]. However, the interpretation of serological results should be made with caution because the cross-reactions were not well characterised at that time [12]. From the first report of ZIKV infection in humans in 1954 (or 1964) through the early 2000s, only a few isolated cases of ZIKV infection disease have been documented. However, the outbreak in Yap State "initiates" the events that would make ZIKV a potential pandemic threat and are briefly described in **Figure 1**.

Other publications describe these milestones in more detail [13–26].

The ZIKV is a member of the Flaviviridae family of viruses, which includes small viruses with a positive single-stranded RNA genome (9000–13000 bases) and which in turn is composed of 4 genera (Flavivirus, Hepacivirus [hepatitis C virus], Pegivirus and Pestivirus) [27]. Although transmission through mosquito bites is the

*Chronology of ZIKV infection and reported cases. \*ZIKV = Zika virus; GBS = Guillain-Barré syndrome;* 

*M-F = Maternal-fetal; PHEIC = Public Health Emergency of International Concern.*

**4**

**Figure 1.**

Possibly the first clinical description of a patient with ZIKV was reported in 1956 [32]. It was a 34-year-old European volunteer who was inoculated subcutaneously with a strain of ZIKV from Nigeria. After an incubation period of 82 hours, he developed a frontal headache associated with mild and short-term fever. On the afternoon of the fifth day, the headache, fever, and malaise increased in severity. It was accompanied by nausea and vertigo (which was attributed to a histamine reaction that responded to a small dose of aspirin). By the seventh day, the patient had fully recovered [32]. The percentage of asymptomatic ZIKV infections is estimated to be 50–80% [16, 33]. Data obtained from the Yap Island seroprevalence study showed that only 19% of those infected had symptoms attributable to ZIKV [34]. However, the retrospective seroprevalence study in French Polynesia showed that, among ZIKV seropositive patients, the percentage of symptomatic infections was 47% in adults [35]. That suggests that the virus strain could influence the proportion of symptomatic ZIKV infections.

For symptomatic infections, the incubation period varies from 3 to 14 days [36], and in most cases, the disease is self-limited [37]. It generally manifests as an itchy rash, mild fever, fatigue, myalgia/arthralgia, conjunctivitis, and headache, with an average duration of 1 week [37, 38]. The clinical characteristics appear to be similar in all age groups, regardless of sex and gestational status. Serious illness requiring hospitalisation is rare [9–48]. As observed, especially in some countries of Latin America, comorbidities would complicated cases [21–48].

The frequency of complications related to ZIKV infection appears to be low, but when they do occur, they are severe and can be fatal [39]. The most commonly reported complications to include complications associated with ZIKV infection during pregnancy such as congenital ZIKV syndrome (CZS), microcephaly, congenital malformations or abnormalities, brain abnormalities (e.g., delayed brain growth), eye disorders, pregnancy, fetal or perinatal death, hearing disorders, cardiovascular damage, neurological complications, intrauterine growth restriction, amniotic fluid abnormalities and epilepsy; neurological complications in adults, such as Guillain-Barré syndrome (https://www.who.int/csr/disease/zika/case-definition/en/); and finally death associated with ZIKV infection [40–49].

#### **4. Diagnosis**

Because the clinical manifestations of acute ZIKV infection are nonspecific, the definitive diagnosis is made by molecular and serological methods [43]. However, clinical and epidemiological criteria justify screening tests.

## **4.1 Clinical**

For ZIKV disease, the clinical criteria for the definition of a suspected case according to the Centers for Disease Control and Prevention (CDC, 2016) corresponds to a person with one or more of the following (not explained by another aetiology) [15–44]:

#### **Suspected case:**


## **Epidemiological link criteria:**


\*An online resource is available through the CDC website (https://wwwnc.cdc. gov/travel/page/zika-information) to recognise areas with reported cases of ZIKV infection.

### **4.2 Laboratory**

The diagnostic approach to ZIKV infection may vary depending on the resources available. For patients with suspected ZIKV disease, molecular detection of ZIKV RNA (e.g. nucleic acid amplification tests or NAAT [by RT-PCR]) is the preferred diagnostic method because they can provide confirmed evidence of infection and distinguish the specific virus [45, 46]. However, nucleic acid tests only show the presence of ZIKV RNA but do not necessarily indicate the presence of infectious viruses. On the other hand, serological tests (e.g. IgM antibodies [employing an immunosorbent assay linked to IgM antibody capture enzymes or MAC-ELISA]) or IgG [by plaque reduction neutralisation test or PRNT] against ZIKV are They are used mainly in patients who present after the viral nucleic acid is no longer detectable [20–48], despite the number of false-positive results due to cross-reactivity with other flaviviruses.

The algorithm for recommendations and the interpretation of the results of the dengue virus and ZIKV diagnostic tests are described in **Figure 2**.

**7**

**Figure 3.**

*and modified from CDC.*

**Figure 2.**

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas*

Pregnant women with a clinically compatible disease and possible exposure to ZIKV or dengue virus should be evaluated as soon as possible (**Figure 3**). Evaluation for ZIKV and dengue infection is performed by performing NAAT and IgM antibody testing on serum sample and NAAT on a urine sample. NAATs can be performed on

*Algorithm of recommendations for testing ZIKV and dengue virus for people with the clinically compatible disease and risk for infection with both viruses. \*And risk for infection with both viruses. Adapted and modified from CDC.*

*Algorithm of recommendations for testing ZIKV and dengue virus for pregnant women with the clinically compatible disease and risk for infection with both viruses. \*And risk for infection with both viruses. Adapted* 

*DOI: http://dx.doi.org/10.5772/intechopen.95537*

*4.2.1 Pregnant women*

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas DOI: http://dx.doi.org/10.5772/intechopen.95537*

#### *4.2.1 Pregnant women*

*Current Concepts in Zika Research*

central nervous system).

**Epidemiological link criteria:**

30 days after the onset of symptoms.

conditions for possible local vector transmission.

For ZIKV disease, the clinical criteria for the definition of a suspected case according to the Centers for Disease Control and Prevention (CDC, 2016) corresponds to a person with one or more of the following (not explained by another

• Clinically compatible disease including acute onset of fever (measured or

• Complications of pregnancy (e.g., loss of a fetus, fetus or newborn with congenital microcephaly, congenital intracranial calcifications, structural abnormalities of the brain or eyes, or structural abnormalities related to the

• Recent residence or travel to areas with known ZIKV transmission, sexual

• Receipt of blood, blood products or organ or tissue transplantation within

• Association in time and place with a confirmed or probable case; and

• Probable vector exposure in an area with suitable ecological and seasonal

\*An online resource is available through the CDC website (https://wwwnc.cdc. gov/travel/page/zika-information) to recognise areas with reported cases of ZIKV

The diagnostic approach to ZIKV infection may vary depending on the resources available. For patients with suspected ZIKV disease, molecular detection of ZIKV RNA (e.g. nucleic acid amplification tests or NAAT [by RT-PCR]) is the preferred diagnostic method because they can provide confirmed evidence of infection and distinguish the specific virus [45, 46]. However, nucleic acid tests only show the presence of ZIKV RNA but do not necessarily indicate the presence of infectious viruses. On the other hand, serological tests (e.g. IgM antibodies [employing an immunosorbent assay linked to IgM antibody capture enzymes or MAC-ELISA]) or IgG [by plaque reduction neutralisation test or PRNT] against ZIKV are They are used mainly in patients who present after the viral nucleic acid is no longer detectable [20–48], despite the number of false-positive results due to cross-reactivity

The algorithm for recommendations and the interpretation of the results of the

dengue virus and ZIKV diagnostic tests are described in **Figure 2**.

contact with a confirmed or probable case within the transmission risk window

reported), maculopapular rash, arthralgia or conjunctivitis; or

• Guillain-Barré syndrome or other neurological manifestations; or

**4.1 Clinical**

aetiology) [15–44]: **Suspected case:**

(2 weeks).

infection.

**4.2 Laboratory**

with other flaviviruses.

**6**

Pregnant women with a clinically compatible disease and possible exposure to ZIKV or dengue virus should be evaluated as soon as possible (**Figure 3**). Evaluation for ZIKV and dengue infection is performed by performing NAAT and IgM antibody testing on serum sample and NAAT on a urine sample. NAATs can be performed on

#### **Figure 2.**

*Algorithm of recommendations for testing ZIKV and dengue virus for people with the clinically compatible disease and risk for infection with both viruses. \*And risk for infection with both viruses. Adapted and modified from CDC.*

#### **Figure 3.**

*Algorithm of recommendations for testing ZIKV and dengue virus for pregnant women with the clinically compatible disease and risk for infection with both viruses. \*And risk for infection with both viruses. Adapted and modified from CDC.*

#### *Current Concepts in Zika Research*

plasma, cerebrospinal fluid whole blood, or amniotic fluid; likewise, IgM antibody tests can be performed on plasma, whole blood, or cerebrospinal fluid. Specimens should be collected as soon as possible and within 12 weeks of the onset of symptoms. A positive NAAT result in any sample provides sufficient evidence of a recent infection. However, suppose the NAAT is only positive for ZIKV in a single sample, and the IgM antibody test is negative. In that case, the NAAT should be repeated with fresh RNA from the same sample to rule out false-positive results. If the NAAT is negative, but the IgM antibody test is positive, confirmatory PRNTs should be performed for dengue, ZIKV, and other flaviviruses endemic to the region (**Figure 3**).

For asymptomatic pregnant women without continued risk of possible Zika virus infection, routine screening for ZIKV infection is not recommended. However, the assessment must be considered in terms of risk and through a shared decision-making model. A more detailed review is described elsewhere [15–39].

The algorithm of recommendations and the interpretation of the results of the dengue virus and ZIKV diagnostic tests in pregnant women are described in **Figure 2**.

## **5. Classification of cases**

The definition of suspected cases was previously described. According to the CDC [44], suspected cases should be classified into:


### **6. Differential diagnostics**

Differential diagnosis includes Dengue fever, Chikungunya virus, West Nile virus, Yellow fever, Malaria infection, Leptospirosis, Rubella, infectious Erythema (parvovirus B19 infection), Rocky Mountain spotted fever, Group A streptococcal infection, alphavirus infections, and coronavirus disease 2019 (COVID-19) [15–41]. Also, Mayaro, and the proposed ChikDenMaZika syndrome should be considered in the differential diagnostics [15–41].

#### **7. Treatment of ZIKV disease**

There is no specific treatment for ZIKV infection [15–43], and current treatment recommendations are based on limited evidence [15–44]. Thus, as with other

**9**

underway.

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas*

mosquito-borne flaviviruses, treatment for ZIKV infection is symptomatic and

• Use of analgesics or antipyretics: Acetaminophen 325–1000 mg orally every 4 to 6 hours when necessary (maximum 4000 mg/day). Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) should be avoided until

The management of pregnant women, congenital Zika syndrome, and Guillain-

Significant efforts have been made to develop safe and effective vaccines against ZIKV. International scientific cooperation has resulted in multiple candidate vaccines that are now in various stages of clinical and preclinical development [22–42]. Several vaccines are being developed, including purified inactivated viral particles (PIV), purified virus-like particles (VLPs) and viral subunit proteins, live attenuated vaccines, chimeric vaccines, and viral and non-viral vectors that encode ZIKV structural proteins [20–43]. The most advanced candidate corresponds to a DNA vaccine (VRC5283) developed by the National Institute of Allergy and Infectious Diseases (NIAID) that is being evaluated in phase 2 clinical study (NCT03110770) that seeks to evaluate the safety and tolerability of the vaccine. (Phase A) and the safety and efficacy compared to placebo (Phase B); that is, the safety, immunogenicity, optimal dose of administration are evaluated, and an attempt will be made to determine the ability of the vaccine to prevent the disease caused by ZIKV infection effectively. At the time of this writing, the results published on www.clinical.trials. gov had not completed the quality control review process. However, its results have been promising [20–44]. However, significant challenges remain in the development of vaccines for ZIKV. Difficulties include the heterogeneity of the incidence of ZIKV infection, difficulties in financing, regulation and authorisation of vaccines, which in turn has limited the conduct of phase 2 and 3 clinical trials and prompted international organisations to consider approaches alternative, as models of infection by controlled human exposure [25–46]. Vaccine development for ZIKV is

Although a large number of existing drugs and novel chemical compounds have been postulated as possible interventions against ZIKV [15–46] and some have shown activity against ZIKV in animal and experimental models [15–48], none have yet demonstrated safety and efficacy in clinical trials [15-49]. A safety and tolerability trial for the monoclonal antibody against ZIKV called Tyzivumab (NCT03443830), and another for a polyclonal antibody against ZIKV (NCT03624946) have completed their recruitment phase and are currently in progress. At the moment, there are no treatments approved by government agencies for ZIKV, and there is no information to suggest that there are treatments in advanced

dengue virus infection has been ruled out, to reduce the risk of

• Calamine lotion can be used for the maculopapular rash.

Barré syndrome is described in detail elsewhere [15–41].

*DOI: http://dx.doi.org/10.5772/intechopen.95537*

**7.1 Symptomatic and supportive treatment**

supportive and includes [15–45]:

• Rest and adequate hydration.

stages of development [20–40].

**8. Advances in vaccination**

bleeding.

\*

mosquito-borne flaviviruses, treatment for ZIKV infection is symptomatic and supportive and includes [15–45]:

## **7.1 Symptomatic and supportive treatment**

• Rest and adequate hydration.

*Current Concepts in Zika Research*

**5. Classification of cases**

• Clinical criteria for ZIKV disease; and

• Laboratory evidence of recent ZIKV or

○ Positive test of IgM antibodies against ZIKV in serum or CSF; and

○ Positive neutralising antibody titers (e.g. PRNT) against ZIKV, dengue or other flaviviruses endemic to the region where

○ Negative test for IgM antibodies against dengue virus, and no neutralising antibody

 *The criteria for WHO/PAHO are similar (www.paho.org).*

• Epidemiological link; and

flavivirus infection by:

**6. Differential diagnostics**

the exposure occurred; or

tests were performed.

in the differential diagnostics [15–41].

**7. Treatment of ZIKV disease**

CDC [44], suspected cases should be classified into:

**Probable case Confirmed case**

plasma, cerebrospinal fluid whole blood, or amniotic fluid; likewise, IgM antibody tests can be performed on plasma, whole blood, or cerebrospinal fluid. Specimens should be collected as soon as possible and within 12 weeks of the onset of symptoms. A positive NAAT result in any sample provides sufficient evidence of a recent infection. However, suppose the NAAT is only positive for ZIKV in a single sample, and the IgM antibody test is negative. In that case, the NAAT should be repeated with fresh RNA from the same sample to rule out false-positive results. If the NAAT is negative, but the IgM antibody test is positive, confirmatory PRNTs should be performed for dengue, ZIKV, and other flaviviruses endemic to the region (**Figure 3**). For asymptomatic pregnant women without continued risk of possible Zika

virus infection, routine screening for ZIKV infection is not recommended.

However, the assessment must be considered in terms of risk and through a shared decision-making model. A more detailed review is described elsewhere [15–39]. The algorithm of recommendations and the interpretation of the results of the dengue virus and ZIKV diagnostic tests in pregnant women are described in **Figure 2**.

The definition of suspected cases was previously described. According to the

• Clinical criteria for ZIKV disease; and

ruses in the region of exposure.

saliva); or

• Laboratory evidence of recent ZIKV infection by: ○ Detection of ZIKV by culture, viral antigen or viral RNA in serum, CSF, tissue or another sample (e.g. amniotic fluid, urine, semen,

○ Positive serum or CSF IgM antibody test against ZIKV with positive ZIKV neutralising antibody titers (≥10) and negative neutralising antibody titers against dengue or other endemic flavivi-

Differential diagnosis includes Dengue fever, Chikungunya virus, West Nile virus, Yellow fever, Malaria infection, Leptospirosis, Rubella, infectious Erythema (parvovirus B19 infection), Rocky Mountain spotted fever, Group A streptococcal infection, alphavirus infections, and coronavirus disease 2019 (COVID-19) [15–41]. Also, Mayaro, and the proposed ChikDenMaZika syndrome should be considered

There is no specific treatment for ZIKV infection [15–43], and current treatment recommendations are based on limited evidence [15–44]. Thus, as with other

**8**

*\**


\* The management of pregnant women, congenital Zika syndrome, and Guillain-Barré syndrome is described in detail elsewhere [15–41].

Although a large number of existing drugs and novel chemical compounds have been postulated as possible interventions against ZIKV [15–46] and some have shown activity against ZIKV in animal and experimental models [15–48], none have yet demonstrated safety and efficacy in clinical trials [15-49]. A safety and tolerability trial for the monoclonal antibody against ZIKV called Tyzivumab (NCT03443830), and another for a polyclonal antibody against ZIKV (NCT03624946) have completed their recruitment phase and are currently in progress. At the moment, there are no treatments approved by government agencies for ZIKV, and there is no information to suggest that there are treatments in advanced stages of development [20–40].

## **8. Advances in vaccination**

Significant efforts have been made to develop safe and effective vaccines against ZIKV. International scientific cooperation has resulted in multiple candidate vaccines that are now in various stages of clinical and preclinical development [22–42]. Several vaccines are being developed, including purified inactivated viral particles (PIV), purified virus-like particles (VLPs) and viral subunit proteins, live attenuated vaccines, chimeric vaccines, and viral and non-viral vectors that encode ZIKV structural proteins [20–43]. The most advanced candidate corresponds to a DNA vaccine (VRC5283) developed by the National Institute of Allergy and Infectious Diseases (NIAID) that is being evaluated in phase 2 clinical study (NCT03110770) that seeks to evaluate the safety and tolerability of the vaccine. (Phase A) and the safety and efficacy compared to placebo (Phase B); that is, the safety, immunogenicity, optimal dose of administration are evaluated, and an attempt will be made to determine the ability of the vaccine to prevent the disease caused by ZIKV infection effectively. At the time of this writing, the results published on www.clinical.trials. gov had not completed the quality control review process. However, its results have been promising [20–44]. However, significant challenges remain in the development of vaccines for ZIKV. Difficulties include the heterogeneity of the incidence of ZIKV infection, difficulties in financing, regulation and authorisation of vaccines, which in turn has limited the conduct of phase 2 and 3 clinical trials and prompted international organisations to consider approaches alternative, as models of infection by controlled human exposure [25–46]. Vaccine development for ZIKV is underway.

## **9. Conclusions**

The impact and burden of Zika in the Americas region have multiple implications. Clinical and epidemiological research has been vital in the understanding and developing of knowledge for the management and prevention of this emerging arboviral disease [30–48]. Still, many challenges exist, including the developing of an effective vaccine, still under developing.

## **Author details**

Carlos Andrés Rosero-Oviedo1 , D. Katterine Bonilla-Aldana2,3, Jaime A. Cardona-Ospina1,2,4, Wilmer E. Villamil-Gómez5,6 and Alfonso J. Rodriguez-Morales1,2,3,4,5\*

1 Grupo de Investigación Biomedicina, Faculty of Medicine, Fundación Universitaria Autónoma de las Américas, Pereira, Risaralda, Colombia

2 Public Health and Infection Research Group, Faculty of Health Sciences, Universidad Tecnológica de Pereira, Pereira, Colombia

3 Semillero de Investigación en Zoonosis, Grupo BIOECOS, Fundación Universitaria Autónoma de las Américas, Pereira, Colombia

4 Emerging Infectious Diseases and Tropical Medicine Research Group, Instituto para la Investigación en Ciencias Biomédicas-Sci-Help, Pereira, Colombia

5 Infectious Diseases and Infection Control Research Group, Hospital Universitario de Sincelejo, Sincelejo, Sucre, Colombia

6 Programa del Doctorado de Medicina Tropical, SUE Caribe, Universidad del Atlántico, Barranquilla, Colombia

\*Address all correspondence to: ajrodriguezmmd@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**11**

mm6503e2

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas*

Colombia. Emerging infectious diseases, 22(5), 927-929. https://doi.org/10.3201/

[7] Sarmiento-Ospina, A., Vásquez-Serna, H., Jimenez-Canizales, C. E., Villamil-Gómez, W. E., & Rodriguez-Morales, A. J. (2016). Zika virus associated deaths in Colombia. The Lancet. Infectious diseases, 16(5), 523-524. https://doi.org/10.1016/

[8] WHO statement on the first meeting of the International Health Regulations

Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. (2016). Retrieved November 23, 2020, from World Health Organization (WHO) website: https://www.who.int/news/ item/01-02-2016-who-statement-onthe-first-meeting-of-the-internationalhealth-regulations-(2005)-(ihr-2005) emergency-committee-on-zika-virus-and-observed-increase-in-neurologicaldisorders-and-neonatal-malformations

(2005) (IHR 2005) Emergency

[9] Zika situation report. (2016). Retrieveded November 23, 2020, from World Health Organization (WHO) website: https://www. who.int/emergencies/zika-virus/ situation-report/31-march-2016/en/

[10] Zika epidemiology update. (2019). Retrieveded November 23, 2020, from World Health Organization (WHO) website: https://www.who. int/emergencies/diseases/zika/ epidemiology-update/en/

[11] Simmonds, P., Becher, P., Bukh, J., Gould, E. A., Meyers, G., Monath, T., Muerhoff, S., Pletnev, A., Rico-Hesse, R., Smith, D. B., Stapleton, J. T., & Ictv Report Consortium (2017). ICTV Virus Taxonomy Profile: Flaviviridae. The Journal of general virology, 98(1), 2-3. https://doi.org/10.1099/jgv.0.000672

S1473-3099(16)30006-8

eid2205.160023

*DOI: http://dx.doi.org/10.5772/intechopen.95537*

[1] DICK, G. W. (1953). Epidemiological

notes on some viruses isolated in Uganda; Yellow fever, Rift Valley fever, Bwamba fever, West Nile, Mengo, Semliki forest, Bunyamwera, Ntaya, Uganda S and Zika viruses. Transactions of the Royal Society of Tropical Medicine and Hygiene, Jan;47(1), 13-48. https://doi. org/10.1016/0035-9203(53)90021-2

**References**

[2] DICK, G. W., KITCHEN, S. F., & HADDOW, A. J. (1952). Zika virus. I. Isolations and serological specificity. Transactions of the Royal Society of Tropical Medicine and Hygiene, Sep;46(5), 509-20. https://doi. org/10.1016/0035-9203(52)90042-4

[3] DICK, G. W. (1952). Zika virus. II. Pathogenicity and physical properties. Transactions of the Royal Society of Tropical Medicine and Hygiene, Sep;46(5), 521-34. https://doi. org/10.1016/0035-9203(52)90043-6

[4] Campos, G. S., Bandeira, A. C., & Sardi, S. I. (2015). Zika Virus Outbreak, Bahia, Brazil. Emerging Infectious Diseases, Oct;21(10), 1885-1886. https://

doi.org/10.3201/eid2110.150847

[5] Schuler-Faccini, L., Ribeiro, E. M., Feitosa, I. M., Horovitz, D. D., Cavalcanti, D. P., Pessoa, A., Doriqui, M. J., Neri, J. I., Neto, J. M., Wanderley, H. Y., Cernach, M., El-Husny, A. S., Pone, M. V., Serao, C. L., Sanseverino, M. T., & Brazilian Medical Genetics Society–Zika Embryopathy Task Force (2016). Possible Association Between Zika Virus Infection and Microcephaly -

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[6] Camacho, E., Paternina-Gomez, M., Blanco, P. J., Osorio, J. E., & Aliota, M. T. (2016). Detection of Autochthonous Zika Virus Transmission in Sincelejo,

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas DOI: http://dx.doi.org/10.5772/intechopen.95537*

## **References**

*Current Concepts in Zika Research*

**9. Conclusions**

**Author details**

Carlos Andrés Rosero-Oviedo1

and Alfonso J. Rodriguez-Morales1,2,3,4,5\*

an effective vaccine, still under developing.

Jaime A. Cardona-Ospina1,2,4, Wilmer E. Villamil-Gómez5,6

Universidad Tecnológica de Pereira, Pereira, Colombia

Autónoma de las Américas, Pereira, Colombia

de Sincelejo, Sincelejo, Sucre, Colombia

provided the original work is properly cited.

Atlántico, Barranquilla, Colombia

1 Grupo de Investigación Biomedicina, Faculty of Medicine, Fundación Universitaria Autónoma de las Américas, Pereira, Risaralda, Colombia

2 Public Health and Infection Research Group, Faculty of Health Sciences,

para la Investigación en Ciencias Biomédicas-Sci-Help, Pereira, Colombia

3 Semillero de Investigación en Zoonosis, Grupo BIOECOS, Fundación Universitaria

4 Emerging Infectious Diseases and Tropical Medicine Research Group, Instituto

5 Infectious Diseases and Infection Control Research Group, Hospital Universitario

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

6 Programa del Doctorado de Medicina Tropical, SUE Caribe, Universidad del

\*Address all correspondence to: ajrodriguezmmd@gmail.com

, D. Katterine Bonilla-Aldana2,3,

The impact and burden of Zika in the Americas region have multiple implications. Clinical and epidemiological research has been vital in the understanding and developing of knowledge for the management and prevention of this emerging arboviral disease [30–48]. Still, many challenges exist, including the developing of

**10**

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[2] DICK, G. W., KITCHEN, S. F., & HADDOW, A. J. (1952). Zika virus. I. Isolations and serological specificity. Transactions of the Royal Society of Tropical Medicine and Hygiene, Sep;46(5), 509-20. https://doi. org/10.1016/0035-9203(52)90042-4

[3] DICK, G. W. (1952). Zika virus. II. Pathogenicity and physical properties. Transactions of the Royal Society of Tropical Medicine and Hygiene, Sep;46(5), 521-34. https://doi. org/10.1016/0035-9203(52)90043-6

[4] Campos, G. S., Bandeira, A. C., & Sardi, S. I. (2015). Zika Virus Outbreak, Bahia, Brazil. Emerging Infectious Diseases, Oct;21(10), 1885-1886. https:// doi.org/10.3201/eid2110.150847

[5] Schuler-Faccini, L., Ribeiro, E. M., Feitosa, I. M., Horovitz, D. D., Cavalcanti, D. P., Pessoa, A., Doriqui, M. J., Neri, J. I., Neto, J. M., Wanderley, H. Y., Cernach, M., El-Husny, A. S., Pone, M. V., Serao, C. L., Sanseverino, M. T., & Brazilian Medical Genetics Society–Zika Embryopathy Task Force (2016). Possible Association Between Zika Virus Infection and Microcephaly - Brazil, 2015. MMWR. Morbidity and mortality weekly report, 65(3), 59-62. https://doi.org/10.15585/mmwr. mm6503e2

[6] Camacho, E., Paternina-Gomez, M., Blanco, P. J., Osorio, J. E., & Aliota, M. T. (2016). Detection of Autochthonous Zika Virus Transmission in Sincelejo,

Colombia. Emerging infectious diseases, 22(5), 927-929. https://doi.org/10.3201/ eid2205.160023

[7] Sarmiento-Ospina, A., Vásquez-Serna, H., Jimenez-Canizales, C. E., Villamil-Gómez, W. E., & Rodriguez-Morales, A. J. (2016). Zika virus associated deaths in Colombia. The Lancet. Infectious diseases, 16(5), 523-524. https://doi.org/10.1016/ S1473-3099(16)30006-8

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[12] Kuno, G., Chang, G. J., Tsuchiya, K. R., Karabatsos, N., & Cropp, C. B. (1998). Phylogeny of the genus Flavivirus. Journal of virology, 72(1), 73-83. https://doi.org/10.1128/ JVI.72.1.73-83.1998

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[14] Gregory, C. J., Oduyebo, T., Brault, A. C., Brooks, J. T., Chung, K. W., Hills, S., Kuehnert, M. J., Mead, P., Meaney-Delman, D., Rabe, I., Staples, E., & Petersen, L. R. (2017). Modes of Transmission of Zika Virus. The Journal of infectious diseases, 216(suppl\_10), S875–S883. https://doi.org/10.1093/ infdis/jix396

[15] Grischott, F., Puhan, M., Hatz, C., & Schlagenhauf, P. (2016). Non-vectorborne transmission of Zika virus: A systematic review. Travel medicine and infectious disease, 14(4), 313-330.

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[18] Duffy, M. R., Chen, T. H., Hancock, W. T., Powers, A. M., Kool, J. L., Lanciotti, R. S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., Guillaumot, L., Griggs, A., Bel, M., Lambert, A. J., Laven, J., Kosoy, O., Panella, A., Biggerstaff, B. J., Fischer, M., & Hayes, E. B. (2009). Zika virus

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[24] Ximenes, R., Ramsay, L. C., Miranda, R. N., Morris, S. K., Murphy, K., Sander, B., & RADAM-LAC Research Team (2019). Health outcomes associated with Zika virus infection in humans: a systematic review of systematic reviews. BMJ open, 9(11), e032275. https://doi.org/10.1136/ bmjopen-2019-032275

**13**

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detail/zika-virus

handle/10665.2/50667

bmj.com/topics/en-us/1302

case-definition/en/

*Introductory Chapter: Zika 2015-2020 - Knowledge and Experience in the Americas DOI: http://dx.doi.org/10.5772/intechopen.95537*

[25] Identification and management of Guillain-Barré syndrome in the context of Zika virus Interim guidance. (2016). Retrieved November 23, 2020, from World Health Organization (WHO) website: https://www.who. int/csr/resources/publications/zika/ guillain-barre-syndrome/en/

*Current Concepts in Zika Research*

JVI.72.1.73-83.1998

s41559-019-0836-z

infdis/jix396

[12] Kuno, G., Chang, G. J., Tsuchiya, K. R., Karabatsos, N., & Cropp, C. B. (1998). Phylogeny of the genus Flavivirus. Journal of virology, 72(1), 73-83. https://doi.org/10.1128/

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2014-2015. Emerging infectious diseases, 23(4), 669-672. https://doi.

[20] Krow-Lucal, E. R., Biggerstaff, B. J., & Staples, J. E. (2017). Estimated Incubation Period for Zika Virus Disease. Emerging infectious diseases, 23(5), 841-845. https://doi.org/10.3201/

[21] Musso, D., & Gubler, D. J. (2016). Zika Virus. Clinical microbiology

org/10.3201/eid2304.161549

eid2305.161715

reviews, 29(3), 487-524.

[22] Plourde, A. R., & Bloch, E. M. (2016). A Literature Review of Zika Virus. Emerging infectious diseases, 22(7), 1185-1192. https://doi.

org/10.3201/eid2207.151990

[23] Cardona-Ospina, J. A., Henao-SanMartin, V., Acevedo-Mendoza, W. F., Nasner-Posso, K. M., Martínez-Pulgarín, D. F., Restrepo-López, A., Valencia-Gallego, V., Collins, M. H., & Rodriguez-Morales, A. J. (2019). Fatal Zika virus infection in the Americas: A systematic review. International journal of infectious diseases, 88, 49-59. https:// doi.org/10.1016/j.ijid.2019.08.033

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bmjopen-2019-032275

Research Team (2019). Health outcomes associated with Zika virus infection in humans: a systematic review of systematic reviews. BMJ open, 9(11), e032275. https://doi.org/10.1136/

[13] Gutiérrez-Bugallo, G., Piedra, L. A., Rodriguez, M., Bisset, J. A., Lourençode-Oliveira, R., Weaver, S. C., Vasilakis, N., & Vega-Rúa, A. (2019). Vectorborne transmission and evolution of Zika virus. Nature ecology & evolution, 3(4), 561-569. https://doi.org/10.1038/

[14] Gregory, C. J., Oduyebo, T., Brault, A. C., Brooks, J. T., Chung, K. W., Hills, S., Kuehnert, M. J., Mead, P., Meaney-Delman, D., Rabe, I., Staples, E., & Petersen, L. R. (2017). Modes of Transmission of Zika Virus. The Journal of infectious diseases, 216(suppl\_10), S875–S883. https://doi.org/10.1093/

[15] Grischott, F., Puhan, M., Hatz, C., & Schlagenhauf, P. (2016). Non-vectorborne transmission of Zika virus: A systematic review. Travel medicine and infectious disease, 14(4), 313-330.

[16] BEARCROFT W. G. (1956). Zika virus infection experimentally induced in a human volunteer. Transactions of the Royal Society of Tropical Medicine

[17] Baud, D., Gubler, D. J., Schaub, B., Lanteri, M. C., & Musso, D. (2017). An update on Zika virus infection. Lancet (London, England), 390(10107), 2099-2109. https://doi.org/10.1016/

and Hygiene, 50(5), 442-448.

S0140-6736(17)31450-2

[18] Duffy, M. R., Chen, T. H., Hancock, W. T., Powers, A. M., Kool, J. L., Lanciotti, R. S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., Guillaumot, L., Griggs, A., Bel, M., Lambert, A. J., Laven, J., Kosoy, O., Panella, A., Biggerstaff, B. J., Fischer, M., & Hayes, E. B. (2009). Zika virus

**12**

[26] Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Velez, J. O., Lambert, A. J., Johnson, A. J., Stanfield, S. M., & Duffy, M. R. (2008). Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerging infectious diseases, 14(8), 1232-1239.

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[29] Clinical Evaluation & Disease. (2019). Retrieved November 23, 2020, from Centers for Disease Control and Prevention (CDC) website: https://www.cdc.gov/zika/ hc-providers/preparing-for-zika/ clinicalevaluationdisease.html

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[31] Epidemiological Update, Zika virus infection. (2015). Recuperado 23 de noviembre de 2020, de Pan American Health Organization (PAHO) website: https://iris.paho.org/ handle/10665.2/50667

[32] LaBeaud, A. D. (2020). Zika virus infection: An overview.

Retrieved November 23, 2020, from UpToDate website: https:// www.uptodate.com/contents/ zika-virus-infection-an-overview

[33] Barrows, N. J., Campos, R. K., Powell, S. T., Prasanth, K. R., Schott-Lerner, G., Soto-Acosta, R., Galarza-Muñoz, G., McGrath, E. L., Urrabaz-Garza, R., Gao, J., Wu, P., Menon, R., Saade, G., Fernandez-Salas, I., Rossi, S. L., Vasilakis, N., Routh, A., Bradrick, S. S., & Garcia-Blanco, M. A. (2016). A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell host & microbe, 20(2), 259-270.

[34] Bernatchez, J. A., Tran, L. T., Li, J., Luan, Y., Siqueira-Neto, J. L., & Li, R. (2020). Drugs for the Treatment of Zika Virus Infection. Journal of medicinal chemistry, 63(2), 470-489.

[35] Gorshkov, K., Shiryaev, S. A., Fertel, S., Lin, Y. W., Huang, C. T., Pinto, A., Farhy, C., Strongin, A. Y., Zheng, W., & Terskikh, A. V. (2019). Zika Virus: Origins, Pathological Action, and Treatment Strategies. Frontiers in microbiology, 9, 3252.

[36] G Viveiros Rosa, S., Fierro, I. M., & C Santos, W. (2020). Repositioning and investigational drugs for Zika virus infection treatment: a patent review. Expert opinion on therapeutic patents, 30(11), 847-862. https://doi.org/10.1080 /13543776.2020.1811854

[37] Zika Virus Response Updates from FDA. (2020). Retrieved November 23, 2020, from United States Food and Drug Administration (FDA) website: https:// www.fda.gov/emergency-preparednessand-response/mcm-issues/ zika-virus-response-updates-fda

[38] Abbink, P., Stephenson, K. E., & Barouch, D. H. (2018). Zika virus vaccines. Nature reviews. Microbiology, 16(10), 594-600.

[39] Pattnaik, A., Sahoo, B. R., & Pattnaik, A. K. (2020). Current Status of Zika Virus Vaccines: Successes and Challenges. Vaccines, 8(2), 266.

[40] Poland, G. A., Kennedy, R. B., Ovsyannikova, I. G., Palacios, R., Ho, P. L., & Kalil, J. (2018). Development of vaccines against Zika virus. The Lancet. Infectious diseases, 18(7), e211–e219.

[41] Gaudinski, M. R., Houser, K. V., Morabito, K. M., Hu, Z., Yamshchikov, G., Rothwell, R. S., Berkowitz, N., Mendoza, F., Saunders, J. G., Novik, L., Hendel, C. S., Holman, L. A., Gordon, I. J., Cox, J. H., Edupuganti, S., McArthur, M. A., Rouphael, N. G., Lyke, K. E., Cummings, G. E., Sitar, S., … VRC 320 study teams (2018). Safety, tolerability, and immunogenicity of two Zika virus DNA vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials. Lancet (London, England), 391(10120), 552-562.

[42] Wilder-Smith, A., Vannice, K., Durbin, A., Hombach, J., Thomas, S. J., Thevarjan, I., & Simmons, C. P. (2018). Zika vaccines and therapeutics: landscape analysis and challenges ahead. BMC medicine, 16(1), 84.

[43] Sharp TM, Fischer M, Muñoz-Jordán JL, Paz-Bailey G, Staples JE, Gregory CJ, Waterman SH. Dengue and Zika Virus Diagnostic Testing for Patients with a Clinically Compatible Illness and Risk for Infection with Both Viruses. MMWR Recomm Rep. 2019 Jun 14;68(1):1-10. doi: 10.15585/mmwr.rr6801a1.

[44] Rodriguez-Morales AJ, Villamil-Gómez WE, Franco-Paredes C. The arboviral burden of disease caused by co-circulation and co-infection of dengue, chikungunya and Zika in the Americas. Travel Med Infect Dis. 2016 May-Jun;14(3):177-179.

[45] Villamil-Gómez WE, Mendoza-Guete A, Villalobos E, González-Arismendy E, Uribe-García AM,

Castellanos JE, Rodríguez-Morales AJ. Diagnosis, management and follow-up of pregnant women with Zika virus infection: A preliminary report of the ZIKERNCOL cohort study on Sincelejo, Colombia. Travel Med Infect Dis. 2016 Mar-Apr;14(2):155-158.

[46] Arzuza-Ortega L, Polo A, Pérez-Tatis G, López-García H, Parra E, Pardo-Herrera LC, Rico-Turca AM, Villamil-Gómez W, Rodríguez-Morales AJ. Fatal Sickle Cell Disease and Zika Virus Infection in Girl from Colombia.Emerg Infect Dis 2016 May; 22(5):925-927.

[47] Villamil-Gomez WE, Sánchez-Herrera ÁR, Hernandez H, Hernández-Iriarte J, Díaz-Ricardo K, Castellanos J, de Jesús Villamil-Macareno W, Rodriguez-Morales AJ. Guillain-Barré syndrome during the Zika virus outbreak in Sucre, Colombia, 2016. Travel Med Infect Dis. 2017;16:62-63.

[48] Paniz-Mondolfi AE, Rodriguez-Morales AJ, Blohm G, Marquez M, Villamil-Gomez WE. ChikDenMaZika Syndrome: the challenge of diagnosing arboviral infections in the midst of concurrent epidemics. Ann Clin Microbiol Antimicrob. 2016 Jul 22; 15:42.

[49] Oduyebo T, Polen KD, Walke HT, Reagan-Steiner S, Lathrop E, Rabe IB, Kuhnert-Tallman WL, Martin SW, Walker AT, Gregory CJ, Ades EW, Carroll DS, Rivera M, Perez-Padilla J, Gould C, Nemhauser JB, Ben Beard C, Harcourt JL, Viens L, Johansson M, Ellington SR, Petersen E, Smith LA, Reichard J, Munoz-Jordan J, Beach MJ, Rose DA, Barzilay E, Noonan-Smith M, Jamieson DJ, Zaki SR, Petersen LR, Honein MA, Meaney-Delman D. Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus Exposure - United States (Including U.S. Territories), July 2017. MMWR Morb Mortal Wkly Rep. 2017 Jul 28;66(29):781- 793. doi: 10.15585/mmwr.mm6629e1.

**15**

**Chapter 2**

*Diana Dimitrova*

Zika virus infection is presented.

**1. Introduction**

conceptual idea, nano-biotechnology

**Abstract**

Risk Management of Zika in

(The Conceptual Idea)

Context of Medical Provision and

Opportunities for Risk Reduction

in Favor of Disaster Medicine: New

There is a need for timely medical care to the population for the risk management of Zika nowadays. Although scientists determine the widespread nature of the worldwide outbreak of Zika virus infection, it seems clear that there is a real need for outside help to deal with this disease. The Zika disease affects predominantly negatively the fetus in pregnant women, but cases of severe clinical manifestations are also reported among adults. Irrespective of age, it is known to affect the nervous system in humans. The vector causes epidemiological data to expand its area of expertise. In this light of expression, specialists define and attribute to this disease the type and significance of a worldwide disaster management. This requires an in-depth study and analysis of risk factors and their management as a fundamental approach for their prevention and for the benefit of disaster medicine. Reducing the risk with existing traditional tools and methods is not enough to meet the growing needs of people and territories at risk of Zika infection. New strategy approaches and technologies are being sought, and new risk reduction (RR) options are being interpreted. A framework for an innovative conceptual idea based on nano-biotechnology for risk reduction and prevention for

**Keywords:** Zika, risk management, risk reduction, prevention measures (with SWOT analysis), medical provision of the population, disaster medicine, innovative

The World Health Organization (WHO) has declared the Zika infection an international threat to public health since the beginning of February 2016 [1–3]. The definition, classification, and analysis of the risk factors of the evolution and spread of Zika virus infection require the proper application in time and space of known preventive measures, as well as measures for the protection and medical provision of the population. The real challenge nowadays is to meet the growing demands of society, expanding the opportunities for highly effective risk reduction (RR) and incorporating new technological and strategic solutions to achieve rapid results.

## **Chapter 2**

*Current Concepts in Zika Research*

[39] Pattnaik, A., Sahoo, B. R., & Pattnaik, A. K. (2020). Current Status of Zika Virus Vaccines: Successes and Challenges. Vaccines, 8(2), 266.

Castellanos JE, Rodríguez-Morales AJ. Diagnosis, management and follow-up of pregnant women with Zika virus infection: A preliminary report of the ZIKERNCOL cohort study on Sincelejo, Colombia. Travel Med Infect Dis. 2016

[46] Arzuza-Ortega L, Polo A, Pérez-Tatis G, López-García H, Parra E, Pardo-Herrera LC, Rico-Turca AM, Villamil-Gómez W, Rodríguez-Morales AJ. Fatal Sickle Cell Disease and Zika Virus Infection in Girl from Colombia.Emerg Infect Dis 2016 May; 22(5):925-927.

[47] Villamil-Gomez WE, Sánchez-Herrera ÁR, Hernandez H, Hernández-Iriarte J, Díaz-Ricardo K, Castellanos J, de Jesús Villamil-Macareno W, Rodriguez-Morales AJ. Guillain-Barré syndrome during the Zika virus outbreak in Sucre, Colombia, 2016. Travel Med Infect Dis. 2017;16:62-63.

[48] Paniz-Mondolfi AE,

Rodriguez-Morales AJ, Blohm G, Marquez M, Villamil-Gomez WE. ChikDenMaZika Syndrome: the challenge of diagnosing arboviral infections in the midst of concurrent epidemics. Ann Clin Microbiol Antimicrob. 2016 Jul 22; 15:42.

[49] Oduyebo T, Polen KD, Walke HT, Reagan-Steiner S, Lathrop E, Rabe IB, Kuhnert-Tallman WL, Martin SW, Walker AT, Gregory CJ, Ades EW, Carroll DS, Rivera M, Perez-Padilla J, Gould C, Nemhauser JB, Ben Beard C, Harcourt JL, Viens L, Johansson M, Ellington SR, Petersen E, Smith LA, Reichard J, Munoz-Jordan J, Beach MJ, Rose DA, Barzilay E, Noonan-Smith M, Jamieson DJ, Zaki SR, Petersen LR, Honein MA, Meaney-Delman D. Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus Exposure - United States (Including U.S.

Territories), July 2017. MMWR Morb Mortal Wkly Rep. 2017 Jul 28;66(29):781- 793. doi: 10.15585/mmwr.mm6629e1.

Mar-Apr;14(2):155-158.

[40] Poland, G. A., Kennedy, R. B., Ovsyannikova, I. G., Palacios, R., Ho, P. L., & Kalil, J. (2018). Development of vaccines against Zika virus. The Lancet. Infectious diseases, 18(7), e211–e219.

[41] Gaudinski, M. R., Houser, K. V., Morabito, K. M., Hu, Z., Yamshchikov, G., Rothwell, R. S., Berkowitz, N., Mendoza, F., Saunders, J. G., Novik, L., Hendel, C. S., Holman, L. A., Gordon, I. J., Cox, J. H., Edupuganti, S., McArthur, M. A., Rouphael, N. G., Lyke, K. E., Cummings, G. E., Sitar, S., … VRC 320 study teams (2018). Safety, tolerability, and immunogenicity of two Zika virus DNA vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials. Lancet (London,

England), 391(10120), 552-562.

BMC medicine, 16(1), 84.

[43] Sharp TM, Fischer M, Muñoz-Jordán JL, Paz-Bailey G, Staples JE, Gregory CJ, Waterman SH. Dengue and Zika Virus Diagnostic Testing for Patients with a Clinically Compatible Illness and Risk for Infection with Both Viruses. MMWR Recomm Rep. 2019 Jun 14;68(1):1-10.

doi: 10.15585/mmwr.rr6801a1.

Villamil-Gómez WE, Franco-Paredes C. The arboviral burden of disease caused by co-circulation and co-infection of dengue, chikungunya and Zika in the Americas. Travel Med Infect Dis. 2016

[45] Villamil-Gómez WE, Mendoza-Guete A, Villalobos E, González-Arismendy E, Uribe-García AM,

[44] Rodriguez-Morales AJ,

May-Jun;14(3):177-179.

[42] Wilder-Smith, A., Vannice, K., Durbin, A., Hombach, J., Thomas, S. J., Thevarjan, I., & Simmons, C. P. (2018). Zika vaccines and therapeutics: landscape analysis and challenges ahead.

**14**

## Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine: New Opportunities for Risk Reduction (The Conceptual Idea)

*Diana Dimitrova*

## **Abstract**

There is a need for timely medical care to the population for the risk management of Zika nowadays. Although scientists determine the widespread nature of the worldwide outbreak of Zika virus infection, it seems clear that there is a real need for outside help to deal with this disease. The Zika disease affects predominantly negatively the fetus in pregnant women, but cases of severe clinical manifestations are also reported among adults. Irrespective of age, it is known to affect the nervous system in humans. The vector causes epidemiological data to expand its area of expertise. In this light of expression, specialists define and attribute to this disease the type and significance of a worldwide disaster management. This requires an in-depth study and analysis of risk factors and their management as a fundamental approach for their prevention and for the benefit of disaster medicine. Reducing the risk with existing traditional tools and methods is not enough to meet the growing needs of people and territories at risk of Zika infection. New strategy approaches and technologies are being sought, and new risk reduction (RR) options are being interpreted. A framework for an innovative conceptual idea based on nano-biotechnology for risk reduction and prevention for Zika virus infection is presented.

**Keywords:** Zika, risk management, risk reduction, prevention measures (with SWOT analysis), medical provision of the population, disaster medicine, innovative conceptual idea, nano-biotechnology

## **1. Introduction**

The World Health Organization (WHO) has declared the Zika infection an international threat to public health since the beginning of February 2016 [1–3]. The definition, classification, and analysis of the risk factors of the evolution and spread of Zika virus infection require the proper application in time and space of known preventive measures, as well as measures for the protection and medical provision of the population. The real challenge nowadays is to meet the growing demands of society, expanding the opportunities for highly effective risk reduction (RR) and incorporating new technological and strategic solutions to achieve rapid results.

## **2. Data and analyses of Zika in favor of risk reduction conception**

### **2.1 Epidemiology and the spread of Zika virus: facts and analyses**

It is a well-known fact that the Zika virus belongs to the *Flaviviridae* family of the genus *Flavivirus* [1]. The data indicate that the Zika virus was isolated in 1947 from Macaque monkeys in Uganda in the Uganda Forest to which it is named [1, 4, 5]. Retrospective analysis shows that the Zika virus was first studied by scientists at the Yellow Fever Institute (founded in 1936). Later, in 1948, the Zika virus was isolated for the second time in the tiger mosquito. Scientists have discovered the first manifestations of the infection by observing the effects of the virus on the Macaque monkey. Subsequently, the virus was isolated from the blood serum [4, 5]. During the next few years, the Zika virus underwent serious studies and in 1952 received its name in the virological nomenclature. In the International Classification of Diseases, ICD-10 is described under the code name A92.8 [6]. In humans, the virus is known to be the cause of the disease known as Zika fever or Zika disease, spreading since 1950 from *Africa* to *Asia* [7, 8].

Firstly, the data indicate that in 2014, the virus was *transmitted* across the *Pacific* to *French Polynesia*, reaching the shores of Easter Island. Secondly, the surveillance of Zika virus infection since the beginning of 2016 indicates an intensive *epidemic boom* with increasing prevalence of the disease in El Salvador, Venezuela, Colombia, Brazil, Suriname, French Guiana, Honduras, Mexico, Panama, and Martinique. Thirdly, in 2015 there were reported cases in Central America, the Caribbean, and South America, where Zika caused the development of a *pandemic*. It is also known that *sporadic* cases in Guatemala, Paraguay, Puerto Rico, Barbados, St. Martin, and Haiti are reported. According to the World Health Organization, Zika is spreading explosively and could affect 3–4 million people in the Americas. Furthermore, it is believed that the Zika virus was unknown to the US region prior to 2015. Finally, a retrospective analysis of the data indicates that nowadays Zika fever is a serious socially significant disease in the *US region* for which the resources available for its prevention, treatment, and control are scarce [1, 4, 5, 9, 10].

In 2013, there have been cases reported of the disease in *Europe* as well. From South America, tourists carry the virus to Europe. Ten Zika fever (ZVD) cases have been reported in Germany since 2013. There are four in Italy, three in the UK, and seven in Spain in the Zika virus case, as well as one case each in Sweden and Denmark. It is possible that the number of Europeans infected with Zika is much larger without themselves knowing it. According to the American Center for Disease Control and Prevention (CDCP), only one in five people infected with the virus develops the disease. The southern part of the European continent is generally of moderate risk for ZVD. The risk of Zika spreading in Europe in late spring and summer is described as "small to moderate," according to the WHO. The Zika virus is "highly likely" to spread to three European regions where there are tiger mosquitoes that carry it. These are the Portuguese island of Madeira in the Atlantic Ocean and the Russian and Georgian Black Sea coasts. For the other 18 predominantly Mediterranean countries, including Spain, France, Italy, Croatia, Greece, and Turkey, the risk is mostly considered moderate. According to the data provided by the WHO on the spread of Zika, the territory of Bulgaria is identified to be a moderate-risk country for this disease. Analysis of data from the World Meteorological Organization (WMO), the National Institute of Meteorology and Hydrology (NIMH) in Bulgaria, and the Institute for Atmospheric, Climate and Water Research (IACWR) at BAS (functioning from January 1, 2019) in Bulgaria about humidity, high summer temperatures, and the average altitude of the entire

**17**

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

terrain of Southern Europe shows the increasing number of mosquitoes spreading

The infection is known to be transmitted to humans through the bite of infected mosquitoes of the *Aedes* family, most commonly the species *Aedes aegypti* and *Aedes albopictus* or the "Asian tiger mosquito." It seems that these two species are widespread in the tropical regions of Asia, Africa, and America. In fact, the tiger mosquitoes are native to Southeast Asia. The Zika virus was also isolated from *A. africanus*, *A. apicoargenteus*, *A. furcifer*, *A. hensilli*, *A. luteocephalus*, and *A. vittatus*. Generally, tiger mosquitoes can transmit dangerous viral and parasitic diseases to humans and pets, including *Zika*, yellow fever, St. Louis encephalitis, West Nile encephalitis, chikungunya, dengue, heartworm (in dogs and cats), and

In *the USA*, it was proven in 1985 in Texas and Maine; in Hawaii in 1986; in Brazil, Argentina, and Mexico in 1988; in the Dominican Republic in 1993; in Paraguay in

1999; in Panama in 2002; and in Uruguay and Nicaragua 2003 [1–5, 17, 18].

It penetrated South *Africa* in 1990, Nigeria in 1991, Cameroon in 1999, and Gabon in 2006 [1, 4, 5, 10, 19] and *in the Middle East—*Lebanon and Israel in 2003

The Asian mosquito originates from tropical and subtropical regions of Southeast *Asia* [1, 9, 10, 19]. In the last few decades, it has spread to many other regions of the world mainly through means of transport and intensive commodity trade. It was brought to *Europe* in 1979 in Albania in goods from China and has begun to spread massively along *the Mediterranean coast* since 1990. In 1990–1991 it was established in Italy, transported in old tires from the USA, and spread throughout the country, including the islands of Sicily and Sardinia. In 1999 it reached southern France and the island of Corsica. Other countries affected were Belgium in 2000–2001; Montenegro in 2003; Switzerland and Greece in 2004; Spain and Croatia in 2005; the Netherlands, Bosnia and Herzegovina, and Slovenia in 2006; Germany in 2007; Greece (in areas close to or adjacent to Bulgaria) in 2008; and Malta in 2010 [1, 4, 5, 9–11, 19].

In 2011, the presence of a tiger mosquito in *Bulgaria*, in Sozopol (Burgas region), was first established. In 2014 it was detected in Burgas and Plovdiv. In January 2016, there were also data on the spread of the disease vector in Bulgaria in the districts of Blagoevgrad, Pazardzhik, Varna, Vratsa, Montana, Plovdiv, and Stara Zagora. The risk of expansion of the territory in the Danube Plain and in the Upper Thracian Plain and the valleys of the Struma and Mesta rivers is real and is defined as high for

According to the WHO, *the Black Sea coast* is among the most endangered areas in Europe in the spread of Asian tiger mosquito. The potential risk is identified in all

It is also known that the spread of mosquitoes, which causes the transmission of the Zika virus, develops in places with an altitude of up to 200 m. An interesting fact is that mosquitoes attack during the day, prefer humans over other warm-

Research shows that mosquitoes are the only carriers of the pathogen, and the Zika virus infection can mainly spread to humans and monkeys. There is a serious risk of spreading the infection only if the viral infection spreads through humans and mosquitoes from areas of normal habitat to the European continent. This spread can be done by sick people arriving from Africa and America or by the direct

the country during the warm months of the year [11, 19, 20].

infestation of mosquitoes from infected areas [1, 11, 19, 20].

blooded animals, and hide and breed close to human homes [19, 20].

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

**2.2 Vectors facts and analyses of spreading ZVD**

the Zika virus [1, 9, 11–14].

others [1, 3–5, 11, 15–19].

and Syria in 2005 [4, 5, 19].

territories [1, 11, 19, 20].

terrain of Southern Europe shows the increasing number of mosquitoes spreading the Zika virus [1, 9, 11–14].

## **2.2 Vectors facts and analyses of spreading ZVD**

*Current Concepts in Zika Research*

*Africa* to *Asia* [7, 8].

**2. Data and analyses of Zika in favor of risk reduction conception**

It is a well-known fact that the Zika virus belongs to the *Flaviviridae* family of the genus *Flavivirus* [1]. The data indicate that the Zika virus was isolated in 1947 from Macaque monkeys in Uganda in the Uganda Forest to which it is named [1, 4, 5]. Retrospective analysis shows that the Zika virus was first studied by scientists at the Yellow Fever Institute (founded in 1936). Later, in 1948, the Zika virus was isolated for the second time in the tiger mosquito. Scientists have discovered the first manifestations of the infection by observing the effects of the virus on the Macaque monkey. Subsequently, the virus was isolated from the blood serum [4, 5]. During the next few years, the Zika virus underwent serious studies and in 1952 received its name in the virological nomenclature. In the International Classification of Diseases, ICD-10 is described under the code name A92.8 [6]. In humans, the virus is known to be the cause of the disease known as Zika fever or Zika disease, spreading since 1950 from

Firstly, the data indicate that in 2014, the virus was *transmitted* across the *Pacific* to *French Polynesia*, reaching the shores of Easter Island. Secondly, the surveillance of Zika virus infection since the beginning of 2016 indicates an intensive *epidemic boom* with increasing prevalence of the disease in El Salvador, Venezuela, Colombia, Brazil, Suriname, French Guiana, Honduras, Mexico, Panama, and Martinique. Thirdly, in 2015 there were reported cases in Central America, the Caribbean, and South America, where Zika caused the development of a *pandemic*. It is also known that *sporadic* cases in Guatemala, Paraguay, Puerto Rico, Barbados, St. Martin, and Haiti are reported. According to the World Health Organization, Zika is spreading explosively and could affect 3–4 million people in the Americas. Furthermore, it is believed that the Zika virus was unknown to the US region prior to 2015. Finally, a retrospective analysis of the data indicates that nowadays Zika fever is a serious socially significant disease in the *US region* for which the resources available for its

In 2013, there have been cases reported of the disease in *Europe* as well. From South America, tourists carry the virus to Europe. Ten Zika fever (ZVD) cases have been reported in Germany since 2013. There are four in Italy, three in the UK, and seven in Spain in the Zika virus case, as well as one case each in Sweden and Denmark. It is possible that the number of Europeans infected with Zika is much larger without themselves knowing it. According to the American Center for Disease Control and Prevention (CDCP), only one in five people infected with the virus develops the disease. The southern part of the European continent is generally of moderate risk for ZVD. The risk of Zika spreading in Europe in late spring and summer is described as "small to moderate," according to the WHO. The Zika virus is "highly likely" to spread to three European regions where there are tiger mosquitoes that carry it. These are the Portuguese island of Madeira in the Atlantic Ocean and the Russian and Georgian Black Sea coasts. For the other 18 predominantly Mediterranean countries, including Spain, France, Italy, Croatia, Greece, and Turkey, the risk is mostly considered moderate. According to the data provided by the WHO on the spread of Zika, the territory of Bulgaria is identified to be a moderate-risk country for this disease. Analysis of data from the World Meteorological Organization (WMO), the National Institute of Meteorology and Hydrology (NIMH) in Bulgaria, and the Institute for Atmospheric, Climate and Water Research (IACWR) at BAS (functioning from January 1, 2019) in Bulgaria about humidity, high summer temperatures, and the average altitude of the entire

**2.1 Epidemiology and the spread of Zika virus: facts and analyses**

prevention, treatment, and control are scarce [1, 4, 5, 9, 10].

**16**

The infection is known to be transmitted to humans through the bite of infected mosquitoes of the *Aedes* family, most commonly the species *Aedes aegypti* and *Aedes albopictus* or the "Asian tiger mosquito." It seems that these two species are widespread in the tropical regions of Asia, Africa, and America. In fact, the tiger mosquitoes are native to Southeast Asia. The Zika virus was also isolated from *A. africanus*, *A. apicoargenteus*, *A. furcifer*, *A. hensilli*, *A. luteocephalus*, and *A. vittatus*. Generally, tiger mosquitoes can transmit dangerous viral and parasitic diseases to humans and pets, including *Zika*, yellow fever, St. Louis encephalitis, West Nile encephalitis, chikungunya, dengue, heartworm (in dogs and cats), and others [1, 3–5, 11, 15–19].

In *the USA*, it was proven in 1985 in Texas and Maine; in Hawaii in 1986; in Brazil, Argentina, and Mexico in 1988; in the Dominican Republic in 1993; in Paraguay in 1999; in Panama in 2002; and in Uruguay and Nicaragua 2003 [1–5, 17, 18].

It penetrated South *Africa* in 1990, Nigeria in 1991, Cameroon in 1999, and Gabon in 2006 [1, 4, 5, 10, 19] and *in the Middle East—*Lebanon and Israel in 2003 and Syria in 2005 [4, 5, 19].

The Asian mosquito originates from tropical and subtropical regions of Southeast *Asia* [1, 9, 10, 19]. In the last few decades, it has spread to many other regions of the world mainly through means of transport and intensive commodity trade. It was brought to *Europe* in 1979 in Albania in goods from China and has begun to spread massively along *the Mediterranean coast* since 1990. In 1990–1991 it was established in Italy, transported in old tires from the USA, and spread throughout the country, including the islands of Sicily and Sardinia. In 1999 it reached southern France and the island of Corsica. Other countries affected were Belgium in 2000–2001; Montenegro in 2003; Switzerland and Greece in 2004; Spain and Croatia in 2005; the Netherlands, Bosnia and Herzegovina, and Slovenia in 2006; Germany in 2007; Greece (in areas close to or adjacent to Bulgaria) in 2008; and Malta in 2010 [1, 4, 5, 9–11, 19].

In 2011, the presence of a tiger mosquito in *Bulgaria*, in Sozopol (Burgas region), was first established. In 2014 it was detected in Burgas and Plovdiv. In January 2016, there were also data on the spread of the disease vector in Bulgaria in the districts of Blagoevgrad, Pazardzhik, Varna, Vratsa, Montana, Plovdiv, and Stara Zagora. The risk of expansion of the territory in the Danube Plain and in the Upper Thracian Plain and the valleys of the Struma and Mesta rivers is real and is defined as high for the country during the warm months of the year [11, 19, 20].

According to the WHO, *the Black Sea coast* is among the most endangered areas in Europe in the spread of Asian tiger mosquito. The potential risk is identified in all territories [1, 11, 19, 20].

It is also known that the spread of mosquitoes, which causes the transmission of the Zika virus, develops in places with an altitude of up to 200 m. An interesting fact is that mosquitoes attack during the day, prefer humans over other warmblooded animals, and hide and breed close to human homes [19, 20].

Research shows that mosquitoes are the only carriers of the pathogen, and the Zika virus infection can mainly spread to humans and monkeys. There is a serious risk of spreading the infection only if the viral infection spreads through humans and mosquitoes from areas of normal habitat to the European continent. This spread can be done by sick people arriving from Africa and America or by the direct infestation of mosquitoes from infected areas [1, 11, 19, 20].

#### **2.3 The Zika fever facts and analyses**

Studies show that the incubation period of the Zika virus is about 10 days; mostly it is known that people get infected first and foremost by tiger mosquito *bites* (Zika virus carrier). Some data indicated that ZVD can spread to other people through *sexual contact* and is directly infused and inoculated in the placenta [1, 2, 19].

The first case of a possible Zika virus *transfusion* in 2014 at the Hematology Center at Campinas University in São Paulo has also been reported [2, 7, 19].

The virus has been shown to damage the human nervous system and infect, damage, and kill the cells of the developing human brain even more prenatally, disrupting the localization of a pTBK1-protein, the protein that helps in cell division of the growing brain. The scientific community also discusses the link between Zika and microcephaly or congenital anomalies in newborns whose mothers became ill with the Zika virus during pregnancy. After 2014, the disease is associated with Guillain-Barré syndrome, which has been reported in some of the patients with ZVD [15–20].

### **2.4 Classification and analysis of Zika-related risk factors (author analysis)**

The epidemiological data and the facts and analyses for the development and spread of the Zika vector and Zika virus are a good basis for elucidating Zika-related risk factors. Knowledge of risk factors, in turn, enables us to group them according to their type and nature and to give an idea of their classification. This is in favor of building a concise idea of a concept for the possibilities of the reduction and prevention of ZVDs at different levels and stages according to the specific needs and according to the stage of development of the disease from the moment of entry to the person in the distribution zone of Zika vector to the incidence of Zika virus in the human body.

#### *2.4.1 From current conclusions to the conceptual models of ZVD (author analysis)*

Nowadays the knowledge that we have about Zika virus infection provides us with some generalizing current conclusions that promise a better understanding of some conceptual models of Zika virus infection (**Figure 1**).


**19**

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

7.A prerequisite for the development of Zika fever besides the vector is the pres-

8.The chain of infection with ZVDs can be represented in a few types. The main chain is source-carrier-human. Another chain is also possible: man-man in a vertical and horizontal direction. This is explained by the transplacental transfer (intrauterine infection) of the virus from the pregnant woman to the embryo and fetus, as well as by the transfusion of biological fluids physiologi-

9.The virus affects the human nervous system, but not necessarily every person

10.Probably the human immune system does not have a good enough response in time and space to stop the damage to the nervous system in every case of

During the study of Zika virus infection, the following several questions arose

1.Is global climate change likely to increase the risk of people with Zika virus

2.Is it possible to reduce the risk of vector spread of Zika virus infection, and can the population of the vector be reduced by a competitive species or other

4.Can the vector be tilted in such a way that it does not affect the person with the

ence of a source in a susceptible population by a few chains.

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

cally or mechanically.

infection.

natural species?

bites?

(**Figure 2**):

**Figure 1.**

*Zika VD and Zika vector.*

infected with Zika virus infection.

infection, and is it possible to reduce this threat?

3.Is it possible a mosquito vector to lose its role as a vector?

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine… DOI: http://dx.doi.org/10.5772/intechopen.92128*

**Figure 1.** *Zika VD and Zika vector.*

*Current Concepts in Zika Research*

the human body.

and parasites.

**2.3 The Zika fever facts and analyses**

Studies show that the incubation period of the Zika virus is about 10 days; mostly it is known that people get infected first and foremost by tiger mosquito *bites* (Zika virus carrier). Some data indicated that ZVD can spread to other people through *sexual contact* and is directly infused and inoculated in the placenta [1, 2, 19]. The first case of a possible Zika virus *transfusion* in 2014 at the Hematology Center at Campinas University in São Paulo has also been reported [2, 7, 19].

The virus has been shown to damage the human nervous system and infect, damage, and kill the cells of the developing human brain even more prenatally, disrupting the localization of a pTBK1-protein, the protein that helps in cell division of the growing brain. The scientific community also discusses the link between Zika and microcephaly or congenital anomalies in newborns whose mothers became ill with the Zika virus during pregnancy. After 2014, the disease is associated with Guillain-Barré syndrome, which has been reported in some of the patients with ZVD [15–20].

**2.4 Classification and analysis of Zika-related risk factors (author analysis)**

*2.4.1 From current conclusions to the conceptual models of ZVD (author analysis)*

some conceptual models of Zika virus infection (**Figure 1**).

1.Zika fever is a vector-borne disease [1–22].

temperature at low altitude.

spread of the vector.

tion in warm and humid weather.

Nowadays the knowledge that we have about Zika virus infection provides us with some generalizing current conclusions that promise a better understanding of

2.The Zika virus infection vector is also a vector of many other pathogenic viruses

3.Practically, the vector spreads under high humidity and moderately high

4.The vector is adaptive and easily enters new habitats through the transport of goods and passengers, mainly by means of water, rapidly increasing its popula-

5.Climate change provides new threats to humans and new territories for the

6.The virus spreads in parallel with the area of development and propagation of its vector but does not necessarily exist in all cases of typical habitat of its vector.

The epidemiological data and the facts and analyses for the development and spread of the Zika vector and Zika virus are a good basis for elucidating Zika-related risk factors. Knowledge of risk factors, in turn, enables us to group them according to their type and nature and to give an idea of their classification. This is in favor of building a concise idea of a concept for the possibilities of the reduction and prevention of ZVDs at different levels and stages according to the specific needs and according to the stage of development of the disease from the moment of entry to the person in the distribution zone of Zika vector to the incidence of Zika virus in

**18**


During the study of Zika virus infection, the following several questions arose (**Figure 2**):


#### **Figure 2.**

*New risk factors and risk reduction option.*


## *2.4.2 From groups of risk factors to the conceptual framework for modern technological approaches about ZVD (author analysis)*

Some of the questions asked seem impossible and even naive in their search for possible solutions. However, the search for an answer, based on risk analysis, led slowly to the idea of a conceptual framework for modern technological approaches, in the hope of increasing the real chance of risk reduction in the process of risk management for the health provision of the population in favor of prevention and in the context of a disaster-prone process.

First of all, some groups of risk factors have emerged from a detailed examination of the available data on Zika virus infection [1–20].

According to the origin of their generation, there are two fundamental groups of risk factors, namely, natural and anthropogenic (**Figure 3**).

**21**

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

Fundamental groups of risk factors can be divided into six main groups accord-

1.Risk factors arising from the environment (territorial risk factors, TRFs).

4.Risk factors resulting from the population (receptivity, resistance, immune

6.Factors related to knowledge and competence (scientific-cognitive) and the realization of ethical-legal and preventive-therapeutic ideas, methods, tech-

According to their mechanism, the risk factors can be selected as mechanical,

2.Risk factors resulting from the vector (VRFs).

3.Time and weather as risk factors (time risk factors).

5.Factors arising from human actions and/or inaction.

**3. The Zika contemporary risk reduction conception**

**3.1 A brief look at preventive risk management measures (with SWOT analysis): from tradition to the future stage (author analysis)**

Zika's strategic approaches about prevention are divided into three groups according to the moment of implementation of the specific tasks: before, during, and after the outbreak of the infection is occurred. In practice, these groups of methods are *strategic time-based approaches* to risk reduction. The timely application of this group of methods is also the responsibility for controlling the risk over time. On the other hand, the application of this approach leads to the reduction of disease territory, regression, and/or complete destruction of the risk areas (the occurrence of a hot and warm zone of infection within a particular territory) at a certain point

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

ing to their type:

*Groups of risk factors.*

**Figure 3.**

response).

nologies, and concepts.

physicochemical, and biochemical.

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine… DOI: http://dx.doi.org/10.5772/intechopen.92128*

**Figure 3.** *Groups of risk factors.*

*Current Concepts in Zika Research*

virus into the human body?

*New risk factors and risk reduction option.*

factors that support this course?

in the context of a disaster-prone process.

tion of the available data on Zika virus infection [1–20].

risk factors, namely, natural and anthropogenic (**Figure 3**).

no damage to the nervous system occurs?

bite of the vector?

virus?

**Figure 2.**

5.Is it possible, if a mosquito bite (as a vector) humans, to stop the entry of Zika

6.Is it possible to stop the Zika virus that has entered the human body during the

8.Is it possible to understand the mechanism of the immune response in humans in which the Zika virus does not affect the nervous system, and are there other

9.Is there a mechanism to support a person's immune response in such a way that

Some of the questions asked seem impossible and even naive in their search for possible solutions. However, the search for an answer, based on risk analysis, led slowly to the idea of a conceptual framework for modern technological approaches, in the hope of increasing the real chance of risk reduction in the process of risk management for the health provision of the population in favor of prevention and

First of all, some groups of risk factors have emerged from a detailed examina-

According to the origin of their generation, there are two fundamental groups of

10.Is it possible to prevent the transplacental passage of the Zika virus?

*2.4.2 From groups of risk factors to the conceptual framework for modern technological approaches about ZVD (author analysis)*

7.Is it possible to reduce and/or completely stop human susceptibility to Zika

**20**

Fundamental groups of risk factors can be divided into six main groups according to their type:


According to their mechanism, the risk factors can be selected as mechanical, physicochemical, and biochemical.

## **3. The Zika contemporary risk reduction conception**

## **3.1 A brief look at preventive risk management measures (with SWOT analysis): from tradition to the future stage (author analysis)**

Zika's strategic approaches about prevention are divided into three groups according to the moment of implementation of the specific tasks: before, during, and after the outbreak of the infection is occurred. In practice, these groups of methods are *strategic time-based approaches* to risk reduction. The timely application of this group of methods is also the responsibility for controlling the risk over time. On the other hand, the application of this approach leads to the reduction of disease territory, regression, and/or complete destruction of the risk areas (the occurrence of a hot and warm zone of infection within a particular territory) at a certain point

in time. This is an important element of the health provision of the population (in a certain territory) over a period of time, not just the groups at risk, and is part of the national security. Ongoing control and extreme control are responsible for sporadic Zika infection and for controlling the risk of an epidemic outbreak. The inclusion of additional medical forces and resources when needed at a given time as a health policy results in a reduced response time to the massive nature of Zika disease in the short term. The specific aspects of this approach depend on the seasonality as well as the manifestations of the daily activity of the vector. The time approach is characterized by some cyclicality. Importantly, health-care decision-making considerations based on this approach require firmness, maneuverability, and agility. This strategic approach is a fundamental guarantor for the good control of the risk of Zika virus infection in a timely manner [1–27].

There are some known threats to invoking only these strategic time approaches. They are derived from the nature of the spread of the ZVD and namely through a vector. This requires that the health care of the population be stepped on the basis of *strategic territorial principle* [22] with emphasis on the environment and territory suitable for the development and dissemination of the vector. The control of the vector is possible and also depends on the close transinstitutional interaction, planning, and development of risk maps, as well as their strict updating and upgrading with new methods. Facilitated by appropriate local meteorological and global climate changes, the risk of a hot zone from a Zika virus is present and varies in extent and varies in space. The reduction and even destruction of the natural range of the vector undergo a health-sanitary control in collaboration with various professionals at every level of territorial division of the country—national, regional, and municipal levels. Cross-border cooperation is a fundamental step in the event of a high risk of spreading the disease outside the country. Migration and transoceanic transport of people and goods are factors facilitating the spread of the Zika disease. The globalization of the Zika disease proves the weaknesses of the territorial principle [1–20, 23–27].

*Virus-based strategy*, a strategic principle based on the viral genesis of the disease, the RNA genome of the causative agent, as well as its genetic variability, is being investigated. Insofar as there is predictability in specific parameters in the previous two principles, it is probabilistic in that. On the other hand, the creation of a specific vaccine and/or specific antiviral drug is a process of unknown duration, and time is proven to be an insufficient resource for the purpose of providing the population with medical care in the face of an epidemic. The laboratory demonstration of the virus both in vectors and in humans is an important step. However, the study and demonstration of Zika virus have its weaknesses, the main one being the recruitment of the necessary and sufficient scope of the study under the specific conditions for the construction of a strictly positive and strictly negative hypothesis for its presence. The process is time-consuming. The reliability of laboratory tests in terms of genomic variability of the virus requires both flexibility and a competitive environment for a prompt response by researchers and experts [1–20, 23–27].

*Health education strategy* for raising the health culture and awareness of both the population and health professionals is an approach complementary to the above, extending levels of responsibility to each individual except the medical services for the control of the spread of the Zika disease. Difficulties in making such information available to certain sections of the population in a country are defined as a weakness, as well as varying degrees of understanding of information submitted by each person at risk of being potentially affected. Access to health services is not equally available in all cases of the spread of the Zika disease. The levels of literacy and responsibility to one's own health are at different levels of manifestation [1–21, 23–27].

**23**

**Figure 4.**

*Contemporary strategy of RR.*

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

combination, they produce significantly more reliable results (**Figure 4**).

These four strategic approaches are classic approaches in nature. When used in

Depending on their type [34], the methods can be grouped into both traditional

(classical) and contemporary as well as progressively innovative. If traditional prophylaxis methods [28–31, 34–36] are environment-oriented [22, 37–39], vectororiented [27, 36–38], or target-specific [21, 22, 35], or targeted at a susceptible population (potentially infected) [22, 38, 39], or a combination thereof, and contemporary to the human body's immune response or to the creation of high public immune status, then all these methods apply to society as a whole and have a

**3.2 Progressive-innovative approaches to reducing the risk of ZVD:** 

Innovative technologies excite science—quantum-based as well as nanotechnologies and related software models—supported by mathematical algorithms and block diagrams to model the framework of an innovative idea. In this regard, the Zika RNA virus, with its unknowns, predisposes us scientists to trying to solve the equation from another angle. It turns out that the actual scientific information on Zika is not satisfactory and requires the search for new approaches beyond the known ones in order to achieve more serious results and greater success in solving the equation with such unknowns. This is because if we apply the always known and recognized methods, we will always arrive at the same results. In this case, it means coming up with a specific vaccine and/or specific antiviral agent to deal with Zika disease. This is, of course, an excellent destination and also a well-known area for

**technologies of the future stage (author's idea)**

Modern approaches, but with limited accessibility, are immunoprophylaxis approaches. They aim to enhance the immune response of the community, except for any member of the at-risk society. Approaches to *primary*, *secondary*, *or tertiary immunoprophylaxis* take wide limits. Immune protectors are gaining popularity. Immunomodulating herbal remedies complement and extend the boundaries of current immunoprophylactic approaches. Clinical homeopathy or allopathic homeopathy, phytotherapy, herbal medicine, etc. supplement the ability to stabilize and prepare the immune system to meet the Zika virus that is aggressive to the nervous

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

system [1–20, 22–39].

group character.

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine… DOI: http://dx.doi.org/10.5772/intechopen.92128*

These four strategic approaches are classic approaches in nature. When used in combination, they produce significantly more reliable results (**Figure 4**).

Modern approaches, but with limited accessibility, are immunoprophylaxis approaches. They aim to enhance the immune response of the community, except for any member of the at-risk society. Approaches to *primary*, *secondary*, *or tertiary immunoprophylaxis* take wide limits. Immune protectors are gaining popularity. Immunomodulating herbal remedies complement and extend the boundaries of current immunoprophylactic approaches. Clinical homeopathy or allopathic homeopathy, phytotherapy, herbal medicine, etc. supplement the ability to stabilize and prepare the immune system to meet the Zika virus that is aggressive to the nervous system [1–20, 22–39].

Depending on their type [34], the methods can be grouped into both traditional (classical) and contemporary as well as progressively innovative. If traditional prophylaxis methods [28–31, 34–36] are environment-oriented [22, 37–39], vectororiented [27, 36–38], or target-specific [21, 22, 35], or targeted at a susceptible population (potentially infected) [22, 38, 39], or a combination thereof, and contemporary to the human body's immune response or to the creation of high public immune status, then all these methods apply to society as a whole and have a group character.

#### **3.2 Progressive-innovative approaches to reducing the risk of ZVD: technologies of the future stage (author's idea)**

Innovative technologies excite science—quantum-based as well as nanotechnologies and related software models—supported by mathematical algorithms and block diagrams to model the framework of an innovative idea. In this regard, the Zika RNA virus, with its unknowns, predisposes us scientists to trying to solve the equation from another angle. It turns out that the actual scientific information on Zika is not satisfactory and requires the search for new approaches beyond the known ones in order to achieve more serious results and greater success in solving the equation with such unknowns. This is because if we apply the always known and recognized methods, we will always arrive at the same results. In this case, it means coming up with a specific vaccine and/or specific antiviral agent to deal with Zika disease. This is, of course, an excellent destination and also a well-known area for

**Figure 4.** *Contemporary strategy of RR.*

*Current Concepts in Zika Research*

rial principle [1–20, 23–27].

of Zika virus infection in a timely manner [1–27].

in time. This is an important element of the health provision of the population (in a certain territory) over a period of time, not just the groups at risk, and is part of the national security. Ongoing control and extreme control are responsible for sporadic Zika infection and for controlling the risk of an epidemic outbreak. The inclusion of additional medical forces and resources when needed at a given time as a health policy results in a reduced response time to the massive nature of Zika disease in the short term. The specific aspects of this approach depend on the seasonality as well as the manifestations of the daily activity of the vector. The time approach is characterized by some cyclicality. Importantly, health-care decision-making considerations based on this approach require firmness, maneuverability, and agility. This strategic approach is a fundamental guarantor for the good control of the risk

There are some known threats to invoking only these strategic time approaches. They are derived from the nature of the spread of the ZVD and namely through a vector. This requires that the health care of the population be stepped on the basis of *strategic territorial principle* [22] with emphasis on the environment and territory suitable for the development and dissemination of the vector. The control of the vector is possible and also depends on the close transinstitutional interaction, planning, and development of risk maps, as well as their strict updating and upgrading with new methods. Facilitated by appropriate local meteorological and global climate changes, the risk of a hot zone from a Zika virus is present and varies in extent and varies in space. The reduction and even destruction of the natural range of the vector undergo a health-sanitary control in collaboration with various professionals at every level of territorial division of the country—national, regional, and municipal levels. Cross-border cooperation is a fundamental step in the event of a high risk of spreading the disease outside the country. Migration and transoceanic transport of people and goods are factors facilitating the spread of the Zika disease. The globalization of the Zika disease proves the weaknesses of the territo-

*Virus-based strategy*, a strategic principle based on the viral genesis of the disease, the RNA genome of the causative agent, as well as its genetic variability, is being investigated. Insofar as there is predictability in specific parameters in the previous two principles, it is probabilistic in that. On the other hand, the creation of a specific vaccine and/or specific antiviral drug is a process of unknown duration, and time is proven to be an insufficient resource for the purpose of providing the population with medical care in the face of an epidemic. The laboratory demonstration of the virus both in vectors and in humans is an important step. However, the study and demonstration of Zika virus have its weaknesses, the main one being the recruitment of the necessary and sufficient scope of the study under the specific conditions for the construction of a strictly positive and strictly negative hypothesis for its presence. The process is time-consuming. The reliability of laboratory tests in terms of genomic variability of the virus requires both flexibility and a competitive environment for a prompt response by researchers and experts [1–20, 23–27].

*Health education strategy* for raising the health culture and awareness of both the population and health professionals is an approach complementary to the above, extending levels of responsibility to each individual except the medical services for the control of the spread of the Zika disease. Difficulties in making such information available to certain sections of the population in a country are defined as a weakness, as well as varying degrees of understanding of information submitted by each person at risk of being potentially affected. Access to health services is not equally available in all cases of the spread of the Zika disease. The levels of literacy and responsibility

to one's own health are at different levels of manifestation [1–21, 23–27].

**22**

#### *Current Concepts in Zika Research*

dealing with particularly dangerous infections. The high benefits of this approach have been proven, and this is because the world has dealt with diseases such as smallpox. The benefits are undeniable and highly appreciated. However, this is also not an obstacle to look for new approaches that could give another level of solutions. If we manage to integrate a new approach, although it may seem impossible and even absurd in its initial form as an idea, it may 1 day help to fundamentally tackle similar problems.

The thesis of the unrecognizability of Zika virus that has entered the human body appears as a counterbalance (counter thesis) to its recognition. In reality, heretofore, there is no known mechanism to protect the nervous system of the human body that is targeted by the Zika virus. Once penetrated into the body, the Zika virus takes several steps—to multiply, to cover the whole body, to cross the transplacental barrier, and to affect the nervous system and/or destroy it. Although initially recognized as an infectious agent by the immune system, it seems clear that the Zika virus is able to attack the nervous system both in the developing fetus during pregnancy and in some infected adults. The affinity of the virus to the nerve cell leads to its destruction and even to blocking its development in the embryo and fetus in pregnant women during the period of organogenesis. This makes the thesis of the unrecognizability of Zika virus relevant to the study.

The purpose and the tasks set have led us to the idea that biotechnological methods (in their varieties) can work for us in this direction. In reality, biotechnology gives us the opportunity to conduct research in the field of experience, as well as real-world attempts to solve the problem of early specific recognition of the Zika virus incorporated into the human body. The conceptual design covers several stages of its presentation, the most important of which is the targeted recognition of the Zika virus from the moment of the bite (from the mosquito carrier of the virus), through all steps of its incorporation, before it affects the nervous system and/or its development in the fetus.

The different levels of recognition create obstacles due to lack of information about the actual moment of the bite; ambiguity and unrecognition of nonspecific early symptoms; late identification of the disease; inaccessibility to medical services and early medical care; etc.

The invention and production of a nano-biotechnology carrier (as a conveyor) and its intravenous injection in order to determine whether a Zika virus has entered the human body as a key to dealing with the disease, before the virus strikes the nervous system, is a new type of idea. The capture of each virus in the shuttle (at the site of the bite, during their circulation in the body, or during its entry into the human cell) gives us a new opportunity to control the Zika virus. The implementation of this stage is supported by nano-biotechnology. The capture in the Zika shuttle of the virus in the human body requires sufficient durability to allow time for injection of substance "X" solely into the capsule of the virus from the carrier that has captured the Zika virus. This guarantees the destruction of the pathogenic virus only, without affecting the tissue cells in the human body. This is considered within the framework of achieving successful *secondary prophylaxis*, provided that the Zika virus is already incorporated into the human body or has serious clinical and/or laboratory indications of this in humans at risk. The therapeutic result will be available and delivered in a timely manner.

Moreover, the implementation of this approach can also be carried out in advance (as *primary prevention*) for any person who is at risk of being potentially bitten by a Zika virus vector carrier—researchers and scientists working in the field; field medical teams; emergency rescue teams; disaster, accident, and catastrophe field workers; etc. This will generate safety for the teams and enable rescue and rehabilitation activities on the worksite, as well as the successful completion of each

**25**

**Author details**

Diana Dimitrova

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

mission in a Zika fever area, and will increase the chances of victims of emergency, disaster, accident, and catastrophe at risk of Zika fever. The time factor is in any case the basis for the successful completion of a given mission and/or field task at

Preliminary studies of "X" substance on the effectiveness of the action to eradicate the pathogenic virus have been made. As far as one can tell, this substance can also be synthesized, except that it can be extracted from the natural sources of a precursor substance. It is assumed that there may be other variants of this substance and that there may be others that are effective, even with more potent action and with faster effect. However, in vivo and in vitro processes can sometimes show

The idea presented as a theoretical formulation gives new horizons. It is clear that studies are still ongoing in the direction of effective control of the Zika virus. The aim is to demonstrate a mechanism or combination of methods and measures that can reduce the risk of the Zika virus as a possible result in the near future.

Emergency CC, Department of EM and MCS, Stanke Dimitrov, Sofia, Bulgaria

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: d.dimitrova.phd.md@abv.bg

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

Zika's risk.

surprising results.

**4. Conclusion**

**Conflict of interest**

There are no "conflicts of interest."

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine… DOI: http://dx.doi.org/10.5772/intechopen.92128*

mission in a Zika fever area, and will increase the chances of victims of emergency, disaster, accident, and catastrophe at risk of Zika fever. The time factor is in any case the basis for the successful completion of a given mission and/or field task at Zika's risk.

Preliminary studies of "X" substance on the effectiveness of the action to eradicate the pathogenic virus have been made. As far as one can tell, this substance can also be synthesized, except that it can be extracted from the natural sources of a precursor substance. It is assumed that there may be other variants of this substance and that there may be others that are effective, even with more potent action and with faster effect. However, in vivo and in vitro processes can sometimes show surprising results.

## **4. Conclusion**

*Current Concepts in Zika Research*

ability of Zika virus relevant to the study.

and/or its development in the fetus.

be available and delivered in a timely manner.

and early medical care; etc.

similar problems.

dealing with particularly dangerous infections. The high benefits of this approach have been proven, and this is because the world has dealt with diseases such as smallpox. The benefits are undeniable and highly appreciated. However, this is also not an obstacle to look for new approaches that could give another level of solutions. If we manage to integrate a new approach, although it may seem impossible and even absurd in its initial form as an idea, it may 1 day help to fundamentally tackle

The thesis of the unrecognizability of Zika virus that has entered the human body appears as a counterbalance (counter thesis) to its recognition. In reality, heretofore, there is no known mechanism to protect the nervous system of the human body that is targeted by the Zika virus. Once penetrated into the body, the Zika virus takes several steps—to multiply, to cover the whole body, to cross the transplacental barrier, and to affect the nervous system and/or destroy it. Although initially recognized as an infectious agent by the immune system, it seems clear that the Zika virus is able to attack the nervous system both in the developing fetus during pregnancy and in some infected adults. The affinity of the virus to the nerve cell leads to its destruction and even to blocking its development in the embryo and fetus in pregnant women during the period of organogenesis. This makes the thesis of the unrecogniz-

The purpose and the tasks set have led us to the idea that biotechnological methods (in their varieties) can work for us in this direction. In reality, biotechnology gives us the opportunity to conduct research in the field of experience, as well as real-world attempts to solve the problem of early specific recognition of the Zika virus incorporated into the human body. The conceptual design covers several stages of its presentation, the most important of which is the targeted recognition of the Zika virus from the moment of the bite (from the mosquito carrier of the virus), through all steps of its incorporation, before it affects the nervous system

The different levels of recognition create obstacles due to lack of information about the actual moment of the bite; ambiguity and unrecognition of nonspecific early symptoms; late identification of the disease; inaccessibility to medical services

The invention and production of a nano-biotechnology carrier (as a conveyor) and its intravenous injection in order to determine whether a Zika virus has entered the human body as a key to dealing with the disease, before the virus strikes the nervous system, is a new type of idea. The capture of each virus in the shuttle (at the site of the bite, during their circulation in the body, or during its entry into the human cell) gives us a new opportunity to control the Zika virus. The implementation of this stage is supported by nano-biotechnology. The capture in the Zika shuttle of the virus in the human body requires sufficient durability to allow time for injection of substance "X" solely into the capsule of the virus from the carrier that has captured the Zika virus. This guarantees the destruction of the pathogenic virus only, without affecting the tissue cells in the human body. This is considered within the framework of achieving successful *secondary prophylaxis*, provided that the Zika virus is already incorporated into the human body or has serious clinical and/or laboratory indications of this in humans at risk. The therapeutic result will

Moreover, the implementation of this approach can also be carried out in advance (as *primary prevention*) for any person who is at risk of being potentially bitten by a Zika virus vector carrier—researchers and scientists working in the field; field medical teams; emergency rescue teams; disaster, accident, and catastrophe field workers; etc. This will generate safety for the teams and enable rescue and rehabilitation activities on the worksite, as well as the successful completion of each

**24**

The idea presented as a theoretical formulation gives new horizons. It is clear that studies are still ongoing in the direction of effective control of the Zika virus. The aim is to demonstrate a mechanism or combination of methods and measures that can reduce the risk of the Zika virus as a possible result in the near future.

## **Conflict of interest**

There are no "conflicts of interest."

## **Author details**

Diana Dimitrova Emergency CC, Department of EM and MCS, Stanke Dimitrov, Sofia, Bulgaria

\*Address all correspondence to: d.dimitrova.phd.md@abv.bg

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] WHO. Zika virus [Internet]. 2019. Available from: https://www.who.int/ news-room/fact-sheets/detail/zika-virus

[2] WHO. Zika virus disease [Internet]. 2019. Available from: https://www.who. int/emergencies/diseases/zika/en/

[3] PAHO/WHO. Zika virus infection [Internet]. 2019. Available from: https://www.paho.org/hq/index. php%3Foption%3Dcom\_topics%26view %3Darticle%2520%26id%3D427%26Ite mid%3D41484%26lang%3Den

[4] WHO. Countries and territories with current or previous virus transmission [Internet]. 2019. Available from: https:// www.who.int/emergencies/diseases/ zika/countries-with-zika-and-vectorstable.pdf?ua=1

[5] Health Protection Surveillance Centre (HPSC). Zika virus infection— List of affected countries/areas [Internet]. 2019. Available from: https:// www.hpsc.ie/a-z/vectorborne/zika/ factsheet/listofaffectedcountries/

[6] WHO. International Classification of Diseases-ICD-10 [Internet]. 2019. Available from: https://www.who.int/ classifications/icd/en/

[7] Zika virus. John Hopkins medicine [Internet]. 2019. Available from: https:// www.hopkinsmedicine.org/zika-virus/ what-is-zika-virus.html

[8] WHO. Zika virus disease. Interim case definitions [Internet]. 2016. Available from: https://apps.who.int/ iris/bitstream/handle/10665/204381/ WHO\_ZIKV\_SUR\_16.1\_eng.pdf;jsession id=42E72E2D28DC1561ACA0E75F2E4C F639?sequence=1

[9] CDCP. Zika travel information [Internet]. 2019. Available from: https://wwwnc.cdc.gov/travel/page/ zika-travel-information

[10] Center for Disease Control and Prevention (CDCP). Zika Virus, Statistics and Maps [Internet]. 2019. Available from: https://www.cdc.gov/ zika/reporting/index.html

[11] European Center for Disease Prevention and Control. Environmental risk mapping: *Aedes albopictus* in Europe. Proof-of-concept study for the European environment and epidemiological network, Stockholm. Technical Report. 2013. DOI: 10.2900/78239. Available from: www.ecdc.europe.eu. ISBN: 978-92-9133-449-2

[12] World Meteorological Organization (WMO). Climate [Internet]. 2019. Available from: https://public.wmo.int/ en/our-mandate/climate

[13] National Institute of Meteorology and Hydrology (NIMH) in Bulgaria [Internet]. Seasonal forecasts. 2019. Available from: http://www.weather.bg/0index. php?koiFail=seasonal&lng=1

[14] Institute for Atmospheric, Climate and Water Research (IACWR) at BAS in Bulgaria [Internet]. Country overview and assessment. 2019. Available from: https://ec.europa.eu/maritimeaffairs/ sites/maritimeaffairs/files/docs/body/ bulgaria\_climate\_change\_en.pdf

[15] Leonhard SE, Lant S, Jacobs BC, et al. Zika virus infection in the returning traveller: What every neurologist should know. Practical Neurology. 2018;**18**:271-277. Available from: https://pn.bmj.com/content/ practneurol/18/4/271.full.pdf

[16] Center for Disease Control and Prevention (CDCP). Zika Virus, Life Cycle: The Mosquito [Internet]. 2019. Available from: https://www.cdc.gov/ zika/pdfs/MosquitoLifecycle.pdf

**27**

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine…*

assessment [Internet]. 2017. Available from: https://www.cdc.gov/zika/pdfs/ Draft-Environmental-Assessment-Mosquito-Control-for-publication.pdf

[26] Center for Disease Control and Prevention (CDCP). Zika Virus, Prevent Mosquito Bites [Internet]. 2019. Available

from: https://www.cdc.gov/zika/

prevention/prevent-mosquito-bites.html

[27] Guideline: Infant feeding in areas of Zika virus transmission. WHO Guidelines. ISBN: 978 92 4 154966 0

[28] Prevention of sexual transmission of Zika virus: Interim guidance update. WHO/ZIKV/MOC/16.1 Rev.3. Geneva: World Health Organization; 2016

[29] WHO Handbook for Guideline Development. 2nd ed. Geneva: World

[31] MHNIM. Guidelines for Clinical Management, Laboratory Investigation and Surveillance of Patients with Zika Virus Infection. Ministry of Health, Nutrition & Indigenous Medicine, Sri

[32] Zika Virus Testing Guidelines for Healthcare Providers. Updated on

[33] Guidelines for classifying cases of Zika virus disease and related conditions. Global report. 2019

[34] WHO. Zika virus infection— Statutory notification. In: Public Health Management and Guidelines for Public Health Units. Global Overview. World

[35] Epidemiological alert: Neurological syndrome, congenital malformations,

Health Organization; 2019

and Zika virus infection. In:

Health Organization; 2014

Lanka; 2016

9/1/16

[30] WHO. Toolkit for the care and support of people affected by complications associated with Zika virus. ISBN: 978-92-4-151271-8

*DOI: http://dx.doi.org/10.5772/intechopen.92128*

[17] Integrated Mosquito Management (IMM), Center for Disease Control and Prevention (CDCP). Zika Virus [Internet]. 2019. Available from: https:// www.cdc.gov/zika/vector/integrated\_

mosquito\_management.html

zika/vector/range.html

infektsia&lang=bg

2016;**18**(2):59-64

[19] Zika. Bulgarian Red Cross

[20] Zika virus disease. NCIPD in Bulgaria, in Bulgarian [Internet]. 2019. Available from: https://www. ncipd.org/index.php?option=com\_ k2&view=item&id=255:zika-virusna-

[21] Dimitrova D. Medical provision of the population during Zika viral infection—Viral infections with mass medical losses. General Medicine.

[22] World Health Organization. WHO guidelines for the prevention of sexual transmission of Zika virus: Executive summary. WHO/RHR/19.4; 2019

[23] Center for Disease Control and Prevention (CDCP). Zika Virus, Mosquito Control [Internet]. 2019. Available from: https://www.cdc.gov/

[24] Center for Disease Control and Prevention (CDCP). Zika Virus, Controlling Mosquitoes at Home [Internet]. 2019. Available from: https://www.cdc.gov/zika/prevention/ controlling-mosquitoes-at-home.html

[25] CDCP. Mosquito control activities by CDC to combat Zika virus transmission in the US. Draft programmatic environmental

zika/vector/index.html

[Internet]. 2019. Available from: http:// www.redcross.bg/advice/zika.html

[18] Center for Disease Control and Prevention (CDCP). Zika Virus, Potential Range in US [Internet]. 2017. Available from: https://www.cdc.gov/

*Risk Management of Zika in Context of Medical Provision and in Favor of Disaster Medicine… DOI: http://dx.doi.org/10.5772/intechopen.92128*

[17] Integrated Mosquito Management (IMM), Center for Disease Control and Prevention (CDCP). Zika Virus [Internet]. 2019. Available from: https:// www.cdc.gov/zika/vector/integrated\_ mosquito\_management.html

[18] Center for Disease Control and Prevention (CDCP). Zika Virus, Potential Range in US [Internet]. 2017. Available from: https://www.cdc.gov/ zika/vector/range.html

[19] Zika. Bulgarian Red Cross [Internet]. 2019. Available from: http:// www.redcross.bg/advice/zika.html

[20] Zika virus disease. NCIPD in Bulgaria, in Bulgarian [Internet]. 2019. Available from: https://www. ncipd.org/index.php?option=com\_ k2&view=item&id=255:zika-virusnainfektsia&lang=bg

[21] Dimitrova D. Medical provision of the population during Zika viral infection—Viral infections with mass medical losses. General Medicine. 2016;**18**(2):59-64

[22] World Health Organization. WHO guidelines for the prevention of sexual transmission of Zika virus: Executive summary. WHO/RHR/19.4; 2019

[23] Center for Disease Control and Prevention (CDCP). Zika Virus, Mosquito Control [Internet]. 2019. Available from: https://www.cdc.gov/ zika/vector/index.html

[24] Center for Disease Control and Prevention (CDCP). Zika Virus, Controlling Mosquitoes at Home [Internet]. 2019. Available from: https://www.cdc.gov/zika/prevention/ controlling-mosquitoes-at-home.html

[25] CDCP. Mosquito control activities by CDC to combat Zika virus transmission in the US. Draft programmatic environmental

assessment [Internet]. 2017. Available from: https://www.cdc.gov/zika/pdfs/ Draft-Environmental-Assessment-Mosquito-Control-for-publication.pdf

[26] Center for Disease Control and Prevention (CDCP). Zika Virus, Prevent Mosquito Bites [Internet]. 2019. Available from: https://www.cdc.gov/zika/ prevention/prevent-mosquito-bites.html

[27] Guideline: Infant feeding in areas of Zika virus transmission. WHO Guidelines. ISBN: 978 92 4 154966 0

[28] Prevention of sexual transmission of Zika virus: Interim guidance update. WHO/ZIKV/MOC/16.1 Rev.3. Geneva: World Health Organization; 2016

[29] WHO Handbook for Guideline Development. 2nd ed. Geneva: World Health Organization; 2014

[30] WHO. Toolkit for the care and support of people affected by complications associated with Zika virus. ISBN: 978-92-4-151271-8

[31] MHNIM. Guidelines for Clinical Management, Laboratory Investigation and Surveillance of Patients with Zika Virus Infection. Ministry of Health, Nutrition & Indigenous Medicine, Sri Lanka; 2016

[32] Zika Virus Testing Guidelines for Healthcare Providers. Updated on 9/1/16

[33] Guidelines for classifying cases of Zika virus disease and related conditions. Global report. 2019

[34] WHO. Zika virus infection— Statutory notification. In: Public Health Management and Guidelines for Public Health Units. Global Overview. World Health Organization; 2019

[35] Epidemiological alert: Neurological syndrome, congenital malformations, and Zika virus infection. In:

**26**

*Current Concepts in Zika Research*

**References**

[1] WHO. Zika virus [Internet]. 2019. Available from: https://www.who.int/ news-room/fact-sheets/detail/zika-virus [10] Center for Disease Control and Prevention (CDCP). Zika Virus, Statistics and Maps [Internet]. 2019. Available from: https://www.cdc.gov/

zika/reporting/index.html

978-92-9133-449-2

en/our-mandate/climate

and Hydrology (NIMH) in Bulgaria [Internet]. Seasonal forecasts. 2019. Available from: http://www.weather.bg/0index. php?koiFail=seasonal&lng=1

[11] European Center for Disease

Prevention and Control. Environmental risk mapping: *Aedes albopictus* in Europe. Proof-of-concept study for the European environment and epidemiological network, Stockholm. Technical Report. 2013. DOI: 10.2900/78239. Available from: www.ecdc.europe.eu. ISBN:

[12] World Meteorological Organization (WMO). Climate [Internet]. 2019. Available from: https://public.wmo.int/

[13] National Institute of Meteorology

[14] Institute for Atmospheric, Climate and Water Research (IACWR) at BAS in Bulgaria [Internet]. Country overview and assessment. 2019. Available from: https://ec.europa.eu/maritimeaffairs/ sites/maritimeaffairs/files/docs/body/ bulgaria\_climate\_change\_en.pdf

[15] Leonhard SE, Lant S, Jacobs BC, et al. Zika virus infection in the returning traveller: What every neurologist should know. Practical Neurology. 2018;**18**:271-277. Available from: https://pn.bmj.com/content/ practneurol/18/4/271.full.pdf

[16] Center for Disease Control and Prevention (CDCP). Zika Virus, Life Cycle: The Mosquito [Internet]. 2019. Available from: https://www.cdc.gov/ zika/pdfs/MosquitoLifecycle.pdf

[2] WHO. Zika virus disease [Internet]. 2019. Available from: https://www.who. int/emergencies/diseases/zika/en/

[3] PAHO/WHO. Zika virus infection [Internet]. 2019. Available from: https://www.paho.org/hq/index.

php%3Foption%3Dcom\_topics%26view %3Darticle%2520%26id%3D427%26Ite

[4] WHO. Countries and territories with current or previous virus transmission [Internet]. 2019. Available from: https:// www.who.int/emergencies/diseases/ zika/countries-with-zika-and-vectors-

mid%3D41484%26lang%3Den

[5] Health Protection Surveillance Centre (HPSC). Zika virus infection— List of affected countries/areas

[Internet]. 2019. Available from: https:// www.hpsc.ie/a-z/vectorborne/zika/ factsheet/listofaffectedcountries/

[6] WHO. International Classification of Diseases-ICD-10 [Internet]. 2019. Available from: https://www.who.int/

[7] Zika virus. John Hopkins medicine [Internet]. 2019. Available from: https:// www.hopkinsmedicine.org/zika-virus/

[8] WHO. Zika virus disease. Interim case definitions [Internet]. 2016. Available from: https://apps.who.int/ iris/bitstream/handle/10665/204381/ WHO\_ZIKV\_SUR\_16.1\_eng.pdf;jsession id=42E72E2D28DC1561ACA0E75F2E4C

[9] CDCP. Zika travel information [Internet]. 2019. Available from: https://wwwnc.cdc.gov/travel/page/

classifications/icd/en/

what-is-zika-virus.html

F639?sequence=1

zika-travel-information

table.pdf?ua=1

#### *Current Concepts in Zika Research*

Implications for Public Health in the Americas. Washington, DC: Pan American Health Organization, World Health Organization, Regional Office for the Americas; 2015

[36] Haddow AJ, Williams M, Woodall J, et al. Twelve isolations of Zika virus from *Aedes* (*Stegomyia*) *africanus* (*Theobald*) taken in and above a Uganda forest. Bulletin of the World Health Organization. 1964;**31**(1):57-69

[37] Thomas SM, Tjaden NB, van den Bos S, Beierkuhnlein C. Implementing cargo movement into climate based risk assessment of vector-borne diseases. International Journal of Environmental Research and Public Health AJPH. 2014;**11**(3):3360-3374

[38] MacDonald PDM, Wayne Holden E. Zika and public health: Understanding the epidemiology and information environment. Pediatrics. 2018;**141**(Suppl 2):S137-S145

[39] Howard Z. Zika virus. What New York State Clinicians Need to Know, NYS Commissioner of Health. 2016. Available from: https://www.health. ny.gov/diseases/zika\_virus/docs/ zika\_webinar.pdf

**29**

**Chapter 3**

**Abstract**

Animal Models of Zika Virus

*Rafael K. Campos, Erin M. McDonald, Aaron C. Brault* 

ZIKV was first identified in the 1940s as a mosquito-borne virus; however, sexual transmission, which is uncommon for arboviruses, was demonstrated more than 60 years later. Tissue culture and animal models have allowed scientists to study how this transmission is possible. Immunocompromised mice infected with ZIKV had high viral loads in their testes, and infection of immunocompetent female mice was achieved following intravaginal inoculation or inoculation via mating with an infected male. These mouse studies lead researchers to investigate the individual components of the male reproductive system. In cell culture and mouse models, ZIKV can persist in Sertoli and germ cells of the testes and epithelial cells in the epididymis, which may lead to sexual transmission even after ZIKV has been cleared from other tissues. ZIKV has also been studied in nonhuman primates (NHPs), which appears to mimic the limited human epidemiological data, with low rates of symptomatic individuals and similar clinical signs. Although refinement is needed, these animal models have proven to be key in ZIKV research and continue to help uncovering the mechanisms of sexual transmission. This review will focus on the animal models used to elucidate the mechanisms of sexual transmission and

**Keywords:** Zika virus, sexual transmission, animal models, human, primate, mouse,

Zika virus (ZIKV) is a single-stranded, positive-sense virus of the genus *Flavivirus* of the family *Flaviviridae* that was discovered in the Ziika forest of Uganda in 1947 [1]. The virus was isolated during a surveillance campaign to attempt to identify yellow fever virus in the region. Sentinel rhesus macaques were placed in cages in the canopy layer of the trees and monitored daily for spikes in temperature. One of the rhesus macaques became febrile and was bled to isolate the causative agent of the fever. Serum from clarified blood caused illness when injected

intra-cranially in white mice and the brain homogenates from these animals contained the first isolate of ZIKV, MR766. It is noteworthy that this strain, which is used in many contemporaneous studies, was passaged over 100 times in mice to increase its virulence in rodents. A second strain, ZIKV 758, was made from another rhesus macaque injected with homogenates of *Aedes africanus* mosquitoes collected in that same area. These data demonstrate that ZIKV caused febrile disease in NHPs

Sexual Transmission

*and Shannan L. Rossi*

persistence of flaviviruses.

Sertoli cell, testes

**1. Introduction**

## **Chapter 3**

*Current Concepts in Zika Research*

for the Americas; 2015

2014;**11**(3):3360-3374

zika\_webinar.pdf

[38] MacDonald PDM, Wayne Holden E. Zika and public health: Understanding the epidemiology and information environment. Pediatrics.

2018;**141**(Suppl 2):S137-S145

[39] Howard Z. Zika virus. What New York State Clinicians Need to Know, NYS Commissioner of Health. 2016. Available from: https://www.health. ny.gov/diseases/zika\_virus/docs/

Implications for Public Health in the Americas. Washington, DC: Pan American Health Organization, World Health Organization, Regional Office

[36] Haddow AJ, Williams M, Woodall J, et al. Twelve isolations of Zika virus from *Aedes* (*Stegomyia*) *africanus*

(*Theobald*) taken in and above a Uganda forest. Bulletin of the World Health Organization. 1964;**31**(1):57-69

[37] Thomas SM, Tjaden NB, van den Bos S, Beierkuhnlein C. Implementing cargo movement into climate based risk assessment of vector-borne diseases. International Journal of Environmental Research and Public Health AJPH.

**28**

## Animal Models of Zika Virus Sexual Transmission

*Rafael K. Campos, Erin M. McDonald, Aaron C. Brault and Shannan L. Rossi*

### **Abstract**

ZIKV was first identified in the 1940s as a mosquito-borne virus; however, sexual transmission, which is uncommon for arboviruses, was demonstrated more than 60 years later. Tissue culture and animal models have allowed scientists to study how this transmission is possible. Immunocompromised mice infected with ZIKV had high viral loads in their testes, and infection of immunocompetent female mice was achieved following intravaginal inoculation or inoculation via mating with an infected male. These mouse studies lead researchers to investigate the individual components of the male reproductive system. In cell culture and mouse models, ZIKV can persist in Sertoli and germ cells of the testes and epithelial cells in the epididymis, which may lead to sexual transmission even after ZIKV has been cleared from other tissues. ZIKV has also been studied in nonhuman primates (NHPs), which appears to mimic the limited human epidemiological data, with low rates of symptomatic individuals and similar clinical signs. Although refinement is needed, these animal models have proven to be key in ZIKV research and continue to help uncovering the mechanisms of sexual transmission. This review will focus on the animal models used to elucidate the mechanisms of sexual transmission and persistence of flaviviruses.

**Keywords:** Zika virus, sexual transmission, animal models, human, primate, mouse, Sertoli cell, testes

### **1. Introduction**

Zika virus (ZIKV) is a single-stranded, positive-sense virus of the genus *Flavivirus* of the family *Flaviviridae* that was discovered in the Ziika forest of Uganda in 1947 [1]. The virus was isolated during a surveillance campaign to attempt to identify yellow fever virus in the region. Sentinel rhesus macaques were placed in cages in the canopy layer of the trees and monitored daily for spikes in temperature. One of the rhesus macaques became febrile and was bled to isolate the causative agent of the fever. Serum from clarified blood caused illness when injected intra-cranially in white mice and the brain homogenates from these animals contained the first isolate of ZIKV, MR766. It is noteworthy that this strain, which is used in many contemporaneous studies, was passaged over 100 times in mice to increase its virulence in rodents. A second strain, ZIKV 758, was made from another rhesus macaque injected with homogenates of *Aedes africanus* mosquitoes collected in that same area. These data demonstrate that ZIKV caused febrile disease in NHPs

and could be isolated from mosquitoes, solidifying it as an arbovirus [1]. In the years since, many additional isolates have been made and the ones discussed in this review are listed in **Table 1**.

Prior to the major outbreaks in the 2000s, ZIKV had not been detected in the Americas and reported human cases of ZIKV-caused disease were scarce worldwide. Seroprevalence studies in Asia and Africa revealed human populations were exposed to the virus but disease was rarely reported, likely due to a high percentage of asymptomatic infection or because the signs and symptoms were indistinguishable from other known diseases [2]. Data collected during recent outbreaks in Yap Island and across the Americas revealed ZIKV is usually asymptomatic, with symptomatic cases being characterized by flu-like symptoms including self-limiting fever, headache, rash, and myalgia [3].

Worldwide outbreaks in Gabon, Micronesia and French Polynesia expanded the virus' known range and susceptible population, allowing prospective epidemiological studies. ZIKV likely arrived in the Americas in 2014, and in 2015 it quickly spread, starting its largest outbreaks to date [4, 5]. The large number of infected patients and heightened medical and scientific response allowed rare outcomes and transmission routes to be noticed. It was possible to identify that ZIKV causes congenital malformations [6]. Prior to this, cases of miscarriage, microcephaly and other congenital malformations, such as microcephaly or damage to brain and eye tissues, had been identified and were retrospectively observed to be correlated with infection [7, 8]. Infected travelers returning home initiated small traveler-associated transmission cycles, some of which were between sexual partners. Sexual transmission was proposed by Foy and colleagues, who described a probable case of ZIKV sexual transmission of a scientist that visited Senegal in 2008 [9]. Many more cases of sexual transmission were reported after the outbreaks in the Americas, confirming that sexual transmission played a role in the spread of ZIKV, although the full impact of sexual transmission may be underappreciated since it occurs concomitantly with the more efficient transmission by mosquito vectors [10–13].

ZIKV or viral RNA was found to persist in human semen, vaginal secretions and blood [14, 15]. Acquiring samples to study viral persistence in these tissues is difficult because ZIKV infection is frequently asymptomatic and identification of ZIKV-positive samples has to be done by serology or molecular biology, since other viruses that cause similar signs and symptoms are present in the same regions as


**31**

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

ZIKV. In Brazil, for example, there are several other flaviviruses and alphaviruses in circulation which produce clinical pictures that cannot be readily differentiated from the signs and symptoms associated with ZIKV. Therefore, molecular and serological tools are critical for the identification and classification of such samples [16]. In many locations, this requires a concerted collaborative effort between hospitals and laboratories, and many samples may need to be shipped before determining which patients came in contact with ZIKV. Additionally, sample collection is made difficult because once symptoms have rescinded, sample collections can no longer be made by hospitals and people may be opposed to having samples collected from them when they are no longer in need of medical care. In spite of these obstacles, several studies were able to assess ZIKV persistence in human samples. In semen, ZIKV RNA was detected for up to 6 months and ZIKV infectious particles could be isolated up to 1 month after primary infection [17]. Vaginal secretions were also found to be positive for RNA for up to 6 months after onset of symptoms [18]. These observations suggest sexual transmission could potentially happen even long after resolution of primary infection. Caution must be taken when trying to directly interpret the consequences of persistent viral RNA since this does not directly imply infectious viral particles. Regardless, transmission is possible, as demonstrated by a woman infecting her partner 44 days after onset of symptoms [13]. Although sexual transmission has most often been reported from males to females, there have been reports of suspected male-to-female [19], male-to-male [20] and female-to-male [21] transmissions. The actual rate of sexual transmission remains unknown.

Our current knowledge of the potential for sexual transmission among members

Examples of sexual transmission may be more common in the family *Flaviviridae* than in the genus *Flavivirus*, as exemplified by bovine viral diarrhea virus (pestivirus, BVDV) in cattle and hepatitis C virus (hepacivirus, HCV) in humans. Both of

of the *Flavivirus* genus is scarce but it appears that ZIKV is unique among this genus since most members are transmitted between hematophagous arthropods and vertebrate hosts. Although there is limited evidence that other flaviviruses could be sexually transmitted and persist in semen, the currently available body of data does not support this mode of transmission as significant for the spread of these viruses. West Nile virus (WNV) was proposed to have been possibly sexually transmitted in a clinical study of a woman who developed meningo-encephalitis by WNV 2 weeks after intercourse with an infected individual [22]. Other medically important flaviviruses, dengue virus (DENV) [23] and yellow fever virus (YFV) [24], have both been detected in human semen. DENV RNA was detected in a man returning from Thailand to Italy at 37 days post onset of symptoms, when virus RNA was not detectable in the serum or urine anymore [23]. Detection of DENV RNA in the seminal fluid is nevertheless not commonly reported, and another study that performed PCR in the semen of five man with acute DENV infection failed to detect any viral RNA [25]. YFV RNA was detected in urine and semen, but not in the serum, and the virus was isolated from urine on day 21 post onset the symptoms [24]. Another flavivirus, Japanese encephalitis virus (JEV), can be naturally found in porcine semen [26]. JEV was isolated from aborted fetus and semen of pigs which were naturally infected in China from 2004 to 2009 [27]. This raises the question of whether JEV also has the potential to reside in human testes and semen. Experimental approaches have also been used to determine the potential of flaviviruses to be sexually transmitted. Spondweni virus, a close relative to ZIKV, was detected in semen of mice lacking types I and III interferon (AG129) that were inoculated subcutaneously, although this was a rare occurrence when compared to ZIKV-infected AG129 mice [28]. Despite these clinical and experimental reports, the extent of the contribution of sexual transmission to the spread and maintenance

of ZIKV in the human population remains unknown.

**Table 1.** *Commonly used ZIKV strains.*

#### *Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

*Current Concepts in Zika Research*

review are listed in **Table 1**.

**Strain Year** 

**Isolation**

PRVABC59 2015 Asian

BeH815744 2015 Asian

MR766 1947 African Ziika forest,

FSS13025 2010 Asian French

H/PF/2013 2013 Asian French

(American)

(American)

fever, headache, rash, and myalgia [3].

and could be isolated from mosquitoes, solidifying it as an arbovirus [1]. In the years since, many additional isolates have been made and the ones discussed in this

Prior to the major outbreaks in the 2000s, ZIKV had not been detected in the Americas and reported human cases of ZIKV-caused disease were scarce worldwide. Seroprevalence studies in Asia and Africa revealed human populations were exposed to the virus but disease was rarely reported, likely due to a high percentage of asymptomatic infection or because the signs and symptoms were indistinguishable from other known diseases [2]. Data collected during recent outbreaks in Yap Island and across the Americas revealed ZIKV is usually asymptomatic, with symptomatic cases being characterized by flu-like symptoms including self-limiting

Worldwide outbreaks in Gabon, Micronesia and French Polynesia expanded the virus' known range and susceptible population, allowing prospective epidemiological studies. ZIKV likely arrived in the Americas in 2014, and in 2015 it quickly spread, starting its largest outbreaks to date [4, 5]. The large number of infected patients and heightened medical and scientific response allowed rare outcomes and transmission routes to be noticed. It was possible to identify that ZIKV causes congenital malformations [6]. Prior to this, cases of miscarriage, microcephaly and other congenital malformations, such as microcephaly or damage to brain and eye tissues, had been identified and were retrospectively observed to be correlated with infection [7, 8]. Infected travelers returning home initiated small traveler-associated transmission cycles, some of which were between sexual partners. Sexual transmission was proposed by Foy and colleagues, who described a probable case of ZIKV sexual transmission of a scientist that visited Senegal in 2008 [9]. Many more cases of sexual transmission were reported after the outbreaks in the Americas, confirming that sexual transmission played a role in the spread of ZIKV, although the full impact of sexual transmission may be underappreciated since it occurs concomi-

tantly with the more efficient transmission by mosquito vectors [10–13].

ZIKV or viral RNA was found to persist in human semen, vaginal secretions and blood [14, 15]. Acquiring samples to study viral persistence in these tissues is difficult because ZIKV infection is frequently asymptomatic and identification of ZIKV-positive samples has to be done by serology or molecular biology, since other viruses that cause similar signs and symptoms are present in the same regions as

**Lineage Location Notes**

Contemporaneously available strains are heavily passaged in mice, altering their virulence

the American outbreak

Common isolate used collected prior to the American outbreak. Human isolate

Human isolate

Puerto Rico Isolated from human serum during

Brazil Human isolate

Uganda

Polynesia

Polynesia

IBH30656 1968 African Nigeria Isolated from human blood sample

**30**

**Table 1.**

*Commonly used ZIKV strains.*

ZIKV. In Brazil, for example, there are several other flaviviruses and alphaviruses in circulation which produce clinical pictures that cannot be readily differentiated from the signs and symptoms associated with ZIKV. Therefore, molecular and serological tools are critical for the identification and classification of such samples [16]. In many locations, this requires a concerted collaborative effort between hospitals and laboratories, and many samples may need to be shipped before determining which patients came in contact with ZIKV. Additionally, sample collection is made difficult because once symptoms have rescinded, sample collections can no longer be made by hospitals and people may be opposed to having samples collected from them when they are no longer in need of medical care. In spite of these obstacles, several studies were able to assess ZIKV persistence in human samples. In semen, ZIKV RNA was detected for up to 6 months and ZIKV infectious particles could be isolated up to 1 month after primary infection [17]. Vaginal secretions were also found to be positive for RNA for up to 6 months after onset of symptoms [18]. These observations suggest sexual transmission could potentially happen even long after resolution of primary infection. Caution must be taken when trying to directly interpret the consequences of persistent viral RNA since this does not directly imply infectious viral particles. Regardless, transmission is possible, as demonstrated by a woman infecting her partner 44 days after onset of symptoms [13]. Although sexual transmission has most often been reported from males to females, there have been reports of suspected male-to-female [19], male-to-male [20] and female-to-male [21] transmissions. The actual rate of sexual transmission remains unknown.

Our current knowledge of the potential for sexual transmission among members of the *Flavivirus* genus is scarce but it appears that ZIKV is unique among this genus since most members are transmitted between hematophagous arthropods and vertebrate hosts. Although there is limited evidence that other flaviviruses could be sexually transmitted and persist in semen, the currently available body of data does not support this mode of transmission as significant for the spread of these viruses. West Nile virus (WNV) was proposed to have been possibly sexually transmitted in a clinical study of a woman who developed meningo-encephalitis by WNV 2 weeks after intercourse with an infected individual [22]. Other medically important flaviviruses, dengue virus (DENV) [23] and yellow fever virus (YFV) [24], have both been detected in human semen. DENV RNA was detected in a man returning from Thailand to Italy at 37 days post onset of symptoms, when virus RNA was not detectable in the serum or urine anymore [23]. Detection of DENV RNA in the seminal fluid is nevertheless not commonly reported, and another study that performed PCR in the semen of five man with acute DENV infection failed to detect any viral RNA [25]. YFV RNA was detected in urine and semen, but not in the serum, and the virus was isolated from urine on day 21 post onset the symptoms [24]. Another flavivirus, Japanese encephalitis virus (JEV), can be naturally found in porcine semen [26]. JEV was isolated from aborted fetus and semen of pigs which were naturally infected in China from 2004 to 2009 [27]. This raises the question of whether JEV also has the potential to reside in human testes and semen. Experimental approaches have also been used to determine the potential of flaviviruses to be sexually transmitted. Spondweni virus, a close relative to ZIKV, was detected in semen of mice lacking types I and III interferon (AG129) that were inoculated subcutaneously, although this was a rare occurrence when compared to ZIKV-infected AG129 mice [28]. Despite these clinical and experimental reports, the extent of the contribution of sexual transmission to the spread and maintenance of ZIKV in the human population remains unknown.

Examples of sexual transmission may be more common in the family *Flaviviridae* than in the genus *Flavivirus*, as exemplified by bovine viral diarrhea virus (pestivirus, BVDV) in cattle and hepatitis C virus (hepacivirus, HCV) in humans. Both of

these infections can cause persistent infections in their hosts under certain conditions and can be sexually transmitted. For BVDV, persistent infection was found in the testes, and bulls persistently shed viral particles in the seminal fluid which allows for sexual transmission [29–31]. For HCV, sexual transmission has been well-documented [32–35]; however, transmission is relatively inefficient [36] and increases with the number of partners, among human immunodeficient viruspositive (HIV+) individuals or among men who have sex with other men [37, 38]. Although there are similarities between ZIKV and these two viruses, caution must be exercised when drawing comparisons, especially when looking for viral determinants of persistence since there are multiple and substantial genomic differences between flaviviruses, pestiviruses and hepaciviruses.

The fact that ZIKV was shown to be sexually transmitted raised many questions, as many aspects of this mode of transmission are poorly understood for these viruses. Namely, the importance of sexual transmission for ZIKV in different regions of the world, the role of cells of the reproductive tract involved in ZIKV persistence and transmission, mutations in the virus which favor successful sexual transmission, interactions of ZIKV with the immune system in the reproductive tract and molecular interactions between ZIKV and host cells allowing for sexual transmission to take place, are all critical points of study. Although abundant data on human disease was generated during the recent outbreaks, animal experiments are an important tool to answer these questions because they allow controlled and rationally designed experiments which could ultimately lead to the development of new vaccines, therapeutics and prophylactic measures. This review will focus on what is currently known for ZIKV infection in the male reproductive tract.

## **2. Animal models of sexual transmission**

Upon discovery of ZIKV, animal experiments were first conducted by Dick [39]. They found that white mice younger than 2 weeks were susceptible to ZIKV infections via intraperitoneal route, whereas mice older than 2 weeks were only susceptible to intracerebral inoculations [39]. Non-human primates inoculated subcutaneously developed neutralizing antibodies against ZIKV and only one animal presented a slight elevation in temperature [39, 40]. Several decades after this initial study, several other model species were used as animal models for ZIKV, but efforts were mostly focused on mice [41–43] and non-human primates (NHPs) [44–47], with most studies being conducted after the outbreak in the Americas in 2015–2016. The experimental design and analyses should carefully consider the anatomical and physiological differences from the species used to humans as well as confounding factors such as different viral strains and inoculation titer and route.

#### **2.1 Mouse models**

Mouse models of sexual transmission for ZIKV initially utilized either interferon receptor deficient mice lacking type I (A129) [41] and or type I and II (AG129) interferon receptors [42]. Other models included interferon responsive mouse strains for which a transient knock-down of interferon response was induced by exogenous treatment with antibody against murine type I interferon receptor of wild-type mice or Rag1<sup>−</sup>/<sup>−</sup> mice (lacking both B and T lymphocytes) [48]. Several of these models have demonstrated sexual transmission from needle-inoculated male mice to naive female mice following mating. These studies, coupled with the use of surrogate breeding females from which uteri were gavaged, allowed direct assessment of virus and viral RNA shedding efficiency over time. AG129 males infected

**33**

for fetal involvement [57].

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

significantly lower levels of ZIKV RNA [17].

*2.1.1 Sexual transmission murine models*

ZIKV can replicate and induce pathogenic responses [51].

with ZIKV strain PRVABC59 were shown to shed infectious virus from 7 to 21 days post infection (dpi). Vasectomized males also were shown to shed virus; however, the magnitude was shown to be significantly lower compared to non-vasectomized males [42]. This finding was consistent with a previous study reporting ZIKV RNA shedding in symptomatic men infected with ZIKV in which vasectomized men shed

The necessity for the use of mice lacking the ability to respond to interferon is due to the inherent resistance of murine STAT2 from being inhibited by ZIKV NS5 [49]. In contrast, human STAT2 has been demonstrated to be highly susceptible to antagonism by ZIKV NS5 [50]. As such, the subsequent development of humanized STAT2 mice have proven useful as a fully interferon responsive model for which

A number of different models have been developed in order to directly assess sexual transmission Sexual transmission has been modeled via direct intravaginal inoculation. In one model, AG129 male mice euthanized and caudal epididymal lumen (containing sperm) was collected. This suspension was used to inoculate female AG129 mice. In this study, the antioxidant ebselen was used to alleviate testicular pathology caused by ZIKV. Although intravaginally inoculated female AG129 mice with sperm from male mice treated with ebselen demonstrated reduced mortality, sexual transmission was not prevented, as female organs (ovary/fallopian tubes, spleen and brain) were infected [52]. Other studies have used homogenized accessory gland fluid and epididymal lumen fluid from Ifnar1<sup>−</sup>/<sup>−</sup> male mice subcutaneously inoculated with PRVABC59 to intravaginally inoculated female AG129 mice. In this study, females became viremic and succumbed to infection. Furthermore, progesterone pre-treatment of female mice before intravaginal inoculation was shown to increase mortality of females [53]. IFNAR1<sup>−</sup>/<sup>−</sup> male mice inoculated subcutaneously with PRVABC59 and then at 14 dpi or 35 dpi, prostatic and seminal vesicular homogenates and epididymal flushes were collected. Female AG129 mice inoculated intravaginally with this insemination fluid failed to become viremic [54].

Intravaginal inoculation of di-estrus timed AG129 mice or LysMCre<sup>+</sup>

mice (lacking IFNAR in myeloid cells) was shown to result in viremia and virus replication in peripheral organs and in the vaginal tissues measured by viral assay by RT-PCR through 10 dpi [55]. In an alternative study, immunocompetent C57BL/6 N female mice intravaginally inoculated with PRVABC59 showed a slight increase in viral RNA in the lower female reproductive tract (LFRT) from dpi 1 to 2 and mRNA expression of type I and III IFNs, IRF3/7, RIG-I, and MDA-5 was comparable to uninfected controls, suggesting that a dampened antiviral immune response occurs in the LFRT in response to infection with ZIKV. When mice were treated with an enhancer of RIG-1 signaling, the increase in ZIKV RNA in LFRT was not observed [56]. After intravaginal inoculation during the diestrus phase, ZIKV was shown to replicate in vaginal mucosa of wild type (WT) C57BL/6 and Ifnar1<sup>−</sup>/<sup>−</sup> mice. Fetuses from pregnant WT C57BL/6 mice developed intrauterine growth restriction and fetal brains were infected following intravaginal ZIKV inoculation. Fetuses from pregnant IFNAR1<sup>−</sup>/<sup>−</sup> mice developed severe intrauterine growth restriction and fetal death was observed. The pregnant females were found to develop viremia. These data are suggestive of an ascending infection from the vaginal tissues to the uterus

In addition to sexual transmission models that have utilized intravaginal exposure to model sexual transmission potential, several studies have addressed transmission dynamics through direct coitus models. AG129 males were found to

IFNARfl/fl

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

*Current Concepts in Zika Research*

between flaviviruses, pestiviruses and hepaciviruses.

**2. Animal models of sexual transmission**

these infections can cause persistent infections in their hosts under certain conditions and can be sexually transmitted. For BVDV, persistent infection was found in the testes, and bulls persistently shed viral particles in the seminal fluid which allows for sexual transmission [29–31]. For HCV, sexual transmission has been well-documented [32–35]; however, transmission is relatively inefficient [36] and increases with the number of partners, among human immunodeficient viruspositive (HIV+) individuals or among men who have sex with other men [37, 38]. Although there are similarities between ZIKV and these two viruses, caution must be exercised when drawing comparisons, especially when looking for viral determinants of persistence since there are multiple and substantial genomic differences

The fact that ZIKV was shown to be sexually transmitted raised many questions, as many aspects of this mode of transmission are poorly understood for these viruses. Namely, the importance of sexual transmission for ZIKV in different regions of the world, the role of cells of the reproductive tract involved in ZIKV persistence and transmission, mutations in the virus which favor successful sexual transmission, interactions of ZIKV with the immune system in the reproductive tract and molecular interactions between ZIKV and host cells allowing for sexual transmission to take place, are all critical points of study. Although abundant data on human disease was generated during the recent outbreaks, animal experiments are an important tool to answer these questions because they allow controlled and rationally designed experiments which could ultimately lead to the development of new vaccines, therapeutics and prophylactic measures. This review will focus on

what is currently known for ZIKV infection in the male reproductive tract.

factors such as different viral strains and inoculation titer and route.

Upon discovery of ZIKV, animal experiments were first conducted by Dick [39]. They found that white mice younger than 2 weeks were susceptible to ZIKV infections via intraperitoneal route, whereas mice older than 2 weeks were only susceptible to intracerebral inoculations [39]. Non-human primates inoculated subcutaneously developed neutralizing antibodies against ZIKV and only one animal presented a slight elevation in temperature [39, 40]. Several decades after this initial study, several other model species were used as animal models for ZIKV, but efforts were mostly focused on mice [41–43] and non-human primates (NHPs) [44–47], with most studies being conducted after the outbreak in the Americas in 2015–2016. The experimental design and analyses should carefully consider the anatomical and physiological differences from the species used to humans as well as confounding

Mouse models of sexual transmission for ZIKV initially utilized either interferon

receptor deficient mice lacking type I (A129) [41] and or type I and II (AG129) interferon receptors [42]. Other models included interferon responsive mouse strains for which a transient knock-down of interferon response was induced by exogenous treatment with antibody against murine type I interferon receptor of wild-type mice or Rag1<sup>−</sup>/<sup>−</sup> mice (lacking both B and T lymphocytes) [48]. Several of these models have demonstrated sexual transmission from needle-inoculated male mice to naive female mice following mating. These studies, coupled with the use of surrogate breeding females from which uteri were gavaged, allowed direct assessment of virus and viral RNA shedding efficiency over time. AG129 males infected

**32**

**2.1 Mouse models**

with ZIKV strain PRVABC59 were shown to shed infectious virus from 7 to 21 days post infection (dpi). Vasectomized males also were shown to shed virus; however, the magnitude was shown to be significantly lower compared to non-vasectomized males [42]. This finding was consistent with a previous study reporting ZIKV RNA shedding in symptomatic men infected with ZIKV in which vasectomized men shed significantly lower levels of ZIKV RNA [17].

The necessity for the use of mice lacking the ability to respond to interferon is due to the inherent resistance of murine STAT2 from being inhibited by ZIKV NS5 [49]. In contrast, human STAT2 has been demonstrated to be highly susceptible to antagonism by ZIKV NS5 [50]. As such, the subsequent development of humanized STAT2 mice have proven useful as a fully interferon responsive model for which ZIKV can replicate and induce pathogenic responses [51].

#### *2.1.1 Sexual transmission murine models*

A number of different models have been developed in order to directly assess sexual transmission Sexual transmission has been modeled via direct intravaginal inoculation. In one model, AG129 male mice euthanized and caudal epididymal lumen (containing sperm) was collected. This suspension was used to inoculate female AG129 mice. In this study, the antioxidant ebselen was used to alleviate testicular pathology caused by ZIKV. Although intravaginally inoculated female AG129 mice with sperm from male mice treated with ebselen demonstrated reduced mortality, sexual transmission was not prevented, as female organs (ovary/fallopian tubes, spleen and brain) were infected [52]. Other studies have used homogenized accessory gland fluid and epididymal lumen fluid from Ifnar1<sup>−</sup>/<sup>−</sup> male mice subcutaneously inoculated with PRVABC59 to intravaginally inoculated female AG129 mice. In this study, females became viremic and succumbed to infection. Furthermore, progesterone pre-treatment of female mice before intravaginal inoculation was shown to increase mortality of females [53]. IFNAR1<sup>−</sup>/<sup>−</sup> male mice inoculated subcutaneously with PRVABC59 and then at 14 dpi or 35 dpi, prostatic and seminal vesicular homogenates and epididymal flushes were collected. Female AG129 mice inoculated intravaginally with this insemination fluid failed to become viremic [54].

Intravaginal inoculation of di-estrus timed AG129 mice or LysMCre<sup>+</sup> IFNARfl/fl mice (lacking IFNAR in myeloid cells) was shown to result in viremia and virus replication in peripheral organs and in the vaginal tissues measured by viral assay by RT-PCR through 10 dpi [55]. In an alternative study, immunocompetent C57BL/6 N female mice intravaginally inoculated with PRVABC59 showed a slight increase in viral RNA in the lower female reproductive tract (LFRT) from dpi 1 to 2 and mRNA expression of type I and III IFNs, IRF3/7, RIG-I, and MDA-5 was comparable to uninfected controls, suggesting that a dampened antiviral immune response occurs in the LFRT in response to infection with ZIKV. When mice were treated with an enhancer of RIG-1 signaling, the increase in ZIKV RNA in LFRT was not observed [56]. After intravaginal inoculation during the diestrus phase, ZIKV was shown to replicate in vaginal mucosa of wild type (WT) C57BL/6 and Ifnar1<sup>−</sup>/<sup>−</sup> mice. Fetuses from pregnant WT C57BL/6 mice developed intrauterine growth restriction and fetal brains were infected following intravaginal ZIKV inoculation. Fetuses from pregnant IFNAR1<sup>−</sup>/<sup>−</sup> mice developed severe intrauterine growth restriction and fetal death was observed. The pregnant females were found to develop viremia. These data are suggestive of an ascending infection from the vaginal tissues to the uterus for fetal involvement [57].

In addition to sexual transmission models that have utilized intravaginal exposure to model sexual transmission potential, several studies have addressed transmission dynamics through direct coitus models. AG129 males were found to sexually transmit to naïve AG129 females in 50% of all matings as measured by subsequent viremias in mated females. This initial study demonstrated *in utero* infection after sexual transmission [42]. A subsequent study compared sexual transmission with subcutaneous and intravaginal routes on female disease presentation, tropism and fetal infection. Sexual transmission of ZIKV to naïve female AG129 mice increased morbidity and mortality in these females as compared to female mice subcutaneously or intravaginally inoculated. Fetuses from females infected via sexual transmission had higher ZIKV titers compared to fetuses from pregnant females infected subcutaneously or intravaginally [58].

### *2.1.2 Potential sources of sexually transmitted virus/ZIKV infection of the murine testes and epididymides*

The majority of ZIKV detected in the seminal fluid of infected AG129 mice during the peak timing of sexual transmission (10–12 dpi) was from the supernatant fraction, suggesting cell-free ZIKV may be largely responsible for sexual transmission. In this study, the testes and epididymides were determined to be infected concurrently and epididymal epithelial cells were identified as the predominant cell population infected in epididymides and shown to contain replicating ZIKV by *in situ* hybridization. In the testes, interstitial leukocytes and peritubular myoid cells were found to be infected initially, followed by extensive infection of all layers of the seminiferous tubule epithelium [59]. Similar results were also shown in IFNAR1<sup>−</sup>/<sup>−</sup> mice for which epithelial components of epididymides were identified to be infected and that testes and epididymides could be infected concurrently [60]. In another experiment with immune competent C57BL/6 mice treated with anti-IFNAR1 blocking antibody and subcutaneously inoculated with Asian and African genotype ZIKVs, sexual transmission potential was observed for all viruses with infectious virus identified in the epididymides from all groups even when infectious virus was absent from the testes and seminal vesicles. Infection of the epididymides was demonstrated to be critical for establishing sexual transmission potential, as infectious virus and viral RNA was detected in the epididymides and in semen days before infectious virus was detected in the seminal vesicles or testes [61].

Tissue restricted ZIKVs generated through the incorporation of microRNA target sequences within recombinant ZIKVs were utilized to assess the importance of different cell populations for sexual transmission potential. Testes-restricted ZIKVs could still infect the epididymides, demonstrating a hematogenous/ lymphogenous route of infection. Epididymides-restricted ZIKV had high titers in epididymides by plaque assay, but immunohistochemical analysis confirmed epididymides-restricted ZIKV did not replicate in the epididymal epithelium, suggesting that ZIKV is transported from testes to epididymides via excurrent ducts in a cell-free form or transported in sloughed spermatids/infected luminal leukocytes, and ZIKV can infect the epididymides via hematogenous/lymphogenous route of infection [62].

#### *2.1.3 Persistence and tropism of ZIKV on the male reproductive tract*

Tropism and persistence of ZIKV in the male reproductive tract may be the key factor responsible for the presence of the virus in semen even long after initial infection. A model of the male mouse reproductive tract is shown in **Figure 1A**. Based on *in vivo* studies, ZIKV is thought to infect the testes, an immunologically privileged site, via a hematogenous route [62] and by infecting the Sertoli cells (SC, **Figure 1B**), an important cell population responsible for the formation of the blood-testes barrier, as shown using AG129 mouse models [63, 64]. SC are critical

**35**

**Figure 1.**

*tubules. (C) Seminiferous tubules in detail.*

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

source of cell-free ZIKV in the seminal fluid [66].

for spermatogenesis, since they nourish germ cells and help them mature. Tight junctions formed by adjacent SC are functional components of the blood-testes barrier (**Figure 1C**), which prevents molecules from passing between the blood and the lumen of a seminiferous tubule. From the testes, ZIKV can then reach the epididymides by the excurrent testicular route either by infecting germ cells or as free virus particles [62]. ZIKV can also infect the epididymides directly from the hematogenous route [62] (**Figure 1**), and infection of epididymides was observed to happen concurrently with testes infection, indicating that epididymal infection could happen directly and through the testes, or independently of the testes. Infection of germ cells in the testes is not a requirement for sexual transmission as there are reported cases of ZIKV sexual transmission from vasectomized men [65] and in experiments with vasectomized AG129s [42]. Instead, AG129 mouse models indicate that infection of epididymal epithelial cells may the major factor leading to shedding of virus particles in the semen, with these cells being the predominant

Intracellular viral persistence is likely an important component for long-term sexual transmission. Although the cells that act as reservoirs of ZIKV in testes and the reproductive tract are unknown, possible reservoirs in the host are SC [67, 68] germ cells [69], Leydig cells [70] and epididymal epithelial cells [59, 62] which have been shown to support persistent infections of ZIKV (**Figure 1**). When primary SCs persistently infected with two strains of ZIKV (PRVABC59 or MR766) were monitored for a period of 6 weeks, it was found that 15% of the cells were still positive for both strains of ZIKV [67]. In this same study, Leydig cells were not observed to support persistence [67]. The mechanisms underlying persistence of ZIKV in testes are likely multifactorial and represent a complex phenomenon involving interactions between viral and host factors that needs to be studied in depth. The interactions that occur between ZIKV and host factors that are required for long-term infection of the testes/epididymides are poorly understood and viral persistence likely requires a balance between efficient viral replication and damage caused to host cells. The AXL receptor tyrosine kinase, which was previously shown to be required for entry of ZIKV and other flaviviruses into certain cell types, has been shown to be required for ZIKV entry in SCs [71], but may also be involved in negatively regulating SCs innate immune response [72]. ZIKV infection of SCs results in gene expression changes, with upregulation of antiviral pathways, dysregulation of junction and growth pathways [67, 73]. Although the immune

*The male reproductive tract. (A) Overall representation of the male reproductive tract. (B) Seminiferous* 

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

*Current Concepts in Zika Research*

*testes and epididymides*

sexually transmit to naïve AG129 females in 50% of all matings as measured by subsequent viremias in mated females. This initial study demonstrated *in utero* infection after sexual transmission [42]. A subsequent study compared sexual transmission with subcutaneous and intravaginal routes on female disease presentation, tropism and fetal infection. Sexual transmission of ZIKV to naïve female AG129 mice increased morbidity and mortality in these females as compared to female mice subcutaneously or intravaginally inoculated. Fetuses from females infected via sexual transmission had higher ZIKV titers compared to fetuses from

*2.1.2 Potential sources of sexually transmitted virus/ZIKV infection of the murine* 

before infectious virus was detected in the seminal vesicles or testes [61].

*2.1.3 Persistence and tropism of ZIKV on the male reproductive tract*

Tissue restricted ZIKVs generated through the incorporation of microRNA target sequences within recombinant ZIKVs were utilized to assess the importance of different cell populations for sexual transmission potential. Testes-restricted ZIKVs could still infect the epididymides, demonstrating a hematogenous/ lymphogenous route of infection. Epididymides-restricted ZIKV had high titers in epididymides by plaque assay, but immunohistochemical analysis confirmed epididymides-restricted ZIKV did not replicate in the epididymal epithelium, suggesting that ZIKV is transported from testes to epididymides via excurrent ducts in a cell-free form or transported in sloughed spermatids/infected luminal leukocytes, and ZIKV can infect the epididymides via hematogenous/lymphogenous

Tropism and persistence of ZIKV in the male reproductive tract may be the key factor responsible for the presence of the virus in semen even long after initial infection. A model of the male mouse reproductive tract is shown in **Figure 1A**. Based on *in vivo* studies, ZIKV is thought to infect the testes, an immunologically privileged site, via a hematogenous route [62] and by infecting the Sertoli cells (SC, **Figure 1B**), an important cell population responsible for the formation of the blood-testes barrier, as shown using AG129 mouse models [63, 64]. SC are critical

The majority of ZIKV detected in the seminal fluid of infected AG129 mice during the peak timing of sexual transmission (10–12 dpi) was from the supernatant fraction, suggesting cell-free ZIKV may be largely responsible for sexual transmission. In this study, the testes and epididymides were determined to be infected concurrently and epididymal epithelial cells were identified as the predominant cell population infected in epididymides and shown to contain replicating ZIKV by *in situ* hybridization. In the testes, interstitial leukocytes and peritubular myoid cells were found to be infected initially, followed by extensive infection of all layers of the seminiferous tubule epithelium [59]. Similar results were also shown in IFNAR1<sup>−</sup>/<sup>−</sup> mice for which epithelial components of epididymides were identified to be infected and that testes and epididymides could be infected concurrently [60]. In another experiment with immune competent C57BL/6 mice treated with anti-IFNAR1 blocking antibody and subcutaneously inoculated with Asian and African genotype ZIKVs, sexual transmission potential was observed for all viruses with infectious virus identified in the epididymides from all groups even when infectious virus was absent from the testes and seminal vesicles. Infection of the epididymides was demonstrated to be critical for establishing sexual transmission potential, as infectious virus and viral RNA was detected in the epididymides and in semen days

pregnant females infected subcutaneously or intravaginally [58].

**34**

route of infection [62].

for spermatogenesis, since they nourish germ cells and help them mature. Tight junctions formed by adjacent SC are functional components of the blood-testes barrier (**Figure 1C**), which prevents molecules from passing between the blood and the lumen of a seminiferous tubule. From the testes, ZIKV can then reach the epididymides by the excurrent testicular route either by infecting germ cells or as free virus particles [62]. ZIKV can also infect the epididymides directly from the hematogenous route [62] (**Figure 1**), and infection of epididymides was observed to happen concurrently with testes infection, indicating that epididymal infection could happen directly and through the testes, or independently of the testes. Infection of germ cells in the testes is not a requirement for sexual transmission as there are reported cases of ZIKV sexual transmission from vasectomized men [65] and in experiments with vasectomized AG129s [42]. Instead, AG129 mouse models indicate that infection of epididymal epithelial cells may the major factor leading to shedding of virus particles in the semen, with these cells being the predominant source of cell-free ZIKV in the seminal fluid [66].

Intracellular viral persistence is likely an important component for long-term sexual transmission. Although the cells that act as reservoirs of ZIKV in testes and the reproductive tract are unknown, possible reservoirs in the host are SC [67, 68] germ cells [69], Leydig cells [70] and epididymal epithelial cells [59, 62] which have been shown to support persistent infections of ZIKV (**Figure 1**). When primary SCs persistently infected with two strains of ZIKV (PRVABC59 or MR766) were monitored for a period of 6 weeks, it was found that 15% of the cells were still positive for both strains of ZIKV [67]. In this same study, Leydig cells were not observed to support persistence [67]. The mechanisms underlying persistence of ZIKV in testes are likely multifactorial and represent a complex phenomenon involving interactions between viral and host factors that needs to be studied in depth. The interactions that occur between ZIKV and host factors that are required for long-term infection of the testes/epididymides are poorly understood and viral persistence likely requires a balance between efficient viral replication and damage caused to host cells. The AXL receptor tyrosine kinase, which was previously shown to be required for entry of ZIKV and other flaviviruses into certain cell types, has been shown to be required for ZIKV entry in SCs [71], but may also be involved in negatively regulating SCs innate immune response [72]. ZIKV infection of SCs results in gene expression changes, with upregulation of antiviral pathways, dysregulation of junction and growth pathways [67, 73]. Although the immune

#### **Figure 1.**

*The male reproductive tract. (A) Overall representation of the male reproductive tract. (B) Seminiferous tubules. (C) Seminiferous tubules in detail.*

system of the reproductive tract in females is able to eventually clear ZIKV, largely due to interferon signaling [74, 75], such as the type III interferon lambda [76], the microenvironment of the male testes is an immune-privileged site that lacks adequate response for clearance [77].

#### *2.1.4 Viral genetic changes associated with persistence*

Mosquito-vectored flaviviruses that can result in persistent infections are usually associated with an accumulation of adaptive mutations, suggesting that viral genetics may also play an important role for establishing persistence. Examples of mutations in viruses causing persistence were shown with ZIKV, WNV [78–80], JEV [81], YFV [24, 82], and DENV [23]. These mutations happen in different viral proteins and appear to affect various viral functions, but in general attenuate the virus to cause less cytopathic effects, which is likely important for persistence. Identifying the mutations and cellular components that allow flaviviruses to cause long-term infections may elucidate mechanisms by which ZIKV is able to remain stable in seminal fluid long after infection. Although functional mutations in ZIKV may have been found in the male reproductive tract and ejaculates of mice [83], these were do not appear to be specific to these tissues as they were also found after passaging in Vero cells [83], and no mutation has been identified to be required for persistence and/or sexual transmission. Continuing research efforts using animal models are paramount to understand the mechanisms of ZIKV persistence, sexual transmission, reservoirs and interactions with cellular targets.

#### **2.2 Non-human primate (NHP) models**

NHP models are also frequently used to study ZIKV since their biology more closely relates to a human than does mouse biology. Several non-human primates were shown to be susceptible to ZIKV and used to study pathogenesis and infection of the reproductive tract, including cynomolgus macaques (*Macaca fascicularis*), rhesus macaques (*Macaca mulatta*), pigtail macaques (*Macaca nemestrina*) and olive baboon (*Papio anubis*), as well as neotropical non-human primates of the *Callithrix, Saimiri and Aotus* genera [46, 84]. These species presented viremia and variable degrees of clinical signs following ZIKV infection. Although many studies were conducted looking at ZIKV pathogenesis and the effects of infection on reproductive tissues, studies directly testing sexual transmission are lacking due to technical and logistical difficulties. Instead, vaginal or rectal inoculations have been used to simulate a sexual transmission. These and other insights into sexual transmission of non-human primates can also shed light on whether this type of transmission play a role in maintenance of ZIKV in nature, since primates are putative ZIKV reservoirs.

Because NHPs are evolutionarily close to humans, it is not surprising that these animals and their reproductive tract are anatomically and physiologically more similar to humans than other models such as mice; thus, NHPs are often considered to be one of the most relevant animal models [85, 86]. Unlike mice, immunocompetent NHPs are more suitable to study ZIKV infections, although the extent to which disease is mimicked is difficult to assess and varies with NHP species. Most NHP models were similar to humans in that most infections were asymptomatic, with clinical signs observed in some individuals [87, 88]. Considering that many human infections are asymptomatic, it is currently unclear if the clinical signs displayed by NHPs are a good model for disease rate and severity in humans. In a study using four cynomolgus macaques one individual was shown to present reduced body temperature [87]. Studying ZIKV pathogenesis in NHPs is important to understand disease

**37**

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

*2.2.1 Cynomolgus macaque*

*2.2.2 Olive baboon*

*2.2.3 Rhesus macaque*

progression, clinical signs, negative effects to the reproductive tract and outcomes of pregnancy. These data also inform the design of sexual transmission studies as far as titers, timepoints and strain used for infection, tissues that are important for disease and persistence so sexual transmission can take place. It is worth noting that these experimental infections are performed using virus in a needle while most [89] natural infections occur by a mosquito bite; mosquito saliva has immunomodulatory properties that have been shown to enhance disease in other flaviviral infection

Cynomolgus macaques were shown to be suitable as models for ZIKV pathogenesis and sexual transmission [87, 88, 93]. Infection with 5 log10 of plaque forming units (PFU) of ZIKV from various geographical origins resulted in viremia which peaked at 2-4 dpi at 4-7 log10 ZIKV genome copies/ml [87, 88]. The outbreak strain PRVABC59 was more virulent than the Asian FSS13025 and the African IBH30656 ZIKV, with the animals infected with the PRVABC59 strain being viremic for extended periods of time. Bodily fluids checked did not include seminal fluid, and shedding of virus in the urine and saliva was not observed with either the FSS13025 [87] or IBH30656 strains. ZIKV FSS13025 was detected in testes [88], consistent with murine infections with this strain [41]. On the other hand, ZIKV PRVABC59 was detected in urine, saliva and testes [88]. The fact that virus was found in testes suggests this species of NHP could model virus persistence in testes well and may also be a positive feature of this model for sexual transmission in general. The cynomolgus macaque model has also been shown to likely support sexual transmission, as macaques inoculated with 7 log10 PFU of virus intravaginally and intrarectally became viremic 50% and 100% of the time, respectively [93]. To understand the implications of these findings would be important to know the ranges of ZIKV titers in the semen. Although it is not clear what the viral titers are in most human semen samples, studies detected up to 9 log10

As with cynomolgus macaques, infection of olive baboons with different strains of ZIKV did not result in overt clinical signs. Following subcutaneous inoculation of a French Polynesian ZIKV (H/PF/2013), the baboons presented viremia that peaked at 3 and 4 dpi [45]. Around 40 days post infection, tissues were collected and virus was found in lymph nodes and epididymides, suggesting these are the places where the virus can persist even after viral clearance from the blood [45]. This suggests the testes were likely infected at some point and that the epididymides may also play a role in virus persistence [45]. Olive baboons were also used to model ZIKV infection during pregnancy. Infection of 4 log10 of ZIKV H/PF/2013 resulted in vertical transmission in 3 out of 4 pregnant NHPs [94]. Unlike the non-pregnant animals, all dams presented rash and conjunctivitis [94]. Fetal death and defects in the

The rhesus macaque is perhaps the most utilized NHP model to study ZIKV infection. Many studies of ZIKV were done using this species, including pathogenesis analyses in pregnant and non-pregnant animals, immunological and serological studies, and testing of anti-ZIKV vaccines or drugs [44, 95–106]. A study in which ZIKV was inoculated intravaginally in rhesus macaques to mimic sexual

[90–92] and could be another confounding factor in these studies.

RNA copies/ml of virus in semen of patients [17, 89].

frontal cortex of the fetus were observed [94].

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

progression, clinical signs, negative effects to the reproductive tract and outcomes of pregnancy. These data also inform the design of sexual transmission studies as far as titers, timepoints and strain used for infection, tissues that are important for disease and persistence so sexual transmission can take place. It is worth noting that these experimental infections are performed using virus in a needle while most [89] natural infections occur by a mosquito bite; mosquito saliva has immunomodulatory properties that have been shown to enhance disease in other flaviviral infection [90–92] and could be another confounding factor in these studies.

#### *2.2.1 Cynomolgus macaque*

*Current Concepts in Zika Research*

adequate response for clearance [77].

*2.1.4 Viral genetic changes associated with persistence*

transmission, reservoirs and interactions with cellular targets.

**2.2 Non-human primate (NHP) models**

system of the reproductive tract in females is able to eventually clear ZIKV, largely due to interferon signaling [74, 75], such as the type III interferon lambda [76], the microenvironment of the male testes is an immune-privileged site that lacks

Mosquito-vectored flaviviruses that can result in persistent infections are usually associated with an accumulation of adaptive mutations, suggesting that viral genetics may also play an important role for establishing persistence. Examples of mutations in viruses causing persistence were shown with ZIKV, WNV [78–80], JEV [81], YFV [24, 82], and DENV [23]. These mutations happen in different viral proteins and appear to affect various viral functions, but in general attenuate the virus to cause less cytopathic effects, which is likely important for persistence. Identifying the mutations and cellular components that allow flaviviruses to cause long-term infections may elucidate mechanisms by which ZIKV is able to remain stable in seminal fluid long after infection. Although functional mutations in ZIKV may have been found in the male reproductive tract and ejaculates of mice [83], these were do not appear to be specific to these tissues as they were also found after passaging in Vero cells [83], and no mutation has been identified to be required for persistence and/or sexual transmission. Continuing research efforts using animal models are paramount to understand the mechanisms of ZIKV persistence, sexual

NHP models are also frequently used to study ZIKV since their biology more closely relates to a human than does mouse biology. Several non-human primates were shown to be susceptible to ZIKV and used to study pathogenesis and infection of the reproductive tract, including cynomolgus macaques (*Macaca fascicularis*), rhesus macaques (*Macaca mulatta*), pigtail macaques (*Macaca nemestrina*) and olive baboon (*Papio anubis*), as well as neotropical non-human primates of the *Callithrix, Saimiri and Aotus* genera [46, 84]. These species presented viremia and variable degrees of clinical signs following ZIKV infection. Although many studies were conducted looking at ZIKV pathogenesis and the effects of infection on reproductive tissues, studies directly testing sexual transmission are lacking due to technical and logistical difficulties. Instead, vaginal or rectal inoculations have been used to simulate a sexual transmission. These and other insights into sexual transmission of non-human primates can also shed light on whether this type of transmission play a role in maintenance of ZIKV in nature, since primates are puta-

Because NHPs are evolutionarily close to humans, it is not surprising that these animals and their reproductive tract are anatomically and physiologically more similar to humans than other models such as mice; thus, NHPs are often considered to be one of the most relevant animal models [85, 86]. Unlike mice, immunocompetent NHPs are more suitable to study ZIKV infections, although the extent to which disease is mimicked is difficult to assess and varies with NHP species. Most NHP models were similar to humans in that most infections were asymptomatic, with clinical signs observed in some individuals [87, 88]. Considering that many human infections are asymptomatic, it is currently unclear if the clinical signs displayed by NHPs are a good model for disease rate and severity in humans. In a study using four cynomolgus macaques one individual was shown to present reduced body temperature [87]. Studying ZIKV pathogenesis in NHPs is important to understand disease

**36**

tive ZIKV reservoirs.

Cynomolgus macaques were shown to be suitable as models for ZIKV pathogenesis and sexual transmission [87, 88, 93]. Infection with 5 log10 of plaque forming units (PFU) of ZIKV from various geographical origins resulted in viremia which peaked at 2-4 dpi at 4-7 log10 ZIKV genome copies/ml [87, 88]. The outbreak strain PRVABC59 was more virulent than the Asian FSS13025 and the African IBH30656 ZIKV, with the animals infected with the PRVABC59 strain being viremic for extended periods of time. Bodily fluids checked did not include seminal fluid, and shedding of virus in the urine and saliva was not observed with either the FSS13025 [87] or IBH30656 strains. ZIKV FSS13025 was detected in testes [88], consistent with murine infections with this strain [41]. On the other hand, ZIKV PRVABC59 was detected in urine, saliva and testes [88]. The fact that virus was found in testes suggests this species of NHP could model virus persistence in testes well and may also be a positive feature of this model for sexual transmission in general. The cynomolgus macaque model has also been shown to likely support sexual transmission, as macaques inoculated with 7 log10 PFU of virus intravaginally and intrarectally became viremic 50% and 100% of the time, respectively [93]. To understand the implications of these findings would be important to know the ranges of ZIKV titers in the semen. Although it is not clear what the viral titers are in most human semen samples, studies detected up to 9 log10 RNA copies/ml of virus in semen of patients [17, 89].

#### *2.2.2 Olive baboon*

As with cynomolgus macaques, infection of olive baboons with different strains of ZIKV did not result in overt clinical signs. Following subcutaneous inoculation of a French Polynesian ZIKV (H/PF/2013), the baboons presented viremia that peaked at 3 and 4 dpi [45]. Around 40 days post infection, tissues were collected and virus was found in lymph nodes and epididymides, suggesting these are the places where the virus can persist even after viral clearance from the blood [45]. This suggests the testes were likely infected at some point and that the epididymides may also play a role in virus persistence [45]. Olive baboons were also used to model ZIKV infection during pregnancy. Infection of 4 log10 of ZIKV H/PF/2013 resulted in vertical transmission in 3 out of 4 pregnant NHPs [94]. Unlike the non-pregnant animals, all dams presented rash and conjunctivitis [94]. Fetal death and defects in the frontal cortex of the fetus were observed [94].

#### *2.2.3 Rhesus macaque*

The rhesus macaque is perhaps the most utilized NHP model to study ZIKV infection. Many studies of ZIKV were done using this species, including pathogenesis analyses in pregnant and non-pregnant animals, immunological and serological studies, and testing of anti-ZIKV vaccines or drugs [44, 95–106]. A study in which ZIKV was inoculated intravaginally in rhesus macaques to mimic sexual

transmission found ZIKV RNA in the reproductive tract of all 6 animals infected, thus raising the question of whether fetal disease could be more pronounced after sexual transmission when compared to vectored transmission [103]. This study lacked subcutaneous inoculation controls and further studies need to be conducted to confirm these findings. Another study comparing the intravaginal and subcutaneous routes using ZIKV PRVABC59 found peak viremia at 5–8 dpi that were variable in titer (3–7.5 log10 PFUs). Although the types of tissue found to be positive differed between subcutaneous and intravaginal inoculated animals, there was no obvious preference for reproductive tract tissues in the intravaginal route [107]. The data showed that intravaginal infection resulted in less CD11Chi myeloid cells, reduced expression of programmed cell death protein 1 (PD-1) in natural killer cells (NK) and more Ki67+ CD8+ central memory cells, indicating the route of infection may play a role in shaping the immune response [107].

Persistent ZIKV in reproductive tissues may play a role in sexual transmission long after primary infection. After intravenous inoculation of 5 log10 of a Brazilian ZIKV (BeH815744) in female rhesus macaques, ZIKV RNA was detected in multiple tissues of the NHPs, including reproductive tissues 14 dpi [108]. Lymphoid tissues had the highest detectable amount of viral RNA, suggesting these organs, which span many parts of the body, may act as possible viral reservoir [108].

### **2.3 Other animal models**

Several other animal models have been used to study ZIKV but most are not focused on sexual transmission. These models include guinea pigs [109–111], hamsters [112], bats [113], chick embryos, piglets [114, 115] and boars [116]. Porcine fetuses were shown to present mild to severe neuropathology upon ZIKV infection [114, 115]. Boar semen was inoculated with ZIKV, but it was concluded that ZIKV does not appear to cause cell damage and cannot replicate efficiently or persist in the semen of this species [116]. With respect to the neotropical chiropteran model, viral RNA was found in different tissues of fruit bats 28 dpi, suggesting ZIKV can infect bats which may serve as virus reservoirs. Bats did not show any signs of disease [113]. Stat-2 knockout hamsters infected with ZIKV have shown presence on infected cells with morphology of SCs and spermatogonia, suggesting this could be a suitable model to study persistence of the virus in testes [112]. The guinea pig models report conflicting results. One study with ZIKV challenge mid-gestation showed no evidence of infection [109]; however, Kumar et al. show guinea pigs inoculated subcutaneously with PRVABC59 had viremia and presented signs such as fever, hunched posture and detectable viral RNA in the blood [110]. Deng et al. showed that intranasally-infected guinea pigs have virus in the sera, saliva and tears [111].

#### **3. Conclusions**

ZIKV has emerged explosively since 2007, causing an epidemic in the Americas in 2015/16 and become a matter of global health importance. Although data from epidemiologic analyses and case studies helped shed light on the diseases caused by ZIKV, animal models will be important to substantiate and extend these findings under controlled experimental settings. Animal models can be—and have already proven to be—useful as a tool to understand ZIKV transmission and ZIKV-caused illnesses. Animal models do not fully recapitulate diseases as seen in humans; therefore, it is critical to consider the advantages and drawbacks of each model when designing and executing the experiments as well as interpreting the data. Models

**39**

USA

**Author details**

**Acknowledgements**

**Conflict of interest**

chapter and helpful discussions.

We have no conflicts of interest to declare.

Rafael K. Campos1

Branch, Galveston, USA

provided the original work is properly cited.

, Erin M. McDonald2

, Aaron C. Brault<sup>2</sup>

1 Department of Microbiology and Immunology, University of Texas Medical

\*Address all correspondence to: zlu5@cdc.gov and slrossi@utmb.edu

2 Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Centers for Disease Control and Prevention (CDC),

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\* and Shannan L. Rossi1

\*

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

mental conditions so they can be compared between studies.

to study sexual transmission are currently scarce and need further development. Challenges include scarcity of good models to study ZIKV sexual transmission, low number of animals used and the requirement to improve reproducibility of the findings from animal models, which is caused by differences in experimental conditions and the number of animals used. As in certain cases increasing the number of animals used is not possible due to it being prohibitively expensive or posing ethical issues, future experiments assessing sexual transmission of ZIKV should focus on optimizing experimental design and analysis, when possible standardizing experi-

We thank Drs. Sasha Azar and Nikos Vasilakis for their critical reading of the

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

to study sexual transmission are currently scarce and need further development. Challenges include scarcity of good models to study ZIKV sexual transmission, low number of animals used and the requirement to improve reproducibility of the findings from animal models, which is caused by differences in experimental conditions and the number of animals used. As in certain cases increasing the number of animals used is not possible due to it being prohibitively expensive or posing ethical issues, future experiments assessing sexual transmission of ZIKV should focus on optimizing experimental design and analysis, when possible standardizing experimental conditions so they can be compared between studies.

## **Acknowledgements**

*Current Concepts in Zika Research*

**2.3 Other animal models**

transmission found ZIKV RNA in the reproductive tract of all 6 animals infected, thus raising the question of whether fetal disease could be more pronounced after sexual transmission when compared to vectored transmission [103]. This study lacked subcutaneous inoculation controls and further studies need to be conducted to confirm these findings. Another study comparing the intravaginal and subcutaneous routes using ZIKV PRVABC59 found peak viremia at 5–8 dpi that were variable in titer (3–7.5 log10 PFUs). Although the types of tissue found to be positive differed between subcutaneous and intravaginal inoculated animals, there was no obvious preference for reproductive tract tissues in the intravaginal route [107]. The data showed that intravaginal infection resulted in less CD11Chi myeloid cells, reduced expression of programmed cell death protein 1 (PD-1) in natural killer cells (NK) and more Ki67+ CD8+ central memory cells, indicating the route of infection

Persistent ZIKV in reproductive tissues may play a role in sexual transmission long after primary infection. After intravenous inoculation of 5 log10 of a Brazilian ZIKV (BeH815744) in female rhesus macaques, ZIKV RNA was detected in multiple tissues of the NHPs, including reproductive tissues 14 dpi [108]. Lymphoid tissues had the highest detectable amount of viral RNA, suggesting these organs, which

Several other animal models have been used to study ZIKV but most are not focused on sexual transmission. These models include guinea pigs [109–111], hamsters [112], bats [113], chick embryos, piglets [114, 115] and boars [116]. Porcine fetuses were shown to present mild to severe neuropathology upon ZIKV infection [114, 115]. Boar semen was inoculated with ZIKV, but it was concluded that ZIKV does not appear to cause cell damage and cannot replicate efficiently or persist in the semen of this species [116]. With respect to the neotropical chiropteran model, viral RNA was found in different tissues of fruit bats 28 dpi, suggesting ZIKV can infect bats which may serve as virus reservoirs. Bats did not show any signs of disease [113]. Stat-2 knockout hamsters infected with ZIKV have shown presence on infected cells with morphology of SCs and spermatogonia, suggesting this could be a suitable model to study persistence of the virus in testes [112]. The guinea pig models report conflicting results. One study with ZIKV challenge mid-gestation showed no evidence of infection [109]; however, Kumar et al. show guinea pigs inoculated subcutaneously with PRVABC59 had viremia and presented signs such as fever, hunched posture and detectable viral RNA in the blood [110]. Deng et al. showed that intranasally-infected guinea pigs have virus in the sera, saliva and

ZIKV has emerged explosively since 2007, causing an epidemic in the Americas in 2015/16 and become a matter of global health importance. Although data from epidemiologic analyses and case studies helped shed light on the diseases caused by ZIKV, animal models will be important to substantiate and extend these findings under controlled experimental settings. Animal models can be—and have already proven to be—useful as a tool to understand ZIKV transmission and ZIKV-caused illnesses. Animal models do not fully recapitulate diseases as seen in humans; therefore, it is critical to consider the advantages and drawbacks of each model when designing and executing the experiments as well as interpreting the data. Models

span many parts of the body, may act as possible viral reservoir [108].

may play a role in shaping the immune response [107].

**38**

tears [111].

**3. Conclusions**

We thank Drs. Sasha Azar and Nikos Vasilakis for their critical reading of the chapter and helpful discussions.

## **Conflict of interest**

We have no conflicts of interest to declare.

## **Author details**

Rafael K. Campos1 , Erin M. McDonald2 , Aaron C. Brault<sup>2</sup> \* and Shannan L. Rossi1 \*

1 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, USA

2 Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Centers for Disease Control and Prevention (CDC), USA

\*Address all correspondence to: zlu5@cdc.gov and slrossi@utmb.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[27] Liu WJ, Zhu M, Pei JJ, Dong XY, Liu W, Zhao MQ, et al. Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China. Virus Research. 2013;**178**(2):547-552

[28] McDonald EM, Duggal NK, Brault AC. Pathogenesis and sexual transmission of Spondweni and Zika viruses. PLoS Neglected Tropical Diseases. 2017;**11**(10):e0005990

[29] Rikula U, Nuotio L, Laamanen UI, Sihvonen L. Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions. The Veterinary Record.

[30] Newcomer BW, Toohey-Kurth K, Zhang Y, Brodersen BW, Marley MS, Joiner KS, et al. Laboratory diagnosis and transmissibility of bovine viral diarrhea virus from a bull with a persistent testicular

infection. Veterinary Microbiology.

[31] Voges H, Horner GW, Rowe S, Wellenberg GJ. Persistent bovine

2014;**170**(3-4):246-257

2008;**162**(3):79-82

[24] Barbosa CM, Di Paola N, Cunha MP,

2018;**23**(18):18-00197

[17] Mead PS, Duggal NK, Hook SA, Delorey M, Fischer M, Olzenak McGuire D, et al. Zika virus shedding in semen of symptomatic infected men. The New England Journal of Medicine.

[18] Reyes Y, Bowman NM, Becker-Dreps S, Centeno E, Collins MH, Liou GA, et al. Prolonged shedding of Zika virus RNA in vaginal secretions, Nicaragua. Emerging Infectious Diseases. 2019;**25**(4):808-810

[19] Russell K, Hills SL, Oster AM, Porse CC, Danyluk G, Cone M, et al. Male-to-female sexual transmission of Zika virus-United States, January-April 2016. Clinical Infectious Diseases.

[20] Deckard DT, Chung WM, Brooks JT, Smith JC, Woldai S,

Hennessey M, et al. Male-to-male sexual transmission of Zika virus—Texas, January 2016. MMWR. Morbidity and Mortality Weekly Report.

[21] Davidson A, Slavinski S, Komoto K, Rakeman J, Weiss D. Suspected female-

to-male sexual transmission of Zika virus—New York City, 2016. MMWR. Morbidity and Mortality Weekly Report. 2016;**65**(28):716-717

[22] Kelley RE, Berger JR, Kelley BP. West Nile virus Meningo-encephalitis: Possible sexual transmission. Journal of the Louisiana State Medical Society.

[23] Lalle E, Colavita F, Iannetta M, Gebremeskel Tekle S, Carletti F, Scorzolini L, et al. Prolonged detection of dengue virus RNA in the semen of a man returning from Thailand to Italy, January 2018. Euro Surveillance: Bulletin Europeen sur les Maladies Transmissibles = European

2016;**54**(4):860-867

2018;**378**(15):1377-1385

2017;**64**(2):211-213

2016;**65**(14):372-374

2016;**168**(1):21-22

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

threat. Journal of Clinical Microbiology. 2016;**54**(4):860-867

[17] Mead PS, Duggal NK, Hook SA, Delorey M, Fischer M, Olzenak McGuire D, et al. Zika virus shedding in semen of symptomatic infected men. The New England Journal of Medicine. 2018;**378**(15):1377-1385

[18] Reyes Y, Bowman NM, Becker-Dreps S, Centeno E, Collins MH, Liou GA, et al. Prolonged shedding of Zika virus RNA in vaginal secretions, Nicaragua. Emerging Infectious Diseases. 2019;**25**(4):808-810

[19] Russell K, Hills SL, Oster AM, Porse CC, Danyluk G, Cone M, et al. Male-to-female sexual transmission of Zika virus-United States, January-April 2016. Clinical Infectious Diseases. 2017;**64**(2):211-213

[20] Deckard DT, Chung WM, Brooks JT, Smith JC, Woldai S, Hennessey M, et al. Male-to-male sexual transmission of Zika virus—Texas, January 2016. MMWR. Morbidity and Mortality Weekly Report. 2016;**65**(14):372-374

[21] Davidson A, Slavinski S, Komoto K, Rakeman J, Weiss D. Suspected femaleto-male sexual transmission of Zika virus—New York City, 2016. MMWR. Morbidity and Mortality Weekly Report. 2016;**65**(28):716-717

[22] Kelley RE, Berger JR, Kelley BP. West Nile virus Meningo-encephalitis: Possible sexual transmission. Journal of the Louisiana State Medical Society. 2016;**168**(1):21-22

[23] Lalle E, Colavita F, Iannetta M, Gebremeskel Tekle S, Carletti F, Scorzolini L, et al. Prolonged detection of dengue virus RNA in the semen of a man returning from Thailand to Italy, January 2018. Euro Surveillance: Bulletin Europeen sur les Maladies Transmissibles = European

Communicable Disease Bulletin. 2018;**23**(18):18-00197

[24] Barbosa CM, Di Paola N, Cunha MP, Rodrigues-Jesus MJ, Araujo DB, Silveira VB, et al. Yellow fever virus RNA in urine and semen of convalescent patient, Brazil. Emerging Infectious Diseases. 2018;**24**(1):176-178

[25] Molton JS, Low I, Choy MMJ, Aw PPK, Hibberd ML, Tambyah PA, et al. Dengue virus not detected in human semen. Journal of Travel Medicine. 2018;**25**(1):1-3

[26] Althouse GC, Rossow K. The potential risk of infectious disease dissemination via artificial insemination in swine. Reproduction in Domestic Animals. 2011;**46**(Suppl 2):64-67

[27] Liu WJ, Zhu M, Pei JJ, Dong XY, Liu W, Zhao MQ, et al. Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China. Virus Research. 2013;**178**(2):547-552

[28] McDonald EM, Duggal NK, Brault AC. Pathogenesis and sexual transmission of Spondweni and Zika viruses. PLoS Neglected Tropical Diseases. 2017;**11**(10):e0005990

[29] Rikula U, Nuotio L, Laamanen UI, Sihvonen L. Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions. The Veterinary Record. 2008;**162**(3):79-82

[30] Newcomer BW, Toohey-Kurth K, Zhang Y, Brodersen BW, Marley MS, Joiner KS, et al. Laboratory diagnosis and transmissibility of bovine viral diarrhea virus from a bull with a persistent testicular infection. Veterinary Microbiology. 2014;**170**(3-4):246-257

[31] Voges H, Horner GW, Rowe S, Wellenberg GJ. Persistent bovine

**40**

*Current Concepts in Zika Research*

**References**

[1] Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1952;**46**(5):509-520

non-vector-borne transmission of Zika virus, Colorado, USA. Emerging Infectious Diseases. 2011;**17**(5):880-882

2015;**21**(2):359-361

2016;**374**(22):2195-2198

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*Current Concepts in Zika Research*

1998;**61**(3):165-175

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model to study Zika virus. The

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viral RNA shedding in an

A mouse model of Zika virus pathogenesis. Cell Host & Microbe.

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2016;**19**(5):720-730

2016;**7**:12204

2018;**92**(16):e00186-18

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Immunodeficient mouse model. Cell Reports. 2017;**18**(7):1751-1760

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Chaix ML. Detection of hepatitis C virus in the semen of infected men. Lancet.

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couples. Sexually Transmitted Infections. 2003;**79**(2):160-162

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[103] Carroll T, Lo M, Lanteri M, Dutra J, Zarbock K, Silveira P, et al. Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques. PLoS Pathogens. 2017;**13**(7):e1006537

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[106] Best K, Guedj J, Madelain V, de Lamballerie X, Lim SY, Osuna CE, et al.

**47**

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

PLoS Neglected Tropical Diseases.

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2019;**13**(2):e0007071

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Lai K, Delgado-Ortega M, Wheler C, Wilson D, et al. Zika virus causes persistent infection in porcine

conceptuses and may impair health in offspring. eBioMedicine. 2017;**25**:73-86

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Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies. Proceedings of the National Academy of Sciences of the United States of America.

2017;**114**(33):8847-8852

Pathogens. 2018;**7**(3):E70

One. 2017;**12**(1):e0171148

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[110] Kumar M, Krause KK, Azouz F, Nakano E, Nerurkar VR. A Guinea pig model of Zika virus infection. Virology

[111] Deng YQ, Zhang NN, Li XF, Wang YQ, Tian M, Qiu YF, et al. Intranasal infection and contact transmission of Zika virus in Guinea pigs. Nature Communications. 2017;**8**(1):1648

[112] Siddharthan V, Van Wettere AJ, Li R, Miao J, Wang Z, Morrey JD, et al. Zika virus infection of adult and fetal STAT2 knock-out hamsters. Virology.

[113] Malmlov A, Bantle C, Aboellail T, Wagner K, Campbell CL, Eckley M, et al. Experimental Zika virus infection

*jamaicensis*) and possible entry of virus into brain via activated microglial cells.

of Jamaican fruit bats (*Artibeus* 

Journal. 2017;**14**(1):75

2017;**507**:89-95

[108] Coffey LL, Pesavento PA, Keesler RI, Singapuri A, Watanabe J, Watanabe R, et al. Zika virus tissue and blood compartmentalization in acute infection of rhesus macaques. PLoS

[107] Woollard SM, Olwenyi OA, Dutta D, Dave RS, Mathews S,

Gorantla S, et al. Preliminary studies on immune response and viral pathogenesis of Zika virus in rhesus macaques.

*Animal Models of Zika Virus Sexual Transmission DOI: http://dx.doi.org/10.5772/intechopen.91256*

Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies. Proceedings of the National Academy of Sciences of the United States of America. 2017;**114**(33):8847-8852

*Current Concepts in Zika Research*

[93] Haddow AD, Nalca A, Rossi FD, Miller LJ, Wiley MR,

2017;**23**(8):1274-1281

2019;**9**(1):12802

Perez-Sautu U, et al. High infection rates for adult macaques after intravaginal or intrarectal inoculation with Zika virus. Emerging Infectious Diseases.

virus infection is associated with persistent abnormalities in brain structure, function, and behavior in infant macaques. Science Translational Medicine. 2018;**10**(435):eaao6975

[100] Martinot AJ, Abbink P, Afacan O, Prohl AK, Bronson R, Hecht JL, et al. Fetal neuropathology in Zika virusinfected pregnant female rhesus monkeys. Cell. 2018;**173**(5):1111-1122

[101] Hirsch AJ, Roberts VHJ, Grigsby PL, Haese N, Schabel MC, Wang X, et al. Zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology. Nature Communications. 2018;**9**(1):263

[102] Abbink P, Larocca RA, Visitsunthorn K, Boyd M, De La Barrera RA, Gromowski GD, et al. Durability and correlates of vaccine protection against Zika virus in rhesus monkeys. Science Translational Medicine. 2017;**9**(420):eaao4163

[103] Carroll T, Lo M, Lanteri M,

[104] Nguyen SM, Antony KM, Dudley DM, Kohn S, Simmons HA, Wolfe B, et al. Highly efficient maternalfetal Zika virus transmission in pregnant rhesus macaques. PLoS Pathogens.

[105] Aliota MT, Dudley DM,

Newman CM, Mohr EL, Gellerup DD, Breitbach ME, et al. Heterologous protection against Asian Zika virus challenge in rhesus macaques. PLoS Neglected Tropical Diseases.

[106] Best K, Guedj J, Madelain V, de Lamballerie X, Lim SY, Osuna CE, et al.

2017;**13**(5):e1006378

2016;**10**(12):e0005168

Dutra J, Zarbock K, Silveira P, et al. Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques. PLoS Pathogens. 2017;**13**(7):e1006537

e10

[94] Gurung S, Reuter N, Preno A, Dubaut J, Nadeau H, Hyatt K, et al. Zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon. PLoS Pathogens. 2019;**15**(1):e1007507

[95] Maness NJ, Schouest B, Singapuri A, Dennis M, Gilbert MH, Bohm RP, et al. Postnatal Zika virus infection of nonhuman primate infants born to mothers infected with homologous Brazilian Zika virus. Scientific Reports.

[96] Valiant WG, Mattapallil MJ,

Lewis MG, et al. Simultaneous

[97] Bidokhti MRM, Dutta D,

2018;**12**(10):e0006811

2018;**9**(1):2414

et al. Intraamniotic Zika virus inoculation of pregnant rhesus macaques produces fetal neurologic disease. Nature Communications.

[99] Mavigner M, Raper J, Kovacs-Balint Z, Gumber S, O'Neal JT, Bhaumik SK, et al. Postnatal Zika

Madduri LSV, Woollard SM, Norgren R Jr, Giavedoni L, et al. SIV/SHIV-Zika co-infection does not alter disease pathogenesis in adult nonpregnant rhesus macaque model. PLoS Neglected Tropical Diseases.

[98] Coffey LL, Keesler RI, Pesavento PA, Woolard K, Singapuri A, Watanabe J,

Higgs S, Huang YS, Vanlandingham DL,

coinfection of macaques with Zika and dengue viruses does not enhance acute plasma viremia but leads to activation of monocyte subsets and biphasic release of pro-inflammatory cytokines. Scientific Reports. 2019;**9**(1):7877

**46**

[107] Woollard SM, Olwenyi OA, Dutta D, Dave RS, Mathews S, Gorantla S, et al. Preliminary studies on immune response and viral pathogenesis of Zika virus in rhesus macaques. Pathogens. 2018;**7**(3):E70

[108] Coffey LL, Pesavento PA, Keesler RI, Singapuri A, Watanabe J, Watanabe R, et al. Zika virus tissue and blood compartmentalization in acute infection of rhesus macaques. PLoS One. 2017;**12**(1):e0171148

[109] Bierle CJ, Fernandez-Alarcon C, Hernandez-Alvarado N, Zabeli JC, Janus BC, Putri DS, et al. Assessing Zika virus replication and the development of Zika-specific antibodies after a midgestation viral challenge in Guinea pigs. PLoS One. 2017;**12**(11):e0187720

[110] Kumar M, Krause KK, Azouz F, Nakano E, Nerurkar VR. A Guinea pig model of Zika virus infection. Virology Journal. 2017;**14**(1):75

[111] Deng YQ, Zhang NN, Li XF, Wang YQ, Tian M, Qiu YF, et al. Intranasal infection and contact transmission of Zika virus in Guinea pigs. Nature Communications. 2017;**8**(1):1648

[112] Siddharthan V, Van Wettere AJ, Li R, Miao J, Wang Z, Morrey JD, et al. Zika virus infection of adult and fetal STAT2 knock-out hamsters. Virology. 2017;**507**:89-95

[113] Malmlov A, Bantle C, Aboellail T, Wagner K, Campbell CL, Eckley M, et al. Experimental Zika virus infection of Jamaican fruit bats (*Artibeus jamaicensis*) and possible entry of virus into brain via activated microglial cells. PLoS Neglected Tropical Diseases. 2019;**13**(2):e0007071

[114] Wichgers Schreur PJ, van Keulen L, Anjema D, Kant J, Kortekaas J. Microencephaly in fetal piglets following in utero inoculation of Zika virus. Emerging Microbes and Infections. 2018;**7**(1):42

[115] Darbellay J, Cox B, Lai K, Delgado-Ortega M, Wheler C, Wilson D, et al. Zika virus causes persistent infection in porcine conceptuses and may impair health in offspring. eBioMedicine. 2017;**25**:73-86

[116] Luplertlop N, Suwanmanee S, Ampawong S, Vongpunsawad S, Poovorawan Y. In vitro study of Zika virus infection in boar semen. Archives of Virology. 2017;**162**(10):3209-3213

**49**

Section 2

Clinical Aspects
