**7. Problems of dengue diagnosis**

A series of multiple variant testing have been carried out for dengue diagnosis depending on a person's infection phase, these tests are conducted with different immunological targets for virus recognition, a part of its structure or a reaction in an infected person or an animal's body indicating an exposure to DENV [35] (**Table 2**).

In a dengue study, diagnostic tests display numerous problems which we will describe briefly. The presence of false positive as a result of contamination and different variants of RT-PCR such as endpoint PCR, nested PCR, real-time PCR as well as the use of different primers and enzymes lead to variations in levels of sensitivity and specificity. Some commercial test enables the identification of different microorganisms by utilizing RT-PCR, PCR tests and the detection of the product in about an hour. The RUO Film Array Global Fever Panel tests are utilized for the detection of 6 bacterias, 4 protozoans and 9 viruses like Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Lassa virus, Marburg virus, West Nile virus, Yellow fever virus, Zika virus and Dengue virus. Whole blood (EDTA) is used as a sample. The use of automation equipment can help to reduce contamination problems and deliver results in a short time as well as providing diagnosis for 19 pathogens. Regarding to improvements for this type of systems the equipment can only process one sample at a time, and the study is expensive. The rapid tests do not need sophisticated equipment or qualified staff, it is feasible in situ and they are inexpensive. Nonetheless, its sensitivity and specificity are not as higher as other techniques. Cell cultures are often used for dengue virus isolation. Many cell lines are used for virus isolation; however, their sensitivity can vary depending on the type of cell line, the clone, the system used for viral isolation and the sample type used for isolation [35, 36]. In order to isolate DENV, it is preferable to use C6/36 cell line obtained from *Aedes albopictus* mosquito salivary glands that is replicated at 28°C as


**25**

*Lessons Learned and Recent Advances in Dengue Research*

this test under an Emergency Use Authorization (EUA) [38].

It is not yet found a suitable animal model expressing all characteristics for dengue disease [39]. Among the most common models used for research are mice showing drawbacks with low-level virus replication of clinical samples, and non-human primates showing a problem for not expressing the clinical disease in the same manner as humans [39]. The use of nursing mice used in intracranial inoculations with high viral load inducing neurological diseases and paralysis are still been used; however, its use continues more for the production of biological

**8. Animal models for dengue**

it has higher sensitivity than Vero or BHK-21 (mammalian lines). Some researchers suggest using a variety of C6/36 HT which grows at 33°C. Such cell is considered to be more sensitive than the traditional C6/36, and it is recommended to use the shell vial or shell vial modified method instead of the traditional or standard isolated system in order to achieve better results. In the modified shell vial method, the cell culture is infected, centrifuged and incubated for 10–15 days, then positive cultures by indirect immunofluorescence are identified [35, 36]. Many laboratories do two or three passages from cell cultures following an assessment of virus presence conducted by IFI. This increases more the number of isolations but the cost of the testing goes up and the time needed to obtain the results raises. The quantification or viral load may be run by real time PCR utilizing a standard reference that in comparison with the sample allow us to assess the number of genetic material copies from the sample [35, 36]. When it is intended to quantify infecting viruses in biological samples for producing antigens or vaccines, is preferred to use PA in Vero or BHK-21 cells [35, 36]. One alternative for viral titration, depending on the study performed and especially when evaluating viral load in mosquitoes, is the use of FFA that is a mixture of plaque assay and immunofluorescence test and it allows the use of C6/36 cells containing higher sensitivity than the above-mentioned cells [35, 36]. IF and ELISAS tests are relatively low cost and they may occur commercially or may be designed. Many of their components as antibodies and antigens can be produced in the laboratory at lower costs but it is necessary a good quality control of production so that there can be variations in job titles of lots that may affect test results [35, 36]. Neutralization tests can be performed under different techniques such as PRNT, microneutralization, microneutralization-ELISA. These tests facilitate the high specificity type IgG neutralizing antibodies detection. However, the levels of sensitivity in relation to the cell line, the strain of virus and the technique used are due to cell cultures. Other problems have yet been overcome are DENV cross-reactions with other flavivirus and within the same serotypes in serological tests like ELISAS, IFI and even considered to be gold standard like neutralization test by plaque reduction (PRNT) exhibiting cross-reactions. Crossreactivity between serotypes is frequent and it occurs particularly in epitope on NS protein for the conservation or in conserved epitopes on E glycoprotein and may vary according to homology between every DENV serotype and genotype. The use of specific monoclonal antibodies for every dengue serotype in the IFI tests improve greatly the specificity of the test as compared to the use of HMAF. The use of tests allows to detect multiple pathogens at the same time like CDC DENV-1-4 rRT-PCR multiplex detecting infecting dengue virus serotype and it was approved by FDA in 2013 [35, 37]. The CDC Trioplex rRT-PCR assay uses real-time (TaqMan®) RT-PCR assays for detection and differentiation of RNA from DENV, CHIKV and ZIKV in serum samples, whole blood (EDTA), cerebrospinal fluid. This also enables the RNA detection of ZIKV in urine and amniotic fluids. FDA has authorized the use of

*DOI: http://dx.doi.org/10.5772/intechopen.92076*

**Table 2.**

*Most commonly used diagnostic tests for diagnosing dengue.*

#### *Lessons Learned and Recent Advances in Dengue Research DOI: http://dx.doi.org/10.5772/intechopen.92076*

*Dengue Fever in a One Health Perspective*

**7. Problems of dengue diagnosis**

(**Table 2**).

2 were DENV-2 positive and 4 were DENV-3 positive in a rubber plantation area [33]. It is necessary to continue with this type of studies in these animals in order to develop a viremia high enough to infect mosquitoes. More than 200 viruses from 27 families, including *flaviviridae*, was isolated or detected in bats and several studies have shown DENV nucleic acids and/or antibodies present in Neotropical wildlife. A study in Costa Rica suggest that bats are infected accidentally by DENV because the RNA quantification in blood was low below the minimum infectious dose of mosquito that is needed to maintain the transmission cycle for the virus. Thus, they

A series of multiple variant testing have been carried out for dengue diagnosis depending on a person's infection phase, these tests are conducted with different immunological targets for virus recognition, a part of its structure or a reaction in an infected person or an animal's body indicating an exposure to DENV [35]

In a dengue study, diagnostic tests display numerous problems which we will describe briefly. The presence of false positive as a result of contamination and different variants of RT-PCR such as endpoint PCR, nested PCR, real-time PCR as well as the use of different primers and enzymes lead to variations in levels of sensitivity and specificity. Some commercial test enables the identification of different microorganisms by utilizing RT-PCR, PCR tests and the detection of the product in about an hour. The RUO Film Array Global Fever Panel tests are utilized for the detection of 6 bacterias, 4 protozoans and 9 viruses like Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Lassa virus, Marburg virus, West Nile virus, Yellow fever virus, Zika virus and Dengue virus. Whole blood (EDTA) is used as a sample. The use of automation equipment can help to reduce contamination problems and deliver results in a short time as well as providing diagnosis for 19 pathogens. Regarding to improvements for this type of systems the equipment can only process one sample at a time, and the study is expensive. The rapid tests do not need sophisticated equipment or qualified staff, it is feasible in situ and they are inexpensive. Nonetheless, its sensitivity and specificity are not as higher as other techniques. Cell cultures are often used for dengue virus isolation. Many cell lines are used for virus isolation; however, their sensitivity can vary depending on the type of cell line, the clone, the system used for viral isolation and the sample type used for isolation [35, 36]. In order to isolate DENV, it is preferable to use C6/36 cell line obtained from *Aedes albopictus* mosquito salivary glands that is replicated at 28°C as

**Diagnostic test Immunological target** Polymerase chain reaction (PCR) RNA detection Rapid tests NS1, IgM, IgG

Virus isolation Virus

*Most commonly used diagnostic tests for diagnosing dengue.*

Immunofluorescence (IF) Virus, IgM, IgG Plaque assay (PA) and fluorescent focus assay (FFA) Virus (titer) Enzyme-linked immunosorbent assay (ELISAS) NS1, IgM, IgG, IgA

Neutralization tests Neutralizing antibodies (IgG)

were considered as dead-end hosts for dengue virus [34].

**24**

**Table 2.**

it has higher sensitivity than Vero or BHK-21 (mammalian lines). Some researchers suggest using a variety of C6/36 HT which grows at 33°C. Such cell is considered to be more sensitive than the traditional C6/36, and it is recommended to use the shell vial or shell vial modified method instead of the traditional or standard isolated system in order to achieve better results. In the modified shell vial method, the cell culture is infected, centrifuged and incubated for 10–15 days, then positive cultures by indirect immunofluorescence are identified [35, 36]. Many laboratories do two or three passages from cell cultures following an assessment of virus presence conducted by IFI. This increases more the number of isolations but the cost of the testing goes up and the time needed to obtain the results raises. The quantification or viral load may be run by real time PCR utilizing a standard reference that in comparison with the sample allow us to assess the number of genetic material copies from the sample [35, 36]. When it is intended to quantify infecting viruses in biological samples for producing antigens or vaccines, is preferred to use PA in Vero or BHK-21 cells [35, 36]. One alternative for viral titration, depending on the study performed and especially when evaluating viral load in mosquitoes, is the use of FFA that is a mixture of plaque assay and immunofluorescence test and it allows the use of C6/36 cells containing higher sensitivity than the above-mentioned cells [35, 36]. IF and ELISAS tests are relatively low cost and they may occur commercially or may be designed. Many of their components as antibodies and antigens can be produced in the laboratory at lower costs but it is necessary a good quality control of production so that there can be variations in job titles of lots that may affect test results [35, 36]. Neutralization tests can be performed under different techniques such as PRNT, microneutralization, microneutralization-ELISA. These tests facilitate the high specificity type IgG neutralizing antibodies detection. However, the levels of sensitivity in relation to the cell line, the strain of virus and the technique used are due to cell cultures. Other problems have yet been overcome are DENV cross-reactions with other flavivirus and within the same serotypes in serological tests like ELISAS, IFI and even considered to be gold standard like neutralization test by plaque reduction (PRNT) exhibiting cross-reactions. Crossreactivity between serotypes is frequent and it occurs particularly in epitope on NS protein for the conservation or in conserved epitopes on E glycoprotein and may vary according to homology between every DENV serotype and genotype. The use of specific monoclonal antibodies for every dengue serotype in the IFI tests improve greatly the specificity of the test as compared to the use of HMAF. The use of tests allows to detect multiple pathogens at the same time like CDC DENV-1-4 rRT-PCR multiplex detecting infecting dengue virus serotype and it was approved by FDA in 2013 [35, 37]. The CDC Trioplex rRT-PCR assay uses real-time (TaqMan®) RT-PCR assays for detection and differentiation of RNA from DENV, CHIKV and ZIKV in serum samples, whole blood (EDTA), cerebrospinal fluid. This also enables the RNA detection of ZIKV in urine and amniotic fluids. FDA has authorized the use of this test under an Emergency Use Authorization (EUA) [38].

### **8. Animal models for dengue**

It is not yet found a suitable animal model expressing all characteristics for dengue disease [39]. Among the most common models used for research are mice showing drawbacks with low-level virus replication of clinical samples, and non-human primates showing a problem for not expressing the clinical disease in the same manner as humans [39]. The use of nursing mice used in intracranial inoculations with high viral load inducing neurological diseases and paralysis are still been used; however, its use continues more for the production of biological

supplies. DENV can slightly replicate in mice A/J, BALB/c and C57BL/6. Mice A/J and BALB/c can die of paralysis [39, 40]. For animal studies, it is considered that an infection with DENV 104 –106 PFU (plaque-forming units) concentrations imitate inoculum concentrations of a mosquito bite [39]. The mouse model is generally used initially to assess vaccines being the most suitable the immunocompetent mice but when it exhibits low viremia may result in the underestimation of the results. The use of nursing mice is a good way to assess possible candidate vaccines inducing neurovirulence. Although the NHP do not develop the disease, the antibody seroconversion produced is human-like [39, 40].

### **9. Antivirals for treating dengue**

At the moment, there is no antiviral therapy for DENV treatment. It is believed that the compounds working as inhibitors of RNA-dependent RNA polymerase have shown low efficiency. Moreover, there is a possibility that viral epitopes of DENV may trigger an immune cell response, preceded by development of severe disease. Thus, these epitopes are examined as targets for antiviral productions and they are known as DENV entry inhibitors and can be used in combination with inhibitors of the virus replication in order to increase efficiency.

Inhibition of DENV attachment and entry into the host cell can inhibit immune activation.

Various compounds as peptide entry inhibitors, DN59 and 1OAN1 may inhibit the antibody-dependent enhancement (ADE) in vitro. The doxorubicin antibiotic SA-17 is structured similarly to tetracycline and it has an antiviral activity against DENV serotype 1, 2 and 3 in Vero and C6/36 cells interfering with viral entry by binding to the hydrophobic pocket of the E-protein without exhibiting virucidal activity. The glycosidase inhibitors are unpopular due to its toxicity and low specificity but may help understand E-protein glycosylation processes. The binding agents to carbohydrates occur only during the stage of virus adsorption to the host cell. Concanavalin and agglutinins of wheat germ can bind N-acetylglucosamine residues and the percentage of DENV-lytic plaques in BHK cells can be reduced. Plant lectins such as *Hippeastrum hybrid*, *Galanthus nivalis*, and *Urtica dioica* inhibit DENV-2 infection in Raji/DC-SIGN cells. The compounds of heparan sulfate are potential recipients for DENV. Dengue E protein domain III is responsible for the interaction of heparan sulfate, and it is believed to be in all DENV serotypes and to have epitopes that are recognized by neutralizing antibodies. The suramin is similar to heparan sulfate and persulfated glycosaminoglycan, they bind to a polyanionbinding site of DENV E protein by inhibiting infection. The sulfated polysaccharides extracted from red algae, carrageenans and DL-galactan presented antiviral activity against 4 serotypes of dengue; however, the antiviral activity of DENV-4 DENV-1 weakened in Vero cells and in human Hepatocytes [41, 42].

#### **10. Dengue vaccine**

In May 2019, the U.S. Food and Drug Administration (FDA) approved the use of Dengvaxia®, a vaccine against DENV that may be used in people aged from 9 to 45 years old [43]. This vaccine utilizes a live attenuated chimeric yellow fever/dengue virus based on a Yellow fever 17D vaccine virus backbone chimerized with prM and E proteins from DENV1–4 replacing the YF prM and E, and 3 doses are administered every 6 months. In phase III trials the overall protective efficacy was 56.7% and 60.8% in South East Asia and Latin America countries, respectively. In 2017, the

**27**

*Lessons Learned and Recent Advances in Dengue Research*

vaccine manufacturer, Sanofi Pasteur, announced that some people getting the vaccine without having been previously infected by DENV may be at risk for developing severe dengue if the disease is acquired after immunization [43]. The vaccine candidates utilize strategies such as vaccines of Live-attenuated virus, Inactivated virus, Recombinant protein, DNA vaccine, Viral vector vaccine, Virus like particles and others. Two vaccine candidates (DENVax and TV003/TV005) are being tested in efficacy trials in both Asia and Latin America. DENVax is a tetravalent recombinant live-attenuated dengue vaccine licensed to Takeda. The live virus vaccine utilizes chimerization with DENV-2 PDK-53 as the backbone with DENV-2/-1, -2/-3, and -2/-4 chimeras are created by replacing the DENV-2 prM and E genes with the respective genes from the other DENV serotypes. Other vaccine candidate is TV003/TV00 with a whole attenuated virus for three of four serotypes (attenuated by deletion of 30 nucleotides from 3′ UTR of DENV-1, DENV-3, DENV-4, and a chimeric DENV-2/DENV-4) [44]. In spite of having already a vaccine available to prevent dengue, there is a need to further research in order to improve the vaccine for dengue or to produce a new vaccine which enables improvements in certain aspects, for example, a single dose that may immunize for extended periods of time even lifelong, the vaccine effectiveness no longer relying on previous exposure to flavivirus or DENV, the age of group to provide protection being the broadest possible including children under 9 years old, and its administration decreasing the chances of getting the most aggressive dengue disease at no point post vaccination.

Dengue is still a major public health concern worldwide. Several natural and social factors have contributed to the number of cases increased in recent decades. The efforts attained in the search of new antivirals and vaccines, and prevention and control strategies for this disease have not proved sufficient. However, the science and knowledge development acquired up to now provide us the tools we need

The views expressed in this chapter are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department

The author of this manuscript is an employee of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that 'Copyright protection under this title is not available for any work of the United States Government'. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part

to, and mark the way to follow, in order to achieve a control of this disease.

*DOI: http://dx.doi.org/10.5772/intechopen.92076*

**11. Conclusions**

**Conflict of interest**

**Copyright statement**

of that person's official duties.

**Disclaimer**

The authors declare no conflict of interest.

of Defense, nor the U.S. Government.

*Lessons Learned and Recent Advances in Dengue Research DOI: http://dx.doi.org/10.5772/intechopen.92076*

vaccine manufacturer, Sanofi Pasteur, announced that some people getting the vaccine without having been previously infected by DENV may be at risk for developing severe dengue if the disease is acquired after immunization [43]. The vaccine candidates utilize strategies such as vaccines of Live-attenuated virus, Inactivated virus, Recombinant protein, DNA vaccine, Viral vector vaccine, Virus like particles and others. Two vaccine candidates (DENVax and TV003/TV005) are being tested in efficacy trials in both Asia and Latin America. DENVax is a tetravalent recombinant live-attenuated dengue vaccine licensed to Takeda. The live virus vaccine utilizes chimerization with DENV-2 PDK-53 as the backbone with DENV-2/-1, -2/-3, and -2/-4 chimeras are created by replacing the DENV-2 prM and E genes with the respective genes from the other DENV serotypes. Other vaccine candidate is TV003/TV00 with a whole attenuated virus for three of four serotypes (attenuated by deletion of 30 nucleotides from 3′ UTR of DENV-1, DENV-3, DENV-4, and a chimeric DENV-2/DENV-4) [44]. In spite of having already a vaccine available to prevent dengue, there is a need to further research in order to improve the vaccine for dengue or to produce a new vaccine which enables improvements in certain aspects, for example, a single dose that may immunize for extended periods of time even lifelong, the vaccine effectiveness no longer relying on previous exposure to flavivirus or DENV, the age of group to provide protection being the broadest possible including children under 9 years old, and its administration decreasing the chances of getting the most aggressive dengue disease at no point post vaccination.

#### **11. Conclusions**

*Dengue Fever in a One Health Perspective*

–106

seroconversion produced is human-like [39, 40].

the virus replication in order to increase efficiency.

**9. Antivirals for treating dengue**

infection with DENV 104

activation.

supplies. DENV can slightly replicate in mice A/J, BALB/c and C57BL/6. Mice A/J and BALB/c can die of paralysis [39, 40]. For animal studies, it is considered that an

inoculum concentrations of a mosquito bite [39]. The mouse model is generally used initially to assess vaccines being the most suitable the immunocompetent mice but when it exhibits low viremia may result in the underestimation of the results. The use of nursing mice is a good way to assess possible candidate vaccines inducing neurovirulence. Although the NHP do not develop the disease, the antibody

At the moment, there is no antiviral therapy for DENV treatment. It is believed that the compounds working as inhibitors of RNA-dependent RNA polymerase have shown low efficiency. Moreover, there is a possibility that viral epitopes of DENV may trigger an immune cell response, preceded by development of severe disease. Thus, these epitopes are examined as targets for antiviral productions and they are known as DENV entry inhibitors and can be used in combination with inhibitors of

Inhibition of DENV attachment and entry into the host cell can inhibit immune

Various compounds as peptide entry inhibitors, DN59 and 1OAN1 may inhibit the antibody-dependent enhancement (ADE) in vitro. The doxorubicin antibiotic SA-17 is structured similarly to tetracycline and it has an antiviral activity against DENV serotype 1, 2 and 3 in Vero and C6/36 cells interfering with viral entry by binding to the hydrophobic pocket of the E-protein without exhibiting virucidal activity. The glycosidase inhibitors are unpopular due to its toxicity and low specificity but may help understand E-protein glycosylation processes. The binding agents to carbohydrates occur only during the stage of virus adsorption to the host cell. Concanavalin and agglutinins of wheat germ can bind N-acetylglucosamine residues and the percentage of DENV-lytic plaques in BHK cells can be reduced. Plant lectins such as *Hippeastrum hybrid*, *Galanthus nivalis*, and *Urtica dioica* inhibit DENV-2 infection in Raji/DC-SIGN cells. The compounds of heparan sulfate are potential recipients for DENV. Dengue E protein domain III is responsible for the interaction of heparan sulfate, and it is believed to be in all DENV serotypes and to have epitopes that are recognized by neutralizing antibodies. The suramin is similar to heparan sulfate and persulfated glycosaminoglycan, they bind to a polyanionbinding site of DENV E protein by inhibiting infection. The sulfated polysaccharides extracted from red algae, carrageenans and DL-galactan presented antiviral activity against 4 serotypes of dengue; however, the antiviral activity of DENV-4

DENV-1 weakened in Vero cells and in human Hepatocytes [41, 42].

In May 2019, the U.S. Food and Drug Administration (FDA) approved the use of Dengvaxia®, a vaccine against DENV that may be used in people aged from 9 to 45 years old [43]. This vaccine utilizes a live attenuated chimeric yellow fever/dengue virus based on a Yellow fever 17D vaccine virus backbone chimerized with prM and E proteins from DENV1–4 replacing the YF prM and E, and 3 doses are administered every 6 months. In phase III trials the overall protective efficacy was 56.7% and 60.8% in South East Asia and Latin America countries, respectively. In 2017, the

PFU (plaque-forming units) concentrations imitate

**26**

**10. Dengue vaccine**

Dengue is still a major public health concern worldwide. Several natural and social factors have contributed to the number of cases increased in recent decades. The efforts attained in the search of new antivirals and vaccines, and prevention and control strategies for this disease have not proved sufficient. However, the science and knowledge development acquired up to now provide us the tools we need to, and mark the way to follow, in order to achieve a control of this disease.

### **Conflict of interest**

The authors declare no conflict of interest.

## **Disclaimer**

The views expressed in this chapter are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

#### **Copyright statement**

The author of this manuscript is an employee of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that 'Copyright protection under this title is not available for any work of the United States Government'. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.

*Dengue Fever in a One Health Perspective*
