**4. Pathogenesis of arboviral infections**

Arboviral diseases start with a bite from an arthropod creature carrying infectious virus. The pathogen may be considered an innocent bystander or an unnecessary byproduct from an infected vertebrate host. The arthropod imbibes this blood for its own purposes, to facili‐ tate ovulation, and takes up the accompanying virus in the meal. The presence of pathogen is not a critical event in the life cycle of the insect and may or may not cause it harm. The persistence of disease is none of these creatures' fault, since survival is the game plan for all organisms on earth. In many instances, arboviruses are capable of surviving inside the com‐ ing host without inducing any visible adverse effects. Given the opportunity, the pathogens will reentry and challenge a new host. If the host is capable of implementing a "survival strategy" in response to the viral infection, the host will be fine. Occasionally, these crea‐ tures may enter a host, such as human beings, in which the environment may not be as friendly as others, and a hostile survival race is engaged. The race tactics instigated by both sides are normally controllable and do not result in overt disease. But in some cases, the reg‐ ulatory programs in the host do not coordinate well with each other or could also be distur‐ bed and/or handcuffed by substances released from the pathogens. This can result in dysfunctional operational systems that are harmful to the host, leading to detrimental out‐ comes, including death. As a whole, the occurrence of the severe consequences is very rare. For instance, with JEV infection, the overall global incidence of cases annually is at 1.8 per 100,000 people [60].

Timing is critical in the diagnosis of acute arboviral encephalitis. The progression and varia‐ tion in clinical manifestations among infected subjects may differ, depending on the individ‐ ual's age and geographical habitat, the arthropod's feeding behavior, genetic differences in the viral strain, and the immune status of the affected patients. One of the common clinical features in arboviral infections is viremia. However, the duration and level of this viremia in humans is significantly different with each and every family of viruses. In a commensal ar‐ boviral-host relationship, one may expect high levels of viremia to cause too much patho‐ genesis in the host but too low levels to not facilitate transmission. One may expect a consistent middle range in viremia to be obtained. Extreme variation in or high titers of vi‐ rus in the blood may be a sign that humans are an accidental or dead-end host to most arbo‐ viruses. Identifying the cellular sources responsible for viremia will likely help us uncover the underlying mechanisms leading to arboviral encephalitis and aid in the development of vaccines and anti-viral drugs. Because of this, finding the permissive cell lineages account‐ ing for circulating virus in infected patients has been the central focus for several decades. In spite of these efforts, the answer remains elusive.

Under normal circumstances, lymphocytes constantly enter the CNS, but in small numbers [56]. However their presence in the CNS may increase in response to viral infections [57]. In fact, infected PBMCs can be isolated in brains from mice inoculated with JE virus as early as 3 days post-infection [58]. Moreover, leukocytes were observed moving between endothelial cells of capillaries at sites in the BBB where tight junctions had been dissociated [49]. This suggests that at least some inflammatory leukocytes that had become infected in the periph‐ ery move along in the blood current and migrate to the CNS tissues [58, 59]. Furthermore, infection and resultant apoptosis of astrocytes, which serve as a protective component of the BBB and can defend against penetrated virions or virus-infected leukocytes, are frequently seen in the brain. This probably results in severe impairment of the BBB, facilitating the pas‐

Arboviral diseases start with a bite from an arthropod creature carrying infectious virus. The pathogen may be considered an innocent bystander or an unnecessary byproduct from an infected vertebrate host. The arthropod imbibes this blood for its own purposes, to facili‐ tate ovulation, and takes up the accompanying virus in the meal. The presence of pathogen is not a critical event in the life cycle of the insect and may or may not cause it harm. The persistence of disease is none of these creatures' fault, since survival is the game plan for all organisms on earth. In many instances, arboviruses are capable of surviving inside the com‐ ing host without inducing any visible adverse effects. Given the opportunity, the pathogens will reentry and challenge a new host. If the host is capable of implementing a "survival strategy" in response to the viral infection, the host will be fine. Occasionally, these crea‐ tures may enter a host, such as human beings, in which the environment may not be as friendly as others, and a hostile survival race is engaged. The race tactics instigated by both sides are normally controllable and do not result in overt disease. But in some cases, the reg‐ ulatory programs in the host do not coordinate well with each other or could also be distur‐ bed and/or handcuffed by substances released from the pathogens. This can result in dysfunctional operational systems that are harmful to the host, leading to detrimental out‐ comes, including death. As a whole, the occurrence of the severe consequences is very rare. For instance, with JEV infection, the overall global incidence of cases annually is at 1.8 per

Timing is critical in the diagnosis of acute arboviral encephalitis. The progression and varia‐ tion in clinical manifestations among infected subjects may differ, depending on the individ‐ ual's age and geographical habitat, the arthropod's feeding behavior, genetic differences in the viral strain, and the immune status of the affected patients. One of the common clinical features in arboviral infections is viremia. However, the duration and level of this viremia in humans is significantly different with each and every family of viruses. In a commensal ar‐ boviral-host relationship, one may expect high levels of viremia to cause too much patho‐ genesis in the host but too low levels to not facilitate transmission. One may expect a consistent middle range in viremia to be obtained. Extreme variation in or high titers of vi‐

sage of more virus-infected PBMCs, using a "Trojan horse" strategy.

**4. Pathogenesis of arboviral infections**

100,000 people [60].

78 Encephalitis

**Figure 3.** The possible route of the virus in vertebrates from peripheral tissues to the brain. Arboviral infections start with the bite of insects carrying an infectious virus. The exact location where the virus is deposited remains poorly un‐ derstood. There are multiple ways a virus may spread and circulate before reaching to the brain. Please refer to the text for more details.

Arboviral infections are introduced into the hosts during the blood meals of arthropods car‐ rying infectious virus. The first obstacle that the arthropod encounters is the physical barrier of the skin, which is composed of several layers of keratinocytes interspersed with a net‐ work of capillaries (Figure 3). There are two possible routes that the virus may use as a res‐ ervoir to amplify the progeny after its deposition by the mosquito. One passage way may be released into the blood pools of lacerated capillaries. In this situation, it is generally as‐ sumed that the initial target cell supporting the viral replication is Langerhans dendritic cells of the skin (Figure 3, route 1) [61]. The infected Langerhans dendritic cells migrate to draining lymph nodes where a brief viral replication may occur and the virus is considered to enter the blood stream through the lymphatic and thoracic ducts [61]. The virus may en‐ ter the bone marrow [62] or liver [63] where a secondary amplification may occur or directly disseminate to the brain inducing inflammation.

hans dendritic cells and the cycle of illness will resume. If this is the case, then we would observe a sinusoidal wave-like pattern for viremia in infected dengue patients. But in reali‐ ty, this is not the case. Thus, this evidence indicates that an alternate route could exist, such as direct deposition of virus into the blood stream. Interestingly, it has been suggested that during imbibing, approximately 50% of the fascicle penetrates into the skin [68], suggesting that the location of the blood drawn by the vector is from the capillary-rich dermis layer,

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One of the puzzling issues is what cellular constituents are the protective components in asymptomatic cases. Interestingly, apoptotic keratinocytes and dendritic cells are observed in human skin explants when dengue virus is directly injected into the epidermis with a fine needle [69]. Considering the fact that a majority of dengue virus infections are asymptomat‐ ic, this evidence suggests that the role of dendritic cells at the site of fascicle penetration is to eliminate or temporarily contain the intruders and thereby prevent or reduce the dissemina‐ tion of dengue virus. However, the role of keratinocytes and dendritic cells in clearance of

Although most persons bitten by an infected mosquito will experience no symptoms or will have a very mild presentation of the disease, approximately 1 to 2 percent will develop a recognizable illness. The clinical symptoms for the initial phase of arboviral encephalitis are very similar and similarly variable from person-to-person for all the virus families. Some in‐ dividuals may have mild symptoms, such as a fever and headache, while others may have a more severe presentation. In this case, symptoms may include a rapid onset of severe head‐ ache, high fever, muscle aches, stiffness in the back of the neck, and problems with muscle coordination, disorientation, photophobia, convulsions and coma. The illness will usually occur five to 15 days after the bite of an infected mosquito or tick. However, the symptoms may resemble other common febrile illnesses. Thus, in order to diagnose correctly and de‐ termine the proper treatment in a timely manner, it is important to seek professional help

In order for an affected subject to have a risk for neurological disorder, the virus entering the human host has to possess two major criteria: neuroinvasiveness and neruovirulence. The term "neuroinvasiveness" means that the virus is capable of passing or crossing through the BBB, a structure that separates the immune privileged compartment of the brain from the peripheral system. The term "neruovirulence" refers to the capacity of viral replica‐ tion in the CNS tissues. There are several mechanisms involved on the induction of neuroin‐ vasion. The virus can either replicate and induced damage of the nearby endothelial cells [70] in the cerebral capillary or in striated muscle [71] surrounding the BBB. Alternatively, virus may enter the CNS by endocytosis via the olfactory bulb or the choroid plexus, for ex‐ ample, JEV [43], CHIKV [72] and VEEV [73]. In addition, high viremia is a major feature of only some of the arboviral infections, thus some viruses can cross the BBB via the vascular route by passive transfer carried by infected leukocytes [74]. Spreading virus to the CNS through the trigeminal nerve after local amplification of the virus has been proposed as well [73]. The neurological symptoms induced by some of these arboviruses, which are able to increase the permeability of vasculature and spillover into the CNS, are capable of disrupt‐

implicating that pathogens may be directly injected into the blood.

dengue virus remains to be further investigated.

immediately or as soon as clinical signs appear.

An alternate route would be direct deposition of the viruses into the blood stream (Figure 3, route 2), or so-called capillary feeding, during the engorgement of the arthropod. Results from RVFV suggest that the liver seems to be an early and dominant target of the virus [63]. The damage to the hepatocytes of the RVFV-infected liver is likely a result of apoptosis [63]. The evidence suggests that this virus may get deposit directly into the capillary and take a ride through the circulation to the liver compartment where permissive cells, likely hepato‐ cytes, provide RVFV a means to produce progeny (Figure 3). In addition, studies investigat‐ ing mosquito imbibing behavior with *Aedes aegypti* revealed that the mosquito's proboscis is flexible and predominantly obtains blood directly from the capillary and only occasionally from the blood pools formed in the tissues by the leakage from previously lacerated capilla‐ ries [64]. These results were later confirmed with the mouse's ear and human beings imple‐ menting the same experimental designs [65, 66]. In this route (Figure 3, route 2), the virus may gain access directly to the bone marrow where a brief viral replication can occur, ex‐ travasates into the circulation, disseminates to other parts of the body, and penetrates the brain via mechanisms discussed in Figure 2.

However, determining the first cells infected by the viruses subsequent to the bite remains a challenging event to investigators. The scenario via route 1 (Figure 3) is complicated by a number of issues. Keratinocytes on the outermost epidermal layer of the skin are endowed with toll-like-receptors (TLR) [67] and may be considered a component of the primary in‐ nate immune system. Langerhans cells mainly reside in the thin layer of the epidermis, which does not contain capillaries, while the dendritic cells are predominantly in the thicker dermis layer, which is filled with capillaries. Route 1 has been extensively investigated with diseases derived from mosquito-borne viruses. This pathway could be the true route for those viruses belonging to the human-is-dead-end-host group, since the virus titers from these cells are too low to permit transmission to new mosquitoes. In contrast, if human be‐ ings are the host for the virus, such as dengue virus, then the assumption that this virus takes this route should be reconsidered. Experiments have revealed that only a very short window period is available for dengue virus to be transmitted, during the high viremic stage, usually within 3-5 days after the onset of the clinical fever. Thus, if the mosquitoes imbibe the blood meal during this stage, the virus will spillover and infect the local Langer‐ hans dendritic cells and the cycle of illness will resume. If this is the case, then we would observe a sinusoidal wave-like pattern for viremia in infected dengue patients. But in reali‐ ty, this is not the case. Thus, this evidence indicates that an alternate route could exist, such as direct deposition of virus into the blood stream. Interestingly, it has been suggested that during imbibing, approximately 50% of the fascicle penetrates into the skin [68], suggesting that the location of the blood drawn by the vector is from the capillary-rich dermis layer, implicating that pathogens may be directly injected into the blood.

Arboviral infections are introduced into the hosts during the blood meals of arthropods car‐ rying infectious virus. The first obstacle that the arthropod encounters is the physical barrier of the skin, which is composed of several layers of keratinocytes interspersed with a net‐ work of capillaries (Figure 3). There are two possible routes that the virus may use as a res‐ ervoir to amplify the progeny after its deposition by the mosquito. One passage way may be released into the blood pools of lacerated capillaries. In this situation, it is generally as‐ sumed that the initial target cell supporting the viral replication is Langerhans dendritic cells of the skin (Figure 3, route 1) [61]. The infected Langerhans dendritic cells migrate to draining lymph nodes where a brief viral replication may occur and the virus is considered to enter the blood stream through the lymphatic and thoracic ducts [61]. The virus may en‐ ter the bone marrow [62] or liver [63] where a secondary amplification may occur or directly

An alternate route would be direct deposition of the viruses into the blood stream (Figure 3, route 2), or so-called capillary feeding, during the engorgement of the arthropod. Results from RVFV suggest that the liver seems to be an early and dominant target of the virus [63]. The damage to the hepatocytes of the RVFV-infected liver is likely a result of apoptosis [63]. The evidence suggests that this virus may get deposit directly into the capillary and take a ride through the circulation to the liver compartment where permissive cells, likely hepato‐ cytes, provide RVFV a means to produce progeny (Figure 3). In addition, studies investigat‐ ing mosquito imbibing behavior with *Aedes aegypti* revealed that the mosquito's proboscis is flexible and predominantly obtains blood directly from the capillary and only occasionally from the blood pools formed in the tissues by the leakage from previously lacerated capilla‐ ries [64]. These results were later confirmed with the mouse's ear and human beings imple‐ menting the same experimental designs [65, 66]. In this route (Figure 3, route 2), the virus may gain access directly to the bone marrow where a brief viral replication can occur, ex‐ travasates into the circulation, disseminates to other parts of the body, and penetrates the

However, determining the first cells infected by the viruses subsequent to the bite remains a challenging event to investigators. The scenario via route 1 (Figure 3) is complicated by a number of issues. Keratinocytes on the outermost epidermal layer of the skin are endowed with toll-like-receptors (TLR) [67] and may be considered a component of the primary in‐ nate immune system. Langerhans cells mainly reside in the thin layer of the epidermis, which does not contain capillaries, while the dendritic cells are predominantly in the thicker dermis layer, which is filled with capillaries. Route 1 has been extensively investigated with diseases derived from mosquito-borne viruses. This pathway could be the true route for those viruses belonging to the human-is-dead-end-host group, since the virus titers from these cells are too low to permit transmission to new mosquitoes. In contrast, if human be‐ ings are the host for the virus, such as dengue virus, then the assumption that this virus takes this route should be reconsidered. Experiments have revealed that only a very short window period is available for dengue virus to be transmitted, during the high viremic stage, usually within 3-5 days after the onset of the clinical fever. Thus, if the mosquitoes imbibe the blood meal during this stage, the virus will spillover and infect the local Langer‐

disseminate to the brain inducing inflammation.

80 Encephalitis

brain via mechanisms discussed in Figure 2.

One of the puzzling issues is what cellular constituents are the protective components in asymptomatic cases. Interestingly, apoptotic keratinocytes and dendritic cells are observed in human skin explants when dengue virus is directly injected into the epidermis with a fine needle [69]. Considering the fact that a majority of dengue virus infections are asymptomat‐ ic, this evidence suggests that the role of dendritic cells at the site of fascicle penetration is to eliminate or temporarily contain the intruders and thereby prevent or reduce the dissemina‐ tion of dengue virus. However, the role of keratinocytes and dendritic cells in clearance of dengue virus remains to be further investigated.

Although most persons bitten by an infected mosquito will experience no symptoms or will have a very mild presentation of the disease, approximately 1 to 2 percent will develop a recognizable illness. The clinical symptoms for the initial phase of arboviral encephalitis are very similar and similarly variable from person-to-person for all the virus families. Some in‐ dividuals may have mild symptoms, such as a fever and headache, while others may have a more severe presentation. In this case, symptoms may include a rapid onset of severe head‐ ache, high fever, muscle aches, stiffness in the back of the neck, and problems with muscle coordination, disorientation, photophobia, convulsions and coma. The illness will usually occur five to 15 days after the bite of an infected mosquito or tick. However, the symptoms may resemble other common febrile illnesses. Thus, in order to diagnose correctly and de‐ termine the proper treatment in a timely manner, it is important to seek professional help immediately or as soon as clinical signs appear.

In order for an affected subject to have a risk for neurological disorder, the virus entering the human host has to possess two major criteria: neuroinvasiveness and neruovirulence. The term "neuroinvasiveness" means that the virus is capable of passing or crossing through the BBB, a structure that separates the immune privileged compartment of the brain from the peripheral system. The term "neruovirulence" refers to the capacity of viral replica‐ tion in the CNS tissues. There are several mechanisms involved on the induction of neuroin‐ vasion. The virus can either replicate and induced damage of the nearby endothelial cells [70] in the cerebral capillary or in striated muscle [71] surrounding the BBB. Alternatively, virus may enter the CNS by endocytosis via the olfactory bulb or the choroid plexus, for ex‐ ample, JEV [43], CHIKV [72] and VEEV [73]. In addition, high viremia is a major feature of only some of the arboviral infections, thus some viruses can cross the BBB via the vascular route by passive transfer carried by infected leukocytes [74]. Spreading virus to the CNS through the trigeminal nerve after local amplification of the virus has been proposed as well [73]. The neurological symptoms induced by some of these arboviruses, which are able to increase the permeability of vasculature and spillover into the CNS, are capable of disrupt‐ ing cognitive biological processes. In order to differentiate the evasion strategies employed, animal models are required. Currently, there are only a limited number of animal models available for a few arboviruses; JEV [59], EEEV [75], LACV [71], WNV [76] and CHIKV [72]. However, the cardinal features of human clinical encephalitis induced by these arboviruses are hardly reproduced in these models. Therefore, what the exact mechanisms by which ar‐ boviruses cross the BBB remains poorly understood, as well as the precise mechanisms by which circulating peripheral pathogens induce the inflammation of the brain remain largely unknown.

netic resonance imaging (MRI) and X-ray computed tomography (CT). These tests allow for a scan of the head to detect abnormalities, such as swelling (edema) and bleeding (hemor‐ rhage) [86]. These sophisticated instruments are likely available in very advanced clinics and may not be very convenient or available for the majority of patients affected by arboviral en‐ cephalitis. Thus, alternate diagnostic methods are applied. These are biological approaches, which include virus isolation from cerebrospinal fluid, blood, and biopsy specimens, detec‐ tion of viral genetic and/or antigenic materials, and specific antibodies to the virus. Howev‐ er, there are pros and cons for each of these diagnostic assays. Sensitivity and specificity,

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*Currently used drugs to treat arboviral encephalitis.* There is no cure for arboviral encephalitis and treatment is generally supportive, with maintenance of respiratory and circulatory sys‐ tems while the infection runs its course. The purpose of the palliative care is to reduce the malfunctioning of critical organs and to relieve symptoms, while the body fights the infec‐ tion. The priority of the treatment is to ensure the alleviation of pain, as well as to mitigate the swelling in the brain, reduce the fever and prevent dehydration and other chemical im‐ balances by administration of intravenous fluids. As a whole, the treatment for arboviral en‐ cephalitis depends on the cause. Some clinical cases of arboviral encephalitis can be mitigated successfully if medication is started as soon as possible. A number of therapeutic drugs specific to arboviral infections are under investigation for their potential antiviral and neuroprotective effects: minocycline and curcumin for JEV and other arboviruses [87-89], ribavirin for LACV [90], interferon (Omr-IgG-aM) and humanized monoclonal antibody (Mab E16) as a potential candidate for WNV treatment [61, 91, 92]. However, currently there is limited information available on the effectiveness of these therapeutic modalities in the clinical setting. Additionally, there are a number of reliable medicines that are commonly prescribed to treat the symptoms mentioned above; administration of benzodiazepines (*e.g.*, lorazepam [Ativan®) to prevent seizure, diuretics drugs (*e.g.*, furosemide or mannitol) to re‐ duce brain swelling, sedatives to relieve irritability, antibiotics to prevent secondary infec‐ tions, and acetaminophen to control fever and headache. For those patients whose brain functions may be severely affected, interventions like physical therapy and speech therapy

The life cycle of arboviruses *in vivo* is not well understood, even though a great amount of detail on the comprehensive biology of these viruses *in vitro* has been intensively investigat‐ ed and uncovered. As aforementioned, the genetic material for a majority of the arbovriuses is positive-sense single-stranded RNA, which can function as mRNA and be infectious by itself. It has been proposed that this genomic viral RNA can become encapsulated within the

and antibody cross-reactivity are always a concern.

**5. Treatment of arboviral infections**

may be needed after the illness is controlled.

**6. New drug development**

Nevertheless, the best systems available that have been used to characterize the biological properties of arboviruses in animal models are the WNV [76, 77], LACV [71], EEEV [75], and CHIKV [72, 78]. Results revealed that viral strain variations, in addition to the host age and immune conditions, contribute a significantly to neuroinvasiveness and neurovirulence. In‐ fection of the mice intradermally or subcutaneously leads to the robust replication of WNV, LACV, and CHIKV in the brain, particularly in newborn mice. But the mechanisms contri‐ buting to neurotropism of other viruses are less clear since suitable models are not available.

When viruses enter the CNS, a variety of cells are permissive for infection [46, 79]; some cells may be more susceptible than others, and the viruses may have their differential prefer‐ ences [74, 75, 78, 80]. Regardless, the net consequence is the activation and/or damage to res‐ idential cells. This results in the recruitment of defense cells with immune system functions to the damaged site. An inflammatory response occurs due to the presence of an overpro‐ duction of multiple functional cytokines from the infiltrating cells [81-83]. The nature of the privileged environment of the brain bestows it with characteristics that make restoration to the default normal status far more complicated than other parts of the body. The most sali‐ ent feature of the brain is that a large proportion of the cells are terminally differentiated. These cells are very difficult to renew and replace. Therefore, affected encephalitic patients suffer long-term neurological impairment as a result from the infection [18, 28]. These symp‐ toms include short-term or long-term memory loss, seizures, and impaired judgment [28, 84, 85]. A neurological exam is performed to evaluate the mental status, detect neurological problems, such as motor dysfunction and seizures, and help determine which area of the brain is affected [18].

The causes of the dysfunctional circuitry in neurons are likely different among the arbovi‐ ruses. Some viruses have the capacity of direct engagement with neurons by infection, while others may induce cell death or apoptosis in nearby cells, which shed releasates, likely trig‐ gering a cascade of events that damages the neuronal tissue [81, 82]. This may be why some viruses can be recovered from the CNS easier than others in autopsy specimens. For those viruses capable of infecting small animals, results also suggest the observed scenarios. In contrast, for the viruses with limited capacity to replicate in animal models, the actual caus‐ es of neurological symptoms are less clear.

The initial symptoms of the arbovirus infections that induced encephalitis are very similar, especially for those mild cases of encephalitis, which makes the correct diagnosis a challenge to physicians. In order for accurate diagnosis, in addition to the routine examination on the physical performance, specific tests are required, such as electroencephalogram, brain mag‐ netic resonance imaging (MRI) and X-ray computed tomography (CT). These tests allow for a scan of the head to detect abnormalities, such as swelling (edema) and bleeding (hemor‐ rhage) [86]. These sophisticated instruments are likely available in very advanced clinics and may not be very convenient or available for the majority of patients affected by arboviral en‐ cephalitis. Thus, alternate diagnostic methods are applied. These are biological approaches, which include virus isolation from cerebrospinal fluid, blood, and biopsy specimens, detec‐ tion of viral genetic and/or antigenic materials, and specific antibodies to the virus. Howev‐ er, there are pros and cons for each of these diagnostic assays. Sensitivity and specificity, and antibody cross-reactivity are always a concern.
