Flaviviruses, an expanding threat in public health: focus on Dengue, West Nile, and Japanese encephalitis virus

Abstract

General Introduction

The genus Flavivirus is composed of approximately 73 arthropod-borne viruses, or arboviruses, that infect rodents, pigs, birds, non-human primates, humans, and other mammalian hosts. Several members of this virus family which include the dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus, and Yellow Fever virus are associated with important human diseases which are transmitted by arthropod vectors. Hepatitis C virus is a notable exception as while it is related to the other medially important flaviviruses, it is one of the few members of this viral family that is not vector-borne. Most of these viruses can cause a wide variety of clinical manifestations and complications such as undifferentiated fever, capillary leakage-hemorrhagic disease, and encephalitis which can potentially lead to death. Most flaviviruses are zoonotic and depend upon non-human animal vectors for their survival, replication, and dispersal with the exception of DENV which propagates mainly in humans. While the evolutionary event that led to the increased spread of the viruses is still unknown, population movements, rapid urbanization, and widespread deforestation have contributed to the expansion of the pathogens into previously non-endemic areas (Bhatt et al, 2013; Petersen and Marfin, 2005). In addition, burgeoning travel to endemic areas and the slow increase in global temperatures due to climate changes have allowed the expansion of WNV and DENV into new territories (Beatty et al, 2005; Caminade et al, 2012; Hansen, 2006; Rahmstorf et al, 2007). Despite the adaptation, wild reservoirs of DENV are still maintained in subtropical and tropical areas that support a mosquito-monkey-mosquito transmission cycle (Gubler, 2002). While most flaviviruses are endemic in tropical areas, the distribution of several members of the viral family such as WNV has extended to temperate areas including the United States of America within the past decade (Caminade et al, 2012).

Flavivirus epidemiology and global dispersal

Flaviviruses are important human pathogens that have plagued mankind, accounting for millions of mortality worldwide (Table 1). The first recorded epidemic of dengue-like disease was reported between 1779 and 1780 when outbreaks occurred in Asia, Africa, and North America (Gubler, 1998; Gubler, 2002). Since its emergence, four DENV closely related but genetically distinct serotypes have been identified (Zanotto et al, 1996). Each serotype is believed to have emerged from a common ancestor and evolved separately (Kawaguchi et al, 2003) despite possessing different antigenicity and causing different degrees of illness severities but often potentially leading to dengue hemorrhagic fever (DHF; currently being changed to severe dengue as indicated by WHO). Variations within each serotype have been genetically detected, though not limited to, within the envelope protein (E) and non-structural protein, NS5, within the viral genome. WNV is being transmitted within in the U.S. population since the first reported case in 1999 (see Table 1 for details). Since then, the total number of West Nile virus infection cases has been increasing annually within the United States of America(http://www.cdc.gov/westnile/resources/pdfs/cummulative/99_2013_cummulativeHumanCases.pdf). Currently, DENV is endemic in at least 100 countries throughout Asia, the Pacific, the Americas, Africa, and the Caribbean (Figure 1). Approximately one million infected individuals suffer from either dengue fever or dengue hemorrhagic fever annually with Dengue-related deaths estimated to occur between 1 to 5 percent of those infected as reported by WHO (http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf) (see Table 1). DENV infections are often self-limiting and range from asymptomatic to relatively mild, undifferentiated illness with the patient eventually making full recovery. However, approximately 5% of symptomatic cases progress to a more severe disease whose manifestations include fever, myalgia, vomiting, and acute abdominal pain. The symptoms then progress further to hypotension, tachycardia, decreased peripheral perfusion, perisoreositis, and myocarditis (Krishnamurti et al, 2001; Malavige et al, 2004). In addition to these symptoms, hemorrhage develops approximately 7 days post-infection with no observed circulatory collapse (Krishnamurti et al, 2001). Factors influencing disease severity are not well understood. A predisposition to severe illness in secondary infections due to antibody-dependent enhancement of infection is often cited (Kliks et al, 1989).

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Figure 1

Global distribution of DENV in relation to its arthropod vectors. DENV is endemic in parts of Asia, Australia, Africa, and Latin America (area between the solid lines) and slowly expanding to other parts of the globe. Currently, DENV is located within the distribution range of A. aegypti and A. albopictus (dotted line). The presences of these mosquito vectors in non-DENV endemic areas suggest the potential for the expansion of the virus into new regions if optimal conditions are present.

In the United States and its territories, DENV transmission primarily occurs in tropical and subtropical areas such as Puerto Rico, the US Virgin Islands, American Samoa, and the US-Affiliated Pacific Islands (Imrie et al, 2006; Mohammed et al, 2010a; Rigau-Perez et al, 2001; Tomashek et al, 2009), where dengue is endemic. Dengue has been found to be the most frequent cause of febrile illness among US travelers returning from Asia, Latin America and the Caribbean (Freedman et al, 2006; Mohammed et al, 2010b; Sharp et al, 2012). In addition, outbreaks of dengue occur sporadically in non-endemic areas where the mosquito vectors exist (Brunkard et al, 2007; Graham et al, 2011; Radke et al, 2012; Ramos et al, 2008), Hawaii (Effleret al, 2005), and Florida, (Radke et al, 2012). Meanwhile, WNV infections have been reported throughout the continental United States. While the risk of WNV infection within various age groups remain the same, the potential for developing neurologic complications due to WNV infection increases with age. In the past 10 years approximately 40,000 individuals have become infected with WNV in the U.S. (http://www.cdc.gov/westnile/resources/pdfs/cummulative/99_2013_cummulativeHumanCases.pdf) , of which ~20% developed neuroinvasive diseases (i.e. encephalitis and meningitis) with 12% fatality rate (Lindsey et al, 2010). Interestingly, ~80% of WNV-infected individuals are asymptomatic.

Within days following infection with JEV, the host begins to exhibit clinical manifestations of the disease beginning with cephalalgia, vomiting, and pyrexia which last approximately 1 week (Sarkari et al, 2012b). After which, neurologic disorders ensues hallmarked by Parkinsonism, flaccid paralysis, and coma. Up to one third of infected patients acquire acute encephalitis which can often lead to complications and patient fatality (Lee et al, 2012; Sarkari et al, 2012a; Sarkari et al, 2012b). Indeed, up to fifty percent of patients who contract the virus often die due to complications. Interestingly, patients who enter convalescence remain seropositive against JEV despite persistent viremia (Ravi et al, 1993). Currently, there are no reported JEV cases within the U.S.

Distribution of viral vectors

DENV is transmitted to humans by the mosquito vectors, Aedes aegypti and Aedes albopictus. Similarly, the global dissemination of WNV (Figure 2) and JEV (Figure 3) rely on the Culex species of mosquitoes for their dispersal, in particular C. quinquefasciatus. Unlike WNV, however, JEV is currently localized in parts of Asia and surrounding islands and the northern region of Queensland, Australia. These mosquitoes, in particular A. aegypti, have become widely distributed across tropical and subtropical areas, including vast areas of the United States (Figure 1and ​and2).2). The spread of the DENV in the U.S. is exacerbated by the expanded range of A. albopictus in recent years, reaching as far as New England (Figure 2) (Mousson et al, 2005). In addition, mosquito species capable of transmitting WNV have also been discovered to be as far north as Canada (www.hc-sc.gc.ca). The spread of WNV as well as DENV is facilitated by the dispersal of suitable arthropod vectors which has been accelerated by rapid urbanization, increased travel into endemic countries, and adaptation to climate changes. Together, these events are expected to contribute to increases in the number of individuals affected by these pathogens (Anders et al, 2011; Petersen and Marfin, 2005).

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Figure 2

Distribution of WNV and DENV infections have been reported United States. To date, WNV infections (green states) have been documented throughout the United States with the exception of Alaska (gray). Texas, Florida, and New York are currently the only states in which both WNV and locally acquired DENV (red states) have been reported. The dotted line designates the northernmost distribution of the WNV mosquito vector, Culex sp. while the dashed line illustrates the northern-most territory of the DENV mosquito vectors, Aedes sp, in the United States.

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Figure 3

Distribution of JEV. JEV is currently endemic within South, East, and South East Asia (highlighted in blue) and its surrounding islands indicated by the red border. To date, there are no reported cases of JEV outside of this region; however, the expansion of its mosquito vector, Culex sp, to other countries worldwide make to spread of this dangerous pathogen a significant threat.

Typically, flaviviral transmission into humans occurs within two weeks of viremia following the initial feeding from an infected host. After entering a naϊve mosquito in the blood meal, the virus will require an additional 8-12 days of incubation before it can be re-transmitted to another human. Once infected with the virus, the mosquito remains infectious for the remained of its life. Symptoms among infected human hosts typically develop within seven days after the mosquito bite and lasts between 3-14 days. While some individuals do not develop any significant symptoms, they can still successfully transmit these viruses to others via mosquitoes. DENV is unique among other flaviviruses as they are the only virus within the family which utilizes humans as its amplifying host. Among WNV and JEV, viral amplification have been documented to occur within various mammalian hosts including horses, sheep, pigs, and goats; in the case of JEV, the virus has also been reported in bats as detected by qPCR (Liu et al, 2013; Olaleye et al, 1990; Pauvolid-Correa et al, 2011; Peiris et al, 1992). The expansion of WNV and JEV across different geographical regions is believed to be mediated by the seasonal migrations of birds (Reed et al, 2003).

Dengue is endemic to many parts of the tropics and subtropics, with outbreaks closely correlated with the completion of the annual monsoon season as reported by the WHO. During this time, an increase in the Aedes sp. mosquito population is also typically observed. Furthermore, the risk of contracting DHF also increases as more humans become infected with the virus during this short time period. A combination of optimal environmental conditions that facilitate an increase in infected arthropod vectors, greater presence of individuals with no immunity to one of the four virus types (DENV 1-4), and an opportunity for infected vector-host contact are required for the onset of DENV and other flavivirus epidemics. Interestingly, despite the induction of lifelong protective immunity against one DENV serotype, protection against the virus is partial and transient against other serotypes (Chen et al, 2004). Secondary infection by a different DENV serotype often leads to increased severity and carries a higher risk of susceptibility to DHF and patient mortality. DHF is a life-threatening illness characterized by internal bleeding, serious brain compromise, organ failure, and eventually death. In some cases dengue shock syndrome is observed among DF and DHF patients. While it is generally accepted that tertiary and quartenary DENV infections are asymptomatic, the lack of a clinical data that accurately determine multiple DENV infections among affected individuals and the importance of ADE make the surveillance of the virus and its treatment important. There are currently no approved vaccines or therapies for the disease. Treatment of infected patients is mostly palliative, with an administration of fluids and an occasional blood transfusion in the event of severe hemorrhage. The lack of treatment for DENV and related flaviviruses (i.e WNV and JEV) make these diseases debilitating and deadly.

In the past, DHF cases in the United States were only observed among travelers who have returned from visits to dengue-endemic areas. However, data from the Centers for Disease Control (CDC, http://www.cdc.gov/dengue/) and the National Resources Defense Council (NRDC, http://www.nrdc.org/health/dengue/) indicate that localized cases of DENV infection have already been detected in Hawaii, Texas, Florida, and New York along with the mosquitoes that are responsible for harboring the virus (Figure 2). While the U.S population currently has no immunity to the virus due to infrequent interactions between infected individuals and viral vectors which are necessary for successful dissemination of DENV, the presence of DENV within the U.S. suggests the imminent risk of infection among millions of Americans. Furthermore, the rapid spread of the WNV throughout the U.S. suggests the possibility for DENV to become prevalent within the U.S. given the proper existing conditions.

Flavivirus genetics and life cycle

Flaviviruses are composed of a single-stranded positive-sense RNA genome (~11 kilobases) packaged into a 40-60 nm virion comprising of a spherical nucleocapsid core coated in an icosahedral envelope (Harris et al, 2006; Rodenhuis-Zybert et al, 2010). The entire flaviviral genome consists of 10 genes encoding a large polyprotein that is post-translationally cleaved by host and viral proteases to produce three structural (protein capsid, C; prM/M protein, and envelope protein, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Figure 4) (Leyssen et al, 2000). The NS region encodes for various important proteases, in particular NS3 and NS2B, which are important in the auto-processing of the virus, mediation of viral genome replication, and virus packaging (Clyde and Harris, 2006). The transcription of these genes is controlled by the 5’- and 3’-untranslated regions (UTR) flanking the viral genome (Appaiahgari and Vrati, 2012b; Leyssen et al, 2000; Lindenbach et al, 2007).

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Figure 4

Flavivirus genome. The flavivirus genome consists of a single stranded positive-sense RNA encoding a polyprotein post-translationally cleaved by host proteases (sites designated by the blue arrows) and viral proteases (sites designated by green arrows). The site designated by the red arrow is cleaved by a yet to be identified protease. Processing of the polyprotein produces 3 structural (white boxes) and 7 non-structural genes (grey boxes). The prM protein is then later cleaved within the Golgi to release the M protein important for the maturation of the virus. Translation of viral RNA is controlled by the untranslated regions (UTR) located at the 5’ and 3’-ends of the RNA.

The mechanisms of flaviviral infection of the host cell and its life cycle are not fully understood. The current consensus is that endocytosis of the viral particle is important in the successful infection of the cell and the production of progeny viruses (Figure 5). Attachment of the DENV and other flaviviruses to cells utilizes multiple potential receptors that facilitate the attachment and internalization of the virus (i.e. CD14, R80, heparin sulfate, C-type lectin receptors, DC-SIGN, and mannose receptors) (Rodenhuis-Zybert et al, 2010); however, the exact mechanism by which DENV and other flaviviruses use these molecules for cellular internalization is still under investigation. The attached virus is internalized into an endosomal compartment which acidifies to facilitate the fusion of the viral envelope with the endosomal compartment (Figure 5). This fusion of the viral envelope is due to the rearrangement of the capsid proteins resulting in the release of the virus into the host cell (van der Schaar et al, 2008). The viral RNA is released into the host cytoplasm and transported to the endoplasmic reticulum (ER) where it undergoes two different fates: first, the positive sense RNA is translated to produce a polyprotein that is post-translationally cleaved into structural and non-structural proteins (listed above) or second, the genetic material is converted into a negative sense RNA by viral NS5 RNA dependent RNA polymerases (RdRp) and used to produce positive-stranded RNA copies (Figure 5). The viral genome is then packaged within the cytoplasm by the action of the protein C to form the nucleocapsid while the prM and E proteins heterodimerize within the lumen of the ER and initiate viral budding (Kuhn et al, 2002; Zhang et al, 2004). Nascent virion particles formed within the ER travel through the secretory pathway and into the Golgi apparatus. Changes in pH within the trans-Golgi network trigger the dissociation of the prM/E heterodimers activating the cellular endo-protease furin. Activation of this protease leads to the cleavage the prM protein to generate protein M (membrane associated) and the peptide pr (Rauscher et al, 1997; Yu et al, 2008; Zybert et al, 2008). The cleavage of this protein complex results in a mature, fully infectious virion.

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Figure 5

Flavivirus life cycle. Following attachment to the host extracellular surface (1), the virus is endocytosed (2) and encapsulated inside an endosomal vacuole. Acidification of the endosomal compartment alters the E protein causing the fusion of the virus with the endosome (3) facilitating virion release into the intracellular compartment where it is unpackaged (4). The released viral genome undergoes two different fates: the viral genome is either transported to the endoplasmic reticulum where it is translated into a polyprotein (5a) or converted into a negative-sense RNA to make positive-sense RNA copies (5b). The large polyprotein is post-translationally processed (6) producing structural and non-structural components important for virus assembly and maturation. The viral genome is packaged into a capsid and transported to the Golgi where it is coated by the E/M protein complex (7) to produce a mature virion (8).

Immuno and central nervous system pathogenesis

Studies of DENV immunopathogenesis are highly limited due to the nature of the disease and lack of animal models that recapitulate the disease. However, results obtained from humanized mice show some similarities in the development and progression of the disease which is typically observed in humans. Thus, these models will play an important role in elucidating flaviviral pathogenesis now and in the future (Mota and Rico-Hesse, 2011). Epidemiological studies have identified some of the risk factors important for disease development and severity following flaviviral infection. These factors include age (younger people are most susceptible to DENV and JEV infection while older or immunocompromised individuals are affected by WNV), high body mass index, viral strain, gender, genetic variation of the major histocompatibility complex (MHC)-class I-related sequence B, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), tumor necrosis factor-α (TNF-α) and phospholipase C epsilon 1 genes, environmental conditions such as mosquito spread and temperature, and secondary infection with a different viral strain (in the case of dengue) (Anders et al, 2011; Khor et al, 2011; Nguyen et al, 2005; Simmons et al, 2012).

In DENV infection, the serotype-specific life immunity acquired following the initial infection by the virus does not provide complete protection to other infecting serotypes. In fact, subsequent DENV infections increase the development of more severe dengue illnesses such DHF (Halstead et al, 2002; Kliks et al, 1988). The increased disease severity during secondary DENV infections is potentially due to the role of memory cells and antibodies. This is the concept for antibody-dependent enhancement (ADE) of secondary dengue infections (Halstead and O'Rourke, 1977). During ADE, antibodies generated after the initial infection recognize different DENV serotypes during secondary infections and facilitate increased binding and internalization of the virus via the Fc receptors on leukocytes and blood-brain barrier (BBB) cells. The adherence and infection of the BBB by DENV leads to inflammatory cytokine and chemokine production (i.e TNF-α, MCP-1/CCL2, IL-2, IL-6, IL-8, IL-10, and IL-12) as well as other inflammatory factors that lead to BBB compromise and CNS dysfunction (Anderson et al, 2011; Chaturvedi et al, 2000; Chen, 2012; Gubler, 1998; Malavige et al, 2004; Priyadarshini et al; Restrepo et al, 2008a; Restrepo et al, 2008b). This provides potential targets for therapeutic intervention against succeeding DENV infections.

Among leukocyte populations, dendritic and natural killer (NK) cells are the first ones to become infected. The infection of these cells occurs through pathogen recognizing receptors, such as toll-like receptors and DC-SIGN, and elicit inflammatory signals that lead to a Th₂ immune response and eventual vascular barrier dysfunction (Chaturvedi et al, 2000). Monocyte/macrophages and lymphoid cells (T and B cells) have also been described as major preferential targets for flaviviruses due to the presence of viral RNA or proteins in infected cells which have been collected from lymphoid tissues (Durbin et al, 2008; Jessie et al, 2004; King et al, 1999; Lin et al, 2002; Mentor and Kurane, 1997; Srikiatkhachorn et al, 2012). The high viral titers within the germinal centers of these tissues also suggest that these sites can harbor viral particles that can disseminate and infect other sites within the host. Interestingly, flaviviruses have developed strategies to circumvent the host immune response limiting clearance either by inhibiting IFN secretion (Munoz-Jordan et al, 2003; Umareddy et al, 2008) or by reducing antigen presentation through the limitation of MHC and co-stimulatory molecule expression (Palmer et al, 2005). The loss of these molecules will lead to an impairment of CD4⁺ T lymphocyte activation and CD8⁺ T lymphocyte response and thus leading to the loss of an immune response against the viruses and disease development (Aleyas et al, 2009; Aleyas et al, 2010; Chase et al, 2011).

The in vivo cellular targets of DENV and other flaviviruses in the CNS remain to be fully characterized; however, our laboratory and others have observed that primary human astrocytes and brain microvascular endothelial cells can be infected by DENV (Daep, Munoz-Jordan and Eugenin, unpublished data). Given the potential importance of astrocytes as HIV reservoirs within the central nervous system (CNS) (Eugenin and Berman, 2007; Eugenin et al, 2011), these cells may also function to maintain DENV reservoirs within the brain. To date, the full mechanism of flavivirus infection of the CNS remains poorly understood due to early studies which did not detect pathological signs of viral invasion within the CNS (Burke, 1968; Nathanson and Cole, 1970). Deeper examination of CNS pathology following DENV infection, however, will provide groundbreaking information that will elucidate the role of the blood brain barrier, leukocytes, and CNS inflammation following flaviviral infection in the development of dengue fever (DF) and DHF.

Older manuscripts indicate that DENV antigens can be found in several tissues including neurons in the cerebrum, Purkinje's and granular cells in the cerebellum, astrocyte, microglia and cells of the choroid plexus (Bhoopat et al, 1996a). However, earlier reports describing DHF pathology indicates no brain compromise and clearly stated that minimal damage was observed (Burke, 1968; Nathanson and Cole, 1970) despite the devastation of the CNS following infection by DENV and other flaviviruses. In addition, positive detection of flaviviral antigens without viral RNA (Jessie et al., 2004) indicated viral uptake by endocytosis and/or phagocytosis and not by productive infection that result in progeny virions. Recent improvements in research techniques could provide detection assays with increased sensitivity and improved accuracy. In agreement, recent publications have demonstrated the presence of dengue antigens in the CNS due to the detection of viral proteins, immunoglobulin, and RNA (Araujo et al, 2011; Lima et al, 2011; Miagostovich et al, 1997a; Miagostovich et al, 1997b). Viral infiltration and subsequent infection of the CNS have been linked directly to CNS viral replication. Thus, the mechanism of invasion of the CNS requires reexamination and further studies. Later in this review, we will discuss all these mechanisms.

Role of leukocytes in CNS disease

Although there is a general consensus that flaviviruses can infect mononuclear cells in vivo, only few cell types have been identified to be targeted for infection in vitro. Indeed, autopsy studies clearly indicated that presence of flaviviruses within the Langerhan's cells located in the skin (Clyde et al, 2006). However, the participation of these or other cell types in the flaviviral pathogenesis has yet to be fully explored. Below we will discuss the evidence of leukocyte infections by flaviviruses.

Blood-brain barrier (BBB) dysfunction and CNS compromise

The BBB is a physical and highly specialized barrier separating the peripheral circulation from the CNS. This barrier is composed of multiple cell types, including brain microvascular endothelial cells (BMVEC), astrocytic end feet, pericytes, and perivascular macrophages, all of which are in close contact to neurons, glial cells and microglia (Spindler and Hsu, 2012b). Due to the high expression of tight junction proteins, the BBB is normally impermeable to most peripheral molecules, cells and restricts the diffusion of ions and small molecules.

Several viruses alter the BBB integrity and function, including HIV-1, human T lymphotropic viruse 1 (HTLV-1), lymphocytic choriomeningitis (LCMV), WNV, and simian immunodeficiency virus (SIV). The mechanism of BBB compromise involves transmigration of leukocytes and these viruses into the CNS parenchyma leading to BBB disruption, inflammation, and leukocyte transmigration into the CNS (Buckner et al, 2006; Eugenin and Berman, 2003; Eugenin et al, 2006b; Spindler and Hsu, 2012a; Williams et al, 2012). HIV transmigration and BBB disruption is the best examined cases of CNS viral invasion. Our laboratory and others have demonstrated that HIV infection alters BBB function by three different mechanisms: 1) alteration of the migratory properties of leukocytes, 2) changes in BBB permeability and 3) secretion of factors that compromise leukocyte migration and BBB integrity. We and others have also shown that HIV infection of leukocytes alters the expression of several adhesion molecules involved in leukocyte transmigration and increases the levels of important chemokine receptors such as CCR2 which increases the sensitivity to CCL2, a key chemokine involved in the pathogenesis of HIV CNS disease (Eugenin et al, 2006b; Roberts et al, 2012b). In addition, we demonstrated that BBB permeability among neuroAIDS patients is compromised at two stages: first, during productive HIV infection of leukocytes and second, following increased expression of CCL2 in the CNS. This combination results in BBB disruption potentially due to enhanced secretion of MMP's and others factors that highly compromise the expression and function of key barrier tight junction proteins such as occludin, claudin-1 and ZO-1 (Roberts et al, 2012a; Roberts et al, 2012b). Some factors involved in HIV neuropathogenesis include cleaved forms of adhesion molecules, such as PrP^c and PECAM-1, with the latter competing with the PECAM-1-PECAM-1 or PrP^C-PrP^C interactions between endothelial cells. This direct competition leads to destabilization of the endothelium, compromising BBB integrity thereby facilitating leukocyte migration into the CNS (Eugenin et al, 2006a; Roberts et al, 2012a). However, our experiments using primary BMVEC's, indicate that CCL2 binding to its corresponding receptor alone has profound effects on tight junction protein expression and localization, resulting in BBB compromise by altering β-catenin distribution and interaction with adherence junctions and PECAM-1 (Roberts et al, 2012a; Roberts et al, 2012b). Thus, HIV CNS invasion is a highly regulated process and involves several host and viral components. But whether these mechanism are similar in flaviviruses is totally unknown.

Among the best described cases of CNS infection involves WNV. Due to the close relation of WNV with DENV and JEV, we expected that the mechanism of neuropathogenesis of these viruses to be similar (Figure 6). WNV attacks the brain causing neurotropic effects that include encephalitis and fever. In mouse models, WNV-associated neurologic disease is characterized by BBB disruption, increased leukocyte infiltration, inflammation, and neuronal loss (Glass et al, 2005; Samuel and Diamond, 2006). Some of these effects have been associated with the loss of BBB function due to degradation of junctional proteins such as ZO-1, claudin-1, occludin, JAM-A, β-catenin, VE-cadherin by secreted metalloproteinases (MMP) including −1, −3 and −9 (Roe et al, 2012a; Roe et al, 2012b). In addition, WNV infects human BMVEC's which could lead to a compromised BBB by inflammatory mechanisms described previously (Verma et al, 2009). Experiments using WNV-infected endothelial cells indicate an up-regulation of MHC–I and II, ICAM-1, VCAM-1, E- and P-selectin (King et al, 2003) which can contribute to leukocyte adhesion to the BBB and leukocyte infiltration into the CNS. During WNV infection, CD19⁺B220-BST-2⁺ leukocytes have been described as a major leukocyte population that transmigrates into the CNS and contributes to the development of encephalitis (Brehin et al, 2008). However, the mechanisms by which these cells transmigrate into the CNS remain unknown.

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Figure 6

Infection of CNS by flaviviruses. (1) Infection of the CNS occurs either through the adherence of the virus to molecules present on the surface of brain microvascular endothelial cells (BMVEC's) or infiltration of infected monocytes across the BBB. Viral infiltration then leads to infection of the BBB and CNS cell populations. (2) Infection of human astrocytes leads to chemokine production facilitating further recruitment of monocytes and macrophages. (3) Neurons infected by flaviviruses undergo apoptosis and activates the resident microglia population which produces an inflammatory response. Production of inflammatory cytokines (e.g. TNF-α, IL1β, INF-γ and IL-4), chemokines (e.g. CCL2, CCL5, CXCL9, CXCL10), inflammatory enzymes (COX2) and matrix-metalloproteinases (MMPs) leads to degradation of the endothelial barrier and the release of inflammatory factors (5) recruiting CD4⁺ and CD8⁺ T lymphocytes into the CNS parenchyma. Infiltration of CD4⁺/CD8⁺ T lymphocytes leads to further inflammation and eventually CNS damage.

Flavivirus infection of the CNS, especially with DENV, has been reported by a few studies with contradictory results. Viral RNA has been detected in the cerebrospinal fluid obtained from encephalitic individuals (Kumar et al, 2008; Miagostovich et al, 1997b). In addition, DENV antigens have been detected in neurons, astrocytes, and microglia (Bhoopat et al, 1996b; Ramos et al, 1998). While in situ hybridization for viral particles in neurons, BMVEC's, and glial cells were negative (Jessie et al, 2004), perivascular macrophages were positive for flaviviral NS3 protein. Further studies of these and other CNS cells are required to clarify their role in the neuropathogenesis of DENV and other flaviviruses. Thus, it is unclear whether proteins or RNA are product of replication, viral uptake or both.

Therapeutic approaches to flaviviral infections

Concluding remarks and future directions

The emergence of WNV in the United States, the world-wide expansion of DENV, the continuous threats of JEV, and the rapid dispersal of suitable mosquito vectors make research involving these pathogens imperative. It is clear that drastic global changes are affecting the spread of flaviviruses and their vectors. Further, with the rapidly expanding human population and the encroachment of these viruses in previously non-endemic areas, a global health problem involving these viruses is imminent. Thus, understanding how these viruses cause human diseases such as DHF is imperative. In addition, the ever increasing threats of new infections make the development of novel, effective vaccines and therapeutic agents vital. The sparse, conflicting information available regarding the pathogenesis of these viruses and the cell types, tissues, and organs they infect, however, make it difficult to produce successful drugs against any of these pathogens. Thus, until successful treatments and more effective vaccines are available, the best method of eliminating new infections in public and medical institutions is to stop viral vector spread and prevent becoming bitten by mosquitoes through the copious use of repellents.

The mechanisms of CNS dysfunction induced by flavivirus infection are poorly explored or ignored. The cell targets of flaviviruses and the replication pattern used by these viruses are still a matter of debate and require further investigation. Identifying how flaviviruses alter host cell physiology will provide the basis for understanding their pathogenesis and will subsequently lead to the design of more effective therapeutic agents against the viruses within the CNS. Recent advancements in research techniques and animal models and our data using primary human astrocytes and BMVEC's provided critical evidence suggesting that flaviviruses are indeed neurotropic. Further, identifying how these viruses regulate leukocyte activation and CNS transmigration as well as dysregulation of BBB function leading to CNS compromise and human disease is critically important.

Acknowledgements

Footnotes

References