Acute disseminated encephalomyelitis following Saint Louis encephalitis virus infection.

Saint Louis encephalitis virus (SLEV) infection is an arbovirosis associated with a broad spectrum of neurological complications. We present a case of a 55-year-old man hailing from Manaus, a city situated in the heart of the Amazon Rainforest, who exhibited symptoms of vertigo, tremors, urinary and fecal retention, compromised gait, and encephalopathy 3 weeks following SLEV infection. Neuroaxis MRI revealed diffuse, asymmetric, and poorly defined margins hyperintense lesions with peripheral and ring enhancement in subcortical white matter, as well as severe spinal cord involvement. Serology for SLEV was positive both on serum and cerebrospinal fluid. To the best of our knowledge, the present report is the first to show brain lesions along with myelitis as a post-infectious complication of SLEV infection.

Reemergence of St. Louis Encephalitis Virus in the Americas.

We summarize and analyze historical and current data regarding the reemergence of St. Louis encephalitis virus (SLEV; genus Flavivirus) in the Americas. Historically, SLEV caused encephalitis outbreaks in the United States; however, it was not considered a public health concern in the rest of the Americas. After the introduction of West Nile virus in 1999, activity of SLEV decreased considerably in the United States. During 2014-2015, SLEV caused a human outbreak in Arizona and caused isolated human cases in California in 2016 and 2017. Phylogenetic analyses indicate that the emerging SLEV in the western United States is related to the epidemic strains isolated during a human encephalitis outbreak in Córdoba, Argentina, in 2005. Ecoepidemiologic studies suggest that the emergence of SLEV in Argentina was caused by the introduction of a more pathogenic strain and increasing populations of the eared dove (amplifying host).

West Nile and St. Louis encephalitis viral genetic determinants of avian host competence.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

Development of a model of Saint Louis encephalitis infection and disease in mice.

BACKGROUND: Flaviviruses are a genre of closely related viral pathogens which emerged in the last decades in Brazil and in the world. Saint (St.) Louis encephalitis virus (SLEV) is a neglected flavivirus that can cause a severe neurological disease that may lead to death or sequelae. St. Louis encephalitis pathogenesis is poorly understood, which hinders the development of specific treatment or vaccine. METHODS: To address this problem, we developed a model of SLEV infection in mice to study mechanisms involved in the pathogenesis of severe disease. The model consists in the intracranial inoculation of the SLEV strain BeH 355964, a strain isolated from a symptomatic human patient in Brazil, in adult immunocompetent mice. RESULTS: Inoculated mice presented SLEV replication in the brain, accompanied by tissue damage, disease signs, and mortality approximately 7 days post infection. Infection was characterized by the production of proinflammatory cytokines and interferons and by leukocyte recruitment to the brain, composed mainly by neutrophils and lymphocytes. In vitro experiments indicated that SLEV is able to replicate in both neurons and glia and caused neuronal death and cytokine production, respectively. CONCLUSIONS: Altogether, intracranial SLEV infection leads to meningoencephalitis in mice, recapitulating several aspects of St. Louis encephalitis in humans. Our study indicates that the central nervous system (CNS) inflammation is a major component of SLEV-induced disease. This model may be useful to identify mechanisms of disease pathogenesis or resistance to SLEV infection.

Astrocyte response to St. Louis encephalitis virus.

St. Louis encephalitis virus (SLEV), a flavivirus transmitted to humans by Culex mosquitoes, causes clinical symptoms ranging from acute febrile disorder to encephalitis. To reach the central nervous system (CNS) from circulating blood, the pathogen must cross the blood-brain barrier formed by endothelial cells and astrocytes. Because astrocytes play an essential role in CNS homeostasis, in this study these cells were infected with SLEV and investigated for astrogliosis, major histocompatibility complex (MHC)-I-dependent immune response, and apoptosis by caspase-3 activation. Cultures of Vero cells were used as a positive control for the viral infection. Cytopathic effects were observed in both types of cell cultures, and the cytotoxicity levels of the two were compared. Astrocytes infected with a dilution of 1E-01 (7.7E+08 PFU/mL) had a reduced mortality rate of more than 50% compared to the Vero cells. In addition, the astrocytes responded to the flavivirus infection with increased MHC-I expression and astrogliosis, characterized by intense glial fibrillary acidic protein expression and an increase in the number and length of cytoplasmic processes. When the astrocytes were exposed to higher viral concentrations, a proportional increase in caspase-3 expression was observed, as well as nuclear membrane destruction. SLEV immunostaining revealed a perinuclear location of the virus during the replication process. Together, these results suggest that mechanisms other than SLEV infection in astrocytes must be associated with the development of the neuroinvasive form of the disease.

Consequences of in vitro host shift for St. Louis encephalitis virus.

Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. As arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are still not characterized fully for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrated that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants implicates the NS3 gene as an important host range determinant for SLEV.

Provenance and geographic spread of St. Louis encephalitis virus.

St. Louis encephalitis virus (SLEV) is the prototypic mosquito-borne flavivirus in the Americas. Birds are its primary vertebrate hosts, but amplification in certain mammals has also been suggested. The place and time of SLEV emergence remain unknown. In an ecological investigation in a tropical rainforest in Palenque National Park, Mexico, we discovered an ancestral variant of SLEV in Culex nigripalpus mosquitoes. Those SLEV-Palenque strains form a highly distinct phylogenetic clade within the SLEV species. Cell culture studies of SLEV-Palenque versus epidemic SLEV (MSI-7) revealed no growth differences in insect cells but a clear inability of SLEV-Palenque to replicate in cells from birds, cotton rats, and free-tailed bats permissive for MSI-7 replication. Only cells from nonhuman primates and neotropical fruit bats were moderately permissive. Phylogeographic reconstruction identified the common ancestor of all epidemic SLEV strains to have existed in an area between southern Mexico and Panama ca. 330 years ago. Expansion of the epidemic lineage occurred in two waves, the first representing emergence near the area of origin and the second involving almost parallel appearances of the virus in the lower Mississippi and Amazon delta regions. Early diversification events overlapped human habitat invasion during the post-Columbian era. Several documented SLEV outbreaks, such as the 1964 Houston epidemic or the 1990 Tampa epidemic, were predated by the arrival of novel strains between 1 and 4 years before the outbreaks. Collectively, our data provide insight into the putative origins of SLEV, suggesting that virus emergence was driven by human invasion of primary rainforests. IMPORTANCE St. Louis encephalitis virus (SLEV) is the prototypic mosquito-transmitted flavivirus of the Americas. Unlike the West Nile virus, which we know was recently introduced into North America from the Old World, the provenience of SLEV is obscure. In an ecological investigation in a primary rainforest area of Palenque National Park, Mexico, we have discovered an ancestral variant of SLEV. The ancestral virus was much less active than the epidemic virus in cell cultures, reflecting its incomplete adaptation to hosts encountered outside primary rainforests. Knowledge of this virus enabled a spatiotemporal reconstruction of the common ancestor of all SLEVs and how the virus spread from there. We can infer that the cosmopolitan SLEV lineage emerged from Central America in the 17th century, a period of post-Columbian colonial history marked by intense human invasion of primary rainforests. Further spread followed major bird migration pathways over North and South America.

Structure of the St. Louis encephalitis virus postfusion envelope trimer.

St. Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest virus, an alphavirus. Domains II and III (DII and DIII) of SLEV E also adopt different angles relative to DI, which suggests that the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved among flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variations in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis.

Effects of virus dose and extrinsic incubation temperature on vector competence of Culex nigripalpus (Diptera: Culicidae) for St. Louis encephalitis virus.

Culex nigripalpus Theobald is a primary vector of St. Louis encephalitis virus in the southeastern United States. Cx. nigripalpus females were fed blood containing a low (4.0 +/- 0.01 log10 plaque-forming unit equivalents (PFUeq) /ml) or high (4.7 +/- 0.1 log10 PFUeq/ml) St. Louis encephalitis virus dose and maintained at extrinsic incubation temperatures (EIT) of 25 or 28 degrees C for 12 d. Vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reaction to estimate PFUeq using rates of infection, dissemination, and transmission. There were no differences in infection rates between the two EITs at either dose. The low dose had higher infection rates at both EITs. Dissemination rates were significantly higher at 28 degrees C compared with 25 degrees C at both doses. Virus transmission was observed (<7%) only at 28 degrees C for both doses. The virus titer in body tissues was greater at 28 degrees C compared with 25 degrees C at both doses. The difference between the EITs was greater at the low dose, resulting in a higher titer for the low dose than the high dose at 28 degrees C. Virus titers in leg tissues were greater in mosquitoes fed the high versus low dose, but were not influenced by EIT. Further investigations using a variety of environmental and biological factors would be useful in exploring the complexity of vector competence.

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes.

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

Silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis.

West Nile (WN) and St. Louis encephalitis (SLE) viruses can cause fatal neurological infection and currently there is neither a specific treatment nor an approved vaccine for these infections. In our earlier studies, we have reported that siRNAs can be developed as broad-spectrum antivirals for the treatment of infection caused by related viruses and that a small peptide called RVG-9R can deliver siRNA to neuronal cells as well as macrophages. To increase the repertoire of broad-spectrum antiflaviviral siRNAs, we screened 25 siRNAs targeting conserved regions in the viral genome. Five siRNAs were found to inhibit both WNV and SLE replication in vitro reflecting broad-spectrum antiviral activity and one of these was also validated in vivo. In addition, we also show that RVG-9R delivers siRNA to macrophages and dendritic cells, resulting in effective suppression of virus replication. Mice were challenged intraperitoneally (i.p.) with West Nile virus (WNV) and treated i.v. with siRNA/peptide complex. The peritoneal macrophages isolated on day 3 post infection were isolated and transferred to new hosts. Mice receiving macrophages from the anti-viral siRNA treated mice failed to develop any disease while the control mice transferred with irrelevant siRNA treated mice all died of encephalitis. These studies suggest that early suppression of viral replication in macrophages and dendritic cells by RVG-9R-mediated siRNA delivery is key to preventing the development of a fatal neurological disease.

Severe winter freezes enhance St. Louis encephalitis virus amplification and epidemic transmission in peninsular Florida.

Mosquito-borne arboviral epidemics tend to strike without warning. The driving force for these epidemics is a combination of biotic (vector, amplification host, and virus) and abiotic (meteorological conditions, especially rainfall and temperature) factors. Abiotic factors that facilitate the synchronization and interaction of vector and amplification host populations favor epidemic amplification and transmission. In Florida, epidemics of St. Louis encephalitis (SLE) virus (family Flaviviridae, genus Flavivirus, SLEV) have been preceded by major freezes one or two winters before the onset of human cases. Here, we analyze the relationship between severe winter freezes and epidemic SLEV transmission in peninsular Florida and show that there is a significant relationship between the transmission of SLEV and these severe freezes. We propose that by killing cold-sensitive understory vegetation in the mid-peninsular region of Florida, freezes enhance the reproductive success of ground-feeding avian amplification hosts, especially mourning doves and common grackles. In conjunction with other appropriate environmental signals, increased avian reproductive success may enhance SLEV and West Nile (WN) virus amplification and result in SLE and WN epidemics during years when all of the biological cycles are properly synchronized. The knowledge that winter freezes in Florida may enhance the amplification and epidemic transmission of SLE and WN viruses facilitates arboviral tracking and prediction of human risk of SLE and WN infection during the transmission season.

Environmental and biological factors influencing Culex pipiens quinquefasciatus Say (Diptera: Culicidae) vector competence for Saint Louis encephalitis virus.

Complex interactions between environmental and biological factors influence the susceptibility of Culex pipiens quinquefasciatus to St. Louis encephalitis virus and could affect the epidemiology of virus transmission. Similar interactions could have epidemiologic implications for other vector-virus systems. We conducted an experiment to examine four such factors in combination: mosquito age, extrinsic incubation temperature (EIT), virus dose, and colony. The proportion of mosquitoes with body infections or disseminated infections varied between colonies, and was dependant on age, EIT, and dose. We also show that the probability of a body or leg infection interacted in complex ways between colonies, ages, EITs, and doses. The complex interactive effects of environmental and biological factors must be taken into account for studies of vector competence and epidemiology, especially when laboratory studies are used to generalize to natural transmission dynamics where the extent of variation is largely unknown.

Comparative thermostability of West Nile, St. Louis encephalitis, and western equine encephalomyelitis viruses during heat inactivation for serologic diagnostics.

During the monitoring of arbovirus seroprevalence in wild birds collected in California, we inadvertently made two isolates of western equine encephalomyelitis virus (WEEV) from California quail sera being tested by plaque reduction neutralization assay for antibodies against St Louis encephalitis (SLEV) and West Nile (WNV) viruses despite heating the sera at 56 degrees C for 30 minutes. These data prompted us to examine the thermostability of these viruses during heat treatment. The flaviviruses, SLEV and WNV, at titers up to 10(6) plaque-forming units (PFU), were readily inactivated by the standard protocol of heating at 56 degrees C for 30 minutes. In contrast, solutions containing 10(5) and 10(6) PFU of WEEV required 2 hours for complete inactivation. Occasional presence of live virus within sera could lead to false negatives using standard plaque reduction neutralization test protocols.

Does variation in Culex (Diptera: Culicidae) vector competence enable outbreaks of West Nile virus in California?

Since the invasion of California by West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in 2003, we have annually monitored vector competence for the NY99 strain in Culex tarsalis Coquillett, Culex pipiens quinquefasciatus Say, Culex p. pipiens L., and Culex stigmatosoma Dyar populations from four areas: deserts of Coachella Valley, densely urbanized maritime Los Angeles, southern San Joaquin Valley in Kern County, and southern Sacramento Valley near Davis in Sacramento County. Overall, Cx. stigmatosoma was the most competent vector species, followed by Cx. tarsalis and the Cx. pipiens complex. The median infectious dose (ID50) of WNV required to infect 50% of the F1 female progeny reared from wild-caught females, a measure of mesenteronal susceptibility, ranged between 5 and 8 log10 plaque forming units/ml and was not correlated with annual human case incidence or summer maximum likelihood mosquito infection estimates. Odds ratios comparing nonoutbreak years with referent outbreak years were variable and failed to show a distinct pattern for Cx. tarsalis or Cx. pipiens complex females. Apparently factors other than midgut susceptibility within the ranges we measured enabled WNV outbreaks in California. Culex populations remained competent for St. Louis encephalitis virus, indicating that the disappearance of this virus was not related to a loss of vector competence.

Reduced infection in mosquitoes exposed to blood meals containing previously frozen flaviviruses.

The increased difficulty and expense of using live animals for delivering infectious blood meals in arthropod-borne virus vector competence experiments has resulted in an increase in the use of artificial feeding systems. Compared to live hosts, artificial systems require higher viral titers to attain mosquito infection, thereby limiting the utility of such systems with low or moderate titer virus stocks. Based on the report that freshly propagated virus is more infectious than previously frozen virus, we determined whether such a preparation would enhance the ability to use artificial feeding systems. Culex quinquefasciatus and Aedes aegypti mosquitoes were offered blood in artificial membrane feeders containing freshly collected or previously frozen St. Louis encephalitis and dengue serotype-2 viruses (family Flaviviridae), respectively. Infection rates and estimates of vector competence were significantly lower (P<0.05) for mosquitoes feeding on blood meals containing frozen-thawed compared to freshly collected virus. We indicate that the use of freshly propagated virus in artificial feeding systems can be an effective blood delivery method for low-titer viruses and viruses that are otherwise inefficient at infecting vectors in such systems. Fresh viruses used in artificial feeding systems may be a viable alternative to the heavily regulated and expensive use of live animals.

Previous infection with West Nile or St. Louis encephalitis viruses provides cross protection during reinfection in house finches.

House finches are competent hosts for both West Nile and St. Louis encephalitis viruses and frequently become infected during outbreaks. In the current study, House finches were infected initially with either West Nile or St. Louis encephalitis viruses and then challenged 6 weeks post infection with either homologous or heterologous viruses. Although mortality rates were high during initial infection with West Nile virus, prior infection with either virus prevented mortality upon challenge with West Nile virus. Prior infection with West Nile virus provided sterilizing immunity against both viruses, whereas prior infection with St. Louis encephalitis virus prevented viremia from St. Louis encephalitis virus, but only reduced West Nile virus viremia titers. Immunologic responses were measured by enzyme immunoassay and plaque reduction neutralization tests. Heterologous challenge with West Nile virus in birds previously infected with St. Louis encephalitis virus produced the greatest immunologic response, markedly boosting antibody levels against St. Louis encephalitis virus. Our data have broad implications for free-ranging avian serological diagnostics and possibly for the recent disappearance of St. Louis encephalitis virus from California.

Effects of temperature on the transmission of west nile virus by Culex tarsalis (Diptera: Culicidae).

Culex tarsalis Coquillett females were infected with the NY99 strain of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) and then incubated under constant temperatures of 10-30 degrees C. At selected time intervals, transmission was attempted using an in vitro capillary tube assay. The median time from imbibing an infectious bloodmeal until infected females transmitted WNV (median extrinsic incubation period, EIP50) was estimated by probit analysis. By regressing the EIP rate (inverse of EIP50) as a function of temperature from 14 to 30 degrees C, the EIP was estimated to require 109 degree-days (DD) and the point of zero virus development (x-intercept) was estimated to be 14.3 degrees C. The resulting degree-day model showed that the NY99 WNV strain responded to temperature differently than a lineage II strain of WNV from South Africa and approximated our previous estimates for St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). The invading NY99 WNV strain therefore required warm temperatures for efficient transmission. The time for completion of the EIP was estimated monthly from temperatures recorded at Coachella Valley, Los Angeles, and Kern County, California, during the 2004 epidemic year and related to the duration of the Cx. tarsalis gonotrophic cycle and measures of WNV activity. Enzootic WNV activity commenced after temperatures increased, the duration of the EIP decreased, and virus potentially was transmitted in two or less gonotrophic cycles. Temperatures in the United States during the epidemic summers of 2002-2004 indicated that WNV dispersal and resulting epicenters were linked closely to above-average summer temperatures.

Previous infection protects house finches from re-infection with St. Louis encephalitis virus.

Antibody titers against St. Louis encephalitis virus (SLE) measured by a plaque reduction neutralization test (PRNT) decreased rapidly in house finches (Capodacus mexicanus) after initial infection, whereas antibodies measured by enzyme immunoassay (EIA) remained detectable in all birds for the length of the experiment, indicating long-term persistence and greater assay sensitivity of the EIA. After 52 wk, birds were challenged by subcutaneous inoculation with the same strain of SLE virus. Virus was not detected for 1-4 d postchallenge in blood samples tested by plaque assay and RT-PCR or by xenodiagnosis in Culex tarsalis fed concurrently and then held for 11 d at 26 degrees C. Virus was detected by all three methods in control birds infected concurrently for the first time. Challenge with SLE produced a rapid and marked ananmestic rise in both neutralizing and EIA antibody titers that exceeded the primary response in the same birds or in concurrently inoculated control birds. At necropsy 4 wk postchallenge, 3 of 7 challenged and 1 of 2 positive control birds were chronically infected, with viral RNA detected by RT-PCR in brain, spleen, lung, and/or kidney tissues. Our results indicated that persistence of protective antibody prevents reinfection during the following season and may prevent the recrudescence of infectious virus in chronically infected birds.