St. Louis Encephalitis Virus Disease in the United States, 2003-2017.

St. Louis encephalitis virus (SLEV), an arthropod-borne flavivirus, can cause disease presentations ranging from mild febrile illness through severe encephalitis. We reviewed U.S. national SLEV surveillance data for 2003 through 2017, including human disease cases and nonhuman infections. Over the 15-year period, 198 counties from 33 states and the District of Columbia reported SLEV activity; 94 (47%) of those counties reported SLEV activity only in nonhuman species. A total of 193 human cases of SLEV disease were reported, including 148 cases of neuroinvasive disease. A median of 10 cases were reported per year. The national average annual incidence of reported neuroinvasive disease cases was 0.03 per million. States with the highest average annual incidence of reported neuroinvasive disease cases were Arkansas, Arizona, and Mississippi. No large outbreaks occurred during the reporting period. The most commonly reported clinical syndromes were encephalitis (N = 116, 60%), febrile illness (N = 35, 18%), and meningitis (N = 25, 13%). Median age of cases was 57 years (range 2-89 years). The case fatality rate was 6% (11/193) and all deaths were among patients aged > 45 years with neuroinvasive disease. Nonhuman surveillance data indicated wider SLEV activity in California, Nevada, and Florida than the human data alone suggested. Prevention depends on community efforts to reduce mosquito populations and personal protective measures to decrease exposure to mosquitoes.

Ilheus and Saint Louis encephalitis viruses elicit cross-protection against a lethal Rocio virus challenge in mice.

Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.

Saint Louis Encephalitis Virus, another re-emerging arbovirus: a literature review of worldwide research.

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Tissue tropism of Saint Louis encephalitis virus: Histopathology triggered by epidemic and non-epidemic strains isolated in Argentina.

Saint Louis encephalitis virus (SLEV) reemerged in South America, and caused encephalitis outbreaks at the beginning of the 21st century. To enhance our knowledge about SLEV virulence, we performed comparative pathogenesis studies in Swiss albino mice inoculated with two different variants, the epidemic strain CbaAr-4005 and the non-epidemic strain CorAn-9275. Only the infection of mice with SLEV strain CbaAr-4005 resulted in high viremia, invasion of peripheral tissues including the lungs, kidney, and spleen, and viral neuroinvasion. This was associated with inflammatory pathology in the lungs, spleen, and brain as well as morbidity and mortality. In contrast, neither signs of desease nor viral replication were observed in mice infected with strain CorAn-9275. Interestingly, important loss of B cells and development of altered germinal centers (GC) were detected in the spleen of mice infected with strain CbaAr-4005, whereas mice infected with SLEV CorAn-9275 developed prominent GC with conserved follicular architecture, and neutralizing antibodies.

Exploring Genomic, Geographic and Virulence Interactions among Epidemic and Non-Epidemic St. Louis Encephalitis Virus (Flavivirus) Strains.

St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America. In 2005, an encephalitis outbreak caused by SLEV was reported in Argentina. The reason for the outbreak remains unknown, but may have been related to virological factors, changes in vectors populations, avian amplifying hosts, and/or environmental conditions. The main goal of this study was to characterize the complete genome of epidemic and non-epidemic SLEV strains from Argentina. Seventeen amino acid changes were detected; ten were non-conservative and located in proteins E, NS1, NS3 and NS5. Phylogenetic analysis showed two major clades based on geography: the North America and northern Central America (NAnCA) clade and the South America and southern Central America (SAsCA) clade. Interestingly, the presence of SAsCA genotype V SLEV strains in the NAnCA clade was reported in California, Florida and Texas, overlapping with known bird migration flyways. This work represents the first step in understanding the molecular mechanisms underlying virulence and biological variation among SLEV strains.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina.

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

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.

Seroprevalence of St. Louis encephalitis virus and West Nile virus (Flavivirus, Flaviviridae) in horses, Uruguay.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) belong to the Japanese encephalitis antigenic complex (Flavivirus genus, Flaviviridae family). They show antigenic close relationships and share many similarities in their ecology. Both are responsible for serious human diseases. The aim of this study was to investigate the presence of neutralizing antibodies to these viruses in horses from Uruguay. To do this, 425 horse sera were collected in 2007 and analyzed by plaque reduction neutralization tests. As a result, 205 sera (48.2%) were found positive for SLEV, with titers ranging between 10 and 80. Two sera remained inconclusive, since they showed low titers to WNV and SLEV (10 and 20), not allowing us to demonstrate activity of WNV in our territory. This is the first report of circulation of SLEV in horses in Uruguay.

Comparison of argentinean saint louis encephalitis virus non-epidemic and epidemic strain infections in an avian model.

St. Louis encephalitis virus (SLEV, Flavivirus, Flaviviridae) is an emerging mosquito-borne pathogen in South America, with human SLEV encephalitis cases reported in Argentina and Brazil. Genotype III strains of SLEV were isolated from Culex quinquefasciatus mosquitoes in Cordoba, Argentina in 2005, during the largest SLEV outbreak ever reported in South America. The present study tested the hypothesis that the recent, epidemic SLEV strain exhibits greater virulence in birds as compared with a non-epidemic genotype III strain isolated from mosquitoes in Santa Fe Province 27 years earlier. The observed differences in infection parameters between adult House sparrows (Passer domesticus) that were needle-inoculated with either the epidemic or historic SLEV strain were not statistically significant. However, only the House sparrows that were infected with the epidemic strain achieved infectious-level viremia titers sufficient to infect Cx. spp. mosquitoes vectors. Furthermore, the vertebrate reservoir competence index values indicated an approximately 3-fold increase in amplification potential of House sparrows infected with the epidemic strain when pre-existing flavivirus-reactive antibodies were present, suggesting the possibility that antibody-dependent enhancement may increase the risk of avian-amplified transmission of SLEV in South America.

Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology.

A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 10(1) to 10(6) plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT(50) or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT(50) was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.

Noninfectious recombinant antigen for detection of St. Louis encephalitis virus-specific antibodies in serum by enzyme-linked immunosorbent assay.

Proper surveillance of virus activity and a timely response to viral outbreaks depend upon the rapid diagnosis of viral infections. The immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a fast, sensitive test routinely used for the diagnosis of the medically important West Nile and St. Louis encephalitis flaviviruses. However, the suckling mouse brain-derived (SMB) antigen used in this assay is tedious to prepare and has a risk of exposing personnel to live virus and hazardous chemicals. We report the development of a St. Louis encephalitis virus (SLEV) noninfectious recombinant antigen that is a safe and easily produced alternative antigen for use in diagnostic assays. The expression plasmid pCB8SJ2, containing the premembrane and envelope structural protein-encoding regions of SLEV, was constructed to express secreted extracellular virus-like particles (VLPs) from CHO cells. Blind-coded human serum panels were assembled from patients having recent SLEV, West Nile virus (WNV), Powassan virus, or La Crosse encephalitis virus infections to assess the sensitivity and specificity of assays with SLEV VLP or SMB antigen. MAC-ELISAs with either antigen had comparable sensitivity for the detection of IgM antibodies against SLEV. Importantly, when these two antigens were tested against a human serum panel from patients having recent WNV or Powassan virus infections, the SLEV VLPs were less likely than SMB antigen to detect flavivirus cross-reactive IgM antibodies. An optimized IgG antibody capture ELISA (GAC-ELISA) with both WNV and SLEV VLPs was developed to circumvent the frequently observed higher background in the antigen-capture IgG-ELISA (ACG-ELISA). For the detection of IgG antibodies against WNV, the GAC-ELISA resulted in a statistically significant higher performance accuracy (P = 0.003) than the ACG-ELISA when the WNV VLP antigen was used in both assays. However, no statistical difference was observed in the assay performance of the GAC-ELISA with SLEV VLP or the ACG-ELISA with SLEV SMB antigen.

St. Louis encephalitis virus in wild birds during the 1990 south Florida epidemic: the importance of drought, wetting conditions, and the emergence of Culex nigripalpus (Diptera: Culicidae) to arboviral amplification and transmission.

We analyzed the prevalence of hemagglutination inhibition (HI) antibodies to St. Louis encephalitis (SLE) virus in wild birds during the 1990 SLE epidemic in Indian River County. The initial presence of SLE HI antibody was associated significantly with modeled drought 15 wk prior, wetting conditions 1 wk prior, and the emergence of the Florida SLE virus vector, Culex nigripalpus, 5 wk prior. Our findings indicated that three factors conspired to create the 1990 epidemic: (1) a large population of susceptible wild birds; (2) severe springtime drought, which facilitated amplification of the SLE virus among the Cx. nigripalpus and a portion of the wild bird population; and (3) continued rainfall and wetting of the land surface in the summer and early fall, which sustained a large, host-seeking Cx. nigripalpus population. The continued biting and reproductive activity of Cx. nigripalpus maintained epizootic transmission throughout the summer and early fall in Indian River County. The high level of SLE virus amplification resulted in spillover transmission to humans. We hypothesize that without the continued reproductive activity of the vector mosquito, brought about by excessive summer and fall wetness, the unprecedented SLE virus amplification and consequent transmission to humans would not have been realized in 1990.

St. Louis encephalitis virus induced pathology in cultured cells.

Apoptosis is a highly regulated process of cellular self-destruction with diverse functions in multicellular organisms. It is known to be one of the mechanisms of viral pathogenesis. St. Louis encephalitis virus (SLEV), an arthropod-borne flavivirus, causes encephalitis disease of varying severity mostly in North America and in some regions of South America. This virus induces cytopathic effects in vertebrate cell lines, however, the mechanism by which this occurs is yet to be elucidated. SLEV induced cytopathic effects in K562 cells, a human mononuclear cell line, and in Neuro 2a cells, a mouse neuroblastoma cell line. SLEV-infected K562 and Neuro 2a cells underwent apoptotic cell death, whereas neither the cells inoculated with UV-inactivated virus nor the mock-infected cells developed cytopathic effects. The gene expression of regulators of apoptosis was investigated in K562 cells. A rise in the expression of the pro-apoptotic bax gene was detected specifically in the SLEV-infected K562 cells. These findings suggest that up-regulation of bax mRNA is correlated with cytopathic effects in SLEV-infected K562 cells.

Relationships among weather, mosquito abundance, and encephalitis virus activity in California: Kern County 1990-98.

The summer abundance of Culex tarsalis in Kern County, California, during 1990-98 was related quantitatively to rainfall, snow depth and water content, and runoff of the Kern River. Total monthly rain that fell during winter, lagged by 4-6 months, explained only 13% of the variability in the number of host-seeking females collected per trap night per month during summer. In contrast, regression analysis showed that river runoff 1 month earlier explained 67% of the variability in mosquito abundance. The water content of snowpack measured within the Kern River watershed during winter explained 70% of the variation in average mosquito abundance during the following summer. After being absent from Kern County since 1983, western equine encephalomyelitis virus (WEE) returned during the wet years of 1996-98 after the flow of the Kern River exceeded 150,000 acre-ft (450 hectare-meters) per month. Water content of snow in the Sierra Nevada during winter provided an excellent early warning of vernal river runoff, mosquito abundance, and enzootic WEE activity levels on the floor of the San Joaquin Valley.

Response of house finches to infection with sympatric and allopatric strains of western equine encephalomyelitis and St. Louis encephalitis viruses from California.

Adult house finches from Kern County were inoculated subcutaneously with recent sympatric and allopatric isolates of western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses made from Culex tarsalis Coquillett collected in Kern County and Coachella Valley, CA, respectively. Virulence, as measured by the amplitude of the viremia response during days 1 and 2 postinfection, varied significantly among strains, but independently of geographic origin. The intensity of the immune response, as measured by an enzyme immunoassay and a plaque reduction neutralization test, seemed to be independent of virulence, especially for SLE where some strains failed to produce a detectable viremia but elicited a strong antibody response. Our preliminary data indicated that strain virulence may be associated with the level of enzootic activity during the year of isolation.

Method of infection does not alter response of chicks and house finches to western equine encephalomyelitis and St. Louis encephalitis viruses.

The effects of method of infection and virus dose on the viremia and antibody responses of 1-wk-old chicks and after-hatching-year house finches to infection with western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were studied under laboratory conditions. Using a capillary tube technique, females from 2 strains of Culex tarsalis Coquillett mosquitoes were estimated to expectorate from 1.0 to 1.7 log10 plaque forming units (PFU) of WEE and from 1.9 to 2.2 log10 PFU of SLE. Based on the proportion of parenterally infected females that transmitted and the number that blood fed during each experiment, virus doses per bird were estimated to be 1.0-1.9 log10 PFU for WEE and 1.4-2.3 log10 PFU for SLE. When infected with comparable doses of WEE by subcutaneous inoculation, there was no significant difference in the duration or magnitude of the viremia response between birds infected by mosquito bite or syringe; few birds developed a viremia response after infection with SLE, precluding analysis. In chickens, increasing the syringe dose of WEE from 0.3 to 1.7 log10 PFU/0.1 ml shortened the time when viremia first appeared from 3 to 1 d postinfection and increased the duration of the viremia period from 1 to 3 d, but did not alter the maximum viremia titer. In house finches, increasing the syringe dose of WEE from 2.6 to 3.3 log10 PFU/0.1 ml did not alter markedly the viremia response. Most birds developed antibody detected by enzyme immunoassay (EIA) or plaque reduction neutralization test (PRNT). In chickens, WEE EIA levels and PRNT titers were higher for birds infected by syringe than by mosquito bite, whereas in house finches the pattern was reversed. For birds infected with SLE, there was overlap among groups infected by mosquito bite or syringe. These results indicate that subcutaneous syringe inoculation provides a biologically sound mode of infection that did not alter viremia and antibody responses when compared with infection by mosquito bite.

Genotypic and phenotypic variation of selected Saint Louis encephalitis viral strains isolated in California.

The mechanism for long-term maintenance of St. Louis encephalitis (SLE) virus in California is unknown. Two possibilities are 1) that the virus is maintained locally in discrete enzootic foci by one or more reservoir mechanisms, and/or 2) that the foci are ephemeral in nature and virus is reintroduced periodically from other enzootic areas by migratory birds or movement of vectors. We have investigated these epidemiologic alternatives by studies of genetic variation within a 277 nucleotide portion of the envelope-encoding region among 17 strains of SLE virus isolated since 1952 from different geographic locations in California. Three lineages of virus were detected. One lineage, Group A, consisted of four SLE virus strains isolated in California since 1972 from the Coachella, Sacramento, and San Joaquin Valleys. The group A strains were closely related to strain MSI-7 of SLE virus isolated in Mississippi in 1975. The 13 other strains formed the second and third lineages (Groups B1 and B2) that had geographically overlapping distributions. Group A (BFN 4585) and Group B2 (BFN 4820) appeared to be sympatric in the Sacramento Valley in 1972. Strains from the San Joaquin Valley isolated prior to 1989 (Groups B1 and B2) differed markedly from a 1989 isolate from the same location, Kern 373 (Group A). These results suggest that virus introduction(s) led to changes in genotype, or alternatively that the enzootic virus was subjected to selective pressure leading to rapid emergence of a new genotype. Nucleotide sequences of the envelope and 5' untranslated region of the viral genome of these virus strains did not correlate with virulence as measured by mortality in weanling mice, nor viremia levels and duration in chickens.

Immunity to St. Louis encephalitis virus by sequential immunization with recombinant vaccinia and baculovirus derived PrM/E proteins.

St. Louis encephalitis (SLE) is an important mosquito-borne disease of great public health concern in parts of the United States. South America and Canada. Protective immunogens of flaviviruses produced in different expression systems have been shown to be effective against virulent virus infection in laboratory animal models. Here we show that the pre-membrane and envelope (PrM/E) of SLE virus expressed in insect and mammalian cell systems using baculovirus and vaccinia virus, respectively, are processed correctly and showed similar antigenic characteristics as the authentic proteins. Immunization with the recombinant proteins individually or in combination resulted in neutralizing and protective immune responses. A schedule consisting of initial immunization with recombinant vaccinia virus followed by a secondary boost with recombinant baculovirus protein resulted in higher levels of neutralizing and protective immune responses. The advantages of the use of such a combined approach as a general immunization strategy are discussed.