Louis Encephalitis Virus Research Articles

Mutagen resistance and mutation restriction of St. Louis encephalitis virus.

The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus, is a mosquito-borne flavivirus that has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modelling of the E416K polymerase mutation indicated its location in the pinky finger domain of the RdRp, distant from the active site. Deep sequencing of the E416K mutant revealed lower genetic diversity than wild-type SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed that E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with West Nile virus high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference response, suggesting an impaired capacity to escape RNA interference could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and give further insight into the consequences of altered fidelity of flaviviruses.

Effects of Organic Amendments on Microbiota Associated with the Culex nigripalpus Mosquito Vector of the Saint Louis Encephalitis and West Nile Viruses.

Pollution from nutrients in aquatic habitats has been linked to increases in disease vectors, including mosquitoes and other pestiferous insects. One possibility is that changes in mosquito microbiomes are impacted by nutrient enrichments and that these changes affect various traits, including larval development, susceptibility to larval control agents, and susceptibility of the adult mosquitoes to pathogens. We tested this hypothesis using field mesocosms supplemented with low- and high-organic-nutrient regimens and then sampled microbial communities associated with the naturally colonizing Culex nigripalpus mosquito vector. By high-throughput sequencing of 16S rRNA gene sequences, we found no significant differences in overall microbial communities associated with sampled mosquitoes, despite detecting discernible differences in environmental variables, including pH, dissolved oxygen, and nutrient amendments. Nevertheless, indicator species analysis revealed that members of the Clostridiales were significantly associated with mosquitoes that originated from high-nutrient enrichments. In contrast, members of the Burkholderiales were associated with mosquitoes from the low-nutrient enrichment. High bacterial variability associated with the life stages of the C.nigripalpus was largely unaffected by levels of nutrient enrichments that impacted larval microbial resources, including bacteria, ciliates, and flagellates in the larval environments. IMPORTANCE Mosquito microbiota provide important physiological and ecological attributes to mosquitoes, including an impact on their susceptibility to pathogens, fitness, and sensitivity to mosquito control agents. Culex nigripalpus mosquito populations transmit various pathogens, including the Saint Louis and West Nile viruses, and proliferate in nutrient-rich environments, such as in wastewater treatment wetlands. Our study examined whether increases in nutrients within larval mosquito developmental habitats impact microbial communities associated with C.nigripalpus mosquitoes. We characterized the effects of organic enrichments on microbiomes associated with C.nigripalpus mosquitoes and identified potential bacterial microbiota that will be further investigated for whether they alter mosquito life history traits and for their potential role in the development of microbial-based control strategies.

Exploring genetic variation in haplotypes of the filariasis vector Culex quinquefasciatus (Diptera: Culicidae) through DNA barcoding

Culex quinquefasciatus (Diptera: Culicidae) is a vector of many pathogens and parasites of humans, as well as domestic and wild animals. In urban and semi-urban Asian countries, Cx. quinquefasciatus is a main vector of nematodes causing lymphatic filariasis. In the African region, it vectors the Rift Valley fever virus, while in the USA it transmits West Nile, St. Louis encephalitis and Western equine encephalitis virus. In this study, DNA barcoding was used to explore the genetic variation of Cx. quinquefasciatus populations from 88 geographical regions. We presented a comprehensive approach analyzing the effectiveness of two gene markers, i.e. CO1 and 16S rRNA. The high threshold genetic divergence of CO1 (0.47%) gene was reported as an ideal marker for molecular identification of this mosquito vector. Furthermore, null substitutions were lower in CO1 if compared to 16S rRNA, which influenced its differentiating potential among Indian haplotypes. NJ tree was well supported with high branch values for CO1 gene than 16S rRNA, indicating ideal genetic differentiation among haplotypes. TCS haplotype network revealed 14 distinct clusters. The intra- and inter-population polymorphism were calculated among the global and Indian Cx. quinquefasciatus lineages. The genetic diversity index Tajima’ D showed negative values for all the 4 intra-population clusters (G2-4, G10). Fu’s FS showed negative value for G10 cluster, which was significant and indicated recent population expansion. However, the G2-G4 (i.e. Indian lineages) had positive values, suggesting a bottleneck effect. Overall, our research firstly shed light on the genetic differences among the haplotypes of Cx. quinquefasciatus species complex, adding basic knowledge to the molecular ecology of this important mosquito vector.

Thiosemicarbazones and Phthalyl-Thiazoles compounds exert antiviral activity against yellow fever virus and Saint Louis encephalitis virus.

Arboviruses, arthropod-borneviruses, are frequency associated to human outbreak and represent a serious health problem. The genus Flavivirus, such as Yellow Fever Virus (YFV) and Saint Louis Encephalitis Virus (SLEV), are important pathogens with high morbidity and mortality worldwide. In Brazil, YFV is maintained in sylvatic cycle, but many cases are notified annually, despite the efficiency of vaccine. SLEV causes an acute encephalitis and is widely distributed in the Americas. There is no specific antiviral drugs for these viruses, only supporting treatment that can alleviate symptoms and prevent complications. Here, we evaluated the potential anti-YFV and SLEV activity of a series of thiosemicarbazones and phthalyl-thiazoles. Plaque reduction assay, flow cytometry, immunofluorescence and cellular viability were used to test the compounds in vitro. Treated cells showed efficient inhibition of the viral replication at concentrations that presented minimal toxicity to cells. The assays showed that phthalyl-thiazole and phenoxymethyl-thiosemicarbazone reduced 60% of YFV replication and 75% of SLEV replication.

Rearing of Culex spp. and Aedes spp. Mosquitoes.

Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.

Reemergence of St. Louis Encephalitis Virus, California, 2015.

St. Louis encephalitis virus infection was detected in summer 2015 in southern California after an 11-year absence, concomitant with an Arizona outbreak. Sequence comparisons showed close identity of California and Arizona isolates with 2005 Argentine isolates, suggesting introduction from South America and underscoring the value of continued arbovirus surveillance.

Climate Change Influences on the Global Potential Distribution of the Mosquito Culex quinquefasciatus, Vector of West Nile Virus and Lymphatic Filariasis.

Rapid emergence of most vector-borne diseases (VBDs) may be associated with range expansion of vector populations. Culex quinquefasciatus Say 1823 is a potential vector of West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. We estimated the potential distribution of Cx. quinquefasciatus under both current and future climate conditions. The present potential distribution of Cx. quinquefasciatus showed high suitability across low-latitude parts of the world, reflecting the current distribution of the species. Suitable conditions were identified also in narrow zones of North Africa and Western Europe. Model transfers to future conditions showed a potential distribution similar to that under present-day conditions, although with higher suitability in southern Australia. Highest stability with changing climate was between 30°S and 30°N. The areas present high agreement among diverse climate models as regards distributional potential in the future, but differed in anticipating potential for distribution in North and Central Africa, southern Asia, central USA, and southeastern Europe. Highest disparity in model predictions across representative concentration pathways (RCPs) was in Saudi Arabia and Europe. The model predictions allow anticipation of changing distributional potential of the species in coming decades.

Molecular detection of flaviviruses and alphaviruses in mosquitoes (Diptera: Culicidae) from coastal ecosystems in the Colombian Caribbean.

Arboviruses belonging to the genera Flavivirus and Alphavirus were detected in mosquitoes in a rural area of San Bernardo del Viento (Córdoba, Colombia). A total of 22,180 mosquitoes were collected, sorted into 2,102 pools, and tested by generic/nested reverse transcription-polymerase chain reaction. Venezuelan equine encephalitis virus, dengue virus, West Nile virus, St. Louis encephalitis virus, yellow fever virus, and Culex flavivirus were detected and identified by sequencing. The detection of arboviral pathogens in this zone represents possible circulation and indicates a human health risk, demonstrating the importance of virological surveillance activities.

Green-Synthesized Mosquito Oviposition Attractants and Ovicides: Towards a Nanoparticle-Based “Lure and Kill” Approach?

Mosquitoes are key vectors of malaria, dengue, yellow fever, chikungunya, West Nile, Japanese encephalitis, lymphatic filariasis, Zika virus and St. Louis encephalitis virus. Eco-friendly control tools of Culicidae vectors are a priority. Green nanotechnologies may help to boost the effectiveness of mosquito vector control. We proposed a facile fabrication of poly-disperse and stable silver nanoparticles (AgNPs) using the Aganosma cymosa leaf extract. Nanoparticles were characterized by UV–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, atomic force microscopy, scanning electron microscopy and transmission electron microscopy. Nanoparticles showed high toxicity on eggs and larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. LC50 values were 12.45, 13.58 and 14.79 μg/mL, respectively. No egg hatchability was noted post-treatment with 40, 50 and 60 μg/mL, respectively. Nanoparticles were found safer to non-target mosquito predators Anisops bouvieri, Diplonychus indicus and Gambusia affinis, LC50 values ranged from 673.36 to 2247.43 µg/mL. Notably, AgNPs showed high oviposition attractiveness towards the three mosquito species. Overall, the oviposition attractiveness of the A. cymosa extract coupled with the ovicidal action of AgNPs can help to develop “lure and kill” tools to be used at mosquito breeding sites.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1:20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.

Vertical Transmission of Zika Virus in Aedes aegypti Mosquitoes.

Previous experimental studies have demonstrated that a number of mosquito-borne flavivirus pathogens are vertically transmitted in their insect vectors, providing a mechanism for these arboviruses to persist during adverse climatic conditions or in the absence of a susceptible vertebrate host. In this study, designed to test whether Zika virus (ZIKV) could be vertically transmitted, female Aedes aegypti and Aedes albopictus were injected with ZIKV, and their F1 adult progeny were tested for ZIKV infection. Six of 69 Ae. aegypti pools, comprised of a total of 1,738 F1 adults, yielded ZIKV upon culture, giving a minimum filial infection rate of 1:290. In contrast, none of 803 F1 Ae. albopictus adults (32 pools) yielded ZIKV. The MFIR for Ae. aegypti was comparable to MFIRs reported for other flaviviruses in mosquitoes, including dengue, Japanese encephalitis, yellow fever, West Nile, and St. Louis encephalitis viruses. The results suggest that vertical transmission may provide a potential mechanism for the virus to survive during adverse conditions.

Activity Patterns of St. Louis Encephalitis and West Nile Viruses in Free Ranging Birds during a Human Encephalitis Outbreak in Argentina.

St. Louis encephalitis virus (SLEV) (Flavivirus) is a reemerging arbovirus in the southern cone of South America. In 2005, an outbreak of SLEV in central Argentina resulted in 47 human cases with 9 deaths. In Argentina, the ecology of SLEV is poorly understood. Because certain birds are the primary amplifiers in North America, we hypothesized that birds amplify SLEV in Argentina as well. We compared avian SLEV seroprevalence in a variety of ecosystems in and around Córdoba city from 2004 (before the epidemic) and 2005 (during the epidemic). We also explored spatial patterns to better understand the local ecology of SLEV transmission. Because West Nile virus (WNV) was also detected in Argentina in 2005, all analyses were also conducted for WNV. A total of 980 birds were sampled for detection of SLEV and WNV neutralizing antibodies. SLEV seroprevalence in birds increased 11-fold from 2004 to 2005. Our study demonstrated that a high proportion (99.3%) of local birds were susceptible to SLEV infection immediately prior to the 2005 outbreak, indicating that the vertebrate host population was primed to amplify SLEV. SLEV was found distributed in a variety of environments throughout the city of Córdoba. However, the force of viral transmission varied among sites. Fine scale differences in populations of vectors and vertebrate hosts would explain this variation. In summary, we showed that in 2005, both SLEV and to a lesser extent WNV circulated in the avian population. Eared Dove, Picui Ground-Dove and Great Kiskadee are strong candidates to amplify SLEV because of their exposure to the pathogen at the population level, and their widespread abundance. For the same reasons, Rufous Hornero may be an important maintenance host for WNV in central Argentina. Competence studies and vector feeding studies are needed to confirm these relationships.

Tridentate Schiff base (ONO) transition metal complexes: Synthesis, crystal structure, spectroscopic and larvicidal studies

A series of four new Schiff base transition metal complexes [Co(II), Ni(II), Cu(II) and Zn(II)] derived from N-(salicylidene)-L-alanine and N,N,N’,N’-tetramethylethylene-1,2-diamine (tmen) were designed, synthesized and tested for larvicidal activity against Culex quinquefasciatus, the southern house mosquito, which is the primary vector of St. Louis encephalitis virus and West Nile virus. All the complexes were characterized by physicochemical and spectral studies such as UV-Visible, FTIR, and EPR. The X-ray crystallographic analysis of Ni(II) complex revealed that, Ni(II) cation is surrounded by nitrogen and oxygen atoms from the Schiff base ligand, the oxygen atom of a water molecule, and two nitrogen atoms from tmen. Intermolecular hydrogen bonding stabilizes the Ni(II) complex. Results indicated that all the complexes exhibited higher mosquito larvicidal activity against C. quinquefasciatus. A series of four new Schiff base transition metal complexes [Co(II), Ni(II), Cu(II) and Zn(II)] derived from N-(salicylidene)-L-alanine and N,N,N’,N’-tetramethylethylene-1,2-diamine (tmen) were designed, synthesized, characterized and tested for mosquito larvicidal activity against C. quinquefasciatus.

Widespread Detection of Antibodies to Eastern Equine Encephalitis, West Nile, St. Louis Encephalitis, and Turlock Viruses in Various Species of Wild Birds from Across the United States.

Wild birds serve as amplifying hosts for many arboviruses, and are thought to be responsible for introducing these viruses into new areas during migration as well as reintroducing them to places where winter temperatures disrupt mosquito-borne transmission. To learn more about four mosquito-borne arboviruses of concern to human or animal health, we tested sera from 997 wild birds of 54 species and 17 families across 44 states of the United States collected from January 1, 2013, through September 30, 2013. Samples were tested for antibody against eastern equine encephalitis, St. Louis encephalitis, West Nile, and Turlock viruses using plaque reduction neutralization tests with an endpoint of 80% or greater. Of the 333 (33.4%) birds that tested positive for antibody to at least one arbovirus, 29.7% were exposed to two or more arboviruses. Exposure to all four arboviruses was detected in Canada geese, double-crested cormorants, mallards, mute swans, laughing gulls, and American coots. Our results suggest that exposure to arboviruses is widespread in the United States across a diversity of wild bird species.

Surveys for Antibodies Against Mosquitoborne Encephalitis Viruses in California Birds, 1996-2013.

From 1996 through 2013, 54,546 individual birds comprising 152 species and 7 orders were banded, bled, and released at four study areas within California, from which 28,388 additional serum samples were collected at one or more recapture encounters. Of these, 142, 99, and 1929 birds from 41 species were positive for neutralizing antibodies against western equine encephalomyelitis virus (WEEV), St. Louis encephalitis virus (SLEV), or West Nile virus (WNV) at initial capture or recapture, respectively. Overall, 83% of the positive serum samples were collected from five species: House Finch, House Sparrow, Mourning Dove, California Quail, and Western Scrub-Jay. Temporal data supported concurrent arbovirus surveillance and documented the disappearance of birds positive for WEEV in 2008 and SLEV in 2003 and the appearance of birds positive for WNV after its invasion in 2003. Results of these serosurveys agreed well with the host selection patterns of the Culex vectors as described from bloodmeal sequencing data and indicated that transmission of WNV seemed most effective within urban areas where avian and mosquito host diversity was limited to relatively few competent species.

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.

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses.

The Yin and Yang of Antiviral Innate Immunity in Central Nervous System.

The innate immune system provides protection against invading neurotropic viruses. It acts as the first line of defense against invading viruses and plays an elementary role in their pathogenesis. The list of viruses capable of infecting human central nervous system (CNS) is quite long, most important of them are Japanese Encephalitis virus (JEV), rabies virus, West Nile virus (WNV), herpes simplex virus (HSV), St. Louis encephalitis virus (SLEV), La Crosse virus, tick borne encephalitis virus (TEBE) and polio virus. Germ line pattern recognition receptors (PRRs) such as Toll like receptors (TLRs), nucleotide binding oligomerization domain (NOD) - like receptors (NLRs), retinoic acid-inducible gene I (RIG-I) -like helicases or RIG-I-like receptors (RLRs) and cytosolic DNA sensors recognize the pathogen associated molecular patterns (PAMPs) and initiate an immune response against invading pathogen. Although PRRs were originally characterized in peripheral immune cells but accumulating evidence also suggest their crucial roles in CNS to combat against neurotropic viruses. In this review, we will highlight the recent developments in our understating of the mechanisms by which PRRs in resident brain cells provide protection against invading neurotropic viruses.

Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification.

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.

Concurrent Outbreaks of St. Louis Encephalitis Virus and West Nile Virus Disease - Arizona, 2015.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that can cause outbreaks of acute febrile illness and neurologic disease. Both viruses are endemic throughout much of the United States and have the same Culex species mosquito vectors and avian hosts (1); however, since WNV was first identified in the United States in 1999, SLEV disease incidence has been substantially lower than WNV disease incidence, and no outbreaks involving the two viruses circulating in the same location at the same time have been identified. Currently, there is a commercially available laboratory test for diagnosis of acute WNV infection, but there is no commercially available SLEV test, and all SLEV testing must be performed at public health laboratories. In addition, because antibodies against SLEV and WNV can cross-react on standard diagnostic tests, confirmatory neutralizing antibody testing at public health laboratories is usually required to determine the flavivirus species (2). This report describes the first known concurrent outbreaks of SLEV and WNV disease in the United States.