Western Equine Encephalitis Virus Strains Research Articles

Outbreak of Western Equine Encephalitis Virus Infection Associated with Neurological Disease in Horses Following a Nearly 40-Year Intermission Period in Argentina.

Western equine encephalitis virus (WEEV) is a mosquito-borne arbovirus (genus Alphavirus, family Togaviridae) that has re-emerged in South America in late 2023, causing severe disease in both horses and humans after a nearly 40-year intermission period. We here describe the virological, serological, pathological, and molecular features of WEEV infection in horses during the 2023-2024 outbreak in Argentina. WEEV-infected horses developed neurological signs with mild to severe encephalitis associated with minimal to abundant WEEV-infected cells, as demonstrated by WEEV-specific in situ hybridization. The distribution of viral RNA was multifocal, with predominance within neuronal bodies, neuronal processes, and glial cells in the medulla oblongata and thalamic regions. Phylogenetic analysis of partial nsP4 sequences from three viral isolates obtained from three different provinces of Argentina support grouping with other temporally current WEEV strains from Uruguay and Brazil under a recently proposed novel lineage.

Shifts in receptors during submergence of an encephalitic arbovirus

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1–3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2–E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Identification of salivary gland escape barriers to western equine encephalitis virus in the natural vector, Culex tarsalis.

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Complete Coding Sequence of Western Equine Encephalitis Virus Strain Fleming, Isolated from a Human Case.

We sequenced the complete coding genome of the western equine encephalitis virus (WEEV) strain Fleming. This strain was originally isolated in 1938 from a human WEEV case.

Susceptibility and Lethality of Western Equine Encephalitis Virus in Balb/c Mice When Infected by the Aerosol Route.

Western equine encephalitis virus (WEEV) naturally cycles between mosquitos and birds or rodents, with a case fatality rate of up to 15% in humans during epizootic outbreaks. There are no medical countermeasures to treat WEEV infection, and accidental aerosol exposure increases the case fatality rate up to 40%. Understanding the pathogenesis of infection is required to develop and assess medical countermeasures. This study describes the clinical and pathological findings of mice infected with WEEV by the aerosol route, and use as a model for WEEV infection in humans. Balb/c mice were infected by the aerosol route with a dose range of high-virulence WEEV strain Fleming to establish the median lethal dose (MLD). The disease course was acute, culminating in severe clinical signs, neuroinvasion, and dose-dependent mortality. Further groups of mice were exposed by the aerosol route, periodically sacrificed, and tissues excised for histopathological examination and virology. Viral titres peaked four days post-challenge in the brain and lungs, corresponding with severe bilateral lesions in rostroventral regions of the encephalon, especially in the olfactory bulb and piriform cortex. Recapitulation of the most serious clinical presentations of human WEEV disease in mice may prove a useful tool in the evaluation of medical countermeasures.

A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

Envelope protein E1 as vaccine target for western equine encephalitis virus

Western equine encephalitis virus (WEEV) is a mosquito-borne RNA virus which causes lethal infection in humans and equines. There are no commercial vaccines or anti-WEEV drugs available for humans. We used replication-defective, human adenovirus serotype-5 (HAd5) as a delivery vector for developing WEEV vaccine. Our previous study found delivery of both E1 and E2 envelope proteins of WEEV by HAd5 vector offers complete protection against lethal challenge of WEEV. In this paper, we constructed a HAd5-vectored E1 vaccine, Ad5-E1. Mice given single-dose vaccination of Ad5-E1 were completely protected against both homologous and heterologous WEEV strains. The protection was rapid, which was achieved as early as day 7 after vaccination. In addition, Ad5-E1 induced a strong WEEV-specific T cell response. Our data suggest E1 is a potential target for developing single-dose, fast-acting, HAd5-vectored vaccine for WEEV.

Treatment with cationic liposome–DNA complexes (CLDCs) protects mice from lethal Western equine encephalitis virus (WEEV) challenge

Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome–DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4–5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-γ, TNF-α, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-γ, TNF-α, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.

Chimeric alphavirus vaccine candidates protect mice from intranasal challenge with western equine encephalitis virus

We developed two types of chimeric Sindbis virus (SINV)/western equine encephalitis virus (WEEV) alphaviruses to investigate their potential use as live virus vaccines against WEE. The first-generation vaccine candidate, SIN/CO92, was derived from structural protein genes of WEEV strain CO92-1356, and two second-generation candidates were derived from WEEV strain McMillan. For both first- and second-generation vaccine candidates, the nonstructural protein genes were derived from SINV strain AR339. Second-generation vaccine candidates SIN/SIN/McM and SIN/EEE/McM included the envelope glycoprotein genes from WEEV strain McMillan; however, the amino-terminal half of the capsid, which encodes the RNA-binding domain, was derived from either SINV or eastern equine encephalitis virus (EEEV) strain FL93-939. All chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in 6-week-old mice. Vaccinated mice developed little or no detectable disease and showed little or no evidence of challenge virus replication; however, all developed high titers of neutralizing antibodies. Upon intranasal challenge with high doses of virulent WEEV strains, mice vaccinated with ≥10 5 PFU of SIN/CO92 or ≥10 4 PFU of SIN/SIN/McM or SIN/EEE/McM were completely protected from disease. These findings support the potential use of these live-attenuated vaccine candidates as safe and effective vaccines against WEE.

Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Western Equine Encephalitis submergence: Lack of evidence for a decline in virus virulence

The incidence of Western Equine Encephalitis (WEE) in humans and equids peaked during the mid-20th century and has declined to fewer than 1–2 human cases annually during the past 20 years. Using the mouse model, changes in WEE virus (WEEV) virulence were investigated as a potential explanation for the decline in the number of cases. Evaluation of 10 WEEV strains representing a variety of isolation locations, hosts, and all decades from the 1940's to the 1990's yielded no evidence of a decline in virulence. These results suggest that ecological factors affecting human and equine exposure should be investigated to explain the decline in WEE.

Pre- and post-exposure protection against Western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha

Western equine encephalitis virus ( WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNα) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNα provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNα delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection.

Complete protection of mice against a lethal dose challenge of western equine encephalitis virus after immunization with an adenovirus-vectored vaccine

Western equine encephalitis virus (WEEV) is an important pathogen for both humans and equines. The virus is also listed as a bioterrorism agent due to its ability for aerosol transmission with high mortality. No commercial vaccines or antiviral drugs are available for the prevention and treatment of WEEV infection in humans. In this paper, we constructed a recombinant WEEV vaccine, designated as Ad5-WEEV, using a replication defective, human adenovirus serotype 5 (HAd5) as a delivery vector. Ad5-WEEV contains the E3-E2-6K-E1 structural protein gene of the 71V-1658 strain of WEEV and the E1 and E2 proteins were synthesized in cells inoculated with Ad5-WEEV. After intramuscular immunization of mice with two doses of Ad5-WEEV, neutralizing antibodies against WEEV were generated and the mice were completely protected from a lethal dose challenge of 71V-1658. In addition, we showed that passive transfer of serum from the Ad5-WEEV-immunized mice could partially control WEEV infection. These results demonstrate that HAd5 vectors are promising for WEEV vaccine development.

Infectivity variation and genetic diversity among strains of Western equine encephalitis virus

Variation in infectivity and genetic diversity in the structural proteins were compared among eight strains of Western equine encephalitis virus (WEEV) to investigate WEEV virulence at the molecular level. A lethal intranasal infectivity model of WEEV was developed in adult BALB/c mice. All eight strains examined were 100 % lethal to adult mice in this model, but they varied considerably in the time to death. Based on the time to death, the eight strains could be classified into two pathotypes: a high-virulence pathotype, consisting of strains California, Fleming and McMillan, and a low-virulence pathotype, comprising strains CBA87, Mn548, B11, Mn520 and 71V-1658. To analyse genetic diversity in the structural protein genes, 26S RNAs from these eight strains were cloned and sequenced and found to have > 96 % nucleotide and amino acid identity. A cluster diagram divided the eight WEEV strains into two genotypes that matched the pathotype grouping exactly, suggesting that variation in infectivity can be attributed to genetic diversity in the structural proteins among these eight strains. Furthermore, potential amino acid differences in some positions between the two groups were identified, suggesting that these amino acid variations contributed to the observed differences in virulence.

Peroral susceptibility of Aedes albifasciatus and Culex pipiens complex mosquitoes (Diptera: Culicidae) from Argentina to western equine encephalitis virus

The transmission cycle of western equine encephalitis (WEE) virus in South America is unknown. A WEE virus strain was isolated from Aedes albifasciatus in Argentina during the WEE epizootic of 1982-83. Also, Culex pipiens from Argentina was reported to be able to transmit WEE virus experimentally, but other results indicate that Cx. pipiens from the USA is refractory to this virus. We determined the susceptibility of Argentina strains of Ae. albifasciatus and Culex pipiens complex mosquitos to infection by WEE virus by the oral route. Adult females were fed on chicks infected with a WEE virus strain isolated in Cordoba Province, Argentina, or were fed on a blood/virus suspension. Each mosquito ingested between 10(1.6) to 10(6.4) vero cell plaque-forming units of virus. Each of 28 Ae. albifasciatus was positive for virus from the fourth day postfeeding, and there was evidence for virus replication. In contrast, 0/44 Cx. p. quinquefasciatus and only 1/15 Cx. p. pipiens was positive. Aedes albifasciatus is susceptible to infection by WEE virus and should be considered a potential vector of this virus in Argentina. Both subspecies of Cx. pipiens are refractory to peroral infection by WEE virus and probably do not play a role in the WEE virus cycle in Argentina.