RESUMEN
INTRODUCTION: Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne New World alphaviruses that cause encephalitis in equids and humans. These viruses can cause severe disease and death, as well as long-term severe neurological symptoms in survivors. Despite the pathogenesis and weaponization of these viruses, there are no approved therapeutics for treating infection. AREAS COVERED: In this review, we describe the molecular pathogenesis of these viruses, discuss host-pathogen interactions needed for viral replication, and highlight new avenues for drug development with a focus on host-targeted approaches. EXPERT OPINION: Current approaches have yielded some promising therapeutics, but additional emphasis should be placed on advanced development of existing small molecules and pursuit of pan-encephalitic alphavirus drugs. More research should be conducted on EEEV and WEEV, given their high lethality rates.
Asunto(s)
Alphavirus , Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina , Virosis , Animales , Humanos , Caballos , Virus de la Encefalitis Equina Venezolana/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Encefalomielitis Equina/tratamiento farmacológicoRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Genoma Viral , ARN Viral , Inactivación de Virus , Animales , Células Cultivadas , Chlorocebus aethiops , Efecto Citopatogénico Viral , ADN Complementario/biosíntesis , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/fisiología , Virus de la Encefalitis Equina Venezolana/fisiología , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/fisiología , ARN Viral/química , ARN Viral/fisiología , Ribonucleasas/metabolismo , Células VeroRESUMEN
Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.
Asunto(s)
Barrera Hematoencefálica/virología , Caveolas/virología , Virus de la Encefalitis Equina Venezolana/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Transcitosis , Internalización del Virus , Animales , Caveolina 1/genética , Línea Celular , Sistema Nervioso Central/virología , Células Endoteliales/virología , Masculino , Ratones Endogámicos C57BL , Replicación ViralRESUMEN
RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.
Asunto(s)
Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Interferencia de ARN/fisiología , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/metabolismo , Animales , Cápside , Línea Celular , Virus Chikungunya/fisiología , Drosophila , Virus de la Encefalitis Equina del Oeste/fisiología , Células HEK293 , Humanos , ARN Interferente Pequeño , ARN Viral , Virus Sindbis/fisiología , Virión , Replicación ViralRESUMEN
UNLABELLED: Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE: VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.
Asunto(s)
Barrera Hematoencefálica/virología , Encéfalo/virología , Virus de la Encefalitis Equina Venezolana/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Encefalomielitis Equina Venezolana/virología , Internalización del Virus , Adulto , Animales , Barrera Hematoencefálica/fisiopatología , Encéfalo/patología , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/crecimiento & desarrollo , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/crecimiento & desarrollo , Humanos , Luciferasas , Mediciones Luminiscentes , Ratones , Neuronas Receptoras Olfatorias/virología , Imagen Óptica/métodos , Carga ViralRESUMEN
Serological diagnosis is a critical component for disease surveillance and is important to address the increase in incidence and disease burden of alphaviruses, such as the chikungunya (CHIKV) and Ross River (RRV) viruses. The gold standard for serological diagnosis is the plaque reduction neutralization test (PRNT), which demonstrates the neutralizing capacity of serum samples after the removal of complement activity and adventitious viruses. This procedure is normally performed following inactivation of the virus at 56°C for 30min. Although this protocol has been widely accepted for the inactivation of envelope RNA viruses, recent studies have demonstrated that prolonged heat inactivation is required to completely inactivate two alphaviruses, Western equine encephalitis virus and CHIKV. Incomplete inactivation of viruses poses a laboratory biosafety risk and can also lead to spurious test results. Despite its importance in ensuring the safety of laboratory personnel as well as test integrity, systematic investigation on the thermostability of alphaviruses has not been performed. In this study, the temperature tolerance and heat inactivation profiles of RRV, Barmah Forest, and o'nyong-nyong viruses were determined. Variations in thermostability were observed within the Semliki forest serocomplex. Therefore, evidence-based heat inactivation procedures for alphaviruses are recommended.
Asunto(s)
Alphavirus/fisiología , Inactivación de Virus , Animales , Virus Chikungunya/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Calor , Pruebas de Neutralización , Virus del Río Ross/fisiologíaRESUMEN
While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.
Asunto(s)
Infecciones por Alphavirus/patología , Virus de la Encefalitis Equina del Oeste/patogenicidad , ARN Viral/sangre , Convulsiones/patología , Administración Intranasal , Infecciones por Alphavirus/virología , Animales , Conducta Animal , Cognición , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina del Oeste/fisiología , Especificidad del Huésped , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Convulsiones/virología , Especificidad de la Especie , Carga Viral , Replicación ViralRESUMEN
New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transducción de Señal/fisiología , Replicación Viral/fisiología , Animales , Línea Celular , Supervivencia Celular , Virus de la Encefalitis Equina del Este/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Viral de la Expresión Génica , Fosforilación , Tirfostinos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Encefalitis Equina del Este/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Encefalomielitis Equina/prevención & control , Liposomas/inmunología , Ácidos Nucleicos/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/administración & dosificación , Antígenos Virales/química , Antígenos Virales/genética , Virus de la Encefalitis Equina del Este/inmunología , Virus de la Encefalitis Equina del Oeste/inmunología , Encefalomielitis Equina/inmunología , Encefalomielitis Equina/virología , Femenino , Humanos , Inmunización , Liposomas/administración & dosificación , Liposomas/química , Ratones , Ácidos Nucleicos/administración & dosificación , Ácidos Nucleicos/química , Proteínas Virales/administración & dosificación , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/química , Vacunas Virales/genéticaRESUMEN
Neurotropic alphaviruses, which include western equine encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. We previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted Sindbis virus infection in mice. We describe here further development of this series, resulting in 10-fold improvement in potency in a WEEV replicon assay and up to 40-fold increases in half-lives in mouse liver microsomes. Using a rhodamine123 uptake assay in MDR1-MDCKII cells, we were able to identify structural modifications that markedly reduce recognition by P-glycoprotein, the key efflux transporter at the blood-brain barrier. In a preliminary mouse PK study, we were able to demonstrate that two new analogues could achieve higher and/or longer plasma drug exposures than our previous lead and that one compound achieved measurable drug levels in the brain.
Asunto(s)
Diseño de Fármacos , Virus de la Encefalitis Equina del Oeste/efectos de los fármacos , Virus de la Encefalitis Equina del Oeste/fisiología , Indoles/química , Indoles/farmacología , Replicación Viral/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacocinética , Antivirales/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Línea Celular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/metabolismo , Indoles/farmacocinética , Ratones , Peso Molecular , Solubilidad , Relación Estructura-ActividadRESUMEN
Two strains of the Western equine encephalitis virus (WEEV) were first detected and isolated in China in 2001. The maintenance and transmission cycles of WEEV in China are currently not well understood, and the mosquito vectors involved in these cycles are unknown. To understand the ability of the local mosquitoes in China to transmit WEEV, the vector competence of five mosquito species, namely, Culex pipiens pallens Coquillett, Cx. p. quinquefasciatus Say, Aedes (Stegomyia) albopictus Skuse, Ae. (Stegomyia) aegypti Linnaeus, and C. tritaeniorhynchus Giles, for WEEV were evaluated. Infection rates for Cx. p. pallens, Cx. p. quinquefasciatus, Cx. tritaeniorhynchus, Ae. Albopictus, and Ae. aegypti were 46%, 60%, 80%, 37%, and 25%, respectively. Dissemination rates for the same species were 60%, 61%, 75%, 55%, and 50%, respectively. Transmission rates were 41%, 53%, 57%, and 45% for Cx. p. pallens, Cx. p. quinquefasciatus, Ae. Albopictus, and Ae. Aegypti, respectively. Infection rates were significantly different between species, but the difference between dissemination and transmission rates were nonsignificant. These results suggest that several local mosquito species in China are competent laboratory vectors for WEEV.
Asunto(s)
Aedes/virología , Culex/virología , Virus de la Encefalitis Equina del Oeste/fisiología , Encefalomielitis Equina/transmisión , Insectos Vectores/virología , Animales , China/epidemiología , Encefalomielitis Equina/epidemiología , Encefalomielitis Equina/virología , HumanosRESUMEN
Two western equine encephalomyelitis virus (WEEV) strains have been isolated in China. Our previous studies have verified that the mosquito Culex pipiens pallens Coquillett (Diptera: Culicidae) infected with WEEV was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. In this study, we observed the distribution of WEEV antigen in orally-infected Cx. p. pallens by immunohistochemistry in order to better understand the initial infection, dissemination, and transmission of WEEV in the potential vector. Orally-infected WEEV dissemination varied within the different tissues of Cx. p. pallens, with virus antigen consistently observed in the salivary glands, foregut, midgut epithelial cells, Malpighian tubules, hindgut, and ovarian follicles of some individuals after various days of extrinsic incubation. We suggest that Cx. p. pallens, the potential vector of WEEV, has the ability to harbor the virus through the alimentary system, and the midgut epithelial cell may be the initial site of WEEV replication after ingestion of a viremic blood meal.
Asunto(s)
Culex/virología , Sistema Digestivo/virología , Virus de la Encefalitis Equina del Oeste/fisiología , Insectos Vectores/virología , Animales , Antígenos Virales/metabolismo , Virus de la Encefalitis Equina del Oeste/metabolismo , Femenino , InmunohistoquímicaRESUMEN
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.
Asunto(s)
Virus de la Encefalitis de San Luis/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Calor , Manejo de Especímenes/métodos , Virus del Nilo Occidental/fisiología , Animales , Enfermedades de las Aves/diagnóstico , Enfermedades de las Aves/inmunología , Enfermedades de las Aves/virología , Coturnix , Encefalitis de San Luis/sangre , Encefalitis de San Luis/diagnóstico , Encefalitis de San Luis/veterinaria , Encefalomielitis Equina del Oeste/sangre , Encefalomielitis Equina del Oeste/diagnóstico , Encefalomielitis Equina del Oeste/veterinaria , Pruebas Serológicas , Fiebre del Nilo Occidental/sangre , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/veterinariaRESUMEN
The vector competence of Culex tarsalis Coquillett for the BFS 1703 strain of western equine encephalomyelitis virus (WEEV) changed significantly as a function of time after infection, mosquito genotype, and infectious virus dose. After ingesting a high virus dose (5 log10 plaque-forming units [PFU]/0.1 ml), female of the susceptible high virus producer (HVP) strain rapidly amplified the virus, developed a disseminated infection, and efficiently transmitted WEEV by 4 days postinfection (dpi). The quantity of virus expectorated peaked at 4 dpi (mean 3.4 log10 PFU), and the percentage of females transmitting per os peaked at 7 dpi (80%); both measures of transmission subsequently decreased to low levels throughout the remainder of infected life. HVP females imbibing a low virus dose (3 log10 PFU/0.1 ml) were infected less frequently and took longer to amplify virus to levels recorded for the high virus dose group and did not transmit virus efficiently, thereby indicating midgut infection and escape barriers were dose and time dependent. These data emphasized the importance of elevated avian viremias in Cx. tarsalis vector competence. Females from the WEEV-resistant (WR) strain and two wild-type strains from Kern and Riverside counties were significantly less susceptible to infection at both high and low doses than was the HVP strain. Overall, females with a high virus titer more frequently had a disseminated infection, but there did not seem to be a distinct threshold demarcating this relationship. In marked contrast, all infected females transmitting virus had body titers >4.3 log10 PFU, and most had titers >4.8 log10 PFU. These data indicated that not all females with a disseminated infection transmitted virus because of the presence of one or more salivary gland barriers.
Asunto(s)
Culex/virología , Virus de la Encefalitis Equina del Oeste/fisiología , Insectos Vectores/virología , Animales , Culex/genética , Encefalomielitis Equina/transmisión , Encefalomielitis Equina/virología , Femenino , Ratones , Factores de TiempoRESUMEN
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.
Asunto(s)
Culex/virología , Insectos Vectores/virología , Temperatura , Fiebre del Nilo Occidental/transmisión , Virus del Nilo Occidental/fisiología , Animales , California , Virus de la Encefalitis de San Luis/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Femenino , Análisis de Regresión , Saliva/virología , Estaciones del Año , Factores de Tiempo , Virus del Nilo Occidental/crecimiento & desarrolloRESUMEN
Female Culex tarsalis fed heparinized chicken blood-western equine encephalomyelitis virus (WEEV) mixtures through a biomembrane feeder were compared with females fed sweetened blood-virus mixtures presented in pledgets or as hanging drops or to restrained chickens with natural or artificial viremias. Results indicated that sodium heparin did not adversely affect the infection of Culex tarsalis with WEEV. Overall advantages of the biomembrane system included 1) increased blood feeding frequency, 2) control of the infectious virus dose, and 3) greater or similar infection rates and body titers to females taking blood meals from viremic chickens. Anesthetizing females with triethylamine for in vitro transmission assessment using the capillary tube method produced results similar to immobilization using cold or CO2 + cold. Our research provided insight into tools useful to investigate the infection and transmission of WEEV by Cx. tarsalis.
Asunto(s)
Culex/virología , Virus de la Encefalitis Equina del Oeste/fisiología , Insectos Vectores/virología , Animales , Pollos/parasitología , Femenino , InmovilizaciónRESUMEN
In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 A with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.
Asunto(s)
Cápside/ultraestructura , Virus de la Encefalitis Equina del Oeste/ultraestructura , Proteínas de la Nucleocápside/ultraestructura , Virus del Río Ross/ultraestructura , Ensamble de Virus/fisiología , Animales , Microscopía por Crioelectrón , Citoplasma/metabolismo , Virus de la Encefalitis Equina del Oeste/fisiología , Caballos , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/aislamiento & purificación , Virus del Río Ross/fisiología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/aislamiento & purificación , Proteínas del Núcleo Viral/ultraestructura , Virión/fisiología , Virión/ultraestructuraRESUMEN
Female Culex tarsalis Coquillett in reproductive diapause were infected per os or by intrathoracic inoculation with western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during "fall," maintained over a simulated "winter," and then tested for virus infection and transmission in vitro and in vivo after "vernal" termination. Exposure of F1 progeny of field-collected females to cool temperatures and short daylength produced females in reproductive diapause that were reluctant to imbibe infectious virus from pledgets soaked with suspensions of virus, blood and sucrose (2.5% by volume). Those infected per os maintained virus at very low or undetectable titers. Some females that originally tested negative for WEE by plaque assay on Vero cell culture tested positive by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Vero cell culture after passage in mosquito cells. Few females became infected orally with SLE, but these infected females developed elevated titers. Females inoculated with SLE retained their infection through winter and then transmitted readily in vitro and in vivo. Feeding on a vertebrate host after diapause termination significantly increased the titer of SLE in previously infected females. These experiments simulated how infections acquired either horizontally or vertically may provide mechanisms for WEE and SLE overwintering. Attempts to detect infected females during winter following a summer with enzootic WEE activity were negative by both RT-PCR and plaque assay.
Asunto(s)
Culex/virología , Virus de la Encefalitis de San Luis/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Insectos Vectores/virología , Animales , Culex/fisiología , Virus de la Encefalitis de San Luis/genética , Virus de la Encefalitis Equina del Oeste/genética , Femenino , Insectos Vectores/fisiología , Estaciones del AñoRESUMEN
Field-collected house finches of mixed sex and age were infected experimentally with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during the summer or fall of 1998 and maintained over the winter under ambient conditions. To detect natural relapse during the spring, 32 birds were bled weekly from February through June 1999, and then necropsied 1 yr after infection to detect chronic infections using a reverse transcription polymerase chain reaction (RT-PCR). After 10 mo, 13/14 surviving birds previously infected with WEE were antibody positive by enzyme immunoassay (EIA), and 11/14 had plaque reduction neutralization test (PRNT) antibody titers >1:20, whereas only of 8/13 birds previously infected with SLE were positive by EIA and all had PRNT titers <1:20. When necropsied, 1/14 and 1/13 birds had WEE and SLE RT-PCR positive lung or spleen tissue, respectively; blood, brain, and liver tissues were negative as were all previous blood samples. All tissues from these birds including weekly blood samples tested negative for infectious virus by plaque assay on Vero cell culture. To determine if persistent antibody was protective, birds infected initially with WEE or SLE in November 1998 were challenged 6 mo later with homologous virus. WEE antibody persisted well (5/6 birds remained PRNT positive before challenge) and remained protective, because 0/6 birds were viremic after challenge. In contrast, SLE antibody decayed rapidly (0/6 birds remained PRNT positive before challenge) and was not protective, because 3/6 birds developed an ephemeral viremia on day 1 after infection (mean titer, 10(2.73) plaque forming units/0.1 ml). When necropsied 7 wk after challenge, 1/110 birds infected with WEE and 1/10 birds infected with SLE exhibited an RT-PCR positive spleen, despite the fact that both birds had PRNT antibody titers >1:40 at this time. To determine if immunosuppression would cause a chronic infection to relapse, eight birds initially infected with either WEE or SLE were treated with cyclophosphamide and then tested repeatedly for viremia; all samples were negative for virus by plaque assay. Collectively, our results indicated that a low percentage of birds experimentally infected with WEE or SLE developed chronic infections in the spleen or lung that could be detected by RT-PCR, but not by plaque assay. Birds did not appear to relapse naturally or after immunosuppression. The rapid decay of SLE, but not WEE, antibody may allow the relapse of chronic infections of SLE, but not WEE, to produce viremias sufficiently elevated to infect mosquitoes.
Asunto(s)
Aves/virología , Virus de la Encefalitis de San Luis/fisiología , Virus de la Encefalitis Equina del Oeste/fisiología , Animales , California , Virus de la Encefalitis de San Luis/genética , Virus de la Encefalitis de San Luis/inmunología , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/inmunología , Femenino , MasculinoRESUMEN
Consistent temporal and spatial patterns in the activity of Culex tarsalis Coquillett and western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were delineated that were useful in developing a stratified surveillance program. Vernal increases in Cx. tarsalis abundance typically were associated with flooding of saline marshes along the north shore of the Salton Sea and were followed 6-8 wk later by the onset of WEE and SLE virus activity. Viruses then spread to managed marsh (duck club) and agricultural habitats in the Whitewater Channel flood plain and, depending upon the intensity of amplification, to agricultural and residential areas in the more elevated northwestern portion of the valley. Mean annual Cx. tarsalis abundance was correlated inversely with elevation and distance from the Salton Sea. Abundance was greatest at managed marsh habitats. Although spatially correlated with vector abundance among sites, virus transmission rates to sentinel chickens were asynchronous temporally with vector abundance. Seroconversion rates were related to flock location but not flock size (10 versus 20 chickens). Human cases were not detected during the study period, despite elevated transmission rates of both WEE and SLE viruses to sentinel chickens positioned in peridomestic habitats.