Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.     

Properties of monospecific antibodies to the glycoprotein of western equine encephalitis virus

Monospecific (MSp-) antisera against E1 and E2 glycoproteins of western equine encephalitis (WEE) virus were prepared and examined for binding activities to whole virions, hemagglutination-inhibition (HI), neutralization (NT) and protection. Both anti-E1 and anti-E2 MSp-Abs protected mice against WEE virus challenge. A competition experiment with monoclonal antibodies showed that these MSp-antisera appear to lack the antibody population for some epitopes involved in viral neutralization.     

Purification of the envelope glycoproteins of western equine encephalitis virus by glass wool column chromatography.

Glass wool column chromatography was used for separation of the two glycoproteins of western equine encephalitis virus. Cross-contamination of each protein separated was confirmed to be negligible by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

Antiviral effects of double-stranded RNA from rice dwarf virus on infection of mice with western equine encephalitis virus.

A single administration (1 to 10 mg/kg) of rice dwarf virus RNA (RDV-RNA) prior to virus challenge reduced the mortality of mice infected with western equine encephalitis (WEE) virus. The protective effect of RDV-RNA was significantly higher than that of polyinosinic-polycytidylic acid. However, when these RNAs were given after virus infection, the protective effect was negligible. The titer of circulating interferon in mice reached a peak about 5 hr after injection of these RNAs and remained at this level for about 24 hr. Viremia in mice infected with a lethal dose of WEE virus was markedly suppressed by the treatment of mice with these RNAs. A pathological examination of mice treated with a lethal dose of RDV-RNA revealed marked changes including degeneration and karyorrhexis in the lymphoid tissues of the spleen.