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Abstract Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.

Abstract The virulence of A/Vietnam/1194/2004 (VN1194) in mice attenuated after serial passages in MDCK cells and chicken embryos, because the enriched large-plaque variants of the virus had significantly reduced virulence. In contrast, the small-plaque variants of the virus and the variants isolated from the brain of mice that were infected with the parental virus VN1194 had much higher virulence in mice. The virulence attenuation of serially propagated virus may be caused by the reduced neurotropism in mice. Our whole genome sequence analysis revealed substitutions of a total of two amino acids in PB1, three in PB2, two in PA common for virulence attenuated variants, all or part of which may be correlated with the virulence attenuation and reduced neurotropism of the serially propagated VN1194 in mice. Our study indicates that serial passages of VN1194 in vitro lead to adaptation and selection of variants that have markedly decreased virulence and neurotropism, which emphasizes the importance of direct analysis of original or less propagated virus samples.

Abstract In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.