Anti-WEEV E2 polyclonal antibody

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.

The genes encoding envelope proteins El and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6 x His-tagged WEEV El and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6 x His-tagged proteins showed 50 kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV El and E2. The potential of the recombinant WEEV El and E2 as antigens for serologic tests to detect anti-WEEV antibodies for diagnosis of WEEV infection was assessed by an enzyme-linked immumosorbent assay with anti-WEEV polyclonal. antibodies obtained from the mice infected with WEEV. The anti-WEEV antibodies bound the recombinant WEEV E1 and E2 in a dose dependent manner. On the contrary, antibodies against Venezuelan equine encephalitis virus with a genetic background and a disease spectrum very similar to WEEV, did not bind to the recombinant WEEV El and E2. Our results suggest that the recombinant WEEV El and E2 possess predominant antigenicity of WEEV and have the potential to be used as antigens in immunoassays to detect anti-WEEV antibodies for serological diagnosis of WEEV infection so as to eliminate the need for preparation of cell culture-derived viral antigens, which is time-consuming, expensive, laborious, tedious, and hazardous. Crown Copyright (C) 2007 Published by Elsevier B.V. All rights reserved.