| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
We isolated two variant equine herpesvirus-1 (EHV-1) strains, 5089 and 5586, from the lung of a dead neonatal foal in 2000 and from the lung of an aborted equine fetus in 2002, respectively. 5089 spread in cultured cells mainly by cell-to-cell infection and 5586 spread in cultured cells by direct cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. Cell-free 5586 virus was hardly detected even in supernatants of infected cells that had been disrupted by freezing and thawing. We characterized the molecular biological nature of 5089 and 5586 and searched for the gene(s) responsible for cell-to-cell infection. Sequence analysis of the 5089 and 5586 genomes revealed several amino acid substitutions in 53 and 67 of 76 possible EHV-1 open reading frames (ORFs), respectively. In several ORFs, mutations in nucleotide sequence resulted in position change of stop codon. Length of 5586 glycoprotein D (gD) reduced to about 10% of wild-type gD. gD is an essential protein for EHV-1 infectivity. Real-time PCR analysis revealed that mRNA expressions of ORFs 13 (tegument), 14 (unknown), 15 (virion protein), 16 (gC), 53 (origin-binding protein), 54/57 (proteins associated with a DNA helicase/primase complex), 55 (unknown), 58 (unknown) and 71 (gp2) of 5089 and 5586 were reduced considerably compared to those of wild-type EHV-1. gC was not detected in 5089- and 5586-infected cells by Western blotting. gC is an important protein for EHV-1 adsorption in cells. These results suggested that absence of or reduced amounts of some viral structural proteins, including the lack of gC in 5089 and 5586, and shortened gD in 5586, might be one of the reasons for spread of the virus by cell-to-cell infection.
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