RESUMEN
Tegument protein VP8 encoded by the U(L)47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U(L)47 gene-deleted BHV-1 in cultured cells and its natural host. The U(L)47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U(L)47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U(L)47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U(L)47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U(L)47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U(L)47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.
Asunto(s)
Proteínas de la Cápside/genética , Eliminación de Gen , Herpesvirus Bovino 1/genética , Proteínas Virales/fisiología , Animales , Proteínas de la Cápside/fisiología , Bovinos , Regulación Viral de la Expresión Génica , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 1/patogenicidad , ARN Viral/análisis , Proteínas Virales/análisis , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. RESULTS: Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. CONCLUSION: Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.