Comparative Analysis Of BTV-HbC₃ Strain And BTV-10 Strain

Bluetongue virus (BTV) is the type species (one of twenty different species) of the genus Orbivirus, within the family Reoviridae. BTV can infect most ruminants and even some predatory carnivores. The virus can also infect and is transmitted by certain species of biting midges (Culicoides species) and is divided into 25 distinct serotypes in the world. BTV is a double-shelled virus with a genome consisting of 10 dsRNA segment. The virus has an icosahedral core that contains two major (VP7 and VP3) and three minor (VP1,VP4 and VP6) structural proteins. VP7 is a dominant BTV serogroup-reactive antigen and is the major, constituent of capsomers on the surface of core particles, accounting for 36% of capside protein. And most amino acids of VP7 are conserved being the major research target as vaccine of BTV. The core is surrounding by an outer layer composed of two structural proteins. VP2 and VP5. In addition to seven structural proteins, BTV infected cells contain three virus-specific,non-structural proteins (NS1,NS2 and NS3).BTV-HbC3 strain is isolated by Dr. Dong Changyuan in Xiangfan City Hube Province in 1994. With the systemic analysis of our lab that the replicated characteristics of BTV-HbC3 between BTV-10 in different cells; the genome characteristics; the argrose diffusion test about immune across reaction between BTV-HbC3 and BTV-10; the characteristics of the sequence of RT-PCR of BTV-HbC3 S7 5' -NCR which proved that BTV-HbC3 strain is probably a new genome type of Bluetongue Virus.1. With the replicated and morphologic characteristic of BTV-HbC3 infecting different cells such as Vero, MCF-7 and Hep-3B ,all the cells displayed a rounded, granular phenotype having the same CPE(characteristics of cytopathic effects) with that of BTV-10.Through agar gel immunodiffusion test, the cross-reactivities were detected between BTV-HbC3 and BTV-10 indicating that the common antigenic determinants on the VP7 proteins of BTV-HbC3 and BTV-10 shared some epitopes.2. Using primers corresponding to the conserved sequence of BTV genomic dsRNAsegment previously published, S7 5' -NCR of BTV-HbC3 and BTV-10 were amplified by RT-PCR. And the PCR products is 277bp and 290bp respectively. Comparative sequence analyses of 5' -NCR show that 100% of the nucleotides are conserved between BTV-HbC3 and Reovirus-3 L2, while none of the bases are identical between BTV-HbC3 and BTV-10. In this study, partial sequence of S7 segment permitted us to investigate the role of genetic exchange(reassortment and recombination) in the evolutionary mechanisma deriving the evolution of the genus Orbivirus. Pehaps BTV-HbC3 and BTV-10 originated from two gene pools or some sequences of Reovirus-3 were inserted into BTV-HbC3 undergo genetic drift which contributed to diversification of individual gene segments of field strains of BTV.3. Flow cytometry (FCM) and MTT analysis was used to detect the CPE of MCF-7 which was infected by BTV-HbC3 and BTV-10 strain with 1MOI. And the results demonstrated that the apoptosis rate is 31.9% and 29.1% respectively. The CPE of cells infected by these two BTV strain indicated that BTV can induce tumour cells qpoptosis, andit is the foundation which we use them as oncolytic virus theraping cancer.Conclusions: With the replicated and morphologic characteristic of BTV-HbC3 and BTV-10 infecting different cells, agar gel immunodifrusion test between BTV-HbC3 and BTV-10, the analysis of the sequence results of BTV-HbC3 and BTV-10 S7 5' -NCR showed that BTV-HbC3 is the prototype virus of the Orbivirus genus, probably a new genome type of Bluetongue Virus.