The Study Of Genetic Reassortment Among Hantaviruses

RNA viruses with segmented genomes have the capacity of reassort their RNA segment upon dual infections. This ability is believed to play a key role in the evolution, pathogenesis, and epidemiology of important pathogens such as influenzaviruses, rotaviruses, and arthropod-borne orbiviruses. Genetic reassorment has been demonstrated both in nature and in the laboratory among the family Bunyaviridae. Hantaviruses possess a genome consisting of three segment of single-stranded, negative-sense RNA. In some recently published studies, the ability of Hantaviruses causing Hantavirus Pulmonary Syndrome to undergo genetic reassortment has been demonstrated both in nature and in the laboratory. But the ability of genetic reassortment of other types of Hantaviruses is unknown. In this study, mixed infections were initiated in tissue culture by using Hantaan virus (76-118 strains) and Seoul virus (SR-il strains), and using Hantaan virus (76-118 strains) and Prospect Hill Virus (PHV Strains). Potential reassortant virus plaques were screened by multiplex RT-PCR, using primers specific for individual genome segments of each strain. In the mixed infections using Hantaan virus (76-118 strains) and Seoul virus (SR-li strains), We found that most of the progeny virus plaques (68.19% of 44) had the parental genotype of 76-118 strain or SR-li strain. 2 of 44 plaques had mixed genotypes that yielded RT-PCR bands -4- of both parental strains. Reassortant viruses were detected in 18.19% of 44 progeny plaques tested, involving the M and S segments. In addition, approximately 4.55% of the progeny virus plaques appeared to contain S or M segments originating from both parental virus strains, i.e., they were diploid. In the mixed infections using Hantaan virus (76-118 strains) and Prospect Hill Virus (PHV Strains), We found that most of the progeny virus plaques (72.22% of 36) had the parental genotype of 76-118 strain or PHV strain. 5 of 36 plaques had mixed genotypes that yielded RT-PCR bands for the same segment of both parental strains. Reassortant viruses were detected in 3 of 36 progeny plaques tested. In addition, approximately 8.33% of the progeny virus plaques appeared to contain M segments originating from both parental virus strains, i.e., they were diploid. 7 strains of the progeny plaques contained all three segments from both parental strains. One possible explanation for this would be that these were mixed plaques, originating from wells with a higher plaque density, hence making it difficult to collect individual ones or there was a background of poorly visible virus plaques in the cell monolayer between the visible plaques. Another possible explanation is that there was variance or reassortment occurred. The three strains of the progeny plaques from which the virus antigens could not be detected by immunofluorescence techniques were found to be negative to all three segments from both parental viruses by RT-PCR. One possible explanation for this would be that there were no plaque been collected. Another possible explanation is that the viruses were died in culture or the load of the viruses was too lower to be detected. The reassortant ratio of virus progeny from mixed infections with 76-118 and SR-li strains (20.45%) has been observed higher than that from mixed infections with 76-118 and PHV strains (8.33%). The different frequency of reassortment might be explained by the difference of their genetic divergence of their three seg...