Research On Gene Reassortment And Pathogenicity Of Partly Reassortant Progeny Viruses Of Reovirus (Muscovy Duck) Different Isolates

Avian reovirus is the pathogen of muscovy duck liver white spots disease. Avian reovirus avian reovirus is consisted of 10 gene segments which are divided into three groups, L, M and S. The S-class segments of the viral genome is the key to study the evolution of avian reovirus in the world, and the S-class has two genotypes, one genotype which standard strains are DRV-89026 and DRV-89330 (YB belongs to this genotype), the other genotype which standard strain is ARV-S1133 (YJL belongs to this genotype). When two avian reoviruses infected the same host, gene reassortment could happen and progeny virus may have exchanged gene segments. Compared with the parental strains, the progeny viruses with reassorted segments may have different biological characteristics or pathogenicity to the host or even a new serotype may come into being because of gene reassortment.This experiment is to simulate the natural infection. When two avian reoviruses (YB and YJL) infect the avian embryo fibroblasts at the same time, gene reassortment may probably happen. When we get the reassortant progeny viruses, we will do a study between the parental viruses and the reassortant progeny viruses on the changes in their biological characteristics as well as the differences on the pathogenic.First,we obtained the cloned parental virus by plaque method. Then we designed eight pairs of primers of nucleic acid sequences which encodingσC,σNS,σB,σA protein respectively according to sequences from the Gene bank (the French isolates of DRV-89026 and DRV-89330, the United States isolates of ARV-S1133 and 176, as well as our country's S14 isolate), in order to establish the multiple RT-PCR testing method of avian reovirus S group gene segments. Secondly, after the two parental viruses infected the avian embryo fibroblasts simultaneously, we pick the possible reassortant virus plaque which may have the exchanged gene segments according to shape, size and other cultivate traits of the plaque. Then, we use the two parental viruses multiple RT-PCR to determine the progeny virus which have reassorted gene segments. At last, the parental and progeny virus inoculated the avian embryo fibroblasts, avian embryo sac, one-day old chicken and muscovy ducklings respectively. Studies were made on the pathogenicity differences between the parental and progeny viruses.The results we have made are:⑴the plaque of avian reovirus on avian embryo fibroblasts is round and have a neat edge.⑵the differences on death time of avian embryos: death time of chicken embryos: YB, 9d-14d; YJL ,65-71h; reassortant progeny virusⅠ,56-61h; reassortant progeny virusⅡ, 7d-10d. death time of muscovy duck embryos: YB ,134-229h; YJL ,90-95h; reassortant progeny virusⅠ,66-76h; reassortant progeny virusⅡ,93-117h.⑶hemorrhage of embryos: reassortant progeny virusⅠ> YJL> reassortant progeny virusⅡ> YB.⑷the differences of lesions after the One-day-old chicken and muscovy duckling were inoculated with parental and reassortant progeny viruses: Compared with parental viruses, the ressortant viruses had caused more serious lesions. The major lesions are liver and spleen when chicken and muscovy duckling were inoculated with Parental strains. While there are more lesions in chiken and muscovy duckling when inoculated with reassortant progeny viruses, including the liver and spleen.⑸the TCID50 results of parental and progeny viruses: YB,10-6.5;YJL,10-7.24;reassortant progeny virusⅠ,10-7.77;reassortant progeny virusⅡ,10-7.53.⑹the results of pathological sections showed: Compared with parental viruses, the ressortant viruses had caused more necrotic foci, more serious cell degeneration and necrosis, lymphocytes decreased in lymphoid tissues.⑺analysed by the software, we can see that the amino acid sequence homology of proteinσA of avian reovirus two parental strains YB and YJL is 88%; the amino acid sequence homology of proteinσB is 61%.⑻the secondary structure analysis and hydrophilicity and hydrophobicity analysis of proteinσA andσB showed that: the differences ofσB are more serious thanσA,especially in the amino acid sites between 90 and 200 ofσB.From the results we can concluded that:⑴as this experiment proved , when the two avian (muscovy duck) reoviruses (YB and YJL) infected the avian embryo fibroblast cells simultaneously, the gene recombination (gene reassortment) could occur.⑵according to the results of this experiment, the biological characteristics of reassortant progeny viruses have changed, including plaque forming ability on avian embryo fibroblast, the pathogenicity to avian embryos (death time of avian embryos and hemorrhage of embryos) and the different lesions in organs and tissues of chicken and muscovy duckling after infection. We know that the hosts are more sensitive to reassortant progeny viruses.⑶as this experiment proved, as far as the ressortant progeny viruses are concerned, there are differences on culture character compared with the parental strains. There are significant differences between the parental viruses and reassortant progeny viruses on the size, shape and edge of the plaque. We concluded that avian (muscovy duck) reovirus'plaque forming ability is related to the virulence of the virus on the same cell (avian embryonic fibroblasts)⑷from the secondary structure analysis of proteinσA andσB and the differences between parental viruses and reassortant progeny viruses on pathogenicity, we can see that the spatial structure of virus proteins has an important role on the virulence of the virus. It indicated that the greater the difference in spatial structure of reassorted protein, the greater impact the gene reassortment did to the virulence of the reassortant progeny viruses.