Isolation, Identification And Genome Sequence Analysis Of A Goose Reovirus

In recent years, there has been found a new viral infectious disease characterized by hemorrhagic necrotizing hepatitis in many goose farms in Jiangsu province. We collected the substantive organs of the diseased goose from the goose farms as the epidemic materials and separated the virus after inoculating SPF chicken embryos, and then identified the virus by RT-PCR (one of the molecular biology technology) combined with observation of the clinical and pathological changes. Finally we identified it as the goose reovirus and named it as GRV JS2012 temporarily.In order to determine the taxonomic position of the virus, we did a variety of experiments including the organic solvent sensitive experiment, the temperature sensitive experiment and hemagglutination experiment. It showed that the virus, which identified as RNA virus with resistence to heat, could not agglutinate chicken blood red blood cells and was sensitive to typsin but not to acid as well as chloroform.Then we purified the virus by chloroform extraction, Ultra-high speed differential centrifugation and sucrose density gradient centrifugation. And then through phosphoric acid dyeing by transmission electron microscope to observe the virus particles, the results revealed that the virus particles were approximate spherical without posterior capsule, of which diamerter was about 80 nm. Each layer of the particle was 20 sides symmetry with double capsid structure, which was featured as prototypical reovirus.We inoculated 9-11 days of SPF chicken embryos though chorioallantoic membrane with the virus, After observing the chicken embryos for 2-6 days we collected the allantoic fluid and allantoic membrane of the dead chicken embryo, and inoculated to the third-generation in turn. Then we measured the ELD50 of the allantoic fluid, allantoic membrane and dead chicken embryo from the third-generation. The results indicated that the ELD50 of the allantoic fluid was 10-5.83/0.2mL,while that of allantoic membrane and dead chicken embryo was 10"5633/0.2mL.Therefore, we can concluded that the content of the virus in the allantoic membrane was higher than that in the allantoic fluid. After that, we inoculated the virus in chicken fibroblast cells with the allantoic fluid to the fifth-generation in turn and measured its TCID50as 10-5.75/0.1mL. In addition, the pathogenicity test proved that this strain of virus could be fatal to 1 day cheeper with the mortality of nearly 100%, but could not cause disease of young and middle-aged goose.Moreover, according to the published goose reovirus gene sequences on the NCBI,3 pairs of primers were designed to amplify three parts of gene sequences by RT-PCR. The sequence analysis displayed that this strain was close to the published duck and goose reovirus, and the single gene phylogentic tree indicated that the homology was as high as 80-90% with the 03G strain and J18 strain, but low with the standard stains S1133. Accordingly, this strain should be relegated to the waterfowl reovirus which was different from the chicken reovirus.Finally, in order to know the evolution of the relationship between this strain and ARV, DRV as well as GRV, we analyzed the whole genome sequence. According to the published gene sequences, we designed 13 pairs of primers, amplified the purpose fragments with the method of RT-PCR, sent to sequencing analysis. We also inspected the size and numbers of the ORF and compared them with other members of the genus reovirus to draw into genetic evolutionary tree. It pointed out that the full-length of GRV JS2012 genome is 23,418bp, which could be divided into 10 segments:S1,1568bp; S2,1326bp; S3,1202bp; S4,1191bp; M1, 2283bp; M2,2158bp; M3,1996bp; L1,3958bp; L2,3829bp; L3,3907bp. Unlike other ARV strains whose σC protein is coded by S4 gene, which of GRV JS2012 strain is coded by S1 gene. And the GRV JS2012 conservative sequences on its 5’and 3’,5’-GCUUUU,3’-UCAUC with other AVR strains. The homology analysis showed the homology of nucleotide was as high as 89.7-99.1% compared with GRV 03G strain, while 20.4-96.9% with genetic variability in L1、 L2、M1 and S2 compared with DRV J18 strain and 20.8-77.0% with ARV S1 133 strain. Thus, we summarized that the GRV JS2012 strain should be relegated to the waterfowl reovirus genera which differs from chicken reovirus. Phylogenetic analysis suggested that our strain and goose reovirus are on the same branch which was different from duck reovirus and chicken reovirus. Therefore, we confirmed that there is genetic diversity in different reovirus strains. However, whether there are genetic recombinations between different strains still need further study. This study provides a reliable basis for the further study of the reovirus.