| There was an outbreak of a disease with the characteristic "Porcine diarrhoea" in pig herds in China in2010. The purpose of this study was to investigate the epidemic situation of the MRV in recent China. We also sequencd and analysed the full genome of one predominant MRV strain in China. On the base of these knwoledge, we generated a complete cDNA clone of a MRV. These findings enhance our knowledge of the genetic diversity of pocine reovirus in China and contribute to the development effective strategies for swine disease control.1. Molecular epidemiology of MRV in parts of ChinaTo gain an insight into the molecular epidemiology of MRV in China, we analyzed the S1gene of45Chinese MRV positive samples. Clinical samples were collected between January2010and December2012. MRVs were detected in45out of these samples by RT-nested PCR and were selected for sequencing. Further analysis based on S1sequences revealed that all Chinese isolates belonged to serotype1、2、3. The most significant correlation between genetic and geographical distribution for the isolates in the study, especially for theserotype3strains, was they take up the widest area, since these viruses existed throughout the mainland of China. These findings enhance our knowledge of the genetic diversity of Chinese MRV, and may contribute to the development of reliable diagnostic tests, the epidemiological surveillance, and the development of effective strategies for disease control.2. Isolation and Identification of a Porcine Reovirus in ChinaA strain SHR-A of Reovirus serotype1was isolated from Vero cell line by the method of adding trypsin into the DMEM medium from piglets’diarrhea fecal sample. The stable cell pathogenic effect (CPE) caused by the virus including cell granule increasing, cellular swelling and shedding were observed. Virus purification was conducted based on reovirus physical and chemical properties. Sequence analysis showed that the strain belongs to Reovirus serotype1.S1gene of SHR-A showed higher homology to that of serotype1than that of other serotype viruses. This is the first report of type1Reovirus was isolated from porcine herds in China.3’-UTR of its S1genome segment showed higher homology to that of serotype3than to serotype1. That means the S1gene was a chimerism from that of serotype1to serotype3.Our results provided evidence that RNA virus may have the third way, genomic recombination, for viral evolution, beside genomic reassortment and genomic point mutation.3. Sequencing and cDNA cloning of complete genome of pig MRV SHR-A strain.Complete genome of the isolate(SHR-A) were sequenced and used to study swine MRV. RT-PCR assay was respectively applied to obtain10fragments of SHR-A strain. Sequence of both5’-and3’-ends were performed with the Rapid-amplification of cDNA ends (RACE). All fragments were spliced with Seqman software and the complete genome of the isolate obtained was23608nt. The genomic RNA contained5’-untra nslated region (5’-UTR),3’-untranslated region (3’-UTR), and one open reading frame (ORF) encoding a large polyprotein of3899amino acids but not exogenetic genomic sequences. Complete genome of MRV SHR-A strain was successfully obtained. The study on molecular epidemiology of pig MRV in China, to some extent, was promoted.4. Expression of Reovirus Type1S1、S4Gene in Sf9Insect CellThis research was focused on expressing reovirus serotype1S1and S4gene in Sf9insect cells by BacMagic DNA Baculovirus expression system. S1gene was amplified by reverse transcript-polymerase chain reaction (RT-PCR) and inserted into pBAC-1to obtain recombinant transfer vectors of pB AC-S1and pBAC-S4. Then the pB AC-S1and pBAC-S4were transfected into sf9cells through lipid-mediated transfection with BacMagic-3DNA. Immunoflorescence and western blot analysis confirmed that the recombinant protein was expressed in Sf9cells (50kD and41kD) and had its immunogenicity. This is the first report in China which shows Reovirus σl and σ3proteins with immunogenicity expressed in Sf9insect cells successfully. |
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