| Rotaviruses have been identified as one of the main etiological agents of diarrhea and enteritis in human and animals. Rotavirus-associated enteritis is a major problem in livestock animals, notably in young piglets. Porcine Rotavirus (PoRV) infection is an acute swine disease with intestinal infectious symptoms, causing severe diarrhea and dehydration, piglets are in high mortality, which can cause enormous economic loss in the pig industry. Therefore, it is of great significance to isolate and find the best way to protect and cure this virus. In this study, we detected the diarrhea specimen, from Sichuan Chengdu, using reverse transcription-polymerase chain reaction (RT-PCR) method, then the positive sample was inoculated into MA104 cells for rotavirus isolation. We successfully isolated a porcine rotavirus and investigated the characteristics of this strain.1. Isolation of a porcine rotavirusThe positive sample detected by RT-PCR was inoculated into MA 104 cells. Trypsin was used to sensitize virus,60ug/ml trypsin in the inoculums and 6ug/ml in the maintenance were used to isolate this rotavirus strain. The cytopathic effect (CPE) was observed in the first passage, but it was not typical. Until the third passage CPE could be observed on day 2 post-inoculation, with death cells appeared and accumulated together.3 days after inoculation the significant CPE emerged, consisting of cell rounding, obscure cell borders, cell piling up, cell fusion and cauliflower-like shape. Finally, a large number of cells died and fallen off the surface of tubes.22 passages were conducted and the virus was tested by RT-PCR every 3 passages. The result shown that a strain of porcine rotavirus from the intestinal specimen of diarrheal piglets in Sichuan province was successfully isolated, named SC-C. Through the physicochemicalproperty test we knew that this porcine rotavirus was not sensitive to chloroform and diethylether, but it loses infectivity when in the circumstance of 56℃ or above for 30 min, demonstrating that this strain was not hot-resistant.2. Cloning and analysis of VP7 geneThe outer capsid VP7 protein is the dominant pathogen to elicit neutralizing antibodies, which induce homotypic as well as heterotypic protective immunity in animals. Besides, VP7 protein is thought to have an important role in protecting the host against diarrhea upon challenge with the virulent parental rotaviruses. Therefore, VP7 protein plays an important role in protecting pigs from porcine rotavirus infection. In this research we designed a pair of primer according to the published cDNA sequence of RV’s VP7 gene in GenBank, the full length gene encoding VP7 protein was amplified by RT-PCR. Then the cDNA of VP7 gene was inserted into pMD19-T vector and was identified by sequencing. The function of VP7 protein was predicted by bio informatics software and the phylogenetic tree of VP7 gene was constructed by MEGA software. The results showed that the full length of VP7 gene was 1061bp. Comparing this strain with other 54 porcine rotavirus isolated from Korea, the identity of nuclear sequence and amino acid sequence were all above 99%, indicating that the VP7 gene was of high conversation. Besides, comparing this strain with other G5 serotype porcine rotavirus strains, the identity of nuclear sequence and amino acid sequence were 87.4%-99.8% and 92.6%-99.7% respectively. So we informed that this porcine rotavirus we isolated belongs to G5 serotype. The bioinformatics analysis indicated that 981bp open reading fram was contained, which encoding 326aa. The molecular weight, isoelectric point, estimated half-life, instability index of VP7 protein were 37.018KD,4.86, about 20hours and 35,81 respectively, indicating that the VP7 protein has strong stability. Besides, the aliphatic index and average hydrophily were 95.06 and 0.072 respectively. While the grand average of hydropathicity of VP7 protein was between -2.400 and 3.678. Through the prediction on line, we found that the protein has 9 antigenic determinants and 2 transmembrane regions 4-23 site and 32-54 site, and the N terminal and C terminal were all in the outer region. Moreover, the on line PredicProtein predicted that the secondary structure composition of VP7 protein is consist of 30.67% α-helix,32.52% β-sheet and 36.81β-turn, and this protein was predicted to have 1 N-glycosylation sites,4 protein kinase C phosphorylation sites,3 casein kinase Ⅱ hposphorylation sites,1 tyrosine kinase phosphorylation sites and 3 N-myristoylation sites. The phylogenetic tree showed that the evolution distance of this porcine rotavirus VP7 gene is homogeneous to JL94 strain which was isolated from Dongbei province in 2004, and it is closely to group A bovine rotavirus.3. Animal regress experiment of this strainPathogenicity of porcine rotavirus infection was investigated by inoculating 4 days old suckling pigs and 3.75×106PFU/ml of PoRV solution was used.3 hours post-inoculation, the pigs walked around with instability, shown abdominal pain symptoms and breathe shortness, reaching the rate of 70 per minutes. Until 5 days, the mild diarrhea was observed with yellow watery diarrhea. The lesions were observed in intestines, including each intestinal segments emerged flatulence with a large number of bubbles; The jejunum and mesenteric were obviously haperemia with visible hemorrhagic spots, and lymphadenectasis. Besides, the rotavirus virion was tested in duodenum, jejunum, ileum, colon by RT-PCR, which indicated that there were porcine rotaviruses existing in the intestines. Inoculating the content of intestine to MA 104 cells and mild CPE was observed in day 4. Serum neutralization test indicated that 1:2347 dilution positive serum was capable for neutralizing PoRV. High IgG antibody level of suclking pigs was induced by oral infection of rotavirus, inactivated rotavirus produced higer antibody level than that of live rotavirus, and immunization of every 7 days could induce the best IgG level with the highest level at 21 day.The research successfully isolated a porcine rotavirus strain, obtained the clone of VP7 gene and re-infected it with suckling pigs for pathogenicity research, which lay the foundation for further studying the diagnosis method and pathogenic mechanism of the rotavirus. |
