| Porcine rotavirus(PRV) is a member of Reovirus family, rotavirus genus, it is an important pathogen which can cause viral diarrhea. The disease mainly infects young piglets with severe diarrhea syndrome. High death rate and high morbidity occurs in cases, accompanied with widely spread in the world, which distributed serious damage to livestock industry.With various serum type and serum group, different biology characteristics were exhibited in different PRV strains, it is important to isolate and obtained the epidemic strain for further research not only on biological characteristics, but disease epidemiology and laboratory diagnosis.Some feces was obtained from He Bei Provice, and identified by RT-PCR using PRV VP6nested-primer, amplificated gene sequences was obtained with a period of about 275 bp. Homology of the VP6 gene position from the isolated strain is 93% compared with the published in the GenBank. Moreover, TGEV and PEDV was shown as negative when amplifing the feces sample.All those showing that the diarrhea piglets PR virus existed in feces. The sample was desposed with centrifugal filtration and inoculated on the IEC-2 cell, MA-104 cell and Marc-145 cell respectively. All the three could produce CPE, and with the passage gose on, CPE exhibits more and more promince, CPE appear more and more advanced, showing the titer of isolated PRV was positive relative with passage grows. To prove the existence of PRV, electronmicroscopy ultramicrotomy, negative staining electron microscopy and immuneoelectron microscopy experiment were done to find the virus has no envelope and is about 70 ~ 75 nm in diameter.Moreover, electronmicroscopy ultramicrotomy at different period of virus infected with MA-104 cell showed that 60h’s optimal propagating. Indirect immunofluorescence assay was conducted using the selected virus propagated on MA-104 cell, the results showed that the specificity reaction was displayed in cytoplasm. Otherwise, the same result was presented in the neutralization assay.In order to purify the virus, It was mixed with CH3 Cl for continuous 5 passages in order to eliminate the envelope virus like TGEV and PEDV for preliminary purification. Plague formation assay was then conducted to select the PRV nucleic acid positive..The purified virus partial physicochemical characteristics were measured, it is concluded that the virus was sensitive to heat. When the virus deal with the temperature of 65℃, it can be completely inactivated. The virus is not resistant to acid and base, in PH3 and PH9 virus titer conditions significantly reduced, but not completely inactivated. The virus is insensitive to the organic reagents such as ether chloroform, showing that the purified virus was no envelopedviruses, according with the characteristic of this kind of virus can resistant to organic solvents.Animal experiment showed that the piglet infected with the isolated virus became vomiting,diarrhea and weight loss, piglet died on the 5th day. The autopsy showed significant association with expansion, full of yellow liquid, intestinal wall thinning. Detection of feces and small intestine by RT-nested-PCR was showed the positive result.Purification of the successful isolating of porcine rotavirus, biological characteristics, the disease epidemiology and laboratory diagnosis and virus and porcine rotavirus vaccine preparation and laid the material foundation. |
