Study On The Biological Characteristics Of Subgroup B Avian Leukosis Virus

ALV (avian leukosis, AL) refers to an avian cancer caused by retrovirus virus belonging to the family of Retroviridae, Alpharetrovirus genus. Certain avian hematopoietic cell proliferation are the main characteristics, commonly in poultry broiler lymphoblastic leukemia and myeloid leukemia. This disease causes emaciation undergrowth maldevelopment and evokes nonspecific resistance decline and immune suppression. Coupled with the fact that vaccines or medicines against avian leucosis virus have not been successful up to now, in recent years, some poultry raising developed countries took some measures to purify poultry through eliminating sick chicken and decreasing vertical transmission. In some parts of our country, there are some reports about the outbreak of ALV and the trend of popularity. In this study, we obtained one strain virus from the liver of osteopetrosis egg breeder from Hebei Baoding farm and using Polymerase Chain Reaction (PCR), biological characteristics and PCR / RFLP analysis identified as the B sub-Avian Leukosis virus. In addition, the preliminary research of the effect of the isolated strain on Newcastle disease antibody levels was carried out.The material was collected from osteopetrosis egg adult breeder in asepsis, then the liver and spleen were grinded and diluted 3 times with sterile saline water. After centrifuging at 12 000 rmp for 5 min, the supernatant obtained was inoculated in 11 day-old well-developed SPF chick embryo chorioallantoic membrane. For cytopathic effect did not form in chorioallantoic membrane when avian leukemia virus was inoculated, then the ALV was harvested after inoculating for 7 days and blind passaged for 3 generations. According to the P27 gene sequence of avian leulemia virus published in GenBank, one pair of specific primer of P27 were designed and synthesized, after chick embryo virus DNA extraction and PCR amplification, a 746 bp fragment was detected.In this study, the EID50 of ALV strain was determined by PCR, physicochemical characteristics also be determined by this method. The results showed that: the strain of the virus solution were completely inactivated at pH 3 and 60℃, and the EID50 difference were greater than 2 at pH 12 after 1 hr, indicating the strain are highly sensitive to acid below the pH4.5, heat and alkali; as well as trypsin-sensitive. After treated by chloroform, the EID50 difference was more than 2, indicating that the virus has no envelope. These results are in accordance with ALV biological properties.The gp85 district of ALV's envelope gene is different between various sub-groups, but highly conservative among different strains of the same sub-group. Therefore it can be determined by comparing the gp85 of their respective subsets. On A, B, E subsets gp85 region restriction site polymorphism analysis, A sub-group has SspⅠ, BglⅡrestriction site; B subgroup has BamHⅠrestriction site; E subsets has SspⅠrestriction site. In this study, by using ALV subgroup J-specific primers designed by Smith for pol gene of the 3 'end and the gp85 gene and amplified by PCR, no fragment was amplified which proved that this isolated strain does not belong to the subgroup J avian leukosis virus. Then reference primers were designed for the virus according to those designed by Liu Gongping using theconservative sequence area of the genetic gp85 and amplified the purpose of the 1.2kb fragment.When digestedbyBglⅡ, BamHⅠ, this virus can only be opened by BamHⅠrestriction endonuclease digestion , indicating that the isolated strain was subgroup B avian leukemia virus.Through amplifing the env gene of common chick embryo -phore endogenous avian leukemia virus and by SspⅠ, BglⅡdigestion , homology contrast and phylogenetic tree analysis, this experiment proved that the PCR/RFLP technique can be used to identify ALV sub-group.The isolated virus solution was injected subcutaneously into 1 day old chicks before Newcastle Disease immunity. Experiments showed that Newcastle disease antibody levels are significantly lower in the virus injected group than the control group, indicating that the strain could produce immunosuppressive effect to chickens.