Molecular Epidemiological, Pathological Diagnosis And Whole Genome Sequence Analysis Of Ovine Pulmonary Adenomatosis In Some Regions Of Xinjiang

Ovine pulmonary adenomatosis(OPA) is chronic,infectious lung tumor diseases caused by Jaagsiekte sheep retrovirus(JSRV).OPA is determined as B class infectious diseases by the Office International Des Epizooties(OIE),and which is one of the three types of statutory report infectious disease stipulated by the Ministry of Agriculture.Our research work carried out the research in the OPA field later, and the country has no effective method for rapid detection of OPA test,which not only restricts the epidemiology of the disease in our country, but also lags behind the sustainable development of sheep industry.Diagnosis of the disease relies on clinical symptoms and histopathological examination, but a specific diagnosis using molecular biology techniques needed for diagnosis In this study, we use conventional PCR and real-time PCR method for investigate prevalence of OPA in parts of Xinjiang and we base on veterinary clinical pathology and histopathological, and six pairs of primers were designed by RT-PCR and sequence analysis of genomic JSRV for further study of the disease strain genome sequence with domestic and foreign representative strains between homology and differences, so as to find genetic variation patterns.Which could lay the foundation for the study of biological characteristics and pathogenesis of Xinjiang sheep pulmonary adenoma virus.1、Molecular epidemiological of ovine pulmonary adenomatosis in some regions of XinjiangThe primers were designed by Applied Biosystems primer premier 5.0 according to the exogenous reference virus strain(AF105220).Some stud farm in Xinjiang six areas were investigated and then extract the lung tissue and blood DNA,and then detect the 10 breeding stock market, 2534 sheep, 11 disease sheep by conventional PCR and real-time PCR method which were from Xinjiang six regions(Changji, Manas, Shihezi, Nilka, Urumqi, Altay) in 2013 and 2014.The results showed that positive rate of endogenous blood samples of all varieties of sheep OPA were Suffolk 78.20%(538/688);Hu sheep 84.09%(407/484);Kazak sheep 78.88%(874/1108);Dorset 55.74%(68/122);Merino 85.06%(74/87);also Emil 44.44%(20/45).The positive rate of each region are:Manas 80.57%(813/1009);Changji 68.45%(243/355);Nileke80.25%(829/1033);Shihezi 85.06%(74/87);Urumqi 40.00 %(2/5);Altay 44.44%(20/45),the total positive rate was 78.25%(1981/2534). The positive rate of lung tissue lesions exogenous OPA was36.36%(4/11). As can be seen from the test results Merino highest positive rate, the experiment Merino sheep are from Shihezi, so positive rate is the highest in these areas of Shihezi.But the survey shows that the probability of the risk of pathogenic exogenous OPA is not high.2、Clinicopathological study of OPAWe observed the physiological conditions and living conditions of the herd and dissected the sick sheep so as to observed inside,especially whether the lung lesions,and the lesions of lung tissue sections were observed under optical microscope in tissue sections.Weigh 10 g from disease pulmonary tissue,and cut it into pieces and ground it,after the virus suspension was prepared we observed it by transmission electron microscopy. The results showed that early symptoms was not obvious,on disease of sheep,In the anaphase of the disease the lung cancer increases severely and damaged lungs seriously,the disease sheep showed symptoms of weight loss, listlessness, cough and runny nose liquid.When necropsy the lung, the volume increased significantly, the surface of a large number of different sizes of gray nodules, besides we can see a lot of foamy liquid oozing from the cut surface when cutting the lungs, also can seen lung and pericardium chest wall occur adhesions. Sometimes when have the " wheelbarrow test " we can see the nasal fluid flow, under the microscope we could find lung diseased sheep have different sizes of adenoma lesions and papillary hyperplasia of alveolar epithelial cells, alveolar macrophages filled lesions central karyolysis.Under the perspective glass the diameter was about 88 nm ~ 125.4nm.3、Whole genome sequence Analysis of Jaagsiekte Sheep Retrovirus in XinjiangTotal RNAs from lung of JSRV-infected sheep were extracted and reverse-transcribed by c DNA synthesis kit. Six pairs of primers(389bp、2017bp、1124bp、2541bp、2054bp and 535bp)were designed according to the exogenous reference virus strain(AF105220). RT-PCR was carried out from JSRV-infected tissue and whole genome of JSRV from was sequenced. The full-length of viral genome sequence is 7456 bp. BLAST showed nucleotide homology were 96% and 95%compared with that of the United States representative strain(AF105220) and the United Kingdom representative strain(AF357971), and meanwhile the nucleotide homology were 89.8%and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus(en JSRV) Inner Mongolia strain(DQ838493) and American strains(EF680300). The specific pathogenic " Y××M " amino acid sequence were found in the TM district similar to exogenous Jaagsiekte sheep retrovirus(JSRV). This gene have been reported as an oncogenic gene. We firstly reported the complete genome sequence of the exogenous Jaagsiekte sheep retrovirus from Xinjiang in China and submitted to the Gen Bank sequence to obtain the accession number is KP691837, which lay a foundation for studying biological characteristics and pathogenic mechanism fo Xinjiang sheep pulmonary adenomatosis virus.