Molecular Mechanism Of Bovine Viral Diarrhea Virus Envelope Protein E2 Interact With Bovine Trophoblast Cells

Bovine viral diarrhea virus (BVDV) is an important cattle pathogen and widespread all over the world and cause diarrhea, abortion, respiratory passage disease or immun-function defilade. It generates significant economic losses. In our country, BVDV infections usually cause no or only mild clinical symptoms so as to leading less reconstruction. The emphasis and difficulty of preventing this diease is persistant infection(PI) which is caused by transplacental infection. Diaplacental infection of foetuses with a ncp BVDV strain during first trimester of gestation may result in the birth of immunotolerant PI calves which shed the virus through their lifetime. Therefore, they are the main viral reservoir and source for viral transmission. Binucleate trophoblast giant cells(TGC) is the vinculum between parent and fetus and also the target cell for BVDV vertical transmission. At present, there is less molecular mechanism about BVDV infecting TGC. It will be an useful work to find cellular receptors that interacted with BVDV envelope protein E2 and to definite the role of receptors during infection. This work also provide newly consept to breeding for disease resistance. So our investigation is focus on:1. Epidemiology investigation and genotype distribution of BVDV in Xinjiang. Based on the sequence comparison of the highly conserved 5'-UTR among different genotypes and subgroups of BVDV strains, current primers were designed.274 dejecta samples which were collected from 14 cattle farms in Xinjiang were detected by RT-PCR assay and sequencing.18.61%(51/274) samples were positive for BVDV with RT-PCR.21.34%(51/239) were positive in north area but negative in south area. 23 matched-pairs specimens were detected. The cow-positive rate was 39.13%(9/23) and the calf was 30.43%(97/23).35 cattles showed clinical symptoms, the positive rate was 45.71%(16/35). The positive rate of the remaining normal cattle was 17.16% (35/204). Data shows BVDV vertical transmission was more seriously in this region. Alignment of the 5'-UTR sequences of the isolates showed 2 different sequences. In addition, Phylogenetic analysis revealed that all viruses were found in group BVDV-I b. No BVDV typeâ…¡virus was discovered. These results suggest that the BVDV isolated reflect that the virus predominant genotype is BVDV-I b in Shihezi. And by the biological-type identification, the isolates were non-cytopathic type. Information obtained from this study would also be useful when carrying out epidemiological surveys of domestic BVDV and vaccine application in Chinese cattle population. A BVDV strain originated from Shihezi area in Xinjiang autonomous district, namely BVDV shzl32, was isolated and identified by immunological method, electron microscope observation, physico-chemical property analysis, PCR detection and homology comparison. 2. To establish a convenient method of purification and culture bovine trophoblast cells and BVDV shz132 infected cells. Healthy placental tissues were obtained and digested, then the cells were cultured, purified. The results shown livability was above 90% and typical two nucleus were stained blue. The isolated TGC were positive for cytokeratin and negative for vimentin throughout the duration of the experiment. The purified cells contained 95% TGC and unpurified contained only 45%-50%. The cells could passage four generations and survived for sixty days. Inoculated TGC with BVDV shzl32 for 12h to 60h and detected virions copies by Real-time RT-PCR according to BVDV 5'-UTR. The results shown that virions copies obtained from the latter was obviously higher than the former. That is to say BVDV can generate in TGC.3. The cDNA library of TGC infected with BVDV shzl32 strain was constructed. The target protein were screened and verified. Here we found six cellular proteins interacted with BVDV shz132 E2 protein by yeast two-hybrid:Bos taurus tryptophanyl-tRNA synthetase,similar to WARS protein,actin and beta,p21 protein pyruvate kinase and clathrin. As a results, clathrin heavy-chain linker act as a adapter protein between clathrin and ligand-receptor compound and then entry cells by endocytosis in clathrin-coated vesicles. It is clearly that clathrin-mediated endocytosis is a route for BVDV infecting cells. Then clathrin was verified by immunoprecipitation.The above results show that positive ratio of BVDV infection is highly exist in Xinjiang with seriously vertical transmission. All BVDV isolates in this study have been divided into two genotypes, BVDV1 and BVDV2. One of these was isolated and identified as ncp BVDV which named BVDV shz132. TGC was successfully cultured and identified. The cDNA library of TGC infected with BVDV shz132 strain was successfully constructed. We found six proteins that interacted with BVDV envelope protein E2 and verificated clathrin by immunoprecipitation. These findings were useful to demonstrate molecular mechanism of persistent infection. It is also significent to screen specific candidate drugs and give a upstream work for breeding of antivirus livestock.