Isolation, Whole Genome Sequencing And Bioinformatics Analysis Of Bovine Papular Stomatitis

Bovine Papular Stomatitis (BPS) is a highly contagious, ubiquitous disease of bovines, which is also known as contagious ecthymao scabby mouth. It can cause bovines and human acute, contact, addicted epithelial diseases.BPS is characterized by the presence of papules and ulcers in the skin/mucosae of the lips, dental pad, palate, tongue, udder, muzzle, and, less frequently, the esophagus and forestomach. The disease is usually mild, however, when the udder is affected local pain results in interruption of lactation and decreased milk production. In BPS epidemic areas, incidence of a disease can be as high as 90% above, mortality is low, but can make cows lactation interrupt and milk production decline, affect the quality of beef and leather, still can cause secondary infection. BPS is a zoonotic disease. Due to the scarcity of genome sequence data, the structure of the genome BPSV study more scarce, therefore, through our research, we hope to be able to provide basis for BPSV research.l.Wu detected and isolated the cow BPSV by observing the pathological characteristics, collecting the tissue and making pathological section,Virus inoculating in cells, morphology of virus display ovoid by electron microscop.A three-year old Aberdeen-Angus bull presented for a two-month history of a chronic shoulder infection that at autopsy was found to be an abscess with a draining tract, and reactive lymphadenopathy of multiple superficial lymph nodes. In addition, the bull had a chronic proliferative skin condition with bilaterally swollen distal hind limbs. Limbs lesions consisted of extensive proliferative, confluent skin growths with multifocal areas of ulceration and hemorrhage. Histopathologic examination of limb lesions revealed epidermal papillary hyperplasia, hyperkeratosis and focal ulceration overlying a dermal inflammatory infiltrate. Keratinocytes exhibited ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies characteristic of poxviral infection. TEM examination of lesion homogenates showed typical ovoid parapoxviral particles. Homogenate inoculation of primary ovine (OFTu) cell cultures resulted in viral cytopathic effect upon first blind passage. DNA extracted from virus-infected cells was PCR-amplified with primers directed to BPSV BV-024 gene sequences. DNA sequencing of correctly sized amplified products showed 100% nucleotide identity with reference strain BPSV AR02 (GenBank Ace. no. AY386265).2 Whole genome sequencing of BPSV,Separation and purification of C5,C15 and Cl with plaques screening technology.To assess the genomic nature of the BPSV clinical isolate, next-generation sequencing was conducted on DNA extracted from infected cell cultures. Initial analyses indicated presence of BPSV genomic sequence highly similar to but clearly discrete from, the currently available AR-02 sequence (GenBank Ace. no. AY386265). Notably, clear heterogeneity was observed at several genomic loci of the new BPSV genome, indicating the presence of several predominant viral variants within the clinical isolate.The results of fist time gene sequencing, found that there are several high similarity of the viruses in same sample. Base on the character of special gens we employed restriction enzyme digestion and polymerase chain reaction (PCR) for screening and purification of the virus. We got virus C5, C15, Cl of Bovine Papular Stomatitis through several times screened and purified.The 17 differences open reading frame of C5,C15 and C1 were i listed in table the three virus strains (ORF006, ORF008, ORF009, ORF012, ORF020, ORF029, ORF102, ORF103, ORF104, ORF109, ORF110, ORF118, ORF119, ORF120, ORF123, ORF123, ORF124, ORF127).3.Sequencing and sequence alignmentWe sequenced the 3 stains of Bovine papular stomatitis, and fond the gene sequence open reading frame (ORF) by EMBOSS tool and Tennessee Prodigal program of oak ridge national laboratory at the university. Labeled the function and relative position of gens and table listed the clear difference proteins of the three virus stains. This data provides theory basis for research of Unknown-function genes and possible of virulent genes the viral pathogenesis.4. Animal inoculation test with C5,C15 and C1We used C5, C15 and C1 virus infected cells, collect cells at different time, and drew growth curve after tested TCID50. The results show that the C5, C15 and C1 virus showed similar growth trend. We respectively inoculated rabbit with virus C5, C15 and C1, Rabbit appeared pustules in 7 days of the inoculation,11th day pustules burst, scabby recovered after two weeks, and the corresponding virus was detected in the scabby.5.The preliminary screening C1-ORF118 interaction proteinsBait vector pGBKT7-ORF118 was constructed by C1-ORF118 fragment was amplified and ligated to pGBKT7. The transcriptional activation and toxicity were the tested. The 579 bp fragment of target gene was obtained and identified by PCR and restriction enzyme. The pGBKT7-ORF118 was transformed into Y187 and AH109 yeast competent cells respectively. No white transformant clones grew in high selective medium(SD/-TRP/-His、SD/-Trp/-Ade、SD/-Trp/-His/-Ade/-Leu) but grew in low selective medium(SD/-Trp、SD/-Trp/X-a-gal), It is suggested that the bait vector was inactive. Yeast two-hybrid cDNA library of MDBK and bait vector pGBKT7-ORF118 were applied in yeast two-bride system, and the high selective medium was used to screen.18clones were selected after several screened, the positive clones were sequenced, we got 4 positive clone:RPS20,GRPEL2,LGALS3,ovar-MHCI-F12 gene.