Detection Of Sheep Mouth Sore Virus In Some Large-scale Farms In Southern Xinjiang And Isolation And Identification Of Sheep Mouth Sore Epidemic Strains

ORF is a common bloodthirsty and highly contagious infectious disease in sheep and goats caused by sheep mouth sore virus,also known as infectious pustular dermatitis,infectious pustular stomatitis,contact infectious pustular dermatitis.Clinically,the formation of red papules,blisters,pustules,crusts,etc.on the lips,nose,tongue,udder and other parts of sheep and goats is the main symptom.The high incidence and rapid transmission of the disease,and the lack of safe and effective vaccines,make the prevention and control of the disease more difficult.In this study,in order to study the infection of sheep mouth ulcer virus infection in some large-scale farms in southern Xinjiang,scabs and blood samples of sheep with aphthous lesions were collected from some large-scale farms in Aksu,Bazhou and Kashgar in southern Xinjiang,and diagnosed by PCR detection.A positive sample was randomly selected from large-scale farms in each region,and ORFV 059 gene was cloned and sequenced to study the genetic variation of the epidemic strain of sheep’s mouth sovirus in some large-scale farms in southern Xinjiang.The positive samples randomly selected by each farm were inoculated into sheep testicular cells for 3 generations of blind transmission for virus isolation and identification.The main research contents and results are as follows:1.Detection of sheep’s mouth sovirus in some large-scale farms in southern XinjiangFrom March 2022 to March 2023,the clinical symptoms of sheep with oral ulcer lesions were observed from some large-scale farms in Aksu Prefecture,Bazhou Prefecture and Kashgar Prefecture in southern Xinjiang,and the scabs and blood samples of sick sheep were collected,and the collected samples were detected and confirmed by PCR method.The results showed that the incidence of sheep collected from large-scale farms in Aksu area was 16%(16/100),the incidence of sheep collected from large-scale farms in Bazhou was 6%(9/129),and the incidence of sheep collected from large-scale farms in Kashgar area was 2%(10/500).It showed that the virus was widespread in farms in three regions,and the prevention and control of aphthous virus should be strengthened.2.Cloning sequencing and bioinformatics analysis of the epidemic strain ORFV 059 geneIn order to understand the genetic variation of the epidemic strain of sheep’s mouth strains in some large-scale farms in southern Xinjiang,the positive samples randomly selected from large-scale farms in each region were cloned and sequenced,and the epidemic strains were named as Aksu Prefecture(ORFV059 AK),Bazhou Prefecture(ORFV 059 BZ),and Kashgar Prefecture(ORFV 059 KS).Log on to the NCBI website and screen 25 ORFV 059 gene sequences from the Gen Bank database as reference strains.The nucleotide sequence homology analysis of the nucleotide sequences of the epidemic strain and 25 reference strains was performed by MEGA 7.0 software.The results showed that the epidemic strain(ORFV 059 KS)in Kashgar area was 96.4% homologous with the epidemic strain(ORFV 059 AK)in Aksu area.The prevalence of the circulating strain(ORFV 059 KS)in Kashgar area was 99.9% with that of the circulating strain(ORFV 059 BZ)in Bazhou area.The prevalence of the circulating strain(ORFV 059 AK)in Aksu region was 97.7% with that of the circulating strain(ORFV 059 BZ)in Bazhou region.The nucleotide homology of the three epidemic strains and the reference strains was 94.3～99.9%.The results of phylogenetic tree analysis showed that the endemic strain(ORFV 059 KS)and the reference strain in Jilin(JQ271535.1)in Kashgar area were clustered into one developmental branch,the endemic strain(ORFV059 AK)in Aksu region and the reference strain in Guangdong(JQ271535.1),the reference strain in Guangdong(KC569751.1)and the endemic strain in Kashgar area(ORFV 059 KS)were clustered into one developmental branch,and the endemic strain in Bazhou region(ORFV 059).BZ)is most closely related to the nucleotide relatives of the reference strain(JX142183.1)in Gansu.The nucleotide sequence comparison analysis of the nucleotide sequences of the three epidemic strains was performed using Meg Align software,and the results showed that a total of 26 mutations occurred.The nucleotide sequences of the three epidemic strains were translated into amino acid sequences by Edit Seq software in DNA Star,and the amino acid sequences were compared and analyzed by Meg Align software,and the results showed that the genome length of ORFV 059 was 1 023 bp,the GC content was 64.35%,and 341 amino acids were encoded.A total of 20 mutations occurred.3.Isolation and identification of epidemic strains of sheep’s mouth sore virus Three positive samples randomly selected from farms in each region were pretreated and inoculated into blind passage of sheep testicular cells for 3 passages.The results showed that after inoculation of the virus,the cells appeared dragnet,agglomeration,widening of the intercellular space,and then the refractive properties of the cells were enhanced,and finally a large number of cell shedding and other cytopathies appeared.The cytopathies that appear are consistent with those of the sheep mouth virus;The collected viral fluid was DNA extracted,and PCR was amplified with primers of ORFV 059 gene,and the target band with a size of about 1 023 bp was visible,indicating that three strains of oral ulcer were successfully isolated in this experiment.Conclusion: Oral sore virus is widespread in farms in Aksu,Bazhou and Kashgar in southern Xinjiang.The epidemic strains randomly selected by large-scale farms in each region were named ORFV 059 AK,ORFV 059 BZ,ORFV 059 KS,and there were some genetic variations in the epidemic strains in each regional farm.The successful isolation and identification of three strains of sheep mouth sore virus laid a foundation for further research on sheep mouth sore virus.