Identification Of Orf Virus CL17 Strain And Molecular Characterization Of Partial Virulence-related Genes

Contagious ecthyma,also known as contagious pustular dermatitis or sore mouth,is an high contagious disease caused by Orf virus.It is usually infects goats,sheep,humans and characterized by the formation of papule,nodules,vesicles,ulcers or scabs in the oral mucosa,lips,gums,nostrils,and tails of animals.The outbreak of contagious ecthyma will not only bring serious economic losses to the farms,but also threaten the healthcare of the farmers.Therefore,contagious ecthyma,as zoonotic infectious disease,the detriment of it to human being and animal can never be exaggerated.On November 2017,a sheep farm in Changling County,Jilin Province suffered from typical symptoms of oral sores.A total of 120 small-tailed Han sheep were raised in the farm,and more than 10 of them infrcted,mainly lambs around 2 months old.In the early stages,red spots appeared on the cutis around the lips and nostrils,and slowly develop into pustules.Eventually,the pustules ruptured to form gray-brown scabs,followed by enlargement of the lesions to form a large area of hard scab.In severe cases,blisters and pustules appeared in the oral mucosa.When a large number of blisters and pustules appear around the mouth,it seriously affects their feeding,leading to diseased sheep to lose weight and stunting.In order to clarify the cause of aphthous symptom in this flock,this experiment first systematically analyzed from pathological and etiology diagnosis aspects.1 Pathological diagnosisRoutine pathological necropsy was performed on the sheep sent to pathology laboratory,and found that a large number of gray-brown,hard-edged,and carinated scabs around the lips and nostrils.The mesenteric lymph nodes were swollen and other organs were no obvious pathological changes.And then,the cutis of scabs and other tissues were collected,which were used for preparation of the paraffin sections.The observation results of microscopy are as follows: The stratum spinosum of the epidermis was thickened,the granular degeneration occured in the spinous cells,and some of the severe cases were vacuolar degeneration.The tissue surrounding the hairfollicle showed hemorrhage,and some hair follicles were necrosis.The spleen medulla(red pulp)was scattered with a large amount of hemosiderin particles.Many lymphocytes infiltrated around the lymphatic sheath of the spleen corpuscle,and the lymphatic sheath was thickened.A large number of lymphocytes infiltrated in the paracortical area of lymph node.2 Etiology diagnosisThe bacteriological examination of the diseased sheep was carried out by Gram staining and bacterial culture,and no pathogenic bacteria infection was found.Thereby eliminating the possibility of bacterial infection.For the identification of the disease,specific primers for ORFV,FMDV,PPRV and CaPV were designed and synthesized,and PCR or RT-PCR amplification was performed.The amplification results show that only the primers targeting ORFV can amplify bands that match the expected target fragment.Remove lip scab and grind.Repeatedly frozen and thawed,then centrifuged.Twenty percent double-antibody was add to the supernatant for overnight,and OFTu was inoculated for virus isolation.Typical CPE appeared after 4generations of blind transmission.The cytopathic solution was taken for transmission electron micrograph,and the corpuscle-like and elliptical virions were observed.The cytopathic fluid was inoculated to rabbits at 4 months of age,and typical pustular lesions appeared on the 8th day after inoculation.Through the above experiments,we successfully isolated one ORFV strain,named ORFV-CL17.From the pathological point of view,the case was caused by ORFV infection.In order to further understand the molecular characteristics of the isolated strain,virulence-related genes ORF120,ORF122,ORF125,ORF132 are selected for nucleotide sequencing,and deduced the amino acid sequence by nucleotide sequence.Then analyze the amino acid position mutation,nucleotide sequence and amino acid sequence homology and genetic evolution relationship of the corresponding sequence.There is a mutation site in the amino acid sequence of ORF122 gene of ORFV-CL17 strain.The 102 th amino acid is mutated from A to T(A102T).There are two mutation sites in the amino acid sequence of ORF132 gene,and the 19 th amino acid is mutated from N to D(N19D),amino acid 69 is mutated from G to R(G69R).The ORF120,ORF125,and ORF132 genes of ORFV-CL17 strain have the highest amino acid homology with Orf virus strain SY17(accession number: MG712417),respectively,99.5%,100%,98.5%.ORF122 gene and Orfvirus strain NP(KP010355)has the highest amino acid homology of the corresponding gene,which is 99.7%.The results of genetic evolution analysis indicated that the ORFV-CL17 strain was closely related to the domestic strain Orf virus strain SY17,and it was in the same evolutionary branch,and it was far away from foreign strains.This study provides an important technical means for the diagnosis of infectious pustulosis in sheep.At the same time,the research on the molecular genetic characteristics of the isolated strains of ORFV-CL17 is of great significance for the diagnosis,treatment and prevention of the disease.