Detection Of Enzootic Nasal Tumour Virus In Goat And Full Sequence Analysis Of Epidemic Strains In South China

Enzootic nasal adenocarcinoma of goat(ENAG)is a contagious disease caused by enzootic nasal tumor virus 2(ENTV-2)infecting the ethmoid bone and turbinate epithelial mucosa of goats.ENAG has been reported in many areas of the world,there have been more reports in China in recent years,but not reported in Guangxi and Guangdong.In addition,there is a shortage of virus detection methods,and the whole gene sequence characteristics of local virus strains are unknown,therefore,it is of great significant for the healthy development of goat industry to carry out the investigation and related research of this disease in Guangxi and Guangdong.The following researches are carried out in this paper.1.Epidemiological investigations:During the period of 2018～2019,11250goats from 19 goat farms in Guangxi and Guangdong were investigated.ENAG examination was performed on 210 specimens collected from goats with respiratory symptoms such as runny nose.The results of common PCR showed that 10 goats were nucleic acid positive,with a positive rate of 4.8%.After clinical examination and follow-up,except for 4 slaughtered,the remaining 6 goats died of the disease within one year.Four suspected ENAG goats from goat farms in Duan county,Beihai city,Nanning city,Guangxi Province and Jieyang City,Guangdong province were selected.The disease was confirmed as ENAG by clinical symptom,necropsy,and combined with common PCR.This experiment proves that ENAG was some prevalent in Guangxi and Guangdong,China.2.A SYBR Green I RT-q PCR method for detecting and quantifying ENTV-2 was established,and the target gene of this method was gag gene.The low detection limit was3.36×10~1 copies/μL,100 times more sensitive than the traditional PCR method,there are no specific amplification and primer dimer,and the coefficient of variation within and between batches were 1.58%and 1.82%,respectively.The detection methods established in this study had not cross-react with the common pathogens of goats(Orf virus,Pestedes petits ruminant virus,Goatpox virus,Foot and mouth disease virus)and endogenous retroviruses.The positive rate of aforementioned 210 clinical samples detected by this method and conventional PCR method were 6.2%,4.8%,respectively,indicating that the detection rate of this method was higher.3.By piecewise cloning,sequencing and splicing of the full gene of 4 ENTV-2 strains obtained from Guangxi and Guangdong,their complete gene sequences were obtained,which were named ENTV-2-DA0,BH,MC,and JY strains,respectively.The genome length was 7325 bp,7319 bp,7319 bp,and 7325 bp,respectively.The nucleotide similarity between the full gene sequences of 4 ENTV-2 strains was 96.4～99.2%.These four obtained strains had the highest homology with ENTV-2 FJ strain(96.2～98.9%),and the lowest homology with ENTV-2 UK strain(89.4～89.7%).The differences of viral gene sequences were mainly in U3and U5 regions of LTR gene,VR1 and VR2 regions of gag gene,orf-x region of pol gene and TM protein coding region of env gene.Conclusion:This study confirmed the existence of ENAG in Guangxi,Guangdong,China.A SYBR Green I RT-q PCR method for detecting ENTV-2 with gag as the target gene was established,and complete gene sequences of 4representative ENTV-2 strains were obtained.In the future,a wider range of ENAG survey should be carried out to improve the detection technology,screening and diagnosis of diseased goat,so as to provide technical support for healthy goat farming.