| Enzootic nasal adenocarcinoma(ENA)is caused by sheep endemic intranasal tumor virus(ENTV-1)and Enzootic nasal tumor virus of goats(ENTV-2),it is a chronic,progressive,and contact infectious animal disease.ENTV-1 only infects sheep and ENTV-2 only infects goats.ENTV-2 mainly causes irreversible canceration of the ethmoidal epithelial cells of the nasal passage of goats.The disease has shown a worldwide distribution;China first reported ENA cases in Inner Mongolia at 1984,and it is now widely distributed in China.Since July 2018,suspected goat endemic intranasal tumors have appeared in several goat farms in Chongqing.Ⅰ.Clinical diagnosis and molecular diagnosisObserved the clinical symptoms of the ill goats at a farm in Chongqing.The severely ill goats were thin and had hair loss outside the nasal cavity,secreted some serous nasal fluid in the nasal cavity,but the diseased goats had no significant changes in body temperature.After the diseased goat was put to death,the corpse of the diseased goat was subjected to a necropsy.No obvious pathological changes were observed in the viscera of the sick sheep.The most obvious lesions are concentrated in the nasal cavity,there are a large number of pink or gray hyperplasia tissue.In this study,we collected a total of 27 clinical samples,including 15 goats have shown clinical symptoms,while 12 goats have no clinical symptoms.RNA was extracted and reverse transcribed by Trizol from 5 disordered sheep tissues showing clinical symptoms.Most of the samples were successfully amplified by PCR,which were found to be ENTV-2 gene fragments after sequencing and BLAST.Then we designed primers and successfully amplified the whole genome sequence of ENTV-2 in Chongqing.Ⅱ.Genetic evolution analysisThe genomic sequence of ENTV-2 and the representative strains of ENTV-1 and JSRV were analyzed using the adjacency method using Mega 5.1 software under the condition of Bootstrap confidence value of 500.It can be seen that ENTV-2 and ENTV-1 are obviously different from JSRV.All ENTV-2 strains are clustered on one large branch,while ENTV-1 and JSRV are closely clustered on another branch.In addition,ENTV-2 strains show a high degree of genetic diversity.At the same time,ENTV-2 evolved independently of the two viruses which called ENTV-1 and JSRV.The analysis results also indicate that the phylogeny of ENTV-2 may be related to geographic aggregation.Ⅲ.Establishment of fluorescence quantitative detection methodBy designing specific detection primers and constructing standard plasmids,a real-time quantitative PCR assay using Eva Green as a fluorescent dye was established,and compared with conventional PCR and SYBR Green I in sensitivity and repeatability.The measurement coefficient of Eva Green real-time q PCR has a good linear relationship,where R2=0.996 and slope=-3.327.The amplification efficiency of Eva Green method is 99.8%,and the result is more accurate than that of SYBR Green I q PCR.This method can detect virus particles as low as 3.0×101 copies,which is 10 times more sensitive than conventional PCR.This study was the first time that ENTV-2 was discovered in Chongqing,and the entire genome of the ENTV-2 CQ strain with a total length of 7279 bp was obtained.The phylogenetic tree was obtained by the adjacency method and the results showed that the strain CQ1 formed a separate branch,which was closely related to the evolution of Shaanxi strain and Sichuan strain.The evolutionary relationship of the strains is relatively close,indicating that the phylogeny of ENTV-2 may be related to geographic aggregation.And successfully established a real-time fluorescent quantitative PCR detection method using Eva Green.ENTV-2 is already widely available worldwide,except in Australia and New Zealand.Although the prevalence rate is not high,the post-morbidity mortality rate is close to 100%.Because ENTV-2 has the ability to evade the host immune system,there are no reports of detecting ENTV-2 antibodies;nor can ENTV-2 be isolated and cultured in vitro.In China,there are few ENTV-2 epidemic reports,lack of genetic information and lag of detection methods,which have a negative impact on the prevention and control of ENTV-2 epidemic.The fluorescence quantitative method established in this experiment can be used for early detection of the disease.The above research will also provide important reference for the epidemiology,early diagnosis and disease prevention and control of ENTV-2. |
