| Enterovirus(EV),a member of Enterovirus genus within the family of Picornaviridae,is the etiological agents of human and animal diseases characterized by respiratory,digestive and neurological disorders.In 2014,the first Caprine enterovirus strain(CEV-JL14)in the world was isolated from goat herds with severe diarrhea in our laboratory.Characterization of this virus showed that CEV-JL14 belongs to a novel enterovirus species different from those EV-E,EV-F and EV-G species,which currently is listed as reference strains of EV-G20 by the International Committee on Taxonomy of Viruses(ICTV).As an emerging infection,Caprine/Ovine enterovirus(CEV/OEV)infection in our country was revealed in goat/sheep herds in many regions causing a serious economic loss to the goat/sheep industry.Although certain progress has been made toward the detection and diagnosis to CEV/OEV,it remains largely unknown as such research areas on the epidemiology and the genetic variation among the virus strains in different areas.In this study,we carried out the epidemiological investigation on the enterovirus infection among sheep/goat herds in different areas using the sandwich ELISA established previously in our group,isolated the virus strains,unveiled the complete genome sequence,and revealed the genetic variation for these identified strains.The results are presented as follows:1.Epidemiological investigation on Caprine/Ovine enterovirus infection in different areas in ChinaA sandwich ELISA method was used to examine the enterovirus infection in goat/sheep herds characterized by diarrhea in Ningxia,Jilin,Shandong,Henan province and Inner Mongolia autonomous region.Out of the total 772 goat/sheep feces samples detected,varied samples were revealed as positive including 55/175 in Jilin,70/201 in Inner Mongolia,96/182 in Shandong,and 55/214 samples in Ningxia province.The enterovirus infection rate ranged from 25.7% to 52.7%.These results revealed the wide enterovirus infection in our country,and provided the epidemiological data for future prevention and control of this infection.2.Isolation and characterization of the novel virus strains and the unveiling of complete genome sequenceVero cells were used to isolate the potential virus from some ELISA-positive fecal samples.Six strain of Caprine enteroviruses and one strain of Ovine enterovirus were isolated and characterized.They were named JL-LS34,JL-LS127,JL-LS165,JI-LS174,NMG-F37,SD-S68 and NX-DR26,respectively.NX-DR26 was the first isolated Ovine enterovirus in China.Characterization of these newly isolated virus strains demonstrated they shared the similar biological and physiochemical characteristics with CEV-JL14 virus.Moreover,all strains were cross-reacted with CEV-JL14 antibody,suggesting they are enterovirus.Additionally,SD-S68 was identified as the first Ovine enterovirus strain in China.The complete genome of the 7 virus strains were amplified,sequenced,and analyzed.Results showed that the length of complete genome sequence varied with different strains.While the ORFs of all newly identified strains were relatively conserved,the lengths of noncoding regions were relatively diverse,especially for3'UTR region with a range of 107 bp to 137 bp.3.Genetic variation for Caprine/Ovine enterovirus strainsGenetic variations of the isolates were analyzed by bioinformatics method.The results showed that the identity of the complete sequence and 5'UTR region of these newly isolated strains to CEV-JL14 were 79.2% to 87.8% and 89.0% to 95.1%,respectively.Phylogenetic analysis using the full-length genome,ORF,and 3C+3CD genes showed that these isolated enterovirus strains clustered in the clade different from EV-E,EV-F and EV-G species,indicating that they should be classified into a novel enterovirus species.Analysis of the divergence and p-distance of these caprine/ovine strains with porcine enterovirus(PEV)showed that the p-distance between Caprine/Ovine enterovirus and PEV in P1 and 3CD protein was 0.29 to 0.34 and 0.1 to 0.12,respectively,which were in line with different EV classification standards of ICTV(0.19 to 0.59 and 0.1 to 0.48).It is confirmed that Caprine/Ovine enterovirus strains belong to a novel enterovirus species different from EV-E,EV-F and EV-G.Further investigation on the genetic variations among these caprine/ovine enterovirus strains was performed.Surprisingly,four genotypes of caprine/ovine enterovirus were revealed by a standard approach and the VP1 genotyping method.We named these four genotypes as EV-X1,EV-X2,EV-X3,and EV-X4 respectively.EV-X1 contains JL-LS34,JL-LS127 and JL-LS165,while EV-X2 consisted of CEV-J14 and JL-LS174 strain;EV-X3 included NX-DR26 and SD-S68,and EV-X4 contained NMG-F37.Furthermore,analysis of VP1 gene showed mutation in VP1 including base substitutions,deletions,and point mutations.Some point mutations led to the change of amino acid sequence.These results suggested the diversity and the genetic variation existed among different Caprine/Ovine enterovirus strains.Recombination analysis revealed the potential recombination event in the nonstructural protein region(4500 bp to 7200 bp)in caprine enterovirus strain JL-LS174.In summary,we have revealed the epidemiological background on Caprine/Ovine enterovirus infection and shown a varied CEV-OEV infection in different regions in our country.We also have isolated seven novel caprine/ovine enterovirus and decoded their complete genome sequences.Alignment analysis uncovered the genetic variations for these newly isolated Caprine/Ovine enteroviruses.Phylogenetic analysis clustered the caprine/ovine enteroviruses into a clade different from EV-E,EV-F and EV-G species,indicating they should be classified as a new species.Furthermore,four genotypes were divided for Caprine/Ovine enteroviruses.These results provided the basis for further study and prevention and control of enterovirus infection in sheep/goats. |
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