Diversity Detection And Analysis Of Equine Herpesvirus In Xinjiang

Objective:We applied viral metagenomic to investigate this outbreak and identify potential pathogens involved in this equine respiratory syndrome.Our studies provided a basis for the study of molecular epidemiology of equid herpesvirus(EHV),China.Methods:(1)Nasal swabs and sero of horses with respiratory symptoms and healthy horses in some areas of Xinjiang were collected.The c DNA libraries of the nasal swab samples were prepared.Briefly,samples were grinded and pooled to extract the viral nucleic acids that were reverse transcribed and then randomly amplified;(2)All samples were screened using the specific PCR targeting Mammalian papillomavirus contigs of virome.Then,the genetic variation of EHV was described by sequence analysis;(3)According to the identified EHV-2,EHV-4 and EHV-5gB genes,a more sensitive fluorescent quantitative detection method was established;(4)EHV-2,EHV-4 and EHV-5 were serologically investigated to understand the prevalence of EHV in horses.Results:(1)The metagenomic analysis of nasal swab samples pooled collected from 10 foals with respiratory signs,and 117 clinically healthy thoroughbred horse nasal swabs pooled,generated 75 077 096 and 73664 946 150 bp-sequence reads respectively,of which 17 054(0.023%)and 16 067(0.022%),respectively,were annotated to mammalian viruses(Herpesvirus,Leukemia virus,Papillomavirus,cyclovirus and small RNA virus);(2)The metagenomics data revealed presence of sequences matching to those of EHV-4,-2 and-5 genome sequences.PCR specific primers targeting EHV-4and EHV-2 and EHV-5gB genes revealed that the positive rate of EHV-4 was 2.2%,which shared100%nt identity with published EHV-4 strains from Japan and Australia;the positive rates of EHV-2 and EHV-5 were 2.2%and 11%,respectively.The partial EHV-2gB genes of the 10positive nasal swabs revealed 92.0-100%similarity in the nucleotide.EHV-2 WLMQ-5656 and Poland EHV-2 PL?EHV2?20?? shared 99.9%high nt identity;Comparison ofgB genes from the50 EHV-5 positive samples revealed a nucleotide and amino acid identity of 98.6%-100%,indicating a close genetic relatedness.g B genes of many Chinese EHV-5 and EHV-5 EGHV5.M1,EHV-5 ETH?EHV5 had 100%nt identity;(3)SYBR Green q PCR was established for detection of EHV-2,EHV-4 and EHV-5 were 1×10~1 copies/?L,1×10~2 copies/?L and 1×10~1 copies/?L,respectively,which showed higher sensitivity than ordinary PCR;(4)The positive rate of EHV-2,EHV-4 and EHV-5 antibodies were 11.1%,5.1%,and 16.7%in horses in Urumqi,Changji and Yili,Xinjiang.Conclusion:(1)EHV-2,EHV-4 and EHV-5 were detected in Chinese horses for the first time;(2)The SYBR Green q PCR methods for detection of EHV-2,EHV-4 and EHV-5 were successfully established;(3)Serological tests showed that EHV-2,EHV-4 and EHV-5 were circulated in horses of Urumqi,Changji and Yili.