Isolation And Identification Of Bovine Respiratory Syncytial Virus DQ Strain And Establishment Of Real-time RT-PCR Method

Bovine respiratory syncytial virus(BRSV)is an important causative agent of acute respiratory syndrome in calves and feedlot cattle,causing substantial economic losses to the cattle industry worldwide.In 2018,an outbreak of an acute respiratory disease occurred among 4 to10-month-old calves on many cattle farms in Heilongjiang Province,with a 27.42%morbidity rate(329/1200)and a>25%mortality rate(85/329).Using next-generation sequencing,we comprehensively analyzed microbial diversity in the lung samples of the diseased cattle and found that the causative agent of this epidemic outbreak is mainly a bovine orthopneumovirus named BRSV strain DQ,which was stably subcultured by cell isolation and culture and the isolated BRSV DQ strain was identified by RT-PCR and indirect immunofluorescence assay;Through the phylogenetic analysis of BRSV DQ strain,it was determined that the isolated strain belonged to subgroup ? of BRSV,This is the first report of subgroup ? strain of BRSV presence in China;A universal real-time RT-PCR method for BRSV was established,and the epidemiological investigation of BRSV was carried out based on the established method.The specific contents of this study are as follows:The first part is the isolation and identification of BRSV DQ strain.The microbial diversity in lung tissues of diseased cattle was analyzed by next-generation sequencing.The results showed that the abundance of bovine orthopneumonia virus was 27.23%,which was significantly higher than other microorganisms.Therefore,it is speculated that the epidemic of acute respiratory infectious diseases in calves is mainly caused by BRSV,and the virus is named BRSV DQ strain.The primers were designed with reference to the BRSV gene sequence published in Gen Bank,and the RT-PCR method was used to identify the diseased materials,and the results showed that the samples were BRSV positive;the diseased materials were ground with liquid nitrogen and re-suspended in DMEM,and after centrifugation and filtration,the filtrate was inoculated into MDBK cells for subculture in vitro.The results showed that the infected cells produced stable CPE,and P5-P11 cell cultures were detected by RT-PCR method,and the results showed that BRSV was positive.Using the prepared mouse anti-BRSV N protein polyantibody,the BRSV propagated by MDBK cells was detected by indirect immunofluorescence method.The results showed that MDBK cells in the virus inoculation group had specific gr een fluorescence,while there was no fluorescence in the control group of MDBK cells.The results showed that the prepared polyclonal antibody could specifically recognize BRSV;The results of ultrathin sections of cells showed that the virion was spherical,the diameter was about 100-150 nm,and there were spikes on the virus surface.Based on the results,BRSV DQ strain was successfully identified and isolated in this study.The second part is the phylogenetic analysis of BRSV DQ strains.The phylogenetic analysis of the complete genome sequence and gene sequence of BRSV DQ strain was analyzed by MEGA7.0:BRSV typing was mainly based on its G gene sequence,and the phylogenetic analysis based on G gene showed that BRSV DQ strain had the closest genetic relationship with strains USII/S1,15489,V41 and NY487834 belonging to subgroup III of BRSV.In addition,the phylogenetic analysis of genes Complete,F,SH,M,M2-1,M2-2,P,L,N,NS1 and NS2 of BRSV strain DQ showed that this strain shares the highest genetic similar ity with strains USII/S1.Results confirmed that the BRSV DQ strain isolated in this study belonged to subgroup III of BRSV.This is the first report of subgroup III strain of BRSV presence in China.The third part is the establishment and application of BRSV universal real-time RT-PCR detection method.Using the N gene of BRSV as the target gene,primers were designed for the common conserved region of N gene of subgroup I-VII BRSV,and a universal real-time RT-PCR detection method for BRSV was established.The specificity of this method is strong,and the detection limit of this method is high,the lowest detection limit is 10~1 copies/?L,which is significantly higher than that of conventional RT-PCR method.The method was used to detect 68bovine nasal swabs and lung samples collected from Heilongjiang province.the results showed that the positive rate of BRSV was 27.94%.The establishment of a universal real-time RT-PCR detection method for BRSV provides technical support for the epidemiological investig ation of BRSV.