Molecular Detection And Genomic Characteristics Of Bovine Coronavirus In Dariy Calves And Development Of An ELISA Kit For Detecting Bovine Coronavirus Antibodies

Bovine Coronavirus(BCo V)is the causative agent of diarrhea in newborn calves,winter dysentery in adult cattle,and respiratory tract illnesses in cattle across the world,inflicts in severe economic losses on the global cattle industry.Seroepidemiological data showed a high prevalence of BCo V infection with widely geographical distribution in cattle in China,but the etiological epidemiological data is still limited.The aim of this study was to investigate molecular prevalence and genomic characteristics of BCo V in dairy calves in some provinces in China,as well as to develop an Antibody Detection Kit for BCo V seroepidemiological investigation.The results obtained as follows:1.The molecular prevalence of BCo V in dairy calves in 6 provincesA total of 190 fecal samples from dairy calves with diarrhea were collected at 14 farms from 6 provinces in China during September 2017 and May 2018 for detecting BCo V by specific RT-PCR.The complete S,HE,N and M genes were cloned from representative clinical samples for analysis of molecular characteristics.The results showed that the BCo V detection rate was 18.95%(36/190)and the positive farms reached up to 92.86%(13/14),indicating that BCo V had been widely circulating in cattle in sampling provinces.Complete S,HE,N and M genes were successfully cloned from 13 positive samples from 8 farms in 4 different provinces.Compared with all 163 full-length BCo V S genes available in the Gen Bank database,12/13 S genes cloned in the present study together with 13 other BCo V S genes from China clustered on an independent large branch in phylogenetic tree,and they shared an identical aa variant(N1192Y)in the S2 subunit.The remaining S gene clustered with three North American BCo V S genes on a small independent branch of the tree.Compared with all 115 full-length BCo V HE genes available in the Gen Bank database,10/13 HE genes cloned in the present study together with 2 Chinese BCo V HE genes clustered into an independent large branch in phylogenetic tree,and they shared an identical aa variant(F181V)in the R2-loop.It is note worthy that 10 /13 strains in this study were identified as HE recombinant strains,and these strains had experienced the same recombination event and carried the same recombination sites located between the esterase and lectin domain,which may affect the receptor binding capacity of HE.Moreover,the 10 recombinant strains were recovered from 8 farms in 4 provinces.Thus,these HE recombinant strains have been circulating widely in dairy cattle in China.To our knowledge,this is the first description of HE recombinant event in BCo V in dairy cattle.This is also the first molecular epidemiological study of BCo V in China.The findings will enhance understanding of genetic evolution of BCo V,and contribute to the diagnosis and control of diarrhea in dairy calves.2.Genomic characteristics of the BCo V SWUN/A10/2018 strainIn order to further study the molecular characteristics of BCo V HE recombinant strain,44 pairs of primers were designed according to the BCo Vs gene sequences published in Gen Bank for amplify the SWUN/A10/2018 genomic sequences.The Sequences were assembled using Seq Mansoftware.The result showed that the complete genome of SWUN/A10/2018 is 31028 bp in length,with a 37.0% G+C content.The isolate had the closest genetic relationship with AKS-01 which is the sole chinese BCo V genomic sequences in Gen Bank currently.They share 99.0% nt identity and 98.3% aa identity,respectively.Compared with all 176 full-length BCo V S genes available in the Gen Bank database,including 13 complete S genes cloned in this study,this strain has an unique aa variant(G411A)in the S1.This strain occurred recombination in the esterase and lectin domain of HE,moreover,this strain had 12 nt insertion which result in 4aa insertion in R3-loop of receptor binding domain in HE.The 3D model was established based on the hemagglutinin esterase crystal structure of BCo V HE showed that the R domain of SWUN/A10/2018 significantly different from that of other known BCo V strains.As BCo V HE protein is involved in receptor recognition and induction production of neutralizing antibodies,this unique aa change of HE protein in strain SWUN/A10/2018 may affect its function.To the best of our knowledge,this is the first report of aa insertion in HE gene in BCo V,a finding that augments current understanding about the evolution of BCo V.3.Development of BCo V Antibody Detection ELISA kit based on recombinant N proteinThe codon preference of N gene of BCo V strains in E.coli was optimized.ORF sequence of N gene with Bam H1 and Xho1 enzymatic digestion sites at both ends were synthesized,the expression plasmid of p ET28 H was constructed and transformed into Rosetta(DE3)for the expression of N protein.The results showed that BCo V N gene was expressed in DE3 high level.Western blot analysis showed that the recombination N protein has a good reactiongenicity.Using purified N protein as coating antigen,and optimizing reaction system and conditions,an indirect ELISA assay for detecting BCo V antibody was successfully established.The assay has a good specificity and stability as well as sensitivity.The ELISA kit assembled in this study shared 100% coincidence rate with a commercial kit(Svanova company,Sweden).Out of 571 yak serum samples from Northwest of Sichuan province,98.1% samples were detected as BCo V positive by this assay,showing that BCo V infection of yak in this region was very common,which contributed to the diagnosis and control of diarrhea in yak.