| Rotaviruses pose a great threat globally due to their pathogenicity.There are ten different groups(A-J)with vast spread among all species on the earth planet.They cause appalling cases of diarrhea and gastroenteritis in mammals and birds,which worth deep consideration about how to navigate such situation.There are a huge number of studies in China and other countries reporting that bovine rotavirus(BRV)is the cause of high frequency rate of diarrheal cases in bovine species,either alone or in combination with other viruses or pathogens causing diarrhea.Notwithstanding,there are still many scientific gaps in BRV research,which related to the exact prevalence,and genotypes causing the disease and their pathogenesis.Our research aimed to elucidate the prevalence of BRV associated calf diarrhea in China,isolate and identify some of circulating strains,and investigate the viral pathogenesis related mi RNA response in infected cells and its virulence in mice.The main findings are summarized as following:1.Detection,isolation,and identification of BRV in diarrheal calves.The fecal samples of 1–24-week-old calves(number of samples= 427)experienced serious diarrhea,were collected from different Chinese beef and dairy farms in various geographical districts,within the period(2016-2021).By mean of RT-PCR(conserved region of VP6 gene of bovine rotavirus),155 positive samples of bovine rotavirus group A out of 427 were detected with prevalence 36.3%.The prevalence in Northeast,North,Central,Southwest,Northwest and East of China was 58.3%,40.7%,37.4%,32.8%,36.8%,20 %,respectively.Our trials for complete genome characterization and genotyping of bovine rotaviruses directly from the clinical samples either by PCR or even by next generation sequencing,were failed,therefore,we isolated the virus by propagating it in MA-104 cell line.Briefly,the filtered fecal supernatant was experienced several blind passages(up to ten)until occurrence of the typical cytopathic effect(CPE)of rotavirus,meanwhile,the isolation of the virus in the culture fluid was confirmed after each passage by RT-PCR.The harvested cell free and cell associated virus were confirmed as bovine rotavirus(BRV)by both RT-PCR based on conserved region of VP6 gene and SDS-RNAPAGE.The results of the nucleotide sequence analysis of VP6 gene segment proved the successful isolation of bovine rotavirus group A(RVA),whereas the migration pattern of the rotavirus RNA segments by RNA-PAGE showed isolation of both of group A(RVA)and group B bovine rotaviruses(RVB).What is more,we adopted ESI-LC-MS/MS for whole protein sequencing,the proteins of the virus isolates were sequenced,demonstrating presence of two RVA strains and one RVB strain: RVA/Cow-tc/ CHN/35333/2019/G6P[5]was mixed with oneRVB strain(RVB/Cow-tc/CHN/35334/2019/G5P[3])in two samples of the culture fluid,and RVA/Cow-tc/CHN/10927/2019/G8P[7] was found in one sample of the culture fluid.They are of genotype constellations(G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3),(G8-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1),and(G5-P[3]-I3-R5-C5-A5-N4-H5).Besides,the alignment and phylogenetic analysis of the obtained sequences demonstrated that our strains are identical to some of the previously reported strains,but different from others.G6 and P[5] genotypes had the same protein sequences with those of bovine rotavirus Brazilian strains(100% amino acid identity),and G8 and P[7] genotypes were identical to bovine rotavirus South Korean strains(100% amino acid identity).Meanwhile,group B genotypes were identical to those of bovine rotavirus Indian strains.RVA genotype G6,P[5],G8 and P[7] are the first report in China,furthermore,it is the first report to isolate RVB in MA-104 cells.On the other hand,we isolated other three bovine rotavirus group A from other three fecal samples(one sample from Zhejiang and two samples from Hebei,these three fecal samples experienced four blind passages in the MA-104 cell line until appearance of the typical CPE of rotavirus.The three isolates were confirmed by both of RT-PCR and RNA-PAGE.Therefore,these findings would be useful to get a deep insight to the prevalence and circulating strains of BRV in China.2.Identification of mi RNA response associated with BRV pathogenesis in MA-104 cell line.To elucidate the pathogenesis of BRV would help develop more effective measures to control BRV infection.So far,the role of mi RNAs of MA-104 cells during BRV infection didn’t be reported.Therefore,we performed Illumina RNA sequence analysis of BRV G8P[7] isolate-infected and mock-infected MA-104 cells,at different time points post infection,including 0h,6 h,12 h,24 h,36 h and 48 hours post infection.The total clean reads 74701041 and 74184124 were obtained from BRV-infected and non-infected cells,respectively.From these findings,579 were categorized as known mi RNAs and 144 as novel mi RNAs,160 differentially expressed(DE)mi RNAs in BRV G8P[7]-infected in comparison to non-infected MA-104 cells,were successfully investigated,95 upregulated,while 65 downregulated,further,the target m RNAs of DE mi RNAs were expected by bioinformatics.The functional annotation of the target genes by GO and KEGG suggested that they were mainly contributed to biological pathways,endocytosis,apoptotic process,trans-Golgi membrane and lysosome,the signaling pathways of m TOR(mml-mi R-486-3p and mml-mi R-197-3p),NF-κB(mml-mi R-204-3p and novel＿366),Rap 1(mml-mi R-127-3p),c AMP(mml-mi R-106b-3p),MAPK(mml-mi R-342-5p),T cell receptor(mml-mi R-369-5p),RIG-I-like receptor(mml-mi R-504-5p),AMPK(mml-mi R-365-1-5p),and PI3KAkt(mml-mi R-299-3p)were enriched.What is more,real-time q PCR verified the expression pictures of 23 selected DE mi RNAs which were consistent with the results of the deep sequencing,and 28 from their corresponding target m RNAs mainly of regulatory pathways of the cellular machinery and immune importance.Finally,the impact of mmlmi R-99a-5p on BRV replication was verified,it upregulated the virus replication via increase the expression of VP6 gene,during the first six hours of infection.These results are the first report which uncover the impact of BRV infection on mi RNA expression of MA-104 cells along with the impact of the mi RNA on the viral replication,and this will offer clues for identifying potential target candidates for development of antiviral or vaccine strategies.3.The pathogenicity of the BRV isolate in challenged mice.10-day-old mouse was used for assessment the pathogenicity of BRV G8P[7] isolate.In the first 6 days post infection,no obvious diarrheal signs or any other clinical signs were observed in both inoculated and non-inoculated control mice.However,at day 7 post inoculation,the faeces of 10 out of 12 inoculated mice were softer than the non-inoculated group,and the virus was detected in that faeces by real-time absolute q PCR.Furthermore,some histopathological changes in the architecture of the small intestine parts(Duodenum,jejunum,Ileum)and liver,including atrophy and necrosis,were observed in inoculated mice in comparison with the non-inoculated control mice.These findings proved that the isolate has pathogenic effect on mice.In conclusion,the findings determined the prevalence of BRV related diarrhea in Chinese calves and isolated two RVA strains(RVA G6P[5],RVA G8P[7])and one RVB strain(RVB G5P[3]),besides,other three bovine rotavirus group A were identified.What is more,the pathogenesis related mi RNAs response to RVA G8P[7] infected MA-104 cells,was identified along with its pathogenicity in the laboratory animal model(mice).It would be valuable to develop novel measures to control BRV associated diarrhea. |
