Isolation And Identification Of Porcine Rotavirus G4P[23] Strain And Establishment Of Cell Line Stably Expressing T7 RNA Polymerase

Porcine rotavirus(PRV)is the most important pathogen that can cause severe diarrhea newborn piglets with symptoms including diarrhea,vomiting,anorexia,and dehydration.It is characterized by high infectivity,long incubation period,highly morbidity and wide spreading range.PRV is known to frequently occur to co-infect with other porcine diarrhea-associated viruses such as Transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and Escherichia coli,which can results in significant economic loss to pig industry.In order to identify the causative pathogen of the infection,seven diarrhea fecal samples were collected from pigs suspected to be infected with RVA from a pig farm.The RNAs were extracted and subjected to RT-PCR by utilising specific Rotavirus VP7 primers.Specific band was amplified from one sample and in agreement with the expected size.Then we inoculated the positive fecal samples onto the MA104 cells.The positive samples were inoculated into MA104 cells.The samples experienced ten blind passages.Comparing with the non-inoculated cell sheet,the inoculated cells gradually displayed CPE of partial cell rounding with clustering,from the eighth to the tenth passage.The isolated virus strain was named RVA-He NNY-01.To elucidate the molecular characteristics of its genetic composition at the whole-genome level,the genome of this strain was fully sequenced and characterized by using the software Rota C2.0,MEGA 7.0,DNAstar.This strain was genotyped as G4-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1,which belongs to rotavirus group A.This strain was found to possess the R1-C1-M1-A8-N1-T1-E1-H1 genetic backbone which closed to porcine rotavirus strains Gottfried.The NSP2 genes of the isolated strain showed the highest nt sequence identities(96.54%)to sequences of human rotavirus strains,the VP1,VP2,VP7,and NSP5 genes of the isolated strain showed the nt sequence identities(>94%,respectively)to sequences of human rotavirus strains.Therefore,we hypothesized that this strain might be generated from human and porcine rotavirus reassortment.T7 RNA polymerase(T7 pol)is a promoter specific RNA polymerase that can strictly and specifically recognize T7 promoter,which was widely used in RNA virus rescue.In order to construct a cell line stably expressing T7 RNA polymerase and lay a foundation for virus reverse genetics,the gene sequence of T7 RNA polymerase was amplified from BL21 cells and cloned into lentiviral vector p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro to construct recombinant plasmid.Then the BHK21 cells were infected with recombinant lentivirus.The cell line stably expressing T7 RNA polymerase was obtained under the selection pressure of puromycin.In conclusion,a G4P[23] porcine rotavirus have been identified and isolated in MA104 cell line and the cell line stably expressing T7 RNA polymerase was obtained,which provided a theoretical basis for rotavirus related research and vaccine development,and laid foundation for rotavirus reverse genetics.