Isolation And Identification, Complete Genome CDNA Library Construction, And Molecular Characteristics Of Porcine Reovirus SC-A Strain

The mammalian Orthoreoviruse, which named Reovirus directly, are members of theOrthoreovirus genus. Reovirus has general pathogenicity in human and the mammalian, suchas pig, cattle, dog, horse, cat, mouse et al, and induces respiratory or gastrointestinal illnessesof host. The correct relationship has not final conclusion, which of between reovirusinfection andhuman disease. However, reovirus infection had been reported, which inducedhuman fatal interstitial pneumonia, by Tillotson et al. in 1967. Moreover, the studies ofSARS pathogen indicated the outbreak of SARS had relationship with reovirus coinfection.Three serotypes of porcine reovirus have been identified, which generally reside in pig herdand have mild pathogenicity for pigs. Besides a transient rise in temperature and spreading inhealthy pig herd, the reovirus could been recovered from the faeces of pigs, whichexperimentally infected with a human strain of serotype 1 reovirus. All of above hint that weshould cautiously handle reovirus, especially should research the relationship of the reovirus,animal and human disease, thoroughly.In this thesis, we had isolated and identified a strain porcine reovirus from the diarrheafaeces of pigs successfully at first time, and the virus was named as porcine reovirus SC-Astrain (PReoV SC-A). The results of the studies indicated the best working concentration oftrypsin in the viral culture medium was 5μg/mL. The propagation of PReoV SC-A was stablein Vero cells by serial passage. The cytopathic effect characteristics of PReoV SC-A in Vetocells showed the particle increasing in cells, cellular swelling, and fall off from the cultureflask. Intracytoplasm virions showed typical lattice arrangement, and to form different sizeamorphous viral inclusion body by surround the nucleus. The viral inclusion body consistedof mature virions, which contained dense-core, incomplete assembly virions, whichcontained hollow-core, and few of parallel disposed microtubule-like sturcture. The virionswere nonenveloped spheroidal particle, with many of radiation disposed capsomer in particlesurface.The same as other mammalian reovirus, PReoV SC-A could propagate in many culturecells too, such as Vero cells, ST cells, MDBK cells, BHK21 cells et al. The Vero cells wasthe first selection for isolation or propagation of reovirus. The results of the culture cellsadaptability trial indicated ST cells was the best adapted to propagate PReoV SC-A thanMDBK and BHK21, excepted Vero cells. The results of morphogenesis frail indicatedoriginal transcription, translation and assembly of the PReoV SC-A generated within 4 hoursafter inoculation. And the early viral inclusion body appeared in. 4 to 6 hours after inoculation.After inoculation for 8 to 10 hours, a few virions were observed out of the cells, whichreleased possibly by cell disruption. Moreover, the apoptotic early characteristics orintermediate stage were observed in infectious cells too. Those characteristics includedshrinkage and pyknosis of nucleus, chromatin concentration and margination, and the intact cell profile. The profuse glycogenosome were accumulated in cytoplasm tbr within 2 to 8hours after inoculation. That phenomenon was presumed to viral transcription and replicationoffer necessary energy.At present, there was only a little of the research reports about porcine reovirus. As forthe genome information of porcine reovirus had nothing to be reported. So, it was necessaryto construct porcine reovirus complete genome cDNA library, for revealing the molecularcharacteristics of porcine reovirus genome. According to the characteristics of agarose gelelectrophoresis and terminal conserved sequence of the reovirus genome, this thesis has beenestablished a method of constructing PReoV SC-A complete genome cDNA library at fisttime. The viral genome dsRNA, which recovered by agarose gel electrophoresis, or thecomposite RNA, which contained general cell RNA, were used as template for RT-PCR inthe method. And the specificity primers or random primers were selected to single orcombine together use in reverse transcription. With the method, the porcine reoviruscomplete genome cDNA library was constructed successfully, which contained 10recombinant plasmids. The sequencing results indicated PReoV SC-A complete genome was23.539Kb, and the genome segments size of L1 to L3, M1 to M3 and S1 to S4 were 3854bp,3915bp, 3901bp, 2304bp, 2203bp, 2241bp, 1416bp, 1311, 1198 bp, 1196 bp, respectively.And the Genbank accession No. of L1 to L3, M1 to M3 and S1 to S4 genome segments wereDQ997719, DQ885990, DQ403254, DQ396804, DQ482462, DQ403254, DQ396805,DQ411553, DQ396806, DQ911244, respectively.Genome sequence analysis indicated all of the genome segments of PReoV SC-A wasindependent each other in molecular evolution, and was conservative relatively excepted S1genome segments, which determines reovirus serotype. The difference of the S1 genomesegments of PReoV SC-A with typical strains, which of T1L, T2J and T3D, were veryobviously, and the deduced amino acid sequence similarity of PReoV SC-A with T1L, T2Jand T3D were 21.1%, 22.4% and 91.2%, respectively. The phylogenetic tree based ondeduced amino acid of S1 genome segment supported the same serotype viral strains locatedin the same evolution branch. According to the phylogenetic tree, the serotype of PReoVSC-A was identified as serotype 3. Besides possessed identical 5' and 3' terminal conservedsequence, i.e. 5'-GCTA……TCATC-3', all of genome segments of PReoV SC-A possesseda pair of reverse repeated sequence by the terminal conserved sequence. PReoV SC-Adisplayed obviously codon preference only on codon of Leu, Arg, Ser and terminal codons,and disused TAG as terminal codon always. The eukaryotic expression system, especiallythe yeast expression system was the better suitable than prokaryotic expression system forPReoV SC-A gene expression in vitro.The predicted results of deduced protein indicated all of PReoV SC-A proteins werehydrophilicity proteins, without transbilayer helix structures and signal peptide sequence.And exceptedσ1S, which belonged to all-alpha helix protein, all of others PReoV SC-Aproteins belonged to mixed protein. The alpha helix structure were capital ingredient in thesecondary structure of non-structural protein, and higher than structural proteins obviously.Moreover, besides all proteins of PReoV SC-A had conserved protein family domains byprediction, many of possibly possesses functional domains or motifs were predicted in someproteins, such as cAMP and cGMP dependent protein kinase phosphorylation, leucine zippermotifs inσ1, C2H2 zinc-finger motif inσ3 andλ1, et al.