Genome Sequencing And Comparative Genomics Analysis Of Actinobacillus Pleuropneumoniae And Classical Swine Fever Virus

Porcine contagious pleuropneumonia(PCR),caused by Actinobacillus pleuropneumoniae(APP),is a severe contagious respiratory tract disease of pigs.Swine fever(SF,syn.Classical swine fever),caused by classical swine fever virus(CSFV),is also a highly contagious and often fatal disease of domestic pigs and wild boars.These diseases have caused considerable world wide economic losses in the swine industry.Huge amount of genome sequences have been obtained under the help of high-flux sequencing technology.The genomic comparisons and analysis provide an original insight to explore the genetic variation of microorganism.Based on our bioinformatics approaches,genome information of the above pathogens were comprehensively studied, which shows the relationship between the genomic structure and the mechanism of pathogenesis/metabolism.The main research was described as follows:1.Genome sequencing and analysis of APPAt present,APP has divided into at least 15 serotypes,differing in their surface lipopolysaccharide and capsule polysaccharide.As these serotypic characterizations,it is difficult for us to develop safe and effective vaccine and easy means of diagnosis.Moreover,a better understanding about the molecular mechanism of metabolism and pathogenesis of APP always has broad interesting in the field of veterinary as well as clinical medicine.Lack of comprehensive genomic information has become the major obstacle for tackling the scientific and technical problems in the study of this bacterium and related disease.With these considerations,we believe that complete genomic sequencing of serotype 3 of APP prevalent in China can provide reference sequence for international genome project.It also offers theoretical support for prevention and cure of this disease.In our study,we have sequenced the complete genome of A.pleuropneumoniae strain JL03,an isolate of serotype 3 prevalent in China.The detailed analysis and functional annotation for this organism was firstly performed.Its genome is a single circular chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences,six ribosomal rRNA operons,and 63 tRNA genes(GenBank accession number CP000687).On the other hand,the genome of strain L20 of serotype 5b sequenced by Canada is approximately 2.27 Mbps,containing 2012 CDSs,and the incomplete genome of strain 4074 of serotype 1 is about 2.07 Mbps,consisting of 140 contigs encoding 2132 putative CDSs.Among the three serotypic strains,4074 and L20 were known as highly virulent strains but JL03 was a low virulent strain.Comparative genomics analysis using the available sequences of related bacteria detailedly described the characteristics of the mechanisms of pathogenesis and serotypic diversity of A.pleuropneumoniae.We identified a full spectrum of genes invovled in metabolic pathways and confirmed APP could generate energy via fermentation and respiration(aerobic and anaerobic).A complete metabolic network with various kinds of oxidation-reduction enzymes for catabolism and anabolism was comprehensively illustrated at the genomic level for this genus.Furthermore,several strain-specific genomic islands related to assimilatory sulfate reduction,biosynthesis of Flp fimbrial, capsular polysaccharide and lipopolysaccharide O-antigen were identified in JL03. Notably,strain L20 possesses a strain-specific genomic island of 37.7kb encoding eight phage-related proteins,which is absent in JL03.Discovery of such genomic islands provides a foundation for future research into the mechanisms of serotypic diversity of APP.We also described a complete set of genes related to the virulence of the bacterium, of which,some are either missing genetically or impaired by varies kinds of mutations in JL03.These data reconfirmed the knowledge of virulent determinants of APP,and also provided a foundation to explain the discrepancy of virulence between different serotypic strains.In short,preliminary analysis of the genomic sequence and the functions of the encoded proteins not only confirms the present physiological and pathological knowledge but also offers new insights into the metabolic and virulence characteristics of this important pathogen.2.Genome sequencing and analysis of CSFVCSFV containing only one serotype is a member of the genus Pestivirus within the family Flaviviridae.Here,we have sequenced the full genome of a novel CSFV strain, SWH/CA/2004,isolated from a hog pen in Henan Province,central China.It contains 12296 nucleotides(nt) in length.It is composed of a 373-nt 5' terminal non-translated region(NTR),a 11697-nt open reading frame(ORF) encoding a polyprotein of 3898 amino acids,and a 226-nt 3'-NTR.Genome comparison of the SWH/CA/2004 isolate (GenBank Accession:DQ127910) with other known CSFV isolates was performed and analyzed.Corresponding segments from SWH/CA/2004 and other reported strains shared 80.4%-99.8%identity at the nucleotide level and 89.5%-99.8%identity at the amino acid level.From an evolutionary point of view,isolate SWH/CA/2004 is closely related to the highly virulent isolate cF114/CA/2001,with a pairwise distance of 0.013;and distantly related to the moderately virulent isolate GXWZ02/CA/2003,with pairwise distance 0.170.These analyses constitute a comprehensive study of the phylogenetics of CSF based on distinct regions of the genome and may provide the reference resource for future molecular epidemiology research and genetic variations of CSFV strains.3.Inhibition of expression of RNA polymerase in viruses of the the family FlaviviridaeThe RNA-dependent RNA polymerase(RdRp) in viruses of the family Flaviviridae plays an important role in the viral replication process and in the forming of a replicase complex.In this study,we used small interfering RNAs(siRNAs) corresponding to the highly conservative MotifV of RdRp gene of three viruses to examine their role in modulating the expression of RdRp. Evaluation of the expression of RdRps was performed by the fluorescence,flow cytometry,Western blotting,and real-time PCR.The plasmid-based CSFV siRNA is quite effective in silencing target-GFP fusion gene expression in PK-15 cells,which may provide favorable prevention and immunity of CSFV infection in swine.