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Studies on Genome of Avian Infectious Bronchitis Isolates in China

Author: LiuYuFen
Tutor: LiuBaoQuan
School: Northeast Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Infectious Bronchitis Virus Genome analysis Phylogenetic tree constructed
CLC: S852.65
Type: PhD thesis
Year: 2003
Downloads: 312
Quote: 2
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Abstract


Avian infectious bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious respiratory diseases caused by avian infectious bronchitis virus (Infectious Bronchitis Virus, IBV). Clinical diagnostic features for chickens coughing, sneezing, tracheal rales; decline in young chickens runny nose, laying hens in egg production and egg quality. The age, breed and sex chickens are susceptible. Occur in our IB throughout most of the north, south, northwest, southwest and northeast provinces, municipalities and autonomous regions, has been widely used the IB vaccine, but the occurrence of IB is still widespread, especially in the immune flocks outbreak of IB and often cause serious economic losses to the aquaculture industry. Representative species of coronavirus IBV belonging to the coronavirus family Coronaviridae (coronavirus). Its genome is a single strand of linear positive-stranded RNA, infectious, about the size of 27.6kb, with a 3 'poly A tail and 5' cap-like structure (cap). Six subgenomic mRNAs (1-6) can be detected in infected cells has been identified as the 2,4,6, respectively, mRNA encoding the IBV three main structural proteins - spike protein (Spike S), membrane proteins ( Membrane, M) The nucleocapsid protein (Nucleocapsid.N); mRNA1 contains two open reading frames (Open Reading Frame, ORF) that ORF1a and ORF1b, encoding 440KD and 300KD protein. However, in normal circumstances, Preparation ORF1b throughput ribosomal frameshift (Frameshifting) is formed upstream of ORF1a a molecular weight of approximately 750KD polyglutamine protein (the polyprotein) pplab, then the intracellular protease cleavage of the corresponding functional protein. In this study, gene has been isolated and identified three of IBV strains LH2/01/10 (Heilongjiang renal-type strains), D971 (Dalian glandular stomach strain) and LX4 (Xinjiang kidney strain). First at the age of 10 SPF chicken proliferation of the virus, when the virus allantoic fluid hemagglutination price stability in more than 256, purification and concentration of the virus, and then by reverse transcription - polymerase chain reaction (Reverse Transcription-Polymerase Chain Reaction, RT -PCR) technique to amplify the target gene. This study first major structural gene of the three strains were cloned and sequenced. When designing primers 5a, 5b and N gene, 3a, 3b, 3c, and M expansion. Construct the structural gene N, M, S, and 3C (E) and non-structural genes 3a, 3b, 5a and 5b of the phylogenetic tree. The sequence determination showed that three of the N gene by 1230bp composition, the non-nucleotide deletion and insertion, but the presence of point mutations, encoding 409 amino acid (AA), and domestic isolates homology are high, and in some gene region with H120 and H52 homology of up to 100%. N gene phylogenetic tree display LH2/01/10, LX4 in the same group, while the D971 but with foreign reference strains, indicating that the three different regions experience different evolution, leading to the emergence of different genetic characteristics. M gene of different lengths, LH2/01/10 687bp (228 AA), D971 (226 AA), 681bp the LX4 678bp (225 AA), M gene conservative higher degree, tree indicates that the three kinship and domestic strains in recent, relatively distant and foreign strains and vaccine strains. S protein is the the IBV most important immunogen, especially S1 part can stimulate the body to produce protective antibody S1 gene in the genome variation of the highest frequency. S gene sequence analysis showed that the S gene of LH2/01/10 LX4 3495bp, encoding 1164 AA, the cutting of the S protein recognition sites for five consecutive basic amino acid residue histidine - arginine - fine acid - arginine - arginine (His-Arg-Arg-Arg-Arg, HRRRR), IBV isolates that are unique. Both S1 gene is 1614bp, encoding 539 AA; D971S 3498bp, encoding 1165 AA, cutting recognition sites for NSRRR, the sequence for the selected reference strains most unique one. D971S1 1614bp, encoding 538 AA S2 to 1884bp, encoding 627 AA. S1 gene analysis showed that different countries IBV strains significant difference, LH2/01/10 LX4 within the same group, with H120 and H52 vaccine strain is relatively close, and with foreign Northeast Agricultural University, a doctorate in agriculture papers mutant D1466 and DEO72 group only 36% homology. With foreign reference strains are similar, proving once again that the unique evolutionary history of the D971, D971 unique. E gene is another structural gene of IBV Recent studies found the LHZ/0l/1 OE gene was 336bp, 0971 as 309bp, Lx4, 330bp, all these three performance at the 3 'end of the frequent variation, consistent with the reference strains. Phylogenetic analysis showed LHZIO 1/10 with LX4 same large group, and D971 are far from most of the reference strain, evolution results are consistent with the sl gene non-structural genes 3a, 3b, 5a and 5b of the sequence results show that, the three 5a and 5b, respectively, the same number of genes the sa are 198bp, encoding 65 AA, SB 249bp, encoding 82 ^ ^. The 3a gene both are the same, 174bp, encoding 57 ^. LHzlo / lo3b for x89bP encoding 62 AA; o9713b the 195 bp, encoding 64 AA; LX43b 192bp, encoding 63 AA LH power of 60 1/10 and LX4 lack of D. The 3a gene analysis showed that the LX4 evolution of a unique, single row on a branch, D971 and similar mutants GA/98 abroad, LHZ / 0 1/10 and BJ pro-source relations in the past. 3b gene phylogenetic tree sl and 3c. 5a and 5b evolutionary relationships, domestic reference strains in the same group. The LX4 mRNA 15 'end of the unique region sequence determination of the strain is the first time complete genome sequencing IBV local separation strains LX4 mRNAI composed of 19940bp, contains two ORF that is, ORFla and ORFlb. Which the LX4 ORFla to 11,916 by 3971 AA.LX40RFlb coding 7950bp, encoding 2649 AA. In between LX40RFla and ORFlb of 74 bp interval the Beaudette strain slippage sequence bamboo TAAAC,, to ensure polymerization protein pPlab the formation. LX4 false knot \In summary, this study analyzed the three strains of the structural gene N, M, sl and E gene variation characteristics, but also on the non-structural genes 3a, 3b, 3c, 5a and sb gene were the domestic first building analysis . The LX4 determination of the gene sequence is the first isolates Engineering BV place?

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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