Studies On Isolation, Identification, Genetic Evolution Analysis And Rapid Detection Of H1Subtype Of Avian Influenza Virus

Avian influenza virus (AIV) is a kind of infectious pathogen widely exists and transmits in nature, characterized as various subtypes and high gene mutation and with waterfowls as main hosts. Among subtypes, although the H1subtype of AIV belongs to the low pathogenic influenza A virus, it still causes huge economic losses and damage public health. More and more reports suggest that H1subtype of AIV not only spreads in poultry, but also has an ability to cross species barrier to infect some kind of mammalians (such as swine, equine, even human). In order to understand the ecological trend of H1subtype of AIV in Guangxi region, analyze the genetic evolution characteristics and examine the relationship and difference of each gene of H1subtype of influenza virus between from swine and from human, meanwhile, provide a quick and effective molecular biological detection method for the first-line clinical and laboratories, this study is to perform the isolation, identification, genetic evolution analysis and establishment of clinical rapid detection method of H1subtype of AIV In this study, the preliminary screening was carried on cotton swab samples collected from live poultry markets from2011to2012by serological test and then the candidate virus samples was isolated and purified, further confirmed by the H1subtype AIV of polymerase chain reaction (PCR) and determination of the HA gene sequences. During these two years, six strains of AIV H1subtype have been isolated and purified from different hosts (avian, ducks and birds), and these six isolated Guangxi strains are classified into the low pathogenic avian influenza virus by biological characteristic identification.Complete genomic sequences of eight gene segments of six strains of H1subtype of AIV were determined and compared to, the sequences in GenBank. The comparative analysis showed six strains of H1subtype of AIV only contain one basic amino acid in the splice site of HA1and HA2, comparable to the typical sequence signatures of the low pathogenic AIV. These six Guangxi isolated strains of H1subtype of AIV belong to H1N2subtype. SIX strains of isolates comparison with homology in the eight gene segments of maximum strain and H1subtype influenza strains of poultry, pigs and human nucleotide and amino acid homology, respectively moreover, the genetic relationship analysis of the evolutionary tree of each genome show that eight gene segments of the same virus strain likely come from the genome rearrangement and combination between or among different subtypes of AIV and their relationship source with H1subtype influenza virus genetic of pigs and human further.Based on HA gene conserved region of H1subtype of AIV, four rapid molecular detection methods were established:1) Duel-temperature PCR, with high sensitivity and specificity, which detects pathogens more simply and rapidly and reduces the reaction time by merger of annealing temperature and extension temperature, compared with conventional PCR;2) Nested PCR, significantly increasing product specificity through two rounds of specific amplification,100times sensitive than conventional PCR by the sensitivity comparative analysis;3) Real Time Fluorescent Quantitative PCR, with better specificity and higher amplification efficiency,1000times sensitive than conventional PCR, without gel electrophoresis, real-time monitoring of amplification, saving time and effort, only requiring0.5h to complete the entire process;4) Multiple PCR, including duplex PCR of H1and H3subtype of AIV and Multiple PCR of H1, H3subtype of AIV and M gene, capable to simultaneously detect H1, H3subtypes of AIV, and sixteen kinds of subtypes of AIV-M gene, hasving a good specificity and sensitivity, shortening clinical detection time of samples of mixed infection.Finally, the comparison analysis was performed among these five rapid detection methods established in this study, and also the sensitivities were compared with ordinary PCR and applied on clinical samples. Results showed that among these detection methods, the sensitivity of the real-time fluorescence quantitative PCR is highest, followed by nested RT-PCR, ordinary PCR, duel-temperature PCR, multiple PCR is lowest. Clinical examination can choose suitable detection methods, according to need and conditions, or combine various detection methods for better identification and diagnosis.