Epidemiological Investigation Of Influenza A Viruses In Laboratory Swine And Dogs And Detection Of H5,H7 And H9 Subtypes Of Avian Influenza Viruses

Influenza is an acute respiratory tract infection disease which caused by influenza virus.Influenza virus can be divided into A.B.C three types.Influenza A viruses are enveloped,single stranded RNA viruses in the family Orthomyxoviridae.The other two types of influenza type B and C also occur in humans,but they are unimportant for animals and are beyond the scope of this paper.Influenza A viruses are further separated into subtypes based on the antigenic properties of the external glycoproteins haemagglutinin(HA)and neuraminidase(IMA).The HA and NA are much important for the induction of an antibody response in the host,but they are also highly variable while the "internal" proteins as the nucleoprotein(NP)and matrix(M)proteins are more steady among different influenza viruses.Sixteen antigenically different HA(H1-H16)and 9 different NA(N1-N9)have been recognized already,and they combinat to designate the subtype of the virus.Flu has been threatening the health of human beings for over a century.Pigs are also considered as prime candidates for the generation of reassortants between human and avian influenza viruses.Such reassortants may replicate much better in humans than wholly avian viruses,which are generally unable to spread in the human population.Most swine influenza viruses(SIV)are reassortants with mixtures of human,avian and swine virus genes and most frequently involved in epidemics.So to evaluate the epidemiological situation of swine influenza virus(SIV)infections in laboratory swine gives an important theoretical foundation and direction to support the prevention and control of the animal influenza.Additionally,SIV.CIV and AIV(especially the highly pathogenic avian influenza—HPAI)can spread so fast among their native hosts,and do great threat to the health of people.So concerned with the epidemiological situation of swine influenza virus(SIV)and avian influenza virus(AIV)also it is meaning of public hygiene is the first thing we should do all over the world.There have been many reports about the epidemiological investigation of influenza A viruses,such as electron microscopy,isolation and identification of viruses,agar gel immunodiffusion,hemagglutination and hemagglutination inhibition test,immunofluorescence assay,enzyme-linked immunosorbent assay of traditionally laboratory methods,and other molecular biology methods as RT-PCR,real-time RT-PCR,DNA microarray,Fluid Microbead-Based Assay and so on,but the main method is of all is serological detection in laboratory research.1.Epidemiological investigation of influenza A viruses in laboratory swine and dogs in shanghai.Influenza A viruses surveillance conducted on laboratory swine and dogs in shanghai and surrounding area from January 2010 to March 2011 based on the methods of RT-PCR and ELISA.There were 109 throat swabs and blood samples collected from swine and 35 from dogs.RT-PCR detecting percentage in laboratory swine and dogs as follows.There one out of 109 throat swabs specimen was positive,and the positive rate was 0.9%(1/109);while in the throat swabs of dogs there was no positive specimens.Then through the method of ELISA we found that there was no positive serum specimen both of.swine and dogs.But there were 2 false positive serums of swine specimens out of 109,and the false positive rate was 1.8%(2/109).According to this investigation,we known that the laboratory animals—swine and dogs in shanghai and surrounding area were breeding well and no significant flu outbreak.Compared with these two methods,we found that the way of RT-PCR was more specific,sensitive,accurate and convenient than the technique of ELISA,additionally,there was some false positive rate from ELISA.So RT-PCR technique was more suitable and reliable for detecting influenza viruses and supervising the epidemiological situation of influenza.2.Detection of H5,H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction.This study aimed to develop and optimize for a rapid,specific,sensitive,accurate and convenient method of multiplex reverse transcription-polymerase chain reaction to detection of H5,H7 and H9 subtypes of avian influenza viruses.Three sets of specific oligonucleotide primers were used in this test for type A influenza virus,H5,H7 and H9 heamagglutinin subtypes.The DNA products of RT-PCR were visualized by gelelectrophoresis and consisted of fragments of 499 bp for H5,417 bp for H7,246bp for H9 hemagglutinin subtypes.The common set primers for type A influenza virus were able to amplify a 140 bp DNA band for any of the other subtypes of AIV.The RT-PCR assay developed in this study was found to be sensitive and specific after the optimization.There was no specific amplification bands of the same sizes(499,417and 246 bp)could be amplified for RNA of other influenza hemagglutinin subtypes and also nonspecific fragment,nor specific amplification bands of type A influenza(140 bp)for other viral or bacterial pathogens.The detection limit for PCR amplified DNA products was 1ng for the subtypes H5,100Pg for H7,10 Pg for H9 and 10 pg for type A influenza virus in all subtypes.