| In recent years, the avian flu virus (Avian Influenza, AIV) has lead to seriouslyworldwide havoc, H9N2subtype AIV is one of the most widely distributed Asian subtypecurrently.In the clinical manifestations,although the low pathogenic avian influenza viruseshave low mortality, when infected with other bacteria, viruses, microbes,they easily lead tothe occurrence of epidemic disease and a high morbidity and mortality afterwards. Shi Huoying, who has found the reason that the avian influenza virus subtype H9N2becamepandemic in China's mainland in1998is that the virus has got the characteristics of aerosoltransmission. However,the studies of aerosol transmission and pathogenicity is insignificantin the NA gene level. using reverse genetics technology to construct the recombinant virusand then using air-borne transmission experimental models? HE staining andimmunohistochemical techniques?we will study the difference about air-borne transmissionand pathogenicity of H9N2AIV in this experiment.1? The rescue of the R01/NASS and R01/NASS-381recombinant strainThe Pol?-Pol?system transcription and expression plasmids of SD01eight genesplasmid and SS94NA gene plasmid were established by gene clone method using primerswith BsmBI/AarI/BsaI restriction enzyme site and reverse genetic bidirectional transcription/expression vector pHW2000that Webster donated in this study.The313?381?368and370amino acids of SS94NA gene were mutated by site-directed mutagenesis by PCR andthen we can get the NA gene that contains K313E,D381G, E368D, L370S mutation sitesby sequencing.The transcription/expression plasmids of SD01seven genes and wererecombined and transfected with SS94NA gene(pHWSS6-NA)and SS94NA gene which hasalready site-directed mutation (pHWSS6-NA381),then they interacted with293T cells torescue the virus R01/NASS and R01/NASS-381in the plate. It proofs obtain the recombinantvirus by sequencing contrast. The two recombinant strains were inoculated9~11day-old SPFchicken embryos to enhance the ability to replication and is calculated the EID50with theReed-Muench method to determine its virulence.The EID50of R01/NASS strains was8.17±0.20, the R01/NASS-381strains was9.43±0.40, the virulence of R01/NASS-381was moreserious than R01/NASS strains, but the difference was not significant.2?Studies of air transmission of the R01/NASS and R01/NASS-381recombinant strain virusIn the positive and negative pressure SPF isolators,304-week-old SPF chickens healthyexperimental animals were randomly divided into equal amounts inoculate group?direct contact group and aerosol contact group to determined the transmission of R01/NASS andR01/NASS-381recombinant strains of virus. direct contact group is put in after inoculategroup inoculated after24h, every day we collect oropharyngeal cloacal swab samples until14days from the second day. Cotton swabs were inoculated9-day-old SPF chicken embryos totest cotton swabs AIV virus content by the HA/HI test; In4?6?8?10?14day, AGI-30collected air samples in the isolator,at the same time,9-day-old SPF chicken embryos wereinoculated with air samples to determinate the airborne viruses; we also took blood samples in7day,14day,21day, then the virus titer was measured after treatment by HA/HI test.Theresults showed that aerosol contact group chickens oropharyngeal cloacal swabs and airdetected the AIV, blood samples also detected virus titer in the transmission experiment thatwere inoculated with R01/NASS-381, virus was detected after inoculation six days, theeight days to reach the peak and then reduced gradually.However, the spread of the virusstrains R01/NASS vaccination trials?oropharyngeal cloacal swabs and blood samples weredetected the virus and antibodies only in the inoculate group and direct contact group,oropharyngeal cloacal swabs?antibody and airborne particles was negative in the aerosolcontact group which were inoculated with R01/NASS. Air transmission experiment showsthat R01/NASS recombinant strain was not able to get air-borne transmission?recombinantR01/NASS-381strains gained the ability to transmission, when SS94strain NA gene D381G,K313E, E368D, L370S mutated. Thus it explains that the NA protein381?313?368and370amino acids of H9N2AIV can play an important role in air-borne transmission of poultry.3?Studies of pathogenicity of the R01/NASS and R01/NASS-381recombinant strain virus154-week-old SPF chickens were randomly divided into three groups, SPF chickenswere inoculated with the recombinant virus strains each five, dropped106EID50chick embryoallantoic fluid0.2mLthrough the eye, nasal way every SPF chick, the control group wereinoculated the same amount of the sterile saline with the same way. The control group andinoculate group were dissected to get all the heart, liver, spleen, lung, kidney, brain, trachea,bursa and other organizations that well marked.after rearing isolated three days.Then theywere fixed in4%paraformaldehyde, observed distribution of tissue lesions and antigen by HEstaining and immunohistochemistry. The results showed that the control group has no tissuelesions after HE staining and brown area by immunohistochemistry.two experimental groupsinoculated with R01/NASS and R01/NASS-381recombinant strains have severe renal andhepatic pathology, It is more serious that their respiratory tract, trachea, bronchus and lung arelymphocytes infiltrated, congested and bled of SPF chicken inoculated with R01/NASS-381 recombinant viruses than R01/NASS. However, the former has certain lesions in otherorganizations, the latter has no pathological changes basically; we detected a large number ofthe positive signal in the lung the liver and kidney in the SPF chicken inoculated withR01/NASS-381recombinant strain by immunohistochemical test, but another set ofexperiments only find a small number of positive signals and no signal in the controlgroup.Results showed that two strains of the virus bite renal,the histopathological changescaused by R01/NASS-381recombinant virus strains are more serious than R01/NASS,Maybe it is related to381?313?368and370amino acid mutations of NA gene. |
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