| 1 Four strains of avian influenza viruses (AIV) were isolated from diseased ducks inJiangsu and Zhejiang Province, Which haemagglutination activity test (HA) titers were 29,29, 29 and 28, respectively. The virus hemagglutination were inhibited by positive seraagainst H9 subtype avian influenza viruses (AIV), but not inhibited by positive sera againstNewcastle disease virus (NDV), egg-drop syndrome-76 virus (EDS-76V) and H5 subtypeavian influenza virus (AIV), which HI titers were 211, 29, 210 and 29, respectively.Neuraminidase inhibited test (NI) indicated that the isolates belonged to H9N2 subtype ofinfluenza vires and were named NJ01, ZJ03, XZ05 and XZ07, respectively. The content offour strains virus in 0.1ml allantoic succus were 108.5, 109.0,108.38 and 108.83 ELD50 orEID50.The half neutralize titers against H9 subtype positive sera of the four strains viruswere 1:150, 1:93, 1:150 and＞1:64. The ICPI were 0.26, 0.24, 0.19 and 0.31. TheIVPI were 0, 0, 0 and 0.22, and the MDT/MLD of NJ01 strain is 45.6h/10-7. The resultsshowed that the isolations were low pathogenicity AIV.2 50 six-days-old Yingtaogu ducks were immunized with 0.3ml inactivated vaccineof NJ01 per duck in muscular and subcutaneous ways respectively, while another 50 duckswere not and as the control group. In 7th day, 10th day, 14th day, 21st day and 28th day p.i.,five ducks of every group were challenged i.v. with the dose of 108.7 ELD50 and 107.7 ELD50respectively. The death number of the control group with 108.7 ELD50 was 3, 4, 4, 2 and 1,and the protection ratio of vaccine reached 100% at one week p.i. through i.m. and at 2weeks p.i. through injection in subcutaneous. Then, 20 sixteen-days-old Yingtaogu ducksand 20 sixty one-days-old SPF chickens were inoculate with 0.3ml inactivated vaccine ofNJ01, ZJ03, XZ05 and XZ07 per duck/chicken respectively, while the control groups werenot. Every group were divided into four subgroups and cross-challenged through i.v in 21stday with the dose of 109.5 ELD50 per duck and 108.5 ELD50 per chicken. All the vaccinatedducks were survived,but 4, 3, 3 and 4 ducks died in the control groups challenged by NJ01,ZJ03, XZ05 and XZ07. After 5th day p.i., the virus could be separated from the control group chickens in cloacal swabs, but couldn't from the immunization group. Finally, 6five-week-old ICR mice were challenged with 107.0 EID50 (50μl) NJ01, ZJ03, XZ05 andXZ07 strain virus with intranasal inoculation way, while the control group were treatedwith 50μl sterilized salt water. In 3th day p.i, the weight of the mices was declined indifferent degree except the control group. The cross-HI titer of ZJ03 and NJ01 sera againstthe others strain virus was even higher 21.0～21.3 than that of XZ05 and XZ07. The resultsshowed that: four strain virus had certain pathogenic to duck but lower pathogenicity tochicken and mouse, and had immunogenicity in chicken, duck and mouse, while the muscleimmunization was more effective than subcutaneous immunization.3 The rules of H9N2 subtype AIV reproduction on embryo was studied with NJ01strain virus. It was found that ten-day old SPF embryo or the non-immune embryo, whichHI against H9 were≤26, should be used to reproduce the H9 subtype AIV. The virus wasdilute to 10-3～10-4 and injected into the allantoid of eggs with 0.1 ml per egg, hatched at37℃. Per 6 hours the eggs was checked and the dead embryo eggs was stocked at 4℃.After 96 hours all the eggs were taken out, the allantoic succus and chorioallantoicmembrane were harvest. The alive eggs allantoic fluids can be mixed up with the that ofdead eggs when it's HA was≥27.The research might provide indication for developmentof H9 subtype vaccine.4 10 four-month-old chickens and twenty six-day-old ducks were challenged withNJ01 strain i.v. at the dose of 108.0 ELD50, and the virus were isolated in throat swabs ofducks and cloacal swabs of chickens during 1～6 days and 1～8 days p.i.. The results showedthat the virus in ducks reproducted mainly in lung, trachea, fauces and the other tissueswhich associated with respiration symptom. In 1～2 days after challenged, the separatedratio of virus were highest, and the birds also happened to death mainly. But in chickens theseparated ratio in cloacal swabs appeared to highest in 4～6 days after challenged. Theresults were instructive to the quality control of H9 subtype AIV vaccine.5 Eleven primers were designed according to gene segments sequences published inGenbank, and the cDNA were amplified with RT-PCR from NJ01, ZJ03, XZ05 and XZ07.Some products were linked with pMD18-T Vector. The plasmids testified to be positivewere sequenced. The other PCR products were purified and then sequenced withBECKMAN8000 apparatus. The sequences of HA, NA, NP, PA, PB1 and PB2 genes weresubmit to Genbank successfully. At the base of phylogenetic analysis to the sequences, thetrees were made and the genetic homology of the virus strains were analyzed. HA and NA amino acid analysis showed that the enzyme points, the potentialglycosylation sites and receptor binding sites of four virus strains were consistent with lowpathogenic virus, and different from the human flu virus strains. In XZ07 and NJ01 threeamino acid in NA neck were missed at the site of 63～65, and a additional glycosylationsites NGT was added at 145 site of HA, which ensured the effective reproduction of thevirus. The results gave some reasons that the virulences of XZ05 and ZJ03 had differences.The results of the phylogenetic tree analysis showed that the gene segment of fourstrains H9N2 virus (the M gene of XZ05 and BJ/1/94 belonged to the Y739-like branch)and domestic representative stain SH/F/98 (the NS gene belonged to the G1-like branch)belonged to the one bird branch, which were different from the other domesticrepresentative stain BJ/1/94. External protein gene segments (HA, NA and M gene) of fourstrains originated from the Y280-like branch, while the internal protein gene segments (PA,PB2, NP and NS) originated from the Y439-like branch, and the PB1 gene was exceptive,which originated from the KRO/p/96 branch. The gene segments were unrelated withG1-like branch of human except the NP gene segment had the same evolutionary status.However the NP gene segment of representative stain SH/F/98 belonged to the G1-likebranch. So the four strains belonged to recombinant virus of the Eurasia Y280-like andY439-like branch. The gene segments of the four strains had different evolutionary status,in which the NA, NS and PA gene segments evolutionary status of XZ05 were in ahead ofother virus. The results could accumulated information for the molecular epidemiologicalinvestigations of avian influenza, provided reference to multi-channel selection of vaccinesand the early warning and forecasting human influenza pandemic.6 The monoclonal antibody (McAb) specific to the H9 subtype of avian influenzavirus (AIV) was developed by fusing SP2/0 myeloma cell with spleen cells of miceimmunized with inactivated AIV-H9 (NJ01 strain) subtype vaccine.Positive hybridomaclones were screened by hemagglutination inhibition test (HI)and eighteen were detected tobe positive, and the positive ratio were up to 4.69%. Three of them were cloned three timesand were designated as 1E1, 2E10 and 3G2 respectively, which were specific to AIV-H9.AIV-H9 antibodies were still secreted steadily after in one-month culture in vitro andrefrigerated storage and revivification for twice. The three McAbs were identified as IgG1,and the chromosome number of hybridoma cell were 92～108, 97 averagely. The HI titers ofsupernatants of hybridoma cell culture and ascites reacted with AIV-H9 were 25～28 and212～214, and the PD50 were 10-2～10-1.67 and 10-4～10-3.33.The development of three McAbs may provide a useful tool in the fast diagnosis and the forecast of AIV.7 Rapid diagnosis strips of immune colloidal gold particles specific to the H9subtype of avian influenza virus (AIV) was succussfully developed, which based on theresearches of monoclonal antibody (McAb) and the stable stock of colloidal gold particlesand the influencing factors of colloidal. The minimal stable concentration of the strips was20μg/ml, the appropriate pH of labeled McAb was pH 8.5, the optimal dose of labeledMcAb which on the glass fibre membrane was 15μl/cm2, the optimal dose of antibodywhich on the nitrocellulose membrane was 2μl/cm. The strips had highly specify andsensitivity, moreover the result could be showed in 10 minutes. 25 clinical samples wastested, the agreement ratio between the rapid diagnosis strips and the virus isolation andidentification from chick embryo was 85.7%. The result suggested that the strips weresuitable to the rapid diagnosis on the spot.
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