【作者】 李银；

【导师】 沈永林；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2006， 博士

【摘要】 1．从江苏、浙江发病鸭分离到4株具有血凝性的病毒，血凝价分别为2⁹、2⁹、2⁹和2⁸，新城疫阳性血清、产蛋下降综合征抗血凝素单克隆抗体和H5亚型禽流感阳性血清均不能抑制其血凝性，H9亚型禽流感阳性血清能抑制它们的血凝性，血凝抑制价分别为2¹¹、2⁹、2¹⁰和2⁹，结合NI试验均鉴定为H9N2亚型禽流感病毒，分别命名后简称为NJ01、ZJ03、XZ05和XZ07。它们在鸡胚尿囊液中的病毒含量分别为10^8.5、10^5.38、10^9.0和10^8.83ELD₅₀或EID₅₀（0.1ml）；H9亚型阳性血清对它们的半数中和价分别为1：150、1：93、1：150和＞1：64；它们的ICPI分别为0.26、0.24、0.19和0.31；IVPI分别为0、0、0和0.22；NJ01的MDT／MLD为45.6h／10^-7。表明4个分离株均为低致病性。2．用NJ01毒株油乳剂灭活疫苗以肌肉和皮下途径各免疫6日龄樱桃谷鸭50只，每只0.3ml，对照组50只不免疫，免疫后7d、10d、14d、21d和28d时，三组均用10^8.7ELD₅₀和10^7.7ELD₅₀的NJ01毒株静脉注射攻毒，各时段每一攻毒剂量各攻毒5只，观察2周，结果以10^7.7ELD₅₀攻毒各组均未出现死亡，以10^8.7ELD₅₀攻毒，对照组在上述时段分别死亡3只、4只、4只、2只和1只；肌肉免疫后1周、皮下免疫后2周抗死亡保护率为100％。进一步用NJ01、ZJ03、XZ05和XZ07毒株油乳剂灭活疫苗分别肌肉免疫16日龄樱桃谷鸭各20只，免疫61日龄SPF鸡各20只，每只0.3ml，对照鸡、鸭各20只均不免疫，免疫后21d分别用以上4个毒株静脉注射交叉攻毒，每一毒株攻毒5只鸭，攻毒量为10^9.5ELD₅₀，每一毒株攻毒5只鸡，攻毒量为10^8.5ELD₅₀，观察2周，结果各组免疫鸭均未发生死亡，对照鸭在NJ01、ZJ03、XZ05和XZ07毒株攻毒后，分别死亡4只、3只、3只和4只；从攻毒后5d对照鸡的泄殖腔拭子100％分离到病毒，各组免疫鸡均未分离到病毒。用NJ01、ZJ03、XZ05和XZ07毒株各滴鼻攻毒5周龄左右ICR小鼠6只，攻毒量为10^7.0ELD₅₀（50μl），对照组6只，用相同剂量的生理盐水滴鼻，观察2周，攻毒后15d制备各攻毒组小鼠的血清，进行交叉血凝抑制试验，结果在攻毒后3d内，除对照组外其它各组体重均有不同程度下降。交叉血凝抑制试验结果显示，ZJ03和NJ01血清比XZ05和XZ07血清对各毒株的平均HI高2^1.0～2^1.3。以上各试验结果表明：4个分离株对鸭均有一定的致病力，对鸡和小鼠的致病力均较低，4个分离株在鸡、鸭和小鼠均表现较好的免疫原性，肌肉免疫效果优于皮下免疫。3．通过测定毒种（代次、稀释度）和鸡胚（日龄、级别）等因素对NJ01毒株在鸡胚繁殖上的影响，发现使用SPF鸡胚或抗H9 HI≤2⁶非免疫鸡胚，胚龄为10日龄，毒种10^-3～10^-4稀释，鸡胚尿囊腔接种，每胚0.1ml，在37℃条件下孵化，定时照蛋，取出死胚，至96h取出全部活胚和死胚，分别收获活胚和死胚的尿囊液和尿囊膜，活胚尿囊液HA≥2⁷时才能与死胚的尿袭液混合，这样鸡胚尿囊液的收获量较多，病毒含量较高。这一结果对研制和生产H9亚型禽流感疫苗具有借鉴和指导作用。4．用NJ01毒株静脉注射攻击4月龄鸡和26日龄鸭各10只，攻毒量均为10⁸ELD₅₀，观察2周，采取攻毒后1～6d鸭的喉拭子和1～8d鸡的泄殖腔拭子分离病毒。结果鸭在攻毒后的1～2d之内，肺脏、气管、咽喉等组织器官中病毒的分离率最高，死亡也主要发生在这1～2d之内。鸡在攻毒后的4～6d，泄殖腔拭子病毒的分离率最高，病毒在鸡体和鸭体组织器官中的动态分布结果对H9亚型禽流感疫苗的近期效检有指导意义。5．根据GenBank上发表的H9N2禽流感病毒基因各片段的序列，设计了11对引物，采用RT-PCR方法，从NJ01、ZJ03、XZ05和XZ07毒株中扩增出cDNA，采取连接pMD18-T载体或直接用PCR产物进行测序的方法，测定了4个毒株的各8个基因片段的序列，使用DNAstar 5.0软件对测得的序列进行拼接，将具有完整阅读框的4个毒株的HA、NA、NP、PA、PB1和PB2基因序列提交GenBank并被录用。比较了核苷酸序列和氨基酸序列的的同源性，绘制了各基因片段的进化树，并进行了分子进化分析。HA和NA氨基酸分析表明，4株分离病毒的酶切裂解位点、受体结合位点及糖基化位点均符合低致病力禽源病毒特征，与人源株差别较大。XZ07和NJ01毒株在NA颈部63～65位置缺失了三个氨基酸，在HA的145位有额外糖基化位点NGT，达到最佳组合，保证了病毒的有效复制，初步证明这两株病毒与XZ05和ZJ03毒株的毒力差异。通过对进化树的分析得知，4株H9N2病毒（XZ05与BJ／1／94的M基因属于Y739亚分支）和国内代表株SH／F／98（SH／F／98的NS基因属于G1-like亚分支）的各基因片段分别在某一个禽亚分支内，而与中国的另一代表株BJ／1／94均不在同一个禽亚分支内，其中外部蛋白基因片段（HA、NA和M）来源于Y280-like亚分支，内部蛋白基因片段（PA、PB2、NP和Ns）来源于Y439-like亚分支，PB1基因片段比较特殊，来源于KRO／p／96。除NP基因片段与G1-like处于相同的进化位置外，均与人体分离株所在的G1-like亚分支无关，但代表毒株SH／F／98的NS基因片段属于G1-like亚分支，即4株分离病毒均为欧亚大陆Y280-like亚分支和Y439-like亚分支的重组病毒。在4株分离病毒中，各基因片段具有不同的进化地位，XZ05在NA、NS和PA基因片段的进化地位比其它三株病毒靠前。通过本研究为禽流感的分子流行病学调查积累了资料，为多途径筛选疫苗株提供了参考，为人类流感大流行的预警预报提供依据。6．以灭活的H9亚型禽流感NJ01株作为抗原，按常规方法免疫BALB／c鼠，最后一次免疫后第3d取脾细胞与SP2／0细胞进行融合，采用HI进行筛选，检出18个阳性孔，阳性率为4.69％。对其中的3个HI值较高的孔进行3次克隆，最终获得3株单克隆细胞，分别命名为1E1、2E10和3G2。通过与新城疫病毒、产蛋下降综合征病毒、H5亚型AIV进行HI试验，证明1E1、2E10和3G2分泌的抗体具有特异性。经连续1个月的体外培养和两次冻存复苏仍能稳定地分泌抗H9亚型禽流感病毒HI抗体，3株杂交瘤细胞分泌的单克隆抗体均为IgG1，杂交瘤细胞的染色体数目为92～108条，平均97条。杂交瘤细胞培养上清和腹水对H9亚型AIV的HI值分别为2⁵～2⁸和2¹²～2¹⁴，PD₅₀分别10^-2～10^-1.67和10^-4～10^-3.33。单克隆抗体的制备为禽流感的快速诊断、预警预报和病毒的抗原性分析等奠定了基础。7．在成功制备抗H9亚型禽流感病毒血凝素单克隆抗体和用枸橼酸钠还原法制备出可长时间稳定保存的胶体金的基础上，通过对影响胶体金试纸条几种主要因素的摸索，初步研制成功H9亚型禽流感快速诊断试纸条，该试纸条金标单抗的最低稳定量为20μg／ml，单抗的最适标记pH为8.5，玻璃纤维素膜上的金标单抗最佳用量为15μl／cm²，硝酸纤维素膜上多抗血清的最佳用量为2μl／cm，该诊断试纸条具有特异性强，不与H9亚型以外的其它病毒反应，灵敏度高，可以测出HA为2²的尿囊液毒，需时短，10min就可出结果。检测25份临床样品，与鸡胚病毒分离结果的符合率为85.7％，继续完善后，适合基层用于现场快速诊断。更多还原

【Abstract】 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 10^8.5, 10^9.0,10^8.38 and 10^8.83 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 10^8.7 ELD₅₀ and 10^7.7 ELD₅₀respectively. The death number of the control group with 10^8.7 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 10^9.5 ELD₅₀ per duck and 10^8.5 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 10^7.0 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 2^1.0～2^1.3 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 10^8.0 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.更多还原