新城疫—禽流感H9二联灭活疫苗的研究
摘要
自1998年秋冬始于河北省石家庄地区的低致病性禽流感(LPAI)爆发,传播速度很快,短期内即成燎原之势,在近一年的时间里已波及到包括我国主要养禽地区的20多个省、市、自治区。经鉴定各地分离的毒株主要为H9N2亚型。这次全国范围的大流行,已历经数年,至今尚未平息。发病种鸡群和蛋鸡群损失严重,产蛋率下降10-60%不等,个别鸡群甚至绝产；育雏鸡、育成鸡及肉鸡也可感染发病,表现呼吸道症状,受害鸡群死淘率急剧上升,根据鸡群并发和继发感染及饲养管理状况的不同,死淘率在10-80%之间,一般在20%左右。近年来我国部分地区的禽流感血清学调查发现,H9亚型阳性群占禽流感阳性鸡群总数的93.89%,进一步说明这种禽流感的广泛性和严重危害性。基于上述情况,我国迫切需要针对H9亚型的禽流感疫苗,用于控制日益严重的H9亚型禽流感在我国的大范围流行。目前我国新城疫发病流行也非常严重,有时与H9亚型的禽流感病毒混感,给养鸡业造成更大损失。同时基层养鸡户防疫频繁,应激反应大,免疫程序难以制定,急需一针防两病的鸡新城疫、禽流感(H9亚型)二联灭活疫苗。我们在成功研究出禽流感(H9亚型)灭活疫苗基础上,又研制出鸡新城疫、禽流感(H9亚型)二联灭活疫苗。
一、禽流感(H9亚型)灭活疫苗毒种的筛选培育和鉴定
在1998-1999年期间,我国主要养禽地区5个省、市鸡群分离鉴定出8株H9N2亚型禽流感病毒(AIV),进行了系统的常规生物学和分子生物学特性鉴定,从中筛选、培育出对鸡胚的毒力稳定、产生的毒价高、免疫原性强、交叉保护性好的F株(A/Chicken/Shanghai/F/98)作为研制疫苗的候选毒株F株基因组全长序列测定和各基因的遗传分析表明,其HA、NA、M和NS4个基因均属于CkBj94分支,同源率分别为96.7%、96.4%、97.5%和98%。
1、在1998-1999年期间从我国主要养禽地区5个省、市有产蛋下降的产蛋鸡群和有呼吸症状及超常死亡的鸡群分离8株AIV(F、S、A1、H1、D1、D2、J1、J2),初次分离时鸡胚死亡时间36-60小时,HA滴度22-24不等,血清亚型鉴定均为H9N2亚型,4周龄SPF雏鸡致病性试验确定均为低致病性。
2、测定了这8个毒株的主要保护性抗原HA基因的全长序列,由其推导的氨基酸序列同源性在95%以上,在HA基因遗传发生树上这些毒株与CkBJ94一道形成一个独特的分支,提示有共同来源；8株病毒的交叉HI试验、交叉鸡胚中和试验和雏鸡交叉保护试验均表明,它们之间能提供交叉保护。
3、在8株病毒中,选择初次分离时毒价高、鸡胚死亡时间一致的F株在SPF鸡胚中连续继代培育,第5代(E5)时鸡胚液病毒滴度达HA210,至第10代(E10)时HA滴度大于210。经系统鉴定,它对鸡胚的毒力稳定,(90%以上鸡胚在接种后72-96小时内死亡),产生病毒的滴度高(HA大于210),免疫原性强(7日龄SPF鸡免疫后HI滴度>5log2),交叉保护性好,具有优良的疫苗毒株特性。
4、通过RT-PCR和51/31RACE法扩增、各片段克隆测序和序列拼接,得到了F株的基因组全长序列,8个基因长度分别为PB12341bp、PB22341bp、PA2233bp、HA1742bp、NP1565bp、NA1458bp、M1027bp、NS8906bp,并对各基因进行遗传分析,其HA、NA、M、NS4个基因均属于CkBJ94分支,同源率分别为96.7%、96.4%、CkBj94株。
二、鸡新城疫、禽流感(H9亚型)二联灭活疫苗的研制和生产
用新城疫LASOTA株和H9N2亚型AIV F株为毒种,经实验室研究、田间试验、中间试制和区域试研制出对不同品种和年龄的鸡安全而免疫效力高的新城疫、禽流感(H9亚型)二联灭活疫苗,可以达到与各自单苗具有相同免疫效果。已取得了国家新兽药证书和农业部颁发的生产批准文号,累计生产应用2亿羽份以上,产生了巨大的经济效益和社会效益。
1、使用Millipore超滤浓缩机的4#膜浓缩新城疫和禽流感抗原,各浓缩1/2-1/3,测定HA效价,NDHA>1024,AIHA>512,按1：1比例配苗。
2、二联苗效力试验表明,三批疫苗每羽份新城疫分别含有127、131和127PD50,达到英国兽药典每羽份≥50PD50的要求。禽流感的免疫抗体水平及攻毒保护均达到单苗的要求。免疫鸡日龄太小(小于10日龄),鸡体免疫应答反应较差,效力检验最好采用3-4周龄易感鸡。
3、抗体产生时间试验表明,无论免疫剂量多大,疫苗免疫后前4天都测不出抗体,免疫后7天,抗体开始缓慢上升,11天后,抗体上升加快,至21天基本达到高峰。免疫剂量大的,抗体继续上升；免疫剂量小的,抗体滴度就不再上升。相同剂量免疫时,免疫鸡日龄越小,抗体上升越慢,上升幅度越小。攻毒试验表明,免疫后11天攻毒,只有部分免疫鸡被保护,14天攻毒时,大部分免疫鸡保护,21天后方能完全保护。免疫剂量与免疫期试验表明,在一定免疫剂量范围内,免疫保护期与免疫剂量成正相关。免疫剂量偏低时,免疫保护期短；免疫剂量大时,免疫保护期适当延长。二联苗的免疫剂量和免疫期分别定为：商品肉鸡5日龄免疫,0.2m1/羽,出栏前免疫一次；蛋雏鸡和种雏鸡开产前免疫2次,14日龄首免,0.2m1/羽,可保护至60日龄,50-60日龄二免,0.5m1/羽,可保护至开产；或于21-30日龄免疫一次,0.5m1/羽,也可保护至开产。产蛋鸡开产前免疫一次,0.5m1/羽,可保护整个产蛋期。
4、中间试制：在易邦公司生物制品GMP车间共试制10批次总量为540万羽份,成品检验全部合格。
5、区域试验在江苏、山东、天津、河北等省、市的数十个养禽企业进行区域试验,共免疫800余万羽,临床保护率接近100%,取得很满意的效果。
三、H9亚型禽流感发病机理研究：与大肠杆菌联合感染的致病协同作用
用H9N2亚型AIV和禽源性大肠杆菌进行联合感染实验研究,结果发现两者之间有强烈的致病协同作用：AIV(H9N2亚型)单独感染症状轻微,不死亡或死亡率很低,但是与禽源性大肠杆菌,甚至是低致病性的大肠杆菌联合感染均可发生严重死亡,病变出现早、恢复慢且更为严重。
1、H9N2亚型AIV和禽源性大肠杆菌联合感染时能产生致病协同作用：
AIV单独感染(105.17ELD)时死亡率为6.25%,大肠杆菌(037)单独感染(3x107CFU)时死亡率为62.5%,联合感染时死亡率为81.25%。这种致病协同作用在低致病性的禽源大肠杆菌和AIV之间更为显著,单独感染和联合感染的死亡率分别为20.0%、13.3%和100%。
2、H9N2亚型AIV和禽源大肠杆菌联合感染SPF鸡的动态病理变化：单独接种的两组接种后1-96小时内无死亡,联合感染组死亡率为24%：联合感染组死亡率为24%；联合感染组大体病变和显微病变比单独接种的两组出现得早、恢复慢且更为严重。
An outbreak of low pathogenic avian influenza (LPAI) started in Shijiazhuang district, Hebei province in Autumn1998. The disease spread rapidly and affected the main poultry production areas in twenty provinces, municipalities or autonomous regions in the country within only one year. The isolates from various parts were identified as H9N2subtype. The national wide pandemic has lasted for several years and has not yet completely eliminated. The infected breeding flocks and laying flocks suffered great losses, resulting in10-60%egg production reduction and even complete production loss in some individual flocks. Rearing chickens, reared chickens and broilers may also be infected, manifesting respiratory symptoms. The culling and death rates in the affected flocks are increasing sharply, ranging between10%and80%with different husbandry conditions, complications and secondly infections, and about20%in general. Serological investigation of avian influenza in parts in recent years indicated the positive flocks for H9subtype amounted to93.89%of the total AI positive flocks, thus further explaining its broadness and severity. From the above, an AI vaccine is urgently needed specifically against H9subtype for controlling the wide spread of H9subtype AI in China. Meanwhile, the Newcastle disease epidemic is also serious in China and sometimes mixed infection with ND and H9subtype AI occurs occasionally, causing even greater stress reactions and difficulty to formulate immunization programs. Following successful development of the AI vaccine (inactivated), we have developed the binary vaccine of ND and AI (H9)(inactivated).
1. Screening, cultivation and identification of seed virus for AI vaccine (H9),inactivated.
Between1998and1999,8strains of H9N2subtype AIV were isolated and identified from the main poultry production areas in5provinces and municipalities, and were subjected to systematic normal biological and molecular characterization. And the F Strain (A/Chicken/Shanghai/F/98) was selected and cultivated as the candidate vaccine strain due to the stability in virulence, high titer and strong immunogenicity and sound cross protection. The full length sequencing that the4genes such as HA, NA, M and NS all belong to CkBJ94 branch with homology of96.7%,96.4%,97.5%and98%respectively,
(i)8strains of AIV (F, S, AI, H1,D1,D2, J1and J2) were isolated from egg-laying flocks suffering egg production reduction and chicken flocks with respiratory symptoms and excess deaths in the main poultry production areas in5provinces and municipalities between1998and1999.During the preliminary isolation, the chicken embryo death time was36-60hours and HA titer varied between22and24. It was subtyped as H9N2, and was identified as low pathogenicity using4-weeks old SPF chickens in pathogenicity tests.
(ii) The8strains were tested for the full length sequence of the main protective antigen HA gene, and the homology of the aminoacid sequence was inferred as over95%; on the HA gene genetic evolution tree, these strain, together with CkBj94, formed a unique branch, suggesting they shared the common origin; the cross HI test, the cross chicken embryo neutralization test and the cross chick protection test of the8strains showed that they could provide cross protection.
(iii) Among the8strains of AIV, F strain, which had high virulence titer and consistant chicken embryo death time during the preliminary isolation, was selected to passage in SPF chicken embryos, the HA titer of the chicken embryo fluid in the5th passage (E5) reached210,and the HA titer was over210by the10passage (E10). Through serial identification, it was found that F strain was stable in virulence to chicken embryos,(over90%chicken embryos died between72h and96h after inoculation), high in virus titer (HA>210), strong in immunogenicity (HI titer>5log2) in7days old SPF chickens post immunization) and good in cross protection. It expressed very good characterstics as a vaccine strain.
(iv) The full length sequence of the F strain genosome was obtained by using RT-PCR and51/31RACE amplification, sequencing of each fragment clone and sequence joining. The lengths of the8genes were PB12341bp, PB22341bp, PA2233bp, HA1742bp, NP8906bp,and each gene was genetically analyzed. The4genes such as HA, NA, M, and NS belonged to CkBJ94branch with the homology of96.7%,96.4%,97.5%and98%respectively,suggesting that F strain is originated from CkBJ94.
2. Development and production of the binary vaccine of ND and AI (H9) inactivated
The binary vaccine of ND and AI (H9), inactivated was developed with NDV LASOTA strain and AIV H9N2F strain as seed virus through laboratory studies, field experiments, pilot production and regional field experiments. The vaccine is safe and effective for chickens of different breeds and ages and can obtain the same immunological effect as the respective mono vaccine. It has achieved the National New Veterinary Drug Certificate and the MOA-issued production permit. More than200million doses have been accumulatedly produced, with great economic and social benefit.
(i) NDV and AIV antigens are concentrated through4#filters in the Millipore ultra concentrator to1/2-1/3:NDV HA≧1024and AIV HA≧512, The two antigens are mixed in1:1for preparation of the vaccine.
(ii) Potency tests indicated that three batches of the binary vaccine contained127,131and127PDso/dose of NDV, meeting the requirement of≧50PD50/dose specified in the British Veterinary Pharmacopeia. Both the immune antibody level against AI and challenge protection meet the requirements as for the monovaccine. Too young chicks (<10days old) will have poor immune response.3-4weeks old susceptible chickens are preferred in potency tests.
(iii) Antibody production time tests showed that no antibody can be detected within4days after vaccination, in spite of the dosage. The antibody level started to increase gradually7days post vaccination, to be accelerated21day post vaccination. If the immunizing dosage was bigger, the antibody level continued to increase; if the immunizing dosage was small, the antibody titer did not increase any more. Under the same immunizing dosage, the younger the immunized chickens were, the slower the antibody level increased, and the smaller the increasing amplitude was. Challenge tests showed that only part of the immunized chickens got protected when they were challenged11days after vaccination; most immunized chickens were protected when challenged14days after vaccination; and all the chickens were protected when challenged on21days later. Tests on immunizing dosage and immunity duration showed that within certain limit of the immunizing dosage, the immuno protection period was positively proportional to the immunizing dosage. When the immunizing dosage was low, the immuno protection period was short; when the immunizing dosage was properly bigger, the immuno protection period would be prolonged accordingly. The immunizing dosage and immuno protection period of the binary vaccine are required as follows: commercial broilers:vaccinated at5days old, with0.2ml/bird and boostered once before slaughtering; egg-layer chickens and breeding chickens:vaccinated twice before production, first14days old with0.2ml/bird,(maybe protected until60days old) and boostered on50～60 days old with0.5ml/bird (may be protected until production; and vaccinated once just before production with0.5ml/bird.(maybe protected for the whole production period).
(iv) Pilot production:10batches of vaccine,5.4million doses in total were experimentally produced by Yebio GMP plant for bioproducts, and all passed various tests, including finished product inspection.
(v) Regional field experiments:carried in10poultry production enterprises in Jiangsu, Shandong, and Hebei provinces and Tianjing municipality. About8million bird doses were used, with almost100%protection rate clinically resulting in satisfactory effect.
3. Studies on H9subtype AI pathogenesis:Synergic pathopoiesis in combination with E.coli infection
Experiments on combined infection with H9N2subtype AIV and chicken-derived E-coli showed that the two have strong synergic pathopoiesis:infection with AIV (H9N2subtype) alone induced mild symptoms, no deaths or very low mortality, however, when combined infection with AIV (H9N2subtype) and chicken-derived E.coli, or even low pathogenic E.coli induced severe deaths, lesions appeared earlier, recovered very slowly and more serious,
(i) Synergic pathopoiesis was induced in chicken combinedly infected with AIV(H9N2sybtype) and chicken derived E.coli:Infection with AIV (105.17ELD) alone induced a mortality of6.25%, and infection with E.coli (037)(3×107CFU) alone induced a mortality of62.5%. while combined infection with the two pathogens induced a mortality of81.25%. The synergism in low pathogenic E.coli and AIV was more significant and mortalities induced by individual and combined infection were20.0%,13.3%and100%respectively.
(ii) Dynamic pathologic changes in SPF chickens combinedly infected with AIV H9N2subtype and chicken-derived E.coli:No deaths were observed in the two groups between1and96hours after individual infection, and24%mortality was found in the combinedly infected group. The gross lesions and pathologic changes appeared earlier, recovered more slowly and more seriously in the combinedly infected group than in the individually infected groups.
一、禽流感(H9亚型)灭活疫苗毒种的筛选培育和鉴定
在1998-1999年期间,我国主要养禽地区5个省、市鸡群分离鉴定出8株H9N2亚型禽流感病毒(AIV),进行了系统的常规生物学和分子生物学特性鉴定,从中筛选、培育出对鸡胚的毒力稳定、产生的毒价高、免疫原性强、交叉保护性好的F株(A/Chicken/Shanghai/F/98)作为研制疫苗的候选毒株F株基因组全长序列测定和各基因的遗传分析表明,其HA、NA、M和NS4个基因均属于CkBj94分支,同源率分别为96.7%、96.4%、97.5%和98%。
1、在1998-1999年期间从我国主要养禽地区5个省、市有产蛋下降的产蛋鸡群和有呼吸症状及超常死亡的鸡群分离8株AIV(F、S、A1、H1、D1、D2、J1、J2),初次分离时鸡胚死亡时间36-60小时,HA滴度22-24不等,血清亚型鉴定均为H9N2亚型,4周龄SPF雏鸡致病性试验确定均为低致病性。
2、测定了这8个毒株的主要保护性抗原HA基因的全长序列,由其推导的氨基酸序列同源性在95%以上,在HA基因遗传发生树上这些毒株与CkBJ94一道形成一个独特的分支,提示有共同来源；8株病毒的交叉HI试验、交叉鸡胚中和试验和雏鸡交叉保护试验均表明,它们之间能提供交叉保护。
3、在8株病毒中,选择初次分离时毒价高、鸡胚死亡时间一致的F株在SPF鸡胚中连续继代培育,第5代(E5)时鸡胚液病毒滴度达HA210,至第10代(E10)时HA滴度大于210。经系统鉴定,它对鸡胚的毒力稳定,(90%以上鸡胚在接种后72-96小时内死亡),产生病毒的滴度高(HA大于210),免疫原性强(7日龄SPF鸡免疫后HI滴度>5log2),交叉保护性好,具有优良的疫苗毒株特性。
4、通过RT-PCR和51/31RACE法扩增、各片段克隆测序和序列拼接,得到了F株的基因组全长序列,8个基因长度分别为PB12341bp、PB22341bp、PA2233bp、HA1742bp、NP1565bp、NA1458bp、M1027bp、NS8906bp,并对各基因进行遗传分析,其HA、NA、M、NS4个基因均属于CkBJ94分支,同源率分别为96.7%、96.4%、CkBj94株。
二、鸡新城疫、禽流感(H9亚型)二联灭活疫苗的研制和生产
用新城疫LASOTA株和H9N2亚型AIV F株为毒种,经实验室研究、田间试验、中间试制和区域试研制出对不同品种和年龄的鸡安全而免疫效力高的新城疫、禽流感(H9亚型)二联灭活疫苗,可以达到与各自单苗具有相同免疫效果。已取得了国家新兽药证书和农业部颁发的生产批准文号,累计生产应用2亿羽份以上,产生了巨大的经济效益和社会效益。
1、使用Millipore超滤浓缩机的4#膜浓缩新城疫和禽流感抗原,各浓缩1/2-1/3,测定HA效价,NDHA>1024,AIHA>512,按1：1比例配苗。
2、二联苗效力试验表明,三批疫苗每羽份新城疫分别含有127、131和127PD50,达到英国兽药典每羽份≥50PD50的要求。禽流感的免疫抗体水平及攻毒保护均达到单苗的要求。免疫鸡日龄太小(小于10日龄),鸡体免疫应答反应较差,效力检验最好采用3-4周龄易感鸡。
3、抗体产生时间试验表明,无论免疫剂量多大,疫苗免疫后前4天都测不出抗体,免疫后7天,抗体开始缓慢上升,11天后,抗体上升加快,至21天基本达到高峰。免疫剂量大的,抗体继续上升；免疫剂量小的,抗体滴度就不再上升。相同剂量免疫时,免疫鸡日龄越小,抗体上升越慢,上升幅度越小。攻毒试验表明,免疫后11天攻毒,只有部分免疫鸡被保护,14天攻毒时,大部分免疫鸡保护,21天后方能完全保护。免疫剂量与免疫期试验表明,在一定免疫剂量范围内,免疫保护期与免疫剂量成正相关。免疫剂量偏低时,免疫保护期短；免疫剂量大时,免疫保护期适当延长。二联苗的免疫剂量和免疫期分别定为：商品肉鸡5日龄免疫,0.2m1/羽,出栏前免疫一次；蛋雏鸡和种雏鸡开产前免疫2次,14日龄首免,0.2m1/羽,可保护至60日龄,50-60日龄二免,0.5m1/羽,可保护至开产；或于21-30日龄免疫一次,0.5m1/羽,也可保护至开产。产蛋鸡开产前免疫一次,0.5m1/羽,可保护整个产蛋期。
4、中间试制：在易邦公司生物制品GMP车间共试制10批次总量为540万羽份,成品检验全部合格。
5、区域试验在江苏、山东、天津、河北等省、市的数十个养禽企业进行区域试验,共免疫800余万羽,临床保护率接近100%,取得很满意的效果。
三、H9亚型禽流感发病机理研究：与大肠杆菌联合感染的致病协同作用
用H9N2亚型AIV和禽源性大肠杆菌进行联合感染实验研究,结果发现两者之间有强烈的致病协同作用：AIV(H9N2亚型)单独感染症状轻微,不死亡或死亡率很低,但是与禽源性大肠杆菌,甚至是低致病性的大肠杆菌联合感染均可发生严重死亡,病变出现早、恢复慢且更为严重。
1、H9N2亚型AIV和禽源性大肠杆菌联合感染时能产生致病协同作用：
AIV单独感染(105.17ELD)时死亡率为6.25%,大肠杆菌(037)单独感染(3x107CFU)时死亡率为62.5%,联合感染时死亡率为81.25%。这种致病协同作用在低致病性的禽源大肠杆菌和AIV之间更为显著,单独感染和联合感染的死亡率分别为20.0%、13.3%和100%。
2、H9N2亚型AIV和禽源大肠杆菌联合感染SPF鸡的动态病理变化：单独接种的两组接种后1-96小时内无死亡,联合感染组死亡率为24%：联合感染组死亡率为24%；联合感染组大体病变和显微病变比单独接种的两组出现得早、恢复慢且更为严重。
An outbreak of low pathogenic avian influenza (LPAI) started in Shijiazhuang district, Hebei province in Autumn1998. The disease spread rapidly and affected the main poultry production areas in twenty provinces, municipalities or autonomous regions in the country within only one year. The isolates from various parts were identified as H9N2subtype. The national wide pandemic has lasted for several years and has not yet completely eliminated. The infected breeding flocks and laying flocks suffered great losses, resulting in10-60%egg production reduction and even complete production loss in some individual flocks. Rearing chickens, reared chickens and broilers may also be infected, manifesting respiratory symptoms. The culling and death rates in the affected flocks are increasing sharply, ranging between10%and80%with different husbandry conditions, complications and secondly infections, and about20%in general. Serological investigation of avian influenza in parts in recent years indicated the positive flocks for H9subtype amounted to93.89%of the total AI positive flocks, thus further explaining its broadness and severity. From the above, an AI vaccine is urgently needed specifically against H9subtype for controlling the wide spread of H9subtype AI in China. Meanwhile, the Newcastle disease epidemic is also serious in China and sometimes mixed infection with ND and H9subtype AI occurs occasionally, causing even greater stress reactions and difficulty to formulate immunization programs. Following successful development of the AI vaccine (inactivated), we have developed the binary vaccine of ND and AI (H9)(inactivated).
1. Screening, cultivation and identification of seed virus for AI vaccine (H9),inactivated.
Between1998and1999,8strains of H9N2subtype AIV were isolated and identified from the main poultry production areas in5provinces and municipalities, and were subjected to systematic normal biological and molecular characterization. And the F Strain (A/Chicken/Shanghai/F/98) was selected and cultivated as the candidate vaccine strain due to the stability in virulence, high titer and strong immunogenicity and sound cross protection. The full length sequencing that the4genes such as HA, NA, M and NS all belong to CkBJ94 branch with homology of96.7%,96.4%,97.5%and98%respectively,
(i)8strains of AIV (F, S, AI, H1,D1,D2, J1and J2) were isolated from egg-laying flocks suffering egg production reduction and chicken flocks with respiratory symptoms and excess deaths in the main poultry production areas in5provinces and municipalities between1998and1999.During the preliminary isolation, the chicken embryo death time was36-60hours and HA titer varied between22and24. It was subtyped as H9N2, and was identified as low pathogenicity using4-weeks old SPF chickens in pathogenicity tests.
(ii) The8strains were tested for the full length sequence of the main protective antigen HA gene, and the homology of the aminoacid sequence was inferred as over95%; on the HA gene genetic evolution tree, these strain, together with CkBj94, formed a unique branch, suggesting they shared the common origin; the cross HI test, the cross chicken embryo neutralization test and the cross chick protection test of the8strains showed that they could provide cross protection.
(iii) Among the8strains of AIV, F strain, which had high virulence titer and consistant chicken embryo death time during the preliminary isolation, was selected to passage in SPF chicken embryos, the HA titer of the chicken embryo fluid in the5th passage (E5) reached210,and the HA titer was over210by the10passage (E10). Through serial identification, it was found that F strain was stable in virulence to chicken embryos,(over90%chicken embryos died between72h and96h after inoculation), high in virus titer (HA>210), strong in immunogenicity (HI titer>5log2) in7days old SPF chickens post immunization) and good in cross protection. It expressed very good characterstics as a vaccine strain.
(iv) The full length sequence of the F strain genosome was obtained by using RT-PCR and51/31RACE amplification, sequencing of each fragment clone and sequence joining. The lengths of the8genes were PB12341bp, PB22341bp, PA2233bp, HA1742bp, NP8906bp,and each gene was genetically analyzed. The4genes such as HA, NA, M, and NS belonged to CkBJ94branch with the homology of96.7%,96.4%,97.5%and98%respectively,suggesting that F strain is originated from CkBJ94.
2. Development and production of the binary vaccine of ND and AI (H9) inactivated
The binary vaccine of ND and AI (H9), inactivated was developed with NDV LASOTA strain and AIV H9N2F strain as seed virus through laboratory studies, field experiments, pilot production and regional field experiments. The vaccine is safe and effective for chickens of different breeds and ages and can obtain the same immunological effect as the respective mono vaccine. It has achieved the National New Veterinary Drug Certificate and the MOA-issued production permit. More than200million doses have been accumulatedly produced, with great economic and social benefit.
(i) NDV and AIV antigens are concentrated through4#filters in the Millipore ultra concentrator to1/2-1/3:NDV HA≧1024and AIV HA≧512, The two antigens are mixed in1:1for preparation of the vaccine.
(ii) Potency tests indicated that three batches of the binary vaccine contained127,131and127PDso/dose of NDV, meeting the requirement of≧50PD50/dose specified in the British Veterinary Pharmacopeia. Both the immune antibody level against AI and challenge protection meet the requirements as for the monovaccine. Too young chicks (<10days old) will have poor immune response.3-4weeks old susceptible chickens are preferred in potency tests.
(iii) Antibody production time tests showed that no antibody can be detected within4days after vaccination, in spite of the dosage. The antibody level started to increase gradually7days post vaccination, to be accelerated21day post vaccination. If the immunizing dosage was bigger, the antibody level continued to increase; if the immunizing dosage was small, the antibody titer did not increase any more. Under the same immunizing dosage, the younger the immunized chickens were, the slower the antibody level increased, and the smaller the increasing amplitude was. Challenge tests showed that only part of the immunized chickens got protected when they were challenged11days after vaccination; most immunized chickens were protected when challenged14days after vaccination; and all the chickens were protected when challenged on21days later. Tests on immunizing dosage and immunity duration showed that within certain limit of the immunizing dosage, the immuno protection period was positively proportional to the immunizing dosage. When the immunizing dosage was low, the immuno protection period was short; when the immunizing dosage was properly bigger, the immuno protection period would be prolonged accordingly. The immunizing dosage and immuno protection period of the binary vaccine are required as follows: commercial broilers:vaccinated at5days old, with0.2ml/bird and boostered once before slaughtering; egg-layer chickens and breeding chickens:vaccinated twice before production, first14days old with0.2ml/bird,(maybe protected until60days old) and boostered on50～60 days old with0.5ml/bird (may be protected until production; and vaccinated once just before production with0.5ml/bird.(maybe protected for the whole production period).
(iv) Pilot production:10batches of vaccine,5.4million doses in total were experimentally produced by Yebio GMP plant for bioproducts, and all passed various tests, including finished product inspection.
(v) Regional field experiments:carried in10poultry production enterprises in Jiangsu, Shandong, and Hebei provinces and Tianjing municipality. About8million bird doses were used, with almost100%protection rate clinically resulting in satisfactory effect.
3. Studies on H9subtype AI pathogenesis:Synergic pathopoiesis in combination with E.coli infection
Experiments on combined infection with H9N2subtype AIV and chicken-derived E-coli showed that the two have strong synergic pathopoiesis:infection with AIV (H9N2subtype) alone induced mild symptoms, no deaths or very low mortality, however, when combined infection with AIV (H9N2subtype) and chicken-derived E.coli, or even low pathogenic E.coli induced severe deaths, lesions appeared earlier, recovered very slowly and more serious,
(i) Synergic pathopoiesis was induced in chicken combinedly infected with AIV(H9N2sybtype) and chicken derived E.coli:Infection with AIV (105.17ELD) alone induced a mortality of6.25%, and infection with E.coli (037)(3×107CFU) alone induced a mortality of62.5%. while combined infection with the two pathogens induced a mortality of81.25%. The synergism in low pathogenic E.coli and AIV was more significant and mortalities induced by individual and combined infection were20.0%,13.3%and100%respectively.
(ii) Dynamic pathologic changes in SPF chickens combinedly infected with AIV H9N2subtype and chicken-derived E.coli:No deaths were observed in the two groups between1and96hours after individual infection, and24%mortality was found in the combinedly infected group. The gross lesions and pathologic changes appeared earlier, recovered more slowly and more seriously in the combinedly infected group than in the individually infected groups.
引文
[l].殷震,动物病毒学,科学出版社,1985：742-750
[2].陆承平,兽医微生物学,第三版,北京：中国农业出版社,2001年
[3].中国农业科学院哈尔滨兽医研究所.家畜传染病学.农业出版社,1986
[4]. Hu X R, Ray and R W campans. Functional interaction between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses. J. virol.1992,66:1528-1534
[5]. Langedijk.J P M.F.J Dans. Sequence and structure alignment of paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin. J.Virol.1997,71:6155-6167
[6]. LorioR M. syddall R J.P.et al:Neutralization map of the HN glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition. Journal of Virology,1991,65:4999-5006
[7].B.W卡尔尼卡：禽病学.北京农业大学出版社,1991,第九版
[8].张振兴,姜平,实用兽医生物制品技术,中国农业科技出版社,1996
[9].胡子信,张歧蜀,门常平：鸡新城疫免疫防制研究进展,北京农业学院学报,1996,11：115-123
[10].Kalata,E.F; and C.Baldanf.:Newcastle disease in free-living and pet birds. In D.J.Alexander (ed.). Newcastle Disease. Klvwer Acadmic Publishers, Boston.MA.1988:197-246
[11].Zhiyu wang and Ronald M.Lorio. Amina acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain, Journal of General Virilogy,1999,80:749-753
[12].Millar N S. Cgambers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle disease virus. J Gen Virol,1986,67:1917-1927
[13].Young J K, hicks R P. The role of leucine residues in the structure and funcyion of a leucine zipper peptide inhabitor of Paramyxovirus (NDV) fusion virology 1998,243:21-31
[14].Nagai, Y. Shimokara K. Yoshida T,et al The spread of a parhogenic and the apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins. J Gen Virol,1999.45:263-272
[15], Millar N S, Cgamgers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle diseasevirus. J Gen Virol,1986,67:1917-1927
[16].Millar Ns, Cgambers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle diseasevirus. J Gen Virol,1986,67:1917-1927
[17].赵军,张秀根,表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建。中国病毒学,1999。14(3)：259-264
[18].Iorio R.M, Bratt M A, Monoclonal antibodies as functional probes of the HN glycoprotein of Newcastle disease virus:antigenic separation of the hemagglutinating and neuraminidasesites: Journal of Immunol,1994.133:2215-2219
[19].Umino Y. Kohama t, Kohase M. Biological functions of monospecific antibodies to envelope glycoproteins of Newcastle disease virus. Archives of Virology,1994,81:53-65
[20].吴艳涛,刘秀焚,张如宽：新城疫病毒F48E8株融合蛋白基因序列分析,病毒学报,1999。15(2)：143-146
[21].Toyoda T. Sakaguchi T, Hirota H, et al. NDV evolution:II Lack of gene recombination on generating virulent and a virulent strains. Virlogy,1989,169:273-282
[22].Chambers P.Millar N S. Nucleotide sequence of the gene encoding the Fusion glycoprotein of Newcastle disease virus, J Gen Virol,1986,67:2685-2691
[23].王兴龙,金宇,丁壮等,新城疫病毒(长春株)F蛋白基因的克隆和序列分析,中国兽医学报,1999,19(2)：109-111
[24].Glickman R L Syddall R J. Lorio RM, et al. Quantitative basic redidue requirements in the eleavage-activation site of the fusion glycoprotein as a determinant of virulence fro Newcastle disease virus. J Virol,1988,62:354-356
[25].Gorman J. J Biol, Chem, Mutations in the Newcastle disease virus hemagglutinin-neuraminidase protein that interfere with its ability to interact with the homologous F protein in the promotion of fusion.1988,263:12522-12528
[26]. Serget T A, McFinners L W, Morrison TGA single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion, Journal of Virology,2000,74(11): 5101-5107
[27].曹殿军,刘明,王丽林等,新城疫病毒F18E9株病毒糖蛋白的功能分析,中国兽医学报,1996,16(6)：534-539
[28].Sergel T,McQuain LW, Peeples M E, et al The attachment function of the Newcastle disease virus hemagglutinin-neuraminidase protein can be separated from fusion promotions by mutation. Virology1993,193：717-726
[29]. Sergel Germano T, McQuain C, Morrison T. Mutations in the fusion peptide and heptad repeat regions of the New castle disease virus fusion protein block fusion, Virologu 1993:196:831-834
[30].R.A. Lamb.Paramyxovirus fusion:a hypothesis for changes. Virology 1993.197:1-11
[31].Bulter D, Maurice J. Pracrical development of genetically engineered animals, Nature, 1997:47(2):113-117
[32].Robert, A.I. Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cells. Virology,1993,195:1-11
[33].凌育荣,郭玉强：疫苗免疫在禽病控制上的进展,动物医学进展,2000,21(1)：9-12,34
[34].Karaca K, Sharma J M. Recombinant fowlpox virus copressing chicken typel IFNand Newcastle disease virus HN and F genes, vaccine.1998,16(16):1496-1503
[35].钱建匕,钟声：基因工程技术在畜牧兽医上的应用及展望,畜牧与兽医,1999,31(1)：41-44
[36].吴有金,万常吉：基因工程技术在免疫应用方面的研究进展,江西畜牧兽医杂志,2002.2：325
[37].孟国良,李婉涛：新一代畜禽疫苗研究现状以及前景,四川畜牧兽医,1998.86(6)54-55
[38].Scheid A, Choppin P W. Isolation and purification of the envelope proteins of Newcastle disease virus. J Gen Virol,1973,11:263-271
[39].Nagy E, Derbyshire J B, Dobos P et al. Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system. Vorology,1990,176:426-438
[40].Yilma, T. Expressing of the Newcastle disease virus fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus. Avian Dis.1994,38(4):772-777
[41].Yilma, T. Expressing of the Newcastle disease virus fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus. Avian Dis.1994,38(4):772-777
[42].Taylor J.Edbauer C L. Newcastle disease virus fusion protein expressed in a fowl pox virus recombinant confers protection in chickens [J]. Gen Viral,1990.64:1441-1450
[43].Edbauer C, Weinberg R, J, et al. Protection of chickens with a recombinant fowl pox virus expressing the Newcastle disease virus haemagglutinin-neuraminidase gene. Virology,1990,79:901-904
[44].Ogawa R, Yangdia N, Seaki S, et al. Recombinant fowl pox virus inducing protective immunity against Newcastle disease and fowl pox viruses. Vaccine,1990,8:486-490
[45].Boursnell MEG, Green PF, Sampbell JIA et al. insertion of the fusion gene from Newcastle disease virus into a nonr-essential region in the terminal repeates of fowlpox virus and demonstration of protective immunity induced by the recombinant. Journal of the General Virology,1990,171:621-628
[46].Boursnell M E.Green P F. Samson A C R recombinant fowlpox virus expressing the haemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV Virol.,1990,178:297-300
[47].曹殿军,刘惠莉,于莉林等。新城疫病毒HN基因在重组鸡痘病毒中的表达及保护性的研究。中国免疫学杂志,1998,14(1)：61-64
[48]. Morgan R W, Cantello J L, McDermott C H. Transfection of chicken embryo fibroblasts with Mark's disease virus DNA. Avian Disease,1990,34:345-351
[49].Mittal S K, Perevec L, Grannam F, et al. Protection of cattle from hoving adenovirus type-3 based vector. J gen virol,1995,76:93-102
[50].Choe V H, Huang W L, chi W K, et al. Inprovent of Hepatitis B virus DNA vaccines by lasmids coexpressing Hepatitis B surface antigen and interleukin-2 J of Virology,1997(1):169-178
[51]. Wolfgang W, Leitner HY, Nicholas P.DNA and RNA-based vaccines:principal, progress and prospects. Vaccine,2000,18:765-777
[52].Rolf M. Zinkernagel. HansHengartner. Antivirus immunity, immunology today,1997,Vol 18(6):258-259
[53].王莉林,霍龙飞,曹殿军等。新城疫病毒融合蛋白基因(F)的克隆及鉴定,全国首届禽病分子生物学研讨会论文汇编,1998：28
[54].宋长绪,刘福安,杜伟贤等。新城疫病毒融合蛋白裂解点基因的分离克隆及在原核细胞中的表达。中国兽医学报,1996,16(6)：527-532
[55].刘伟忠,吴艳涛,刘秀梵等。新城疫病毒F48E8株血凝-神经胺酸梅基因的克隆与鉴定,中国兽医学报,1997,27(6)：16-18
[56].丁壮,金定一,王兴龙等。新城疫病毒HN蛋白基因真核表达载体的构建及其表达。中国畜牧兽医学会禽病学会分会第十次学术研讨会论文集,2000：14-17
[57].Bangham A D, Standish M M, Diffusion of univalentions across the lamellar of swollen phospholipids. J Mol Biol,1965,13:1238-252
[58].Szoka F, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA,1988,75:4194-4198
[59].Kim S, Turker M S. Proparation of multivescular liposomoes. Biochem Biophys Acta. 1983,723:339-348
[60].Deamer D W. Bangham A D.Large volume liposomes by another vaporization methed, Biochem Biophys Acta,1986,443:629-635
[61].Ohsawa T, Miura H. Evalution of a new liposmoes preparation technique, the freeze-thawing method, using L-asparaginase as a model drug. Chem. Pharm Bullm 1985,33:3945-3952
[62].Ohsawa T. Miura H. Impovement of encapsulation efficacy of water-soluble drug in liposomes formed by the freeze-thawing methjod. Chempharm Bull,1985,33:3945-3952
[63].张丰德,樊廷玉,现代生物技术,南开大学出版社,1996,67-74
[64].陈吉祥,李广林,薛飞群等,脂质体在生物学中的应用新进展,甘肃畜牧兽医,2000,152(3)：28-32
[65]. Allison A C.Liposomes as immunological adjuvants. Nature,1974,254-254
[66].陈丽凤,张西臣,杨永胜等,脂质体的制备及其在兽医学中的。中国兽医寄生虫病,1999,7(4)：56-60
[67].裴瑾,杨翰仪,白细胞介素-2脂质体的制备及其活性和稳定性的研究。中国生物制品学杂志,1996,9(4)：149-151
[68].费丽华,李力,林星石等。冻干水化(DRV)型脂质体作为免疫佐剂可行性的研究,免疫学杂志,2000,16(3)：193-195
[69].Martorell D, Duust R A,Siehert S T A.Lioposome dehydration on nitrocellulose and its application in a biotinimmunoassay. Analvtical Biochemistry,271,1999(2):177-185
[70].徐葛林,脂质体用作免疫佐剂研究进展,《国外医学》预防、诊断、治疗用生物制品分册,1996,19(1)：10-13
[71].Hopz-Berestein QHopfer R L,Mehta R et al. Liposome-encapsulated amphoteriein B for treatment of disseminated candidiasis in neutropenic mice. Infect Dis,1984,150:278-283
[72].Lopz-Berestein G,Hopfer R L,Mehta R et al. Prophylaxis of candidaalbicans infection in neutropenc mice with liposome-encapsulated amphotericin B.Antimicrob agents Chemother 1984,25:366-367
[73].Ogawa Y,Yagi N,Kodaka M, et al. Synthesis of a novel liposomeptide with alpha-melanocyte-stimulatinghormone peptide ligand and its effect on liposome stability. Lipids,34, 1999(4):387-394
[74].Wilson T. Biologocal properties of poliovirus encapsulated in lipid vesieles antibody resistance and infectivity in virus resistant cells. Proc Natl Acad SCI, USA,1997,74:3471-3473
[75].Ostro M.Evidence for translation of rabit globin mRNA after liposome-mediated insertioin into a human cell line. Nature,1978,274:921-922
[76].Perrie Y,Gregoriadis G Liposome-entrapped plasmid DNA:characterization studies. Biochimica Bt Biophysica Acta,2000(2):125-132
[77].王若世,王兴龙,韩文翰等,现代动物免疫学,长春：吉林科学技术出版社,1996：163
[78].刘玉斌,荀仕金。动物免疫学实验技术,吉林科技出版社,1989
[79].郑艳,梁传金,贺英菊等,脂质体包被粉尘螨变应原的研究,免疫学杂志,1998,14(3)：191-193
[80].陈吉祥,李广林,薛飞群等,阳离子脂质体的制备及其与禽流感HA基因作用的研究。中国兽医科技,1999,29(2)：18-20
[81].肖克党,顾建华,徐敏华等,反胶束穿过界面膜组装单层免疫脂质体。生物化学与生物物理学学报,1997,29(6)：617-620
[82].陈吉祥,李广林,薛飞群等。禽流感HA基因疫苗脂质体的制备及其理化性质。中国兽医学报,1999,19(3)：230-232
[1].卡尔尼克BW主编,2000,禽流感,第十版,高相等译,北京：北京农业出版,465-460。
[2].马文军,于康震,1999,禽流感疫苗,中国预防兽医学报,21(5)：398-400。
[3]. Stone HA.1987. Effieacy of anain inflenza oil emulsion vaccine in chickens of various ages,Avian DiS,31:483-490.
[4]. Crawford J, Wilkinson B, Vosnessensky A, Ct al.1999. Baculonims-derived hemagglutinin vaccines protect againet lethal inflenza infections by anain H7 and H5 subtypes. Vaccine,17:2265-74.
[5].黄庚明,辛朝安,2001,禽流感病毒血凝素(HA)基因的研究进展,广东畜牧兽医科技,28(2)：3-5
[6]. Webster R G, Kawaoka Y, Taylor J, et al.1991. E～cacy of nucleoprotein and hemagglutinin antigens expressed in fowlpox virus as vaccine for inflenza in chickens. Vaccine,9:303-308.
[7]. Beard C W, Schnitzlein W M, Tripathy D N. Protection ofchickens against higmy pathogenic avian infhnza virus(HSN5)by recombinant fowlpox viruses. Avian Dis,1991,35:356-359.
[8]. Webster RG, et al. Immunity to Mexican H5N2 avian influenza viruses induced by a fowl pox. H5 recombinant. Avian Dis,1996,40:461-465.
[9]. B.Rajendra Krishnan.Current status of DNA vaccines in veterinary medicine. AdvancedDrugDefiveryReview,2000,43:3-11.
[10].Robin H L, Hunt L A, Webster R G.1993. Protection against a lethal inflenza virus challenge by immunization with a hemagglutinin-expressing plasmid DNA Vaccine.11(9):957-960.
[11].唐秀英,田国斌,于康震等.1999.禽流感油乳剂灭活苗的研究。中国预防兽医学报,21(6)：401-405
[12].程坚,表达和共表达H9亚型禽流感病毒和鸡Ⅱ型干扰素基因的重组鸡痘病毒,2000届博士论文
[13]. Dennis J. Alexander. A review of avian influenza in different bird species.Vet Microbiol,2000,74:3-13
[14].TAISUKE HORIMOTO, YOSHIHIRO KAWAOKA. Pandemic Threat Posed by Avian Influenza A virus. CLINICAL MICROBIOLOGY REVIEWS. Jan.2001,P.129-149
[15].D. E. Swayne, J. R. Beck, et al. Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines. Avian Pahtol,1999,28:245-255
[16].蔡宝祥.家畜传染病学.第三版.中国农业出版社,1996.122-123
[17].陈化兰,于康震等.DNA免疫诱导鸡对禽流感病毒的免疫保护反映,中国农业科学,1998,31(5)：63-68.
[18].Ifonso Clavijo, Jose Riva, et al. Assessment of the pathogenicity of an emu-orgin influenza A H5 virus in ostriches (strnthio camelus). Avian Pathol,2001,30:83-89
[19].SimonS. Avian influenza:controland prevention. Poutry International,1998 (4):32-35
[20]. Alexander D J. Orthomyxovirus infections. IN Viral infections. In Viral infection of vertebrate. Volume3:Viral infections of birds.287-316
[1].郑明球,鸡新城疫。甘孟候主编,《中国禽病学》第88-24页,中国农业出版社,北京,1999年。
[2].徐秀龙,刘秀梵,1988。鸡新城疫病毒单克隆抗体及测定的毒株的抗原性差异。病毒学报,4(1)：39—44。
[1].中国兽药典委员会编,中华人民共和国兽药典,附录122—125,1990,农业出版社,北京。
[1]. International Animal Health Code-1999 edition.OIE.
[2]. B.C.Easterday.et al.1997.Influenza.In B.W.Calnek,etal.(eds).
Diseases of Poultry.Iowa state University Press,Ames,Iowa,USA.pp583-606
[3]. SmionShane,1998.Avianin fluenza:Control and prevention.Poultry Intemational.April 32-45
[4].唐秀英等。H9亚型禽流感的流行与防制。中国畜牧兽医学会禽病学分会第10次学术研讨会论文集。2000年9月。杭州pp125-17
[5].OIE哺乳动物、禽类和蜜蜂A类和B类疾病诊断试验和疫苗标准手册。1996年OIE编著,农业部畜牧兽医局翻译出版,pp136-139
[6].韦婷等,广西暴发中低毒力禽流感情况报告,中国畜牧兽医学会禽病学分会第10次学术研讨会论文集,2000年9月。杭州pp118-120
[7].刘月焕等,H9N2亚型禽流感病毒的分离鉴定,中国畜牧兽医学会禽病学分会第10次学术研讨论文集。2000年9月杭州pp113-115
[1]. Calnek B W主编(高福等译),禽病学,第9版,北京：北京农业大学出版社,1991,126-132
[2]. Gross W B.The development of'air sac disease".Avian Dis,1961,5:431-439
[3]. Gross W B.Escherichia coli as a complicating factor in Newcastle disease vaccination.Avian Dis,1961.5:132-134
[4]. Smith H W,J K A cook and Z E Passell.The experimental infection of chickens with mixtures of infectious bronchitis and Escherichia coli.J Gen Virol,1985,66:777-786
[5].高崧,刘秀梵,张如宽等。我国部分地区禽病原性大肠杆菌的分离与鉴定,畜牧兽医学报,1999,30(2)：164-171
[6].曹澍泽等主编。兽医微生物学及免疫学技术。北京：北京农业大学出版社,第1版,1992,223-224
[7].唐秀英。小心防范禽流感,中国动物保建,1999,第2期：28
[1]. Smion Shane. Avian influenza:control & prevention. Poultry international. April 1998,32-45
[2]. D.J.Alexander.禽流感的世界流行情况及致病的分子基础。第十一届世界禽病会议资料
[3].甘孟侯主编。禽流感。北京农业大学出版社,45-50
[4]. Gillian M.Air.Sequence relationships among the hemaggiutinin genes of 12 subtype of influenza A virus. Proc Natl Acad Sci(1981),78,7639-73435
[5].E.Nobusawa,T.Aoyama,H.Kato.Comparison of complete aminoacid sequences and receptor-binding properties among 13 serotype of hemagglutinin of influenza Avirus.Virology(1991),182,475-485
[6].吴艳涛,刘秀梵,张如宽等。应用酚-SDS法提纯新城疫病毒基因组RNA[J]。江苏农业学院学报,1996,17(4)：73-74
1、 中国兽药典委员会编,中国人民共和国兽药典,附录122-125,1990,农业出版社,北京
[1].郑明球,鸡新城疫。甘孟候主编,《中国禽病学》第8—24页,中国农业出版社,北京,1999年。
[2].唐秀英等。H9亚型禽流感的流行与防制。中国畜牧兽医学会禽病学分会第10次学术研讨会论文集。2000年9月。杭州pp125-17
[3].韦婷等,广西暴发中低毒力禽流感情况报告,中国畜牧兽医学会禽病学分会第10次学术研讨会论文集,2000年9月。杭州pp118-120
[4].刘秀梵等,禽流感(H9亚型)油乳剂灭活苗的研制,待发表
[5].英国兽药典.1985年版.新城疫灭活疫苗
[1].黄国模等.新城疫油乳剂灭活苗接种鸡HI抗体产生时间与效价变化,中国畜禽传染病,1993(2)：8-10
[2].杨增歧等,鸡新城疫油乳剂灭活苗的免疫效力试验,中国畜禽传染病,1997(6)：34-36
[3].刘秀梵等,禽流感(H9亚型)油乳剂灭活苗的研制,待发表
1杨汉春,1996,动物免疫学 中国农业大学出版社
2张振兴等,1996,实用兽医生物技术,中国农业出版社
3王宗元,1997,动物营养代谢病与中毒病学,中国农业出版社
4张益民等,1996,提高制苗抗原浓度的生物与化学技术研究,畜禽重大疫病免疫防治研究进展,中国农业出版社
5张永光,高浓度口蹄疫病毒抗原制造工艺,畜禽重大疫病免疫防治研究进展,中国农业出版社
6黄兵等,新城疫抗原超滤技术研究,中国预防兽医学报,2000(2)：108-110
[2].陆承平,兽医微生物学,第三版,北京：中国农业出版社,2001年
[3].中国农业科学院哈尔滨兽医研究所.家畜传染病学.农业出版社,1986
[4]. Hu X R, Ray and R W campans. Functional interaction between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses. J. virol.1992,66:1528-1534
[5]. Langedijk.J P M.F.J Dans. Sequence and structure alignment of paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin. J.Virol.1997,71:6155-6167
[6]. LorioR M. syddall R J.P.et al:Neutralization map of the HN glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition. Journal of Virology,1991,65:4999-5006
[7].B.W卡尔尼卡：禽病学.北京农业大学出版社,1991,第九版
[8].张振兴,姜平,实用兽医生物制品技术,中国农业科技出版社,1996
[9].胡子信,张歧蜀,门常平：鸡新城疫免疫防制研究进展,北京农业学院学报,1996,11：115-123
[10].Kalata,E.F; and C.Baldanf.:Newcastle disease in free-living and pet birds. In D.J.Alexander (ed.). Newcastle Disease. Klvwer Acadmic Publishers, Boston.MA.1988:197-246
[11].Zhiyu wang and Ronald M.Lorio. Amina acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain, Journal of General Virilogy,1999,80:749-753
[12].Millar N S. Cgambers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle disease virus. J Gen Virol,1986,67:1917-1927
[13].Young J K, hicks R P. The role of leucine residues in the structure and funcyion of a leucine zipper peptide inhabitor of Paramyxovirus (NDV) fusion virology 1998,243:21-31
[14].Nagai, Y. Shimokara K. Yoshida T,et al The spread of a parhogenic and the apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins. J Gen Virol,1999.45:263-272
[15], Millar N S, Cgamgers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle diseasevirus. J Gen Virol,1986,67:1917-1927
[16].Millar Ns, Cgambers P, Emmerson P T, et al. Nucleotide sequence analysis of haemagglutinin-neuraminidase gene of Newcastle diseasevirus. J Gen Virol,1986,67:1917-1927
[17].赵军,张秀根,表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建。中国病毒学,1999。14(3)：259-264
[18].Iorio R.M, Bratt M A, Monoclonal antibodies as functional probes of the HN glycoprotein of Newcastle disease virus:antigenic separation of the hemagglutinating and neuraminidasesites: Journal of Immunol,1994.133:2215-2219
[19].Umino Y. Kohama t, Kohase M. Biological functions of monospecific antibodies to envelope glycoproteins of Newcastle disease virus. Archives of Virology,1994,81:53-65
[20].吴艳涛,刘秀焚,张如宽：新城疫病毒F48E8株融合蛋白基因序列分析,病毒学报,1999。15(2)：143-146
[21].Toyoda T. Sakaguchi T, Hirota H, et al. NDV evolution:II Lack of gene recombination on generating virulent and a virulent strains. Virlogy,1989,169:273-282
[22].Chambers P.Millar N S. Nucleotide sequence of the gene encoding the Fusion glycoprotein of Newcastle disease virus, J Gen Virol,1986,67:2685-2691
[23].王兴龙,金宇,丁壮等,新城疫病毒(长春株)F蛋白基因的克隆和序列分析,中国兽医学报,1999,19(2)：109-111
[24].Glickman R L Syddall R J. Lorio RM, et al. Quantitative basic redidue requirements in the eleavage-activation site of the fusion glycoprotein as a determinant of virulence fro Newcastle disease virus. J Virol,1988,62:354-356
[25].Gorman J. J Biol, Chem, Mutations in the Newcastle disease virus hemagglutinin-neuraminidase protein that interfere with its ability to interact with the homologous F protein in the promotion of fusion.1988,263:12522-12528
[26]. Serget T A, McFinners L W, Morrison TGA single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion, Journal of Virology,2000,74(11): 5101-5107
[27].曹殿军,刘明,王丽林等,新城疫病毒F18E9株病毒糖蛋白的功能分析,中国兽医学报,1996,16(6)：534-539
[28].Sergel T,McQuain LW, Peeples M E, et al The attachment function of the Newcastle disease virus hemagglutinin-neuraminidase protein can be separated from fusion promotions by mutation. Virology1993,193：717-726
[29]. Sergel Germano T, McQuain C, Morrison T. Mutations in the fusion peptide and heptad repeat regions of the New castle disease virus fusion protein block fusion, Virologu 1993:196:831-834
[30].R.A. Lamb.Paramyxovirus fusion:a hypothesis for changes. Virology 1993.197:1-11
[31].Bulter D, Maurice J. Pracrical development of genetically engineered animals, Nature, 1997:47(2):113-117
[32].Robert, A.I. Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cells. Virology,1993,195:1-11
[33].凌育荣,郭玉强：疫苗免疫在禽病控制上的进展,动物医学进展,2000,21(1)：9-12,34
[34].Karaca K, Sharma J M. Recombinant fowlpox virus copressing chicken typel IFNand Newcastle disease virus HN and F genes, vaccine.1998,16(16):1496-1503
[35].钱建匕,钟声：基因工程技术在畜牧兽医上的应用及展望,畜牧与兽医,1999,31(1)：41-44
[36].吴有金,万常吉：基因工程技术在免疫应用方面的研究进展,江西畜牧兽医杂志,2002.2：325
[37].孟国良,李婉涛：新一代畜禽疫苗研究现状以及前景,四川畜牧兽医,1998.86(6)54-55
[38].Scheid A, Choppin P W. Isolation and purification of the envelope proteins of Newcastle disease virus. J Gen Virol,1973,11:263-271
[39].Nagy E, Derbyshire J B, Dobos P et al. Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system. Vorology,1990,176:426-438
[40].Yilma, T. Expressing of the Newcastle disease virus fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus. Avian Dis.1994,38(4):772-777
[41].Yilma, T. Expressing of the Newcastle disease virus fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus. Avian Dis.1994,38(4):772-777
[42].Taylor J.Edbauer C L. Newcastle disease virus fusion protein expressed in a fowl pox virus recombinant confers protection in chickens [J]. Gen Viral,1990.64:1441-1450
[43].Edbauer C, Weinberg R, J, et al. Protection of chickens with a recombinant fowl pox virus expressing the Newcastle disease virus haemagglutinin-neuraminidase gene. Virology,1990,79:901-904
[44].Ogawa R, Yangdia N, Seaki S, et al. Recombinant fowl pox virus inducing protective immunity against Newcastle disease and fowl pox viruses. Vaccine,1990,8:486-490
[45].Boursnell MEG, Green PF, Sampbell JIA et al. insertion of the fusion gene from Newcastle disease virus into a nonr-essential region in the terminal repeates of fowlpox virus and demonstration of protective immunity induced by the recombinant. Journal of the General Virology,1990,171:621-628
[46].Boursnell M E.Green P F. Samson A C R recombinant fowlpox virus expressing the haemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV Virol.,1990,178:297-300
[47].曹殿军,刘惠莉,于莉林等。新城疫病毒HN基因在重组鸡痘病毒中的表达及保护性的研究。中国免疫学杂志,1998,14(1)：61-64
[48]. Morgan R W, Cantello J L, McDermott C H. Transfection of chicken embryo fibroblasts with Mark's disease virus DNA. Avian Disease,1990,34:345-351
[49].Mittal S K, Perevec L, Grannam F, et al. Protection of cattle from hoving adenovirus type-3 based vector. J gen virol,1995,76:93-102
[50].Choe V H, Huang W L, chi W K, et al. Inprovent of Hepatitis B virus DNA vaccines by lasmids coexpressing Hepatitis B surface antigen and interleukin-2 J of Virology,1997(1):169-178
[51]. Wolfgang W, Leitner HY, Nicholas P.DNA and RNA-based vaccines:principal, progress and prospects. Vaccine,2000,18:765-777
[52].Rolf M. Zinkernagel. HansHengartner. Antivirus immunity, immunology today,1997,Vol 18(6):258-259
[53].王莉林,霍龙飞,曹殿军等。新城疫病毒融合蛋白基因(F)的克隆及鉴定,全国首届禽病分子生物学研讨会论文汇编,1998：28
[54].宋长绪,刘福安,杜伟贤等。新城疫病毒融合蛋白裂解点基因的分离克隆及在原核细胞中的表达。中国兽医学报,1996,16(6)：527-532
[55].刘伟忠,吴艳涛,刘秀梵等。新城疫病毒F48E8株血凝-神经胺酸梅基因的克隆与鉴定,中国兽医学报,1997,27(6)：16-18
[56].丁壮,金定一,王兴龙等。新城疫病毒HN蛋白基因真核表达载体的构建及其表达。中国畜牧兽医学会禽病学会分会第十次学术研讨会论文集,2000：14-17
[57].Bangham A D, Standish M M, Diffusion of univalentions across the lamellar of swollen phospholipids. J Mol Biol,1965,13:1238-252
[58].Szoka F, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA,1988,75:4194-4198
[59].Kim S, Turker M S. Proparation of multivescular liposomoes. Biochem Biophys Acta. 1983,723:339-348
[60].Deamer D W. Bangham A D.Large volume liposomes by another vaporization methed, Biochem Biophys Acta,1986,443:629-635
[61].Ohsawa T, Miura H. Evalution of a new liposmoes preparation technique, the freeze-thawing method, using L-asparaginase as a model drug. Chem. Pharm Bullm 1985,33:3945-3952
[62].Ohsawa T. Miura H. Impovement of encapsulation efficacy of water-soluble drug in liposomes formed by the freeze-thawing methjod. Chempharm Bull,1985,33:3945-3952
[63].张丰德,樊廷玉,现代生物技术,南开大学出版社,1996,67-74
[64].陈吉祥,李广林,薛飞群等,脂质体在生物学中的应用新进展,甘肃畜牧兽医,2000,152(3)：28-32
[65]. Allison A C.Liposomes as immunological adjuvants. Nature,1974,254-254
[66].陈丽凤,张西臣,杨永胜等,脂质体的制备及其在兽医学中的。中国兽医寄生虫病,1999,7(4)：56-60
[67].裴瑾,杨翰仪,白细胞介素-2脂质体的制备及其活性和稳定性的研究。中国生物制品学杂志,1996,9(4)：149-151
[68].费丽华,李力,林星石等。冻干水化(DRV)型脂质体作为免疫佐剂可行性的研究,免疫学杂志,2000,16(3)：193-195
[69].Martorell D, Duust R A,Siehert S T A.Lioposome dehydration on nitrocellulose and its application in a biotinimmunoassay. Analvtical Biochemistry,271,1999(2):177-185
[70].徐葛林,脂质体用作免疫佐剂研究进展,《国外医学》预防、诊断、治疗用生物制品分册,1996,19(1)：10-13
[71].Hopz-Berestein QHopfer R L,Mehta R et al. Liposome-encapsulated amphoteriein B for treatment of disseminated candidiasis in neutropenic mice. Infect Dis,1984,150:278-283
[72].Lopz-Berestein G,Hopfer R L,Mehta R et al. Prophylaxis of candidaalbicans infection in neutropenc mice with liposome-encapsulated amphotericin B.Antimicrob agents Chemother 1984,25:366-367
[73].Ogawa Y,Yagi N,Kodaka M, et al. Synthesis of a novel liposomeptide with alpha-melanocyte-stimulatinghormone peptide ligand and its effect on liposome stability. Lipids,34, 1999(4):387-394
[74].Wilson T. Biologocal properties of poliovirus encapsulated in lipid vesieles antibody resistance and infectivity in virus resistant cells. Proc Natl Acad SCI, USA,1997,74:3471-3473
[75].Ostro M.Evidence for translation of rabit globin mRNA after liposome-mediated insertioin into a human cell line. Nature,1978,274:921-922
[76].Perrie Y,Gregoriadis G Liposome-entrapped plasmid DNA:characterization studies. Biochimica Bt Biophysica Acta,2000(2):125-132
[77].王若世,王兴龙,韩文翰等,现代动物免疫学,长春：吉林科学技术出版社,1996：163
[78].刘玉斌,荀仕金。动物免疫学实验技术,吉林科技出版社,1989
[79].郑艳,梁传金,贺英菊等,脂质体包被粉尘螨变应原的研究,免疫学杂志,1998,14(3)：191-193
[80].陈吉祥,李广林,薛飞群等,阳离子脂质体的制备及其与禽流感HA基因作用的研究。中国兽医科技,1999,29(2)：18-20
[81].肖克党,顾建华,徐敏华等,反胶束穿过界面膜组装单层免疫脂质体。生物化学与生物物理学学报,1997,29(6)：617-620
[82].陈吉祥,李广林,薛飞群等。禽流感HA基因疫苗脂质体的制备及其理化性质。中国兽医学报,1999,19(3)：230-232
[1].卡尔尼克BW主编,2000,禽流感,第十版,高相等译,北京：北京农业出版,465-460。
[2].马文军,于康震,1999,禽流感疫苗,中国预防兽医学报,21(5)：398-400。
[3]. Stone HA.1987. Effieacy of anain inflenza oil emulsion vaccine in chickens of various ages,Avian DiS,31:483-490.
[4]. Crawford J, Wilkinson B, Vosnessensky A, Ct al.1999. Baculonims-derived hemagglutinin vaccines protect againet lethal inflenza infections by anain H7 and H5 subtypes. Vaccine,17:2265-74.
[5].黄庚明,辛朝安,2001,禽流感病毒血凝素(HA)基因的研究进展,广东畜牧兽医科技,28(2)：3-5
[6]. Webster R G, Kawaoka Y, Taylor J, et al.1991. E～cacy of nucleoprotein and hemagglutinin antigens expressed in fowlpox virus as vaccine for inflenza in chickens. Vaccine,9:303-308.
[7]. Beard C W, Schnitzlein W M, Tripathy D N. Protection ofchickens against higmy pathogenic avian infhnza virus(HSN5)by recombinant fowlpox viruses. Avian Dis,1991,35:356-359.
[8]. Webster RG, et al. Immunity to Mexican H5N2 avian influenza viruses induced by a fowl pox. H5 recombinant. Avian Dis,1996,40:461-465.
[9]. B.Rajendra Krishnan.Current status of DNA vaccines in veterinary medicine. AdvancedDrugDefiveryReview,2000,43:3-11.
[10].Robin H L, Hunt L A, Webster R G.1993. Protection against a lethal inflenza virus challenge by immunization with a hemagglutinin-expressing plasmid DNA Vaccine.11(9):957-960.
[11].唐秀英,田国斌,于康震等.1999.禽流感油乳剂灭活苗的研究。中国预防兽医学报,21(6)：401-405
[12].程坚,表达和共表达H9亚型禽流感病毒和鸡Ⅱ型干扰素基因的重组鸡痘病毒,2000届博士论文
[13]. Dennis J. Alexander. A review of avian influenza in different bird species.Vet Microbiol,2000,74:3-13
[14].TAISUKE HORIMOTO, YOSHIHIRO KAWAOKA. Pandemic Threat Posed by Avian Influenza A virus. CLINICAL MICROBIOLOGY REVIEWS. Jan.2001,P.129-149
[15].D. E. Swayne, J. R. Beck, et al. Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines. Avian Pahtol,1999,28:245-255
[16].蔡宝祥.家畜传染病学.第三版.中国农业出版社,1996.122-123
[17].陈化兰,于康震等.DNA免疫诱导鸡对禽流感病毒的免疫保护反映,中国农业科学,1998,31(5)：63-68.
[18].Ifonso Clavijo, Jose Riva, et al. Assessment of the pathogenicity of an emu-orgin influenza A H5 virus in ostriches (strnthio camelus). Avian Pathol,2001,30:83-89
[19].SimonS. Avian influenza:controland prevention. Poutry International,1998 (4):32-35
[20]. Alexander D J. Orthomyxovirus infections. IN Viral infections. In Viral infection of vertebrate. Volume3:Viral infections of birds.287-316
[1].郑明球,鸡新城疫。甘孟候主编,《中国禽病学》第88-24页,中国农业出版社,北京,1999年。
[2].徐秀龙,刘秀梵,1988。鸡新城疫病毒单克隆抗体及测定的毒株的抗原性差异。病毒学报,4(1)：39—44。
[1].中国兽药典委员会编,中华人民共和国兽药典,附录122—125,1990,农业出版社,北京。
[1]. International Animal Health Code-1999 edition.OIE.
[2]. B.C.Easterday.et al.1997.Influenza.In B.W.Calnek,etal.(eds).
Diseases of Poultry.Iowa state University Press,Ames,Iowa,USA.pp583-606
[3]. SmionShane,1998.Avianin fluenza:Control and prevention.Poultry Intemational.April 32-45
[4].唐秀英等。H9亚型禽流感的流行与防制。中国畜牧兽医学会禽病学分会第10次学术研讨会论文集。2000年9月。杭州pp125-17
[5].OIE哺乳动物、禽类和蜜蜂A类和B类疾病诊断试验和疫苗标准手册。1996年OIE编著,农业部畜牧兽医局翻译出版,pp136-139
[6].韦婷等,广西暴发中低毒力禽流感情况报告,中国畜牧兽医学会禽病学分会第10次学术研讨会论文集,2000年9月。杭州pp118-120
[7].刘月焕等,H9N2亚型禽流感病毒的分离鉴定,中国畜牧兽医学会禽病学分会第10次学术研讨论文集。2000年9月杭州pp113-115
[1]. Calnek B W主编(高福等译),禽病学,第9版,北京：北京农业大学出版社,1991,126-132
[2]. Gross W B.The development of'air sac disease".Avian Dis,1961,5:431-439
[3]. Gross W B.Escherichia coli as a complicating factor in Newcastle disease vaccination.Avian Dis,1961.5:132-134
[4]. Smith H W,J K A cook and Z E Passell.The experimental infection of chickens with mixtures of infectious bronchitis and Escherichia coli.J Gen Virol,1985,66:777-786
[5].高崧,刘秀梵,张如宽等。我国部分地区禽病原性大肠杆菌的分离与鉴定,畜牧兽医学报,1999,30(2)：164-171
[6].曹澍泽等主编。兽医微生物学及免疫学技术。北京：北京农业大学出版社,第1版,1992,223-224
[7].唐秀英。小心防范禽流感,中国动物保建,1999,第2期：28
[1]. Smion Shane. Avian influenza:control & prevention. Poultry international. April 1998,32-45
[2]. D.J.Alexander.禽流感的世界流行情况及致病的分子基础。第十一届世界禽病会议资料
[3].甘孟侯主编。禽流感。北京农业大学出版社,45-50
[4]. Gillian M.Air.Sequence relationships among the hemaggiutinin genes of 12 subtype of influenza A virus. Proc Natl Acad Sci(1981),78,7639-73435
[5].E.Nobusawa,T.Aoyama,H.Kato.Comparison of complete aminoacid sequences and receptor-binding properties among 13 serotype of hemagglutinin of influenza Avirus.Virology(1991),182,475-485
[6].吴艳涛,刘秀梵,张如宽等。应用酚-SDS法提纯新城疫病毒基因组RNA[J]。江苏农业学院学报,1996,17(4)：73-74
1、 中国兽药典委员会编,中国人民共和国兽药典,附录122-125,1990,农业出版社,北京
[1].郑明球,鸡新城疫。甘孟候主编,《中国禽病学》第8—24页,中国农业出版社,北京,1999年。
[2].唐秀英等。H9亚型禽流感的流行与防制。中国畜牧兽医学会禽病学分会第10次学术研讨会论文集。2000年9月。杭州pp125-17
[3].韦婷等,广西暴发中低毒力禽流感情况报告,中国畜牧兽医学会禽病学分会第10次学术研讨会论文集,2000年9月。杭州pp118-120
[4].刘秀梵等,禽流感(H9亚型)油乳剂灭活苗的研制,待发表
[5].英国兽药典.1985年版.新城疫灭活疫苗
[1].黄国模等.新城疫油乳剂灭活苗接种鸡HI抗体产生时间与效价变化,中国畜禽传染病,1993(2)：8-10
[2].杨增歧等,鸡新城疫油乳剂灭活苗的免疫效力试验,中国畜禽传染病,1997(6)：34-36
[3].刘秀梵等,禽流感(H9亚型)油乳剂灭活苗的研制,待发表
1杨汉春,1996,动物免疫学 中国农业大学出版社
2张振兴等,1996,实用兽医生物技术,中国农业出版社
3王宗元,1997,动物营养代谢病与中毒病学,中国农业出版社
4张益民等,1996,提高制苗抗原浓度的生物与化学技术研究,畜禽重大疫病免疫防治研究进展,中国农业出版社
5张永光,高浓度口蹄疫病毒抗原制造工艺,畜禽重大疫病免疫防治研究进展,中国农业出版社
6黄兵等,新城疫抗原超滤技术研究,中国预防兽医学报,2000(2)：108-110