3株单抗对新城疫病毒HN抗原变异的分析

以新城疫病毒(NDV)LaSota株为免疫原研制了3株针对NDVHN蛋白的单抗,分别命名为1E5、C3-B7和E6-F11。3株单抗与NDV不同毒株在血凝抑制试验(HI)、ELISA和病毒中和试验中的反应性不同,而同一株单抗与同一个NDV毒株在HI、ELISA以及病毒中和试验中的反应性一致。其中,单抗1E5与NDV标准株(LaSota、F48E8)、8个NDV分离株反应阳性,而与9个NDV分离株反应阴性;单抗C3-B7、E6-F11与所有NDV毒株反应均为阳性。结果表明,3株单抗均是针对HN蛋白上的中和位点,国内部分NDV流行株的HN抗原至少有1个抗原位点发生了变异。     

抗禽流感H9亚型血凝素单克隆抗体的病毒中和作用

通过血凝抑制(HI)和鸡胚中和试验(VN)证实，本实验室制备的抗禽流感H9M2单抗11A5和11B2株可特异性地抑制H9亚型禽流感病毒的血凝特性，而与禽流感H5和H7亚型以及其他具有血凝性感染禽类的病毒(如新城疫等)不反应。两株单抗腹水HI效价均达到15Log2。中和试验表明：上述两株单抗均可有效抑制H9N2病毒在SPF鸡胚中的增殖，使病毒失去血凝活性．测得11A5和11B2株腹水对H9N2禽流感病毒的半数保护量分别为10^-3.35和10^-4.58。该单抗的研制成功对于进一步建立快速鉴别诊断禽流感H9亚型病毒具有重要意义。     

鹅副黏病毒甘肃地方毒株的分离和初步鉴定

鹅副黏病毒病是一种由鹅传播的新型急性传染病,该病与鸡感染新城疫具有相类似的临床症状和病理变化。2019年1月从甘肃某鹅场病鹅体内分离到一株副黏病毒,将病料处理后接种10日龄鸡胚,进行血凝试验(HA)和血凝抑制试验(HI),最后进行动物试验。结果表明:该病毒具有血凝活性,可以用标准的新城疫阳性血清抑制血凝活性,可能导致未接种新城疫免疫鸡胚的100%死亡。动物试验证明该病毒对鸡具有100%的发病率和致死率。     

具有HI活性的抗H5亚型流感病毒特异性单克隆抗体的研制

以H5N1亚型禽流感病毒分离株A/Duck/Zhejiang/11/00(H5N1)(DZJ/1100)作为免疫原,浓缩纯化后免疫BALB/c小鼠,三次免疫后,取免疫小鼠脾细胞与SP2/O细胞融合,融合阳性细胞用血凝抑制(HI)方法进行检测,阳性细胞株经三代克隆纯化后,获得三株能稳定分泌抗血凝素特异性HI单克隆抗体的杂交瘤细胞株,分别命名为DD7、FG10、CG12.三株杂交瘤细胞株产生的小鼠腹水和细胞培养液上清的HI滴度分别是213、215、211和27、8、23.与其他具有血凝活性的禽类病毒以及其他14个HA亚型的禽流感病毒的交叉HI试验表明:这三株单抗具有良好的禽流感病毒亚型特异性;与其他H5亚型流感病毒分离株的HI试验和中和试验证实这三株单抗具有良好的交叉性(分别为7/8、8/8、8/8)和中和活性(6/8、8/8、8/8).该亚型特异性单克隆抗体的研制成功为H5亚型禽流感病毒的疫情病原学快速诊断提供了坚实的物质保障.     

一株鸽新城疫病毒的分离鉴定

为了对广东地区某鸽场疑似鸽新城疫病毒感染的鸽群进行病原学诊断,试验采用血凝试验(HA)、血凝抑制试验(HI)、F基因扩增及序列测定等一系列综合试验对其进行病原学鉴定。结果表明:该分离株具有血凝活性,且这种血凝性可被新城疫病毒(NDV)标准阳性血清抑制,而不能被减蛋综合征病毒(EDSV)、禽流感病毒(AIV)-H5、AIV-H7、AIV-H9阳性血清抑制;用针对NDV F基因设计的特异性鉴定引物对该分离株进行PCR扩增,可扩增出相应的目的片段;该分离株与天津分离株AG/Tianjin/07的核苷酸序列的相似性高达99%。说明该分离株为鸽新城疫病毒,命名为Pigeon/Guangdong/SD54/2006。     

中等毒力鸡新城疫抗原抗体复合物疫苗的免疫效果

用中等毒力新城疫病毒Mukteswar株作为复合物疫苗抗原,与新城疫抗体配制不同比例的4种复合物疫苗,用1日龄SPF雏鸡进行免疫效果试验。试验1～4组分别免疫复合物疫苗,试验5组免疫常规新城疫活疫苗,试验6组为空白对照组。免疫后3周采血测定血凝抑制(HI)抗体效价,同时用新城疫强毒北京株F48E9攻击。试验1～6组HI抗体效价分别为28.38、29.0、25.5、23.85、28.43、22.14,攻毒后1～6组鸡的死亡率分别为0%、0%、20%、40%、0%及80%。结果表明,中等毒力新城疫病毒Mukteswar株与特异性抗体制备的复合物疫苗,可以减轻疫苗的毒副作用,提高疫苗的安全性,其免疫效果比常规活苗好。     

新城疫病毒HN标记疫苗的构建及免疫效力

将新城疫病毒AI4株的HN蛋白胞外区替换成禽副黏病毒2型T4株HN相应部分,构建获得了一株重组病毒r AI4-T4HNECD,该病毒具有较高繁殖性能,且毒力弱。将该病毒制成灭活疫苗免疫2周龄SPF鸡后,定期采血用于血凝抑制试验(HI),免疫后3周以基因Ⅶd亚型NDV强毒株JS-5-05攻毒,攻毒后定期采血用于HI检测。结果显示:免疫后,该疫苗可有效诱导抗体的产生,并能提供较强的免疫保护。血清学测定数据显示:攻毒前,免疫组鸡血清对AI4株的HI平均效价低于2 log_2,但感染后第5、7天免疫组鸡血清对AI4株的HI平均效价可分别达4.3 log_2和9.4 log_2,而免疫未攻毒组对AI4株的HI效价仍低于2 log_2,根据血清对AI4株HI效价的变化可区分免疫动物和感染动物。因此,重组病毒rAI4-T4HNECD可用于新城疫标记疫苗的开发。     

禽流感病毒单克隆抗体的制备及其抗蛋白抗原的分析

以纯化的H9N2亚型禽流感病毒为抗原,免疫BALB/c小鼠,细胞融合后,经间接ELISA和血凝抑制试验(HI)筛选,获得了8株能稳定分泌抗禽流感病毒单克隆抗体的杂交瘤细胞株。特异性试验证明,8株杂交瘤细胞株诱生小鼠腹水的特异ELISA抗体效价可达1∶3.2×103~1∶5.1×106,其中2株HI效价达212。8株单抗与H5亚型血凝素分型抗原不发生血凝,与减蛋综合征(EDS-76)病毒、传染性支气管炎病毒(IBV)、新城疫病毒(NDV)均不反应。亚类鉴定证实,除1C7单抗为IgG2b外,其他7株均为IgG1亚类。Westernblotting试验分析初步表明,8株单抗至少针对纯化病毒粒子3种不同的蛋白抗原,其中3株针对核蛋白(NP),2株针对基质蛋白M1,2株针对血凝素HA/HA1。对感染细胞的Western blotting分析结果与纯化病毒结果基本一致,其中1株未明显沉淀纯化病毒粒子蛋白的单抗可以与感染细胞的M2蛋白多肽反应。     

H3亚型禽流感病毒血凝素特异性单克隆抗体的制备

以H3亚型禽流感病毒(AIV)免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用血凝抑制试验(HI)筛选阳性杂交瘤细胞并克隆化。结果获得了4株针对H3亚型禽流感病毒血凝素的单克隆抗体,分别命名为1D7、1F9、5A4、5F5。这些单克隆抗体小鼠腹水HI效价为212～214。用H1、H6、H10亚型禽流感病毒各2株,H4、H5、H9和新城疫病毒(NDV)各1株进行特异性试验。结果表明:所有这些单抗仅与H3亚型AIV发生特异性HI反应,而不与其他亚型AIV及NDV反应。用28株H3亚型禽流感病毒进行排谱试验,结果证明:4株单抗均具有广谱性,其中1F9、5A4、5F5与受试的28株H3亚型AIV均反应,而1D7只与其中的26株反应。以上单抗将为控制畜禽及人类的流感提供必需的诊断试剂。     

一株鸽源新城疫病毒的分离鉴定

从广东发病鸽群中采集的拭子样本中分离到1株新城疫病毒,用聚合酶链反应(PCR)、血凝抑制试验(HI)及基因测序等对其进行鉴定。结果表明,此分离株具有血凝活性,且可与新城疫病毒标准阳性血清发生特异性抑制反应;用针对新城疫病毒的F基因及P基因特异性鉴定引物对该分离株进行PCR扩增,可扩增出特异性目的片段;测序及BLAST分析表明,其与美国新城疫病毒分离株Pigeon/US(TX)/98的F基因核苷酸序列相似性高达97%以上。毒株被鉴定为鸽源新城疫病毒,并命名为Pigeon/Guangdong/ZQ-17。     

新城疫病毒F48E9株病毒糖蛋白的功能分析

对抗新城疫病毒（ＮＤＶ）ＨＮ和Ｆ糖蛋白的单克隆抗体（ＭｃＡｂ）的生物学活性，应用ＥＬＩＳＡ、ＨＩ、ＨＬＩ、Ｗｅｓｔｅｒｎｂｌｏｔ等进行了分析，结果证明，ＮＤＶＦ４８Ｅ９株ＨＮ蛋白除有ＨＡ和ＮＡ功能区外，还有一个促进（启动）融合的功能区。此外，还筛选到４株ＮＤＶ强毒株特异性ＭｃＡｂ，为ＮＤＶ强弱毒株的鉴别打下了基础     

Vaccination against Newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine

Vaccination of chickens with an oil-emulsion vaccine containing a recombinant baculovirus that expressed the hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV)-induced hemagglutination-inhibition (HI) and virus-neutralizing antibodies against NDV. HI antibody titers obtained in response to vaccination with the live recombinant virus were higher than those obtained when the recombinant was inactivated with beta-propiolactone, and the titers were lower than those obtained in response to the same HN concentrations in live or beta-propiolactone-inactivated NDV strain B1. The serological response to the recombinant baculovirus was differentiated from the response to NDV by an enzyme-linked immunosorbent assay in which purified NDV nucleoprotein was used as antigen. Chickens vaccinated with the live recombinant or with inactivated NDV resisted an oculonasal challenge with the neurotropic velogenic Texas GB strain of NDV, which was lethal in unvaccinated controls. It was concluded that the HN protein of NDV expressed as a subunit by a recombinant baculovirus was protective against Newcastle disease.     

H5亚型禽流感病毒血凝素单克隆抗体的制备及鉴定

以禽流感病毒A/Chicken/Hubei/327/2004(H5N1)免疫Balb/c小鼠,将免疫鼠脾细胞与SP2/0骨髓瘤细胞融合,用血凝抑制试验筛选细胞培养上清,采用有限稀释法对阳性孔进行克隆,3次克隆后获得7株能稳定分泌抗H5亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞株,分别命名为1C4,1D4,1E12,2E11,4C12,4G2和5E12。细胞培养上清HI效价为24~27,腹水HI效价可达210~218。所有单抗与禽流感H7和H9亚型标准血凝抗原,新城疫病毒和鸡传染性支气管炎病毒无交叉反应。在细胞上的中和试验显示具有较高的中和效价,获得的单克隆抗体可在禽流感流行病学的监测中发挥重要作用。     

Identification of a variable epitope on the Newcastle disease virus hemagglutinin–neuraminidase protein

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.     

鸡新城疫重组鸡痘病毒活疫苗的免疫持续期和加强免疫研究

本研究旨在评价表达新城疫病毒（NDV）血凝素-神经氨酸酶（HN）基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗的免疫持续期和加强免疫对疫苗免疫效力的影响。用rFPV-12LSHN活疫苗免疫14日龄SPF鸡,103PFU/羽,7d即可检测到NDV HI抗体应答,对NDV强毒F48E8株攻毒保护率达100%。一次免疫18周后,对NDV强毒攻击依然提供完全保护。鸡痘病毒（FPV）疫苗免疫4周,再接种rFPV-12LSHN活疫苗,攻毒保护率降低至50%。相反,rFPV-12LSHN免疫4周,随后二次免疫可显著提高对NDV的体液免疫应答水平（P〈0.01）,对NDV强毒攻击的保护率仍然为100%。结果表明,表达NDV HN基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗,能够快速建立坚强免疫力,免疫持续期至少可达18周,rFPV-12LSHN的二次免疫可以提高疫苗的免疫力。     

1株广东鸡源新城疫病毒的分离鉴定和毒力基因分析

从广东粤东地区某商品蛋鸡场的发病鸡群中分离到1株病毒（YS株）,经血凝和血凝抑制试验确定为新城疫病毒（NDV）.对该分离株的F蛋白氨基酸序列的分析结果表明,其F0裂解位点的氨基酸序列为1nR-R-Q-K-R-F117,且含有101K和121V,符合典型NDV强毒株的分子特点.遗传分析结果显示分离株属于基因Ⅶd亚型.F和HN基因的氨基酸相似性分析表明,分离株与基因Ⅶ型NDV Chicken/China/Shandong/02/2010株的相似性最高,均达到99％,而与常用疫苗株B1、V4、Clone30、Mukteswar和La Sota的相似性则较低,分别在86.8％～ 90.4％和87.2％～88.3％之间,说明分离株与经典的NDV毒株存在一定的差异.     

Potentiation of cell-mediated immune responses against recombinant HN protein of Newcastle disease virus by recombinant chicken IL-18

In this study, recombinant fowlpox viruses (rFPV/HN) expressing Newcastle disease virus (NDV) HN protein and rFPV/HN/chIL-18 co-expressing chicken IL-18 (chIL-18) and HN protein have been constructed and characterized. The co-expressed rHN/chIL-18 antigen or rchIL-18, expressed by our previous construct rFPV/chIL-18 and co-administered with NDV rHN, was assessed for its immunostimulatory activities and protection against NDV challenge in 2-week-old chickens. Chickens were vaccinated, intramuscularly, with various amounts of rHN or rHN/chIL-18 mixed with mineral oil. Production of hemagglutination-inhibition (HI) antibody depended on the concentration of the injected rHN or rHN/chIL-18. The lower HI antibody titers were obtained in chickens group rHN/chIL-18/6 and rHN/chIL-18/7, receiving 50 ng rHN/16.5 ng chIL-18 with mineral oil and 20 ng rHN/6.6 ng chIL-18 with mineral oil, respectively, compared to those in chickens rHN/6 and rHN/7, respectively receiving 50 ng and 20 ng rHN with mineral oil alone. However, the same protection rates were obtained from chickens in groups rHN/chIL-18/6 and rHN/6. Chicken groups rHN/chIL-18/7 and rHN/chIL-18/8 showed higher protective achievements than those in groups rHN/7 and rHN/8, respectively. When rchIL-18 was co-injected with 20ng rHN plus mineral oil, low level of HI antibody titer was produced; whereas, higher level of IFN-γ production and full protection rates were obtained. On the other hand, lower levels of IFN-γ production and lower protection rate (67%) were obtained in chickens injected with the same amount of rHN with mineral oil alone. Similar results were obtained when 10 ng rHN was used. Thus, when the concentration of rHN decreased to 50 ng or less, rchIL-18 reduced HI antibody production. The increase in IFN-γ production suggested that the enhancement of the cell-mediated immunity might confer the protection from NDV challenge, even accompanied with low HI antibody induction.     

基因Ⅶ型新城疫病毒HN蛋白单克隆抗体的制备及特性研究

为制备基因Ⅶ型新城疫病毒(NDV)血凝素-神经氨酸酶蛋白(HN)的单克隆抗体(MAb),本研究利用JS/17病毒株的HN重组蛋白和活病毒分别免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞(SP2/0)融合,并通过ELISA、间接免疫荧光和western blot方法筛选,制备了3株特异性识别HN蛋白的单克隆抗体(MAb)。其中,MAb1D4和4D9具有病毒中和活性(VN)及血凝抑制作用(HI),可以识别多种基因型的Ⅱ类NDV,但与Ⅰ类NDV病毒株无反应。MAb 2G8识别线性表位131DYIGGIGKE139,该表位在各病毒株中高度保守。获得的3株MAb可以用于NDV的鉴定及HN蛋白的功能研究。     

Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein

Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.