抗猪甲型H1N1流感病毒血凝素蛋白单克隆抗体的制备与特性分析

本研究旨在制备猪甲型H1N1流感病毒血凝素(HA)蛋白的特异性单克隆抗体.采用RT-PCR方法扩增猪甲型H1N1流感病毒的HA基因,将其克隆至真核表达载体pCAGGS上,获得重组质粒pCAGGS-HA,转染293T细胞,通过间接免疫荧光(IFA)检测表明HA蛋白在293T细胞中得到表达.将pCAGGS-HA以100 μg·只-1剂量免疫5周龄BALB/c小鼠,获得4株稳定分泌抗HA蛋白单克隆抗体(MAb)的杂交瘤细胞株,分别命名为11G7、3C10、3G3和2B11.其中11G7诱导小鼠产生的腹水HI效价为14log2,中和效价为1:8 192,HI试验结果进一步表明11G7只与甲型H1N1流感病毒发生反应,而不与其他H1N1、H1N2、H3N2、H5N1及H9N2亚型流感病毒反应.该MAb的制备将为建立猪甲型H1N1流感病毒与传统亚型SIV的鉴别诊断方法奠定基础.     

湖北株H3N8亚型马流感病毒HA基因的序列测定及其HA蛋白遗传特性分析

2008年从湖北省分离到1株H3N8亚型马流感病毒A/equine/Hubei/6/08。以2002年美国KENTUKY株为模板设计HA基因测序引物,进行RT-PCR,然后测定该分离株的HA基因核苷酸序列。经NCBI上Blast同源性比较发现,与A/equine/Newmarket/5/2003(H3N8)同源性较高为98.7%。HA蛋白遗传进化分析表明该毒株隶属于H3N8亚型马流感病毒中的美洲系福罗里达亚系。该株与OIE现在推荐的疫苗候选株A/equine/Kentuck-y/5/2002(H3N8)HA1蛋白氨基酸序列比对发现有3处氨基酸替换位点;与OIE以往推荐的疫苗候选株A/e-quine/Kentucky/1/1994(H3N8)比对发现有11处氨基酸替换位点。研究结果表明该分离株可作为中国研制马流感疫苗的候选株。     

1株H3N8亚型马流感病毒的分离鉴定及全基因进化分析

2013年8月某养马厂出现疑似马流感疫情,经分离鉴定为H3N8亚型马流感病毒。为了了解该H3N8马流感分离株(A/equine/Xuzhou/01/2013(H3N8))的基因进化情况,本试验比较了该病毒株,我国已报道的H3N8马流感毒株以及目前全球所使用的疫苗株的病毒基因型,也比较了与抗原性相关的氨基酸的变化情况,为科学防控马流感提供参考依据。采集病死马的内脏样本,进行了A型禽流感病毒通用荧光RT-PCR检测,将荧光RT-PCR检测阳性样品处理后接种鸡胚尿囊腔,检测分离毒尿囊液血凝性及HA效价。参照已发表的扩增H3N8马流感病毒全基因引物,经RT-PCR扩增出该H3N8亚型马流感病毒(A/equine/Xuzhou/01/2013(H3N8))的8个基因片段,对其测序构建基因进化树,并进行分子特征分析。经过比较分析,该病毒株的HA基因在遗传进化上属于佛罗里达Ⅱ型马流感病毒,其余7个片段都属于马流感病毒中的美洲型。在5个公认的抗原位点中,A/equine/Xuzhou/01/2013与我国2007年的分离株(A/donkey/Xinjiang/2007),目前的疫苗株(Miami/63,Kentucky/81,Suffolk/89,Newmarket/2/93和Avesta/93)相比均发生了一些位点的变异。我国H3N8亚型马流感病毒存在抗原变异,需要进一步对其致病性、免疫原性等特性进行研究,同时还需加强马流感病毒分子流行病学监测。     

H9N2亚型猪流感病毒山东分离株的分离鉴定及其HA、NP、NS基因序列分析

从山东地区疑似流感发病猪分离到 10株流感病毒 ,经国家流感中心鉴定均为 A型流感病毒 H9N2亚型。将其中 1株 Sw/ SD/ 1/ 2 0 0 3(H9N2 )的血凝素基因 (HA)、核蛋白基因 (NP)和非结构蛋白基因 (NS)进行克隆与测序 ,与Gen Bank收录的其他猪流感和禽流感 H9N2亚型的相关基因进行比较 ,推测 Sw/ SD/ 1/ 2 0 0 3(H9N2 )可能源于禽流感病毒 H9N2亚型和 H5 N1亚型的重组病毒 ;Sw/ SD/ 1/ 2 0 0 3的 HA氨基酸裂解位点与其他 H9N2亚型不同 ,Sw/ SD/1/ 2 0 0 3的 HA氨基酸裂解位点是 R- S- L- R- G,而其他猪流感和禽流感 H9N2亚型都是 R- S- S- R- G。     

广西1株H3N8型马流感病毒的分离与鉴定

对具有流感临床症状的病马组织经处理后接种SPF鸡胚,分离到1株流感病毒。该病毒经鸡胚接种7代后,出现稳定的对鸡红细胞凝集效价,效价为25,能被H3亚型血清中和,与H1、H5、H7、H9亚型阳性血清无交叉反应;对HA基因及NA基因进行序列分析,结果显示:HA基因与马流感病毒H3亚型同源性最高,NA基因与马流感病毒N8亚型同源性最高,判断该病毒属于H3N8亚型马流感病毒,命名A/EQ/Guangxi/01/09。     

流感病毒A/Brevig Mission/1918/1(H1N1)片段7和8 mRNAs的低效拼接提高NS1蛋白表达量的研究

A型流感病毒编码的2个片段(7和8)产生的mRNAs拼接后,可潜在地改变NS1蛋白和NS2蛋白及M1蛋白和蛋白M2的比例。本试验研究了A型流感病毒自然发生的序列多态性对其mRNAs拼接的影响,通过对A/Brevig Mission/1918/1(H1N1)和A/Netherlands/178/95(H3N2)及多个H5N1禽流感病毒株片段7和8mRNAs拼接效率进行比较,发现与其他毒株的片段7和8mRNAs相比,A/Brevig Mission/1918/1(H1N1)片段7和8 mRNAs拼接效率低,导致更高水平的NS1功能蛋白的产生,可能有利于A/Brevig Mission/1918/1(H1N1)的致病性。结果还显示,A/Brevig Mission/1918/1(H1N1)片段8 mRNAs对SR蛋白过表达的应答与A/Netherlands/178/95(H3N2)不同。     

规模化驴场中马流感病毒H3N8亚型感染情况调查

[目的]调查山东省聊城市规模化驴场中马流感病毒的感染情况,并分析其可能的来源。[方法]从聊城的规模化驴场采集病料和血清,通过HI试验检测驴血清中的马流感病毒H3N8亚型抗体的阳性率。使用RT-PCR技术扩增肺脏和鼻腔棉拭子样品中的马流感病毒M基因,对获得的马流感病毒M基因与不同流感病毒的M基因进行序列比对,推测其来源。[结果]HI试验表明,120个血清样品中马流感病毒H3N8亚型血清抗体阳性率为33.3%(40/120);其中,母驴的马流感病毒H3N8亚型血清抗体阳性率为42.5%(17/40)、公驴为32.5%(13/40)、驴驹为25.0%(10/40)。通过RT-PCR检测发现,32.3%(21/65)的样品可测出目的条带。通过序列比对得出,该试验获得的流感病毒M基因与马属动物的H3N8亚型流感病毒高度同源(CY032222、CY032318、CY028821等),同源性最高可达99.8%。[结论]马流感病毒在聊城周边的数个规模化养驴场发生流行。该研究从驴体内分离的流感病毒M基因属于马流感病毒H3N8亚型M基因。     

广东1株H3N2亚型犬流感病毒全基因组遗传进化分析

本试验对1株犬源H3N2亚型流感病毒(A/Canine/Guangdong/10/2014)进行了遗传进化分析。结果显示,本毒株8个基因片段与在中国和泰国分离的H3N2亚型犬流感的关系最近,与当前亚洲分离的毒株高度相似(99%)。基因亚型分析显示8个基因片段和目前流行的H3N2CIVs有相同的基因型(K,G,E,3B,F,2D,F,1E)。本毒株为低致病性的禽源H3N2亚型犬流感,从而证实了禽源犬流感亚型在中国的存在,对流感病毒实施广泛的血清学和流行病学检测,以及对该病的防控具有重要意义。     

马流感病毒A/Equine/Huabei/01/07(H3N8)对灵缇犬的实验性感染

为了解灵缇犬对马流感病毒A/Equine/Huabei/01/2007(H3N8)的易感性,将马流感病毒培养液以1.0 mL(含105.7EID50)经静脉途径接种4条灵缇犬,另以2.0 mL(含2×105.7EID50)经滴鼻、点眼方式接种3条灵缇犬。采用临床症状和病理学观察、免疫酶组织化学染色技术、病毒分离、HI抗体检测和流感病毒受体类型检测技术,对病毒在灵缇犬体内的感染情况进行了系统研究。结果表明,感染的7条犬在整个试验观察期间体温等临床指标均无异常变化,未见明显的肉眼和组织学病变。感染后5 d,4条感染犬的气管、支气管和细支气管上皮细胞内流感病毒M蛋白均为阳性,1条犬的咽拭子病毒分离结果为阳性,1条犬的马流感病毒H3亚型HI抗体阳性。感染后14d,剩余3条感染犬中有2条犬马流感病毒H3亚型HI抗体呈阳性。试验证明,目前流行的A/Equine/Huabei/01/2007(H3N8)病毒可以感染灵缇犬,但不导致灵缇犬出现明显的临床症状。灵缇犬的喉头、气管上皮细胞具有与马流感病毒结合的SAα2,3 Gal受体。     

犬流感

早在20世纪70年代,Nikitin A等人通过动物试验证明犬可以感染人H3N2流感病毒,并且在犬血清内检测到相应的抗体[1].之后Romvary J Kilbourne E D Onta T等人也证明了犬可以感染流感病毒,但是一直未从自然发病犬体内分离到流感病毒,或者发现流感病毒在犬群中大量流行[2-7].近10年,关于甲型流感病毒(influenza A virus)感染犬猫的报道频繁出现,包括H3N8、H5N1、H3N2和H1N1共4种亚型流感病毒.     

H3N8亚型马流感病毒间接ELISA抗体检测方法建立及应用

为建立马流感血清学ELISA诊断方法,本研究以马流感病毒中国分离株A/马/新疆/07(H3N8)通过SPF鸡胚培养和增殖,收取含病毒尿囊液经蔗糖密度梯度离心纯化后作为ELISA包被抗原,首次在我国建立了检测H3N8亚型马流感抗体的间接ELISA诊断方法。试验的最佳反应条件为:最佳抗原稀释度7μg/mL,封闭液5%脱脂乳,血清稀释度1∶100,二抗稀释度1∶10000,稀释液PBS(pH7.4),血清反应时间1.5h,二抗反应时间1h。通过本方法对555份临床样品进行检测并与血凝抑制(HI)试验检测结果比较,证明本方法特异、敏感,具有良好的稳定性和可重复性,适于马流感的流行病学调查和监测工作。     

H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。     

Inability of kaolin treatment to remove nonspecific inhibitors from equine serum for the hemagglutination inhibition test against equine H7N7 influenza virus.

The hemagglutination inhibition test is used by many diagnostic and surveillance laboratories for detection of antibodies to influenza viruses. It is well known that the hemagglutination inhibition test is affected by nonspecific inhibitors present in equine serum. Several serum treatments are in use to remove these inhibitors, including treatment with kaolin. Discrepant results were observed in the authors' laboratories when using kaolin treatment before testing equine sera for antibodies against equine influenza virus (EIV) subtype-1 (H7N7). It is demonstrated here that kaolin treatment leads to false positive results when testing for antibodies against EIV subtype-1, as compared to other standard serum treatments (trypsin-periodate, receptor-destroying enzyme). Against EIV subtype-2 (H3N8), however, false positive results were not evident. Trypsinperiodate and receptor-destroying enzyme (RDE) treatments appear to be superior to kaolin for removal of nonspecific inhibitors from equine serum and should be used for serological diagnosis and surveillance of equine influenza virus.     

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.     

A sero-survey of subtype H3 influenza A virus infection in dogs and cats in Japan

A sero-epidemiological survey of human and equine H3 influenza A virus infections in dogs and cats using the hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests was conducted. Serum samples were collected from 582 dogs and 237 cats in Japan during the periods 2002-2008 and 1997-2008, respectively. Although no HI antibodies against equine H3 virus were detected, 9 (3.8%) from cats and 12 (2.1%) from dogs were HI-positive against human H3 virus. Only one serum each from dogs and cats was NI-positive against N2 virus. These findings suggest that although equine H3 influenza virus infections have not been prevalent in companion animals, human H3N2 influenza A virus infections have occurred in dogs and cats in recent years in Japan.     

Evaluation of the ability of canarypox-vectored equine influenza virus vaccines to induce humoral immune responses against canine influenza viruses in dogs

OBJECTIVE: To evaluate canarypox-vectored equine influenza virus (EIV) vaccines expressing hemagglutinins of A/equine/Kentucky/94 (vCP1529) and A2/equine/Ohio /03 (vCP2242) for induction of antibody responses against canine influenza virus (CIV) in dogs. ANIMALS: 35 dogs. PROCEDURES: Dogs were randomly allocated into 4 groups; group 1 (n = 8) and group 2 (9) were inoculated SC on days 0 and 28 with 1.0 mL (approx 10(5.7) TCID(50)) of vCP1529 and vCP2242, respectively. Dogs in group 3 (n = 9) were inoculated twice with 0.25 mL (approx 10(5.7) TCID(50)) of vCP2242 via the transdermal route. The 9 dogs of group 4 were control animals. All dogs were examined for adverse reactions. Sera, collected on days -1, 7, 13, 21, 28, 35, and 42, were tested by hemagglutination inhibition (HI) and virus neutralization (VN) assays for antibodies against CIV antigens A/Canine/FL/43/04-PR and A/Canine/NY/115809/05, respectively. RESULTS: Inoculations were tolerated well. The HI and VN antibodies were detected by 7 days after primary inoculation. Most dogs of groups 1 and 2 and all dogs of group 3 had detectable antibodies by 14 days after initial inoculation. The second inoculation induced an anamnestic response, yielding geometric mean HI titers of 139, 276, and 1,505 and VN titers of 335, 937, and 3,288 by day 42 (14 days after booster inoculation) in groups 1, 2, and 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Canarypox-vectored EIV vaccines induce biologically important antibodies and may substantially impact CIV transmission within a community and be of great value in protecting dogs against CIV-induced disease.     

Serological evidence of H3N8 canine influenza-like virus circulation in USA dogs prior to 2004

H3N8 canine influenza virus (H3N8 CIV) was first reported as a novel canine respiratory pathogen in racing greyhounds and shelter dogs in the U.S.A. in 2004. Phylogenetic analyses determined that this host-adapted pathogen originated from interspecies transmission of an equine influenza virus (EIV), but it is unknown when the transmission occurred prior to discovery in 2004. The objective of this study was to determine if racing greyhound and shelter dog sera collected from 1984 to 2004 had serological evidence of exposure to H3N8 CIV or EIV. Archived sera from 702 racing greyhounds and 1568 shelter dogs were tested for H3 antibodies to the original 2004 CIV isolate, as well as EIV isolates from 1991 to 1999. None of the racing greyhounds from 1984 and 1985 had detectable H3 antibodies. One of the shelter dogs, which entered a north Florida shelter in 2004, was seropositive. For racing greyhounds sampled from 1999 to 2004, 133/520 (26%) dogs had antibodies to both CIV and EIV H3 proteins. The annual seroprevalence was 27% in 1999, 28% in 2000, 10% in 2001, 1% in 2002, 41% in 2003, and 28% in 2004. The odds of H3 seropositivity were greater among dogs that raced > or =6 months, raced on > or =2 tracks, and raced in 1998, 2002, and 2003. Many of the seropositive dogs raced at tracks that were involved in 'kennel cough' epidemics in 1998-1999 and 2002-2003. Based on serological evidence, a H3N8 canine influenza-like virus was circulating in racing greyhounds in the U.S.A. as early as 1999.     

马流感病毒双抗体夹心ELISA检测方法的建立

为建立一种快速、有效的检测马流感病毒(Equine influenza virus,EIV)的方法,以EIV中国分离株A/马/新疆/07(H3N8)制备的多克隆抗体为捕获抗体,原核表达的核蛋白(NP)制备的单克隆抗体为检测抗体,在国内首次建立了检测EIV的双抗体夹心ELISA方法.用该检测方法分别检测EIV、马动脉炎病毒、马疱疹病毒1型、马疱疹病毒4型和马乙型脑炎病毒阳性样品.结果表明,该ELISA方法具有良好的特异性;与常规检测EIV的血凝试验相比,其敏感性是后者的2.5～10倍;同时与H7N7亚型EIV有交叉反应.攻毒试验结果表明该方法可有效检测鼻腔分泌物中的EIV.该方法的建立为EIV的检测及早期防控提供了有效工具.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。