2011-2012年肇庆市禽流感职业暴露人群及外环境病毒分布监测分析

目的了解肇庆市禽类职业暴露人群禽流感病毒感染状况以及外环境禽流感病毒的分布情况。方法采集禽类职业暴露人员血清样本,用红细胞血凝抑制试验（HI）检测H5N1流感抗体;采集外环境样本,用荧光定量PCR法检测禽流感病毒FluA、H5、H7和H9核酸。结果2011-2012年共采集职业暴露人员血清样本400份,检测H5N1抗体均为阴性;共采集外环境有效样本202份,检出FluA阳性25份,阳性率为12．38％,其中AH9亚型阳性14份（56．00％）。AH7亚型阳性1份（4．00％）,A未分型10份（40．00％）,未检出AH5亚型。结论肇庆市职业暴露人群尚未发现感染高致病性禽流感H5N1病毒．夕h环境存在禽流感病毒的污染,H9亚型是主要的病原体。     

禽流感病毒感染BALB／c小鼠的动态病理观察

目的将H5N1亚型禽流感病毒（AIV）鼻腔接种BALB／c小鼠,动态观察小鼠每个时期主要器官组织的病理变化。方法将100μlH5N1亚型禽流感病毒原液滴入经麻醉后BALB／c小鼠鼻腔,观察14d,每天取材1次,固定、包埋、切片后HE染色观察各组织病理变化。结果H5N1亚型禽流感病毒感染BALB／c小鼠后,产生一系列与禽流感病毒感染有关的动态病理改变：第1～2天（前驱期）,肺轻微出血、水肿、炎性细胞浸润;第3～7天（发作期）,肺损伤逐渐严重．大量出血、炎性细胞浸润、严重水肿、淤血、肺泡实变塌陷或者气肿;第8～14天（恢复期）,各种损伤逐渐减轻,出血渗出减少,水肿减轻,肺间质出现纤维化而趋于恢复正常。肝、。肾、脑出现病理改变。结论通过动态观察病理,弄清了禽流感病毒每个时期在BALB／c小鼠体内造成的病理损伤。     

高致病性人禽流感H5N1疫苗滴鼻免疫应答效果的初步研究

目的 通过高致病性人禽流感H5N1全病毒-MF59佐剂疫苗滴鼻免疫Balb/c小鼠,评价该疫苗所诱导的系统免疫与黏膜免疫应答效果.方法 以不同剂量抗原按比例与MF59佐剂配伍制成粘膜疫苗,滴鼻免疫Balb/c小鼠,二免2周采血检测血清IgG、IgM效价及血清中HAI(HA inhibitor)的中和抗体效价,同时收集鼻、肺灌洗液,检测其lgG和slgA抗体效价.结果 H5NI+MF59组血清抗体效价较H5NI组有显著升高(P＜0.01);在各剂量组中,随着剂量的增加抗体效价呈上升趋势.12μg腭后抗体效价呈下降趋势,以HSNI+MF59(12μg)组效价最高;肺鼻灌洗液中,均可检测到特异性分泌型IrA、IsG,其中特异性分泌型IgA效价略高于IgG;抗体亚型的分布以IgG1、IgG2b为主.结论 灭活高致病性禽流感全病毒H5N1在佐剂MF59作用下可诱导机体产生体液免疫应答,同时还可以在黏膜局部产生特异性分泌型IgA、IsG,为高致病人禽流感病毒I-15N1黏膜疫苗的研制奠定了基础.     

遗传重配获得H5N1流感病毒Vero细胞适应株

目的以传统遗传重配技术选育HSN1流感病毒Veto细胞适应株,制备Vero细胞H5N1流感疫苗。方法以流感病毒Vero细胞适应株A／Yunnan／1／2005Va（H3N2）为母株与反向遗传学技术改造的禽流感病毒疫苗株A／Anhui／1／2005（H5N1）共同感染SPF鸡胚和Vero细胞,用羊抗A／Yunnan／1／2005Va（H3N2）抗体筛选,血抑试验和基因测序鉴定病毒型别,并进行重配株的其他相关生物学试验。结果获得了1株在Vero细胞高产的H5N1流感病毒,重配前后的单价灭活疫苗免疫小鼠抗体血清效价差异无统计学意义（F=0．857,P〉0．05）。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H5N1流感病毒Vero细胞适应株。     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

1株对小鼠呈高致病性H5N1亚型禽流感病毒的遴选

目的观察从死禽体内分离的禽流感H5N1病毒株对小鼠的致病情况,筛选一株可用于小鼠攻毒实验的H5N1毒株。方法将17株禽源H5N1病毒分别滴鼻感染6～8周龄Balb/c小鼠,观察其对小鼠的致病和致死情况。通过测定其中毒力较强一株的EID_(50)、TCID_(50)、小鼠LD_(50)及濒死小鼠不同组织中病毒载量,判定其致病力;采用遗传进化分析绘制HA基因进化树,了解其进化特征。结果动物实验表明,17株高致病性禽源H5N1毒株中,仅有一株对小鼠为高致病性。该毒株TCID_(50)/100μl=7.2,EID_(50)/100μl=8.325,小鼠1 LD_(50)/100μl=6.318≈10~2EID_(50)。鼻粘膜感染4～6 d小鼠发病,表现为精神萎靡、食欲减退、竖毛、弓背等临床症状,体重明显减轻。小鼠死亡集中在发病后的2～5 d,耐过死亡者12 d完全恢复正常。从濒死感染小鼠脾、肺、鼻及脑组织中皆可检出流感病毒NS片段,病毒数载量以脑组织中最多,达3.56×10~(10)拷贝/5μl。结论筛选到一株对小鼠呈强致病性H5N1亚型禽流感毒株,可以用于禽流感疫苗交叉保护效果评价的小鼠攻击实验,也可以作为感染模...     

表达H1N1 亚型猪流感病毒三聚体HA 重组慢病毒的免疫原性研究

目的:探讨表达猪H1N1亚型流感病毒三聚体HA的重组慢病毒(rLV-HA-GCN4)对BALB/ c 小鼠的免疫保护效果。方法:将雌性BALB/ c 小鼠随机分为重组慢病毒rLV-HA-GCN4 组、重组慢病毒rLV-HA 组、慢病毒LV 空载体对照组及PBS 对照组,先后进行质粒和慢病毒2 次免疫,间隔为2 周,注射部位均为小鼠大腿内侧肌肉;于初次免疫后的第28天,各组小鼠鼻腔滴注50 l 100TCID50 的H1N1 病毒,采用淋巴细胞转化实验、流式细胞术、间接ELISA 和脾肺指数检测各免疫指标。结果:加强免疫后第14 天,rLV-HA-GCN4 组脾淋巴细胞转化率为0.3±0.11,与PBS 组相比,差异有统计学意义(P<0.01),产生以Th1 型CD4+ T 细胞为主的细胞免疫反应;rLV-HA-GCN4 组小鼠IgG 抗体效价可达1;8 000,攻毒后第14 天约为1 :7 000;攻毒后,rLV-HA-GCN4 组脾肺指数小于PBS 组,小鼠体重在前3 d 略有下降,随后逐渐上升,两个指标与PBS 组相比,差异均有统计学意义(P<0.05)。结论:rLV鄄HA鄄GCN4 能够诱导小鼠良好的细胞与体液免疫应答,从而有效地保护小鼠抵御猪H1N1 流感病毒的感染。     

禽流感与人类健康

禽流感（Avian Influenza，AI）是由禽流感病毒引起的一种从呼吸系统到全身性败血症等多种症状的传染性疾病综合征。禽流感病毒在分类地位上与人的流感病毒一样。属于正粘病毒科，A型流感病毒属，单股负链分节段的RNA病毒。根据表面糖蛋白血凝素（HA）和神经氨酸酶（NA）的抗原性差异可分为不同的亚型。到目前为止，已经鉴定了16种H亚型（H1-H16）和9种N亚型（N1-Ng）。禽流感病毒根据其对鸡致病性的强弱可分为高致病性和低致病性两种。高致病性禽流感病毒可引起鸡群高达100％的死亡率，故被世界动物卫生组织（OIE）列为A类疾病。低致病性禽流感病毒可引起温和的呼吸道疾病，但在混合感染或环境因素的作用下可引起严重的疾病。近年来H5N1和H9N2等亚型的禽流感病毒感染人的事件，表明家禽是禽流感人畜传播潜在的中间宿主。这也使得世界各国对禽流感的关注程度大大提高。因此，开展对禽流感病毒的研究，从分子水平掌握禽流感病毒的流行规律和致病机理，不仅在病毒学、兽医学等学科上有重要的学术意义，而且在公共卫生等方面也具有重大的社会意义。     

高致病性人禽流感H5N1透皮疫苗促渗剂的初步筛选

目的 初步筛选用于高致病性人禽流感H5N1透皮疫苗的较有效的促渗剂.方法 选用乙醇、丙二醇、二甲基亚砜、维甲酸、油酸作为促渗剂,将其应用于BALB/c小鼠,再用灭活的高致病性人禽流感H5N1透皮疫苗免疫小鼠,通过评价透皮免疫应答的效果对促渗剂进行初步筛选.结果 二甲基亚砜、维甲酸组和油酸组血清IgG抗体效价明显高于其他促渗剂组(P＜0.05).结论 在高致病性人禽流感H5N1透皮疫苗的小鼠模型中,二甲基亚砜、维甲酸和油酸是较好的促渗剂.     

禽流感病毒血凝素H5特异性单克隆抗体的制备及ELISA捕获法的建立

目的 制备和鉴定禽流感病毒(H5N1)血凝素(H5)特异性单克隆抗体(mAb),建立H5抗原的双抗体夹心ELISA捕获法.方法 以H5血凝素和携带H5全长基因的质粒免疫Balb/c小鼠制备mAb,利用血凝抑制(HI)实验筛选和鉴定,通过竞争抑制试验分析抗体识别表位,并采用抗体配对试验筛选捕获抗体和检测抗体,建立测定H5抗原的双抗体夹心ELISA捕获法.结果 获得16株特异性针对H5的单克隆抗体,与A型流感病毒H1、H3、H7、H9和B型流感病毒的血凝素无HI交叉反应,对H5血凝素的血凝抑制效价为1:100～1:51 200;通过配对实验,建立以单克隆抗体H5M9为捕获抗体,辣根过氧化物酶标记单克隆抗体H5M11为检测抗体的双抗体夹心ELISA;检测多株H5N1病毒和H5血凝素的最低检出值为1/32血凝单位,检测A型流感病毒H1N1、H3N2、B型流感病毒以及H7、H9血凝素均为阴性.结论 建立了一种灵敏度高、特异性强的测定H5抗原的ELISA捕获法,可应用于禽流感病毒H5N1感染的实验室早期诊断.     

Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP

The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002-2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, "FFWTILKP", identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining.     

An outbreak of avian influenza subtype H9N8 among chickens in South Korea.

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical signs included moderate respiratory distress, depression, mild diarrhoea, loss of appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically, mucopurulent tracheitis and air sacculitis were prominently found with urate renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg (A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV) isolate showed that reassortment had occurred. Its haemagglutinin gene was similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild birds in Korea. The pathogenicity of the isolate was tested on 6-week-old specific pathogen free chickens. The inoculated virus (H9N8) was recovered from most tested organs, including the trachea, lung, kidney, spleen, and caecal tonsil. This is the first report of an outbreak of low pathogenic avian influenza in chickens caused by AIV subtype H9N8.     

H5N1型禽流感病毒动物模型的研究现状

H5N1亚型禽流感病毒在全球多个国家肆虐,已严重威胁人类健康。禽流感病毒动物模型的建立可以为研究病毒的突变、传染性和发病机制提供良好的技术平台。本综述概括了H5N1对几种哺乳动物:食蟹猴、雪貂、小鼠、大鼠、沙鼠、家猫等的致病性,为以后理想动物模型的建立和研究提供一定的帮助。     

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.     

Characterization of low-pathogenic H6N6 avian influenza viruses in central China

Three strains of H6N6 subtype avian influenza virus (AIV) were isolated from live-poultry markets of central China during 2009-2010. A phylogenetic analysis showed that these isolates originated from gene reassortment among different virus lineages of the H6 subtype. In an experimental infection of animals, the selected isolate was non-pathogenic for chickens and low-pathogenic for mice. The wild-type isolate was capable of replication in mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaption in mouse lung. The genomes of viruses of passage 0 (P0), P4, and P8 were sequenced and compared, and virulence-related amino acid substitutions were found in multiple sites during mouse lung passage.     

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.     

Characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006–2008

Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006 to 2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of the HA genes, these isolates belong to clade 2.2 and both the HA and NA are closely related to each other (nucleotide identities above 99.0%) and to other Middle Eastern H5N1 AIV isolates (nucleotide identities above 98.0%). The phylogenetic data suggest that the virus in both epornitics of H5N1 HPAIV in commercial poultry in the Karachi region of Pakistan between 2006 and 2008 were from a very closely related source, however, there is inadequate epidemiological data to determine what the reservoir was for the virus between the 2006 and 2007 outbreaks other than that there was a single introduction into the region.     

Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.