禽流感油乳剂灭活疫苗的研究

将6种不同亚型的禽流感病毒（AIVH2N9、H3N8、H5N1、H5N2、H7N1、H9N2）分别接种鸡胚,收获尿囊液,经甲醛灭活,以矿物油为佐剂制成油乳剂灭活疫苗。疫苗接种4和8周龄SPF鸡,注苗后均无不良反应。每种亚型疫苗免疫后14天和21天攻毒保护率均达90％-100％。分别用H5N1和H9N2亚型灭活疫苗免疫8周龄SPF鸡、25和28周龄健康商品蛋鸡,免疫后7天产生免疫力,14天保护率达100％,21天后抗体达高峰,仔鸡接苗后最高血凝抑制（HI）几何平均滴度（GMT）为7.3-8.0log2,蛋鸡为8.0-10.5log2。免疫后180天,抗体效价不低于6.5log2。免疫后180天分别以AIV攻击,H5亚型疫苗组,用强毒攻击无一发病和死亡,对照鸡全部发病死亡;H9亚型疫苗组,对照鸡在攻毒后72小时停产,免疫鸡无一发病且产蛋正常,对同源攻毒的保护率达100％。     

禽流感病毒CH02株(H9)的鉴定及其NS 基因分子特征分析

从疑似感染H9亚型禽流感病毒的病鸡内脏组织中分离到1株能凝集鸡红细胞的病毒,通过血凝抑制试验、鸡胚中和试验、RT-PCR鉴定,确认其为H9亚型禽流感病毒,并命名为CH02株。该病毒HA效价为2^7.67±0.58;其血凝性可被抗H9亚型禽流感病毒阳性血清完全抑制,HI效价为7log2;鸡胚半数感染量(EID₅₀)为10^-8.68 EID₅₀/0.1 mL;最小致死剂量致鸡胚死亡的平均时间(MDT)为85.6 h;1日龄鸡脑内致病指数(ICPI)为0.625;其NS基因与香港株、南京株、北京株、汕头株、韩国株、巴基斯坦株的核苷酸同源性为88.6%~100%,经进化分析CH02株与香港株(A/duck/Hong Kong/Y280/97)同属一个分支。     

H5亚型禽流感疫苗对特禽及水禽的免疫效果观察

用H5N2亚型禽流感疫苗,分别免疫SPF鸡、乌鸡、珍珠鸡、贵妃鸡、火鸡、鸭及鹅,免疫后采血测定其HI抗体,观察我国现有的H5N2亚型禽流感疫苗对特禽及水禽的免疫效力.结果,免疫后3周,SPF鸡、乌鸡、珍珠鸡、贵妃鸡、火鸡、鸭及鹅的平均HI抗体滴度分别为2 7.2、2 7.6、2 4.3、2 4.83、2 4.6、2 6.2及2 5.3,乌鸡两次免疫,其中一次HI抗体为2 9.65.试验证明,SPF鸡、特禽及水禽用H5N2亚型禽流感疫苗免疫后,均能产生一定水平的HI抗体,但不同种类的家禽所产生的HI抗体滴度存在较大差异,以SPF鸡及乌鸡所产生的HI抗体滴度最高,而珍珠鸡、贵妃鸡及火鸡所产生的HI抗体滴度较低,水禽对H5N2亚型禽流感疫苗可产生一定水平的HI抗体.     

H5N1 亚型禽流感病毒在 MDCK 细胞中增殖条件的研究

目的：探索H5N1亚型禽流感病毒在MDCK中增殖规律,确定最佳增殖条件。方法将H5N1亚型禽流感病毒接种到6孔板培养的MDCK细胞进行增殖试验,检测不同病毒感染量、不同浓度TPCK-胰酶,接毒后不同时间病毒的HA滴度。根据确定的最佳增殖条件将病毒接种到微载体培养的MDCK细胞中进行大规模增殖。结果：最佳病毒增殖条件接毒量MOI为5＆#215;10-4、TPCK-胰酶浓度为4μg/mL,在5 L生物反应器中重复验证,获得稳定的试验结果,病毒血凝价最高为8 log2。结论：本研究为禽流感疫苗的生物反应器规模化生产奠定了基础。     

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.     

Limited susceptibility of pigeons experimentally inoculated with H5N1 highly pathogenic avian influenza viruses

An experimental infection study was performed using pigeons reared for racing or meat production in Japan and clade 2.2 and 2.3.2 isolates of H5N1 highly pathogenic avian influenza virus to evaluate the possible role of pigeons in virus transmission to poultry. In experiment 1, when 20 pigeons were intranasally inoculated with high or low viral doses, no inoculated pigeon exhibited clinical signs for 14 days. Drinking water and almost all swab samples were negative for virus isolation. Virus isolation was positive in 3 oral swab samples from 2 pigeons from day 2 through 4 postinoculation, but viral titers of positive samples were extremely low. Immunohistochemical analysis for virus detection was negative in all tissue samples. Along with seroconversion in a limited number of pigeons postinoculation, these results suggest that pigeons have limited susceptibility to the virus used for experimental infection. In experiment 2, when uninoculated chickens were housed with virus-inoculated pigeons, all pigeons and contact chickens survived for 14 days without exhibiting any clinical signs. According to serological analysis, the chickens did not exhibit seroconversion after close contact with inoculated pigeons. Our data suggest that the risk posed by pigeons with respect to the transmission of the H5N1 highly pathogenic avian influenza virus to poultry would be less than that for other susceptible avian species.     

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets,China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.     

禽流感（H9亚型）油乳剂灭活疫苗的研究

将H9亚型禽流感病毒尿囊液，经甲醛灭活，以矿物油为佐剂制成油乳剂灭活疫苗，疫苗物理性状良好，接种鸡无不良反应，接种疫苗后14d，食流感病毒HIGMT（Log2)为7.7-7.8，21d为8.9-9.3，于接种疫苗后14、21、30、60d，攻击同型禽流感病毒，接种疫苗鸡全部获得保护，现地实验取得了良好地免疫效果。证明制备的油乳剂灭活疫苗安全，免疫原性良好。     

Comparative susceptibility of chickens and turkeys to avian influenza A H7N2 virus infection and protective efficacy of a commercial avian influenza H7N2 virus vaccine

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus caused a major outbreak among commercial poultry in Virginia and adjacent states. The virus primarily affected turkey flocks, causing respiratory distress and decreased egg production. Experimentally, turkeys were more susceptible than chickens to H7N2 virus infection, with 50% bird infectious dose titers equal to 10(0.8) and 10(2.8-3.2), respectively. Comparison of virus shedding from the cloaca and oropharynx demonstrated that recent H7N2 virus isolates were readily isolated from the upper respiratory tract but rarely from the gastrointestinal tract. The outbreak of H7N2 virus raised concerns regarding the availability of vaccines that could be used for the prevention and control of this virus in poultry. We sought to determine if an existing commercial avian influenza (AI) vaccine prepared from a 1997 seed stock virus could provide protection against a 2002 LPAI H7N2 virus isolated from a turkey (A/turkey/Virginia/158512/02 [TV/02]) in Virginia that was from the same lineage as the vaccine virus. The inactivated AI vaccine, prepared from A/chicken/ Pennsylvania/21342/97 (CP/97) virus, significantly reduced viral shedding from vaccinated turkeys in comparison with sham controls but did not prevent infection. The protective effect of vaccination correlated with the level of virus-specific antibody because a second dose of vaccine increased antiviral serum immunoglobulin G and hemagglutination inhibition (HI) reactivity titers in two different turkey age groups. Serum from CP/97-vaccinated turkeys reacted equally well to CP/97 and TV/02 antigens by HI and enzyme-linked immunosorbent assay. These results demonstrate the potential benefit of using an antigenically related 1997 H7N2 virus as a vaccine candidate for protection in poultry against a H7N2 virus isolate from 2002.     

Evaluation of a protective immunity induced by an inactivated influenza H3N2 vaccine after an intratracheal challenge of pigs.

A challenge study was conducted to evaluate the safety and efficacy of an inactivated influenza H3N2 virus vaccine combined with Quil A/Alhydrogel mixture under controlled conditions in piglets. Twenty-four piglets from 12 sows were allocated to 2 groups; injected intramuscularly with 2 doses of the tested vaccine or with PBS at 2 wk intervals and challenged intratracheally with 105TCID50 of the H3N2 swine influenza virus 6 d after the 2nd immunization. Clinical and virological parameters were recorded for 4 d after the challenge. The use of the tested vaccine produced high serum hemagglutination-inhibition titers against the swine H3N2 strain virus. This strong immune response suppressed all clinical signs and viral shedding and reduced pulmonary lesions due to the challenge in the vaccinated group, without causing any secondary effects. Our results suggest that the serum HI titers correlated with the degree of protection induced by an inactivated swine influenza H3N2 vaccine.     

Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.     

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.     

Pandemic H1N1 influenza virus in Chilean commercial turkeys with genetic and serologic comparisons to U.S. H1N1 avian influenza vaccine isolates

Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.     

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.     

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。     

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。     

1株H6N5亚型禽流感病毒A/duck/Yangzhou/013/2008的全基因测序及遗传进化分析

在对华东地区家养水禽中流感病毒的带毒状况进行流行病学监测的过程中,采用常规的血清学试验和特异性RT-PCR方法,分离鉴定出1株H6N5亚型禽流感病毒A/duck/Yangzhou/013/2008(简称Dk/YZ/013/08)。为了探讨该亚型病毒在流感病毒生态分布中的作用,作者对Dk/YZ/013/08进行了全基因序列测定,并结合Gen-Bank中已收录的所有H6N5亚型病毒的基因组序列及其它参考序列进行了遗传进化分析。结果表明Dk/YZ/013/08的血凝素基因(HA)与近年中国台湾分离的鸭源毒株A/duck/Kingmen/E322/2004(H6N2)的核苷酸一致性最高(94%),推导的氨基酸剪切位点序列为"P-Q-I-E-T-R-G",为典型低致病性禽流感病毒的特征序列;神经氨酸酶基因(NA)与瑞士分离株A/mallard/Switzerland/WV4060167/2006(H3N5)的亲缘关系最近(核苷酸一致性96.9%);而碱性聚合酶2(PB2)基因则与A/duck/Zhejiang/11/2000(H5N1)的遗传距离最近,可能由H5N1亚型流感病毒提供,提示该毒株可能是一株重组病毒。     

禽流感病毒河北株的分离鉴定

禽流感（Ａｖｉａｎ Ｉｎｆｌｕｅｎｚａ）是由 Ａ型流感病毒引起的一种禽类的病毒性传染病,被国际兽疫局确定为Ⅰ类烈性传染性病。该病毒属正粘病毒科,几乎所有的野生与家养禽类均可感染。１８７８年在意大利的鸡群中首次暴发,其后许多国家和地区都相继报道ＡＩＶ存在的事实,近几年,我国在不同地区都分离到禽流感病毒,广泛存在的主要是Ｈ９亚型病毒,且多为中等以下毒力的毒株。本试验采集病料主要针对河北省养鸡业较为发达的几个县市的七个小型养鸡场,选择未注过流感疫苗,临床症状出现产蛋下降和呼吸道症状严重的鸡只,剖杀,采集病变明显的脏器,如胰、肝、肺、脾、气管、肾、泄殖腔及其干酪样物等,剪碎研磨,加抗生素处理后,接种９－１０日龄鸡胚,弃掉２４小时内死亡的鸡胚,收集２４－７２小时死亡鸡胚的尿囊液,盲传三代,并做血凝试验,对于ＨＡ阳性的样品,进一步做ＨＩ试验,结果有２株禽流感病毒为Ｈ９亚型;进一步测定其神经氨酸酶活性,结果为Ｎ２。取１： １０稀释的尿液０．２ｍｌ肌肉接种８只８周龄ＳＰＦ鸡测其致病力指数,观察１０天内鸡只发病情况,结果第三天鸡群精神稍有沉郁,其中一只出现歪脖的神经症状,采用英国中心兽医实验室静脉致病指数（ＩＶＰＩ）测定计算方法     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.     

水禽用H5亚型禽流感灭活疫苗对高致病性禽流感病毒流行毒株的免疫保护效果研究

为评价水禽用禽流感灭活疫苗(H5N2亚型,D7株)对2010年以后分离的高致病性禽流感病毒流行毒株的免疫保护效果,将该疫苗免疫3周龄SPF鸭后,21 d采血、分离血清测定HI抗体效价,同时用5株2010年以后分离的高致病性禽流感流行毒进行攻毒保护试验,攻毒后5d采集所有试验鸭喉头和泄殖腔拭子进行病毒分离.结果显示,该疫苗免疫SPF鸭21 d后的HI抗体效价的几何平均滴度达7.4log2,对5株高致病性禽流感病毒的攻击均可产生良好的免疫保护,并有效阻止病毒排泄.该疫苗的推广使用将对我国水禽高致病性禽流感的防控发挥重要作用.