小鼠细胞因子实时定量PCR检测方法的建立及应用

采用荧光SYBR Green I建立小鼠细胞因子mRNA实时定量PCR的检测方法;并利用建立的实时定量PCR的检测方法时感染H5N1禽流感病毒的BALB/c小鼠不同时间采集的肺脏组织中几种重要致炎细胞因子及趋化因子mRNA表达水平进行了检测.对HSN1禽流感病毒感染的BALB/c小鼠肺脏细胞因子的检测具有高度的特异性,检测的灵敏度为10~1～10~2拷贝数.H5N1禽流感病毒感染BALB/c小鼠后,小鼠肺脏中IL-1β、IL-6、TNFα、MIG、IP-10、RANTES、MIF和HMGB1细胞因子mRNA表达水平与时照组小鼠相比均有明显差异.建立的实时定量PCR能在基因转录水平敏感和特异地反应细胞因子的表达水平,该技术在基础和临床免疫研究中,具有良好的应用价值.     

重组H5亚型禽流感病毒三价灭活疫苗免疫效果的评价

为了评价重组H5亚型禽流感病毒三价灭活疫苗的免疫效果,在2016年7月至2017年8月期间,对免疫重组H5亚型禽流感病毒三价灭活疫苗(H5N1,Re-6株+Re-7株+Re-8株)的家禽采集了1 999份血清样品,采用血凝与血凝抑制方法进行检测,结果为H5亚型禽流感病毒Re-6株免疫合格率为86.84%,H5亚型禽流感病毒Re-7株免疫合格率71.44%,H5亚型禽流感病毒Re-8株免疫合格率为67.78%。结果表明,免疫重组H5亚型禽流感病毒三价灭活疫苗后,不同毒株的抗体、不同种类的家禽、不同免疫次数的抗体水平均有差异。     

戊二醛苯扎溴铵溶液对H9N2亚型禽流感病毒的灭活效果分析

采用悬浮杀灭试验,考察戊二醛苯扎溴铵溶液对H9N2亚型禽流感病毒的灭活效果。将戊二醛苯扎溴铵溶液通过不同稀释倍数(1 250、2 500、5 000、10 000、20 000)、在不同作用时间(5、10 min)观察其对H9N2亚型禽流感病毒的灭活效果。结果显示,戊二醛苯扎溴铵溶液在10000倍稀释,与H9N2亚型禽流感病毒作用5 min或10 min,灭活率达99.90%以上;在20 000倍稀释,作用5 min或10 min,灭活率均小于99.90%。表明戊二醛苯扎溴铵溶液在10 000倍稀释与H9N2亚型禽流感病毒(20±1)℃水浴作用5 min,即可对H9N2亚型禽流感病毒达到很好的灭活效果。     

成年珍禽禽流感免疫抗体消长规律及免疫程序的研究

为了弄清动物园成年珍禽的禽流感免疫抗体消长规律,用禽流感(H5+H9)二价灭活疫苗免疫接种动物园的8种珍禽,分别在免疫接种前后不同时间无菌采血,检测血清中H5和H9亚型禽流感病毒血凝抑制(HI)抗体。结果表明,大部分珍禽首次免疫接种后都能产生H5和H9亚型禽流感病毒HI抗体,但不同种类免疫反应不同。孔雀等4种珍禽在首次免疫接种后400d,H5、H9亚型禽流感病毒的抗体仍能维持较高的水平;白冠长尾雉等4种珍禽首次免疫后,H5、H9亚型禽流感病毒抗体水平不高,且维持时间很短,在进行H5+H9二价灭活苗加强免疫后,能有效提高H5、H9亚型禽流感病毒免疫抗体,免疫抗体均能维持7个月。通过对免疫抗体的监测,对两种不同剂量的比较研究,并结合动物园珍禽的实际情况,本研究初步建立了成年珍禽的禽流感的免疫程序。     

H5亚型禽流感病毒实时荧光RT-PCR方法的建立与应用

H5亚型禽流感病毒HA序列差异越来越大。为更准确地检测H5亚型禽流感病毒,对GenBank中发表的H5亚型禽流感病毒HA序列以及本实验室保存病毒序列进行比对,同时设计1对引物和1条探针,建立了H5亚型禽流感病毒实时RT-PCR方法,并对该方法的反应体系和反应参数进行了优化。以本实验室分离测序确定的30份H5亚型禽流感病毒RNA为模板,将该方法与两种商品化H5亚型禽流感病毒检测试剂盒进行比对,发现该方法与两种商品化试剂盒的检出率分别为100%、98%、98%。敏感性试验显示,该方法可以检测出0.1 fg的RNA模板,灵敏度比两种商品化试剂盒均提高了10倍。结果表明,该方法具有更高的特异性和敏感性,可用于H5亚型高致病性禽流感的早期诊断。     

重组鸡IL-2对禽流感灭活苗免疫增强作用试验

使用重组鸡白介素-2作为免疫佐剂，检测chIL-2对禽流感病毒H9亚型灭活疫苗的免疫增强作用。将1日龄的AA肉雏鸡，随机分为7组，每组10只，采用胸部肌肉注射方式进行免疫，对照组仅接种H9亚型禽流感灭活疫苗，试验组分别同时接种H9亚型禽流感灭活疫苗O．5mL和不同剂量（0．01mg-1mg）的鸡IL-2。每7d静脉采血，采用血凝和血凝抑制方法检测机体中抗体水平变化情况。结果表明，接种重组鸡IL-2的试验组动物能在2月内保持高抗体水平，且在免疫后第10周，其平均抗体水平仍高于疫苗对照组3．6个～4．5个滴度，表明了重组鸡IL-2蛋白对H9亚型禽流感灭活疫苗具有免疫增强作用。     

应用荧光RT-PCR方法检测H7N9亚型禽流感病毒

为了验证荧光RT-PCR方法检测H7N9亚型禽流感病毒的可行性,应用建立的荧光RT-PCR方法检测H7N9亚型禽流感抗原、H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原评估检测方法的特异性,检测不同稀释度的H7N9亚型禽流感抗原,并用建立的方法检测采自三鸟批发市场100份泄殖腔、喉拭子。结果是建立的荧光RT-PCR方法,H7体系能检测出1:10-10抗原稀释度,N9体系能检测1:10-9抗原稀释度,对H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原无交叉反应;检出100份泄殖腔、喉拭子结果均为阴性。结果表明,荧光RT-PCR方法H7N9亚型禽流感病毒具有快速、灵敏、特异的特点,适合对禽类的大规模监测。     

不同禽流感疫苗和不同接种剂量免疫麻花鸡的免疫效力试验

对我国目前推广的5种不同禽流感疫苗进行了免疫效果的比对分析;对麻花鸡群以不同免疫剂量进行接种,比较分析了疫苗剂量对禽流感免疫效果的影响;对比结果表明,重组禽流感灭活疫苗（H5N1亚型,Re-1株）、禽流感灭活疫苗（H5N2亚型,N28株）、禽流感（H5＋H9）二价灭活疫苗（H5N1Re-1＋H9N2Re-2株）抗体水平高,效果稳定,这3种疫苗为乌鲁木齐市麻花鸡禽流感防控中的疫苗首选;不同免疫剂量的对比试验结果表明,最佳禽流感疫苗接种剂量为0.5ml。     

湘西自治州规模化鸭场H5、H7亚型禽流感病毒抗体水平的检测与分析

为了研究湘西自治州规模化鸭场的高致病性禽流感的免疫情况和潜在的感染风险,笔者于2008年从湘西自治州各县市采集了规模化鸭场血清标本共480份,采用血凝试验和血凝抑制试验对4个免疫了H5亚型禽流感病毒灭活疫苗的规模化鸭场的不同日龄家鸭进行了H5和H7亚型禽流感病毒抗体的检测。结果表明:4个规模化鸭场H5亚型禽流感病毒抗体合格率分别为65.56%(59/90)、67.78%(61/90)、53.33%(48/90)、47.78%(43/90),说明规模化鸭场的H5亚型免疫抗体水平还有待提高,以对家鸭提供确实的保护;H7亚型禽流感病毒抗体阳性率为0,规模化鸭场没有H7亚型禽流感病毒感染。     

湘西自治州禽流感H5、H7亚型的监测与分析

为监测湘西自治州H5亚型禽流感的免疫抗体水平和H7亚型禽流感的感染情况;对32个规模禽场和8个活禽交易市场进行了抽样检测.从整体水平看,采集的810份禽血清样品,有567份样品的H5亚型禽流感免疫抗体合格,合格率为70%.H7亚型禽流感的非免疫抗体水平全为阴性.     

Effect of a prior exposure to a low pathogenic avian influenza virus in the outcome of a heterosubtypic low pathogenic avian influenza infection in mallards (Anas platyrhynchos)

Wild birds, particularly Anseriformes and Charadriiformes, are considered the natural reservoir of low pathogenic avian influenza (LPAI) viruses. The high prevalence and subtype diversity of avian influenza viruses at premigrational staging areas provide the perfect opportunity for multiple exposures to different LPAI virus subtypes. Natural consecutive and concurrent infections of sentinel ducks with different LPAI virus subtypes have been reported. The protective immune response from different LPAI virus infections is not understood nor is the effect of such repeated exposures. This study experimentally evaluated the effect of a prior exposure to a LPAI virus on the outcome of a heterosubtypic LPAI virus infection in mallards (Anas platyrhynchos). The results of this investigation suggest that recent prior exposure to a LPAI virus may affect the outcome of a subsequent heterosubtypic LPAI infection in mallards by reducing the duration of cloacal and oropharyngeal viral shedding as well as the viral load excreted via the cloaca. Wild mallards are likely exposed to multiple subtypes of LPAI virus during the periods of peak viral circulation, and the results of this study suggest that the duration of viral shedding in subsequent exposures might be reduced.     

A型流感病毒致病性及跨种间传播的分子生物学基础

A型流感病毒在温血动物中广泛存在,也是目前导致人类和各种动物流感疾病的主型。在自然情况下流感病毒感染的宿主范围有一定的特异性,分离自人的流感病毒一般不能在鸡、鸭等禽类体内复制,同样禽流感病毒在灵长类动物体内的复制能力也极差。但近年来不断发生禽流感病毒（包括H5N1,H9N2,H7N7亚型病毒）直接传染给人的事件,而2009年爆发的人类的H1N1流感的病原则是猪源流感病毒重组而来。研究证实,流感病毒的致病性及跨种间传播的深层原因是病毒分子结构差异及其与宿主细胞相互作用。病毒多种结构蛋白和非结构蛋白某些功能位点上氨基酸的差异是病毒致病性及其跨中传播的分子生物学基础。     

Avian influenza virus

Avian influenza viruses do not typically replicate efficiently in humans, indicating direct transmission of avian influenza virus to humans is unlikely. However, since 1997, several cases of human infections with different subtypes (H5N1, H7N7, and H9N2) of avian influenza viruses have been identified and raised the pandemic potential of avian influenza virus in humans. Although circumstantial evidence of human to human transmission exists, the novel avian-origin influenza viruses isolated from humans lack the ability to transmit efficiently from person-to-person. However, the on-going human infection with avian-origin H5N1 viruses increases the likelihood of the generation of human-adapted avian influenza virus with pandemic potential. Thus, a better understanding of the biological and genetic basis of host restriction of influenza viruses is a critical factor in determining whether the introduction of a novel influenza virus into the human population will result in a pandemic. In this article, we review current knowledge of type A influenza virus in which all avian influenza viruses are categorized.     

(Highly pathogenic) avian influenza as a zoonotic agent

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.     

H5-H7双价禽流感核酸疫苗免疫研究

由于禽流感的高度变异性,免疫后不同亚型之间难以得到交叉保护,以致病毒得以逃避宿主免疫系统的监视,多价核酸禽流感疫苗具有可以保护不同亚型病毒攻击的特点。本试验设计并构建了包含禽流感H5HA和H7HA1基因的双价真核表达质粒pV-H5-H7及单独表达H5HA和H7HA1的pV-H5和pV-H7HA1。通过RT-PCR,间接免疫荧光(IFA)等方法验证构建质粒的正确性和其表达蛋白的免疫原性。0,21 d分别免疫6~8周龄的BALB/c小鼠,设立双价疫苗组,单表达免疫组和对照组。免疫后35 d用HPAIV H5N1进行致死性攻击。结果显示免疫组均可刺激机体产生H5特异性抗体,pV-H5-H7诱导产生的抗体对H5N1的攻毒保护率为80%,而pV-H5单表达的攻毒保护率也为80%。表明本实验构建的双价禽流感核酸疫苗的免疫效果与单表达组相当(P>0.05),为多价禽流感核酸疫苗的研制提供了基础。     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

H9N2亚型禽流感流行现状

低致病性禽流感主要是由H9N2亚型禽流感病毒所引起,近几年在世界上许多国家都暴发了H9N2亚型禽流感疫情.研究表明,H9N2亚型禽流感病毒在陆禽中至少可分为北美和欧亚两个种系,该病毒在自然环境中很容易发生变异,通过混合基因组分而形成不同的病毒亚型.H9N2亚型禽流感病毒已经能传播至哺乳动物,包括猪和人类,从而引发对其是...     

广西玉林市规模化禽场禽流感病毒检测

为了解广西玉林市2020年规模禽场禽流感病毒感染状况,采用荧光RT-PCR方法,对广西玉林市7个县(市、区)42个规模化禽场采集的1260份禽喉/泄殖腔棉拭子样品进行了通用型禽流感病毒核酸检测(荧光PCR),并对检测为阳性的样本进行H5、H7亚型(双重荧光PCR)和H9亚型(荧光PCR)分型鉴定。结果显示:在42个规模化禽场中,未检出H5和H7亚型高致病性禽流感病毒阳性样品;在2个鸡场中检出18份H9亚型低致病性禽流感病毒阳性样品,在2个鸡场和4个鸭场中检出115份其他亚型低致病性禽流感病毒阳性样品。结果表明:在高致病性禽流感(H5+H7)三价灭活疫苗强制免疫政策下,广西玉林市规模化禽场的高致病性禽流感病毒感染风险较小,但仍须加强禽流感的免疫、监测,做好综合防控,以降低禽流感病毒由低致病性重组变异为高致病性的风险。本检测为指导广西玉林市禽流感防控提供了依据。     

Seroprevalance and Identification of Influenza A Virus Infection from Migratory Wild Waterfowl in China (2004–2005)

Outbreaks of a highly pathogenic avian influenza (H5N1) were reported in birds in more than eight Asian countries. We sought to identify the origin of this infection, and herein report the results of serological and virological monitoring of migrant wild waterfowl in mainland China. From a total of 493 serum samples, collected from 15 migratory wild waterfowl species for 9 months (from June 2004 to May 2005) in mainland China, we detected only low‐level antibodies against influenza subtypes H2, H9 and H10 in the relict gull, little egret, black‐crowned night heron, bar‐tailed godwit, whimbrel and the common greenshank. No virus was identified from the 1052 cloacal and oropharyngeal swabs except dead bar‐headed geese. These data show that the influenza type A virus subtypes H2–H13 did not circulate at detectable levels within the sampled population.