两株京科猪H1N1亚型流感病毒内部基因来源的研究

目的通过内部基因研究了解两株猪(H1N1)亚型流感病毒内部基因是否含有禽流感病毒基因节段及是否与猪群中H9N2亚型毒株发生了基因重配。方法病毒在鸡胚中传代,从收获的尿囊液中提取RNA,通过逆转录合成cDNA,cDNA用PCR扩增。PCR产物用纯化试剂盒纯化,接着进行核苷酸序列测定,然后用MegAlign(Version1.03)和Editseq(Version3.69)软件进行基因进化树分析。结果两株京科猪H1N1病毒内部基因除PB2基因节段有所不同外,其余5个基因节段均相同,但与猪H1N1流感病毒内部基因相近,然而,与古典型猪H1N1毒株有差异。结论两株京科猪H1N1病毒不是基因重配株,它们的内部基因均属猪H1N1流感病毒基因系。     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

我国猪群中H9N2亚型毒株HA和NA基因特性的研究

目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因，与P^GEM-T Easy Vector4℃过夜连接，重组质粒转化DH-10β细菌，筛选阳性菌落，酶切鉴定，测序。然后，进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第226位上氨基酸为L，这与从人和猪所分离出的H9N2毒株相同，其连接肽属对禽致病的毒株，但它们的序列为R-L-S-R，而不是R-S-S-R；其NA蛋白茎区第62～64位存在掉失，这与A／Shaoguarn/408／98，A／Swine／Hong Kong／9／98及A／Duck／Hong Kong／y280／97(H9N2)毒株相同；HA与NA基因进化树分析表明，两株猪H9N2毒株的HA基因接近于A／Chicken／Hong Kong／G23／97和A／Chicken／Hong Kong／G9／97．而NA基因接近于A／Shaoguan／408／98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换，可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失，造成了它们能直接感染猪。     

从人分离出两株甲型流感H9N2亚型毒株内部基因特性的研究

目的 了解2株从人分离出的H9N2亚型毒株内部基因特性，并弄清其来源。方法 用RT-PCR扩增目的基因，用P^CEM-T Vector(美国Promega公司)，4℃过夜连接，重组质粒转入dH5a细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序。然后进行进化树分析。结果 2株测定毒株内部基因均为G9基因系，它们相互间除PA基因有差异外，其余5个基因均相同。结论 2株测定毒株的基因组均为G9基因系，它们是由携带不同基因特性H9N2毒株的禽群分别直接感染人，而不是来自同一禽的H9N2亚型流感病毒。     

甲型H7N9流感裂解疫苗制备及免疫保护效果的评价

目的构建、拯救重配甲型H7N9流感病毒疫苗候选株并制备甲型H7N9流感裂解疫苗,动物实验评价甲型H7N9流感裂解疫苗的免疫原性及免疫保护性效果。方法采用反向遗传学技术将A/Anhui/1/2013(H7N9)疫苗株的HA、NA基因和A/Puerto Rico/8/34(PR8)毒株的PB2、PB1、PA、NP、M、NS基因进行重配,转染细胞后筛选拯救甲型H7N9流感病毒疫苗候选株,制备rgPR8-H7N9流感裂解疫苗抗原。腹腔注射免疫Balb/c小鼠,检测血清IgG、IgG1、IgG2a、HI效价,进一步用野生株攻毒,评价rgPR8-H7N9流感裂解疫苗的免疫保护效果。结果成功拯救甲型H7N9流感病毒疫苗候选株rgPR8-H7N9。制备的重配甲型H7N9流感裂解疫苗对小鼠产生较高的HI抗体效价。IgG1/IgG2a亚型检测结果表明小鼠体内以诱导体液免疫为主。攻毒实验显示甲型H7N9流感裂解疫苗能够有效降低肺部的病毒载量,肺组织病变显著减轻、体质量下降后趋于稳定,疫苗剂量达到15μg即可全部存活。A/Anhui/1/2013(H7N9)野生株流感病毒攻毒,甲型H7N9流感裂解疫苗能够达到保护小鼠效果。结论成功拯救重配甲型H7N9流感病毒疫苗候选株rgPR8-H7N9,制备的甲型H7N9流感裂解疫苗具有较好的免疫原性及免疫保护性,为H7N9流感裂解疫苗的研发及进入临床研究提供了实验依据。     

一株A/PR/8/34(H1N1)类似毒株基因特性的进一步研究

目的 通过病毒粒７个不同ＲＮＡ节段核苷酸序列测定和分析,进一步排除Ａ／广东／６／９１（Ｈ１Ｎ１）毒株由实验室污染而来,它与Ａ／ＰＲ／８７／３４（Ｈ１Ｎ１）毒株基因组间有哪些ＲＮＡ节段存在有差异。同时它是否是一株基因重配株？方法 病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果 所比较的７个不同ＲＮＡ节段中,测定毒株ＲＮＡ６和７     

猪在禽H9N2亚型流感病毒感染人中的作用

目的　了解猪在禽H9N2亚型流感病毒感染人中的作用。方法　用RT PCR扩增目的基因,用PGEM T Vector(美国Promega公司) 4℃过夜连接,重组质粒转入dH5α细菌,筛选阳性菌落,酶切鉴定,送六合通公司自动测序,然后进行进化树分析。结果　两株山东猪H9N2毒株基因组与人及禽分离出的H9N2病毒均有差异,中国内地从人分离出的H9N2毒株的基因组接近鸡的毒株,而香港特区从人分离出的接近鹌鹑的毒株;禽H9N2毒株不仅宿主范围广,同时其基因组具有多样性。结论　禽H9N2亚型毒株是直接感染人,而不是通过所谓的中间宿主猪,然后再感染人。     

我国人群中一种新重配的H1N2亚型流感病毒株的发现

１９９６年１月太原铁路卫生防疫站从上感患者中分离到３株流感病毒。经血清学鉴定，它们不同于１９８９和１９９２年所发现的Ｈ１Ｎ２亚型毒株，其ＨＡ的抗原性类似于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）病毒，而明显不同于当前人群中流行的Ｈ１Ｎ１亚型毒株。病毒粒不同基因节段迁移率比较表明，它们的１～４基因节段迁移率接近于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）毒株，５～６基因节段迁移率类似于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒，而７～８两节段既不同于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１），又不同于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒。故可认为它们是一种新重配的Ｈ１Ｎ２亚型毒株。     

深圳地区人和鸡群中H9N2亚型流感病毒病原学和血清流行病学调查

目的：了解甲型流感病毒N9N2亚型毒株在深圳地区鸡群和人群中的分布。方法：采用常规的鸡胚双腔法来分离病毒。抗体测定，采用红细胞凝集抑制（HI）试验和中和试验测定法。结果：从深圳地区农贸市场鸡群中分离到27株H9N2亚型流感病毒，但未能从人群中分离到H9N2病毒。约有26％人血清中检测到H9亚型毒株的抗体，（HI滴度≥20），同时还发现抗体阳性率和几何均数随人群年龄增长而增高，同时与职业有关。然而，在鸡群中H9毒株的抗体阳性率仅为7％。结论：禽H9N2毒株不仅能感染人，而且在深圳地区人群和禽类中较为广泛的分布。人H9N2很大可能来源于鸡的H9N2毒株。     

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。     

H9N2禽流感病毒反向遗传系统的构建和鉴定

目的 建立H9N2亚型禽流感病毒反向遗传系统,为人禽流感疫苗研制以及传播和致病机制等方面的研究提供技术平台.方法 使用RT-PCR方法获得禽流感H9N2亚型病毒A/Guangzhou/333/99(H9N2)的8条全长基因节段,然后克隆到双表达载体pCI-pol Ⅰ中,获得H9N2禽流感病毒的8个基因节段的8质粒系统.将构建好的8质粒共转染293T细胞后,收获上清接种鸡胚,然后对鸡胚尿囊液进行鉴定;对拯救的病毒进行鉴定.结果 8质粒系统转染293T细胞后可以成功拯救出H9N2禽流感病毒,血凝效价可达到29/50μl,生长特性与野生型病毒类似.结论 成功建立了H9N2禽流感病毒反向遗传系统.     

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.     

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.     

甲型流感病毒（N1N2）亚型毒株血凝素和神经氨酸酶基 …

目的 研究新分离到的Ｈ１Ｎ２亚型毒株血凝素（ＨＡ）和神经氨酸酶（ＮＡ）基因的来源。方法 病毒通过鸡胚增殖后提取其ＲＮＡ,通过逆转录合成ｃＤＮＡ,经ＰＣＲ扩增和产物纯化,用双脱氧链终止法进行核苷酸序列测定,并用ＭｅｇＡｌｉｇｎ（１．０３版）和Ｅｄｉｔｓｅｑ（３．６９版）软件进行种系发生学分析。结果 新分离到Ｈ１Ｎ２毒株ＨＡ１区氨基酸序列与Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）和Ａ／Ｇｕａｍｇｄｏｎｇ／６／９     

Comparison of the virologic and immunologic responses of volunteers to live avian-human influenza A H3N2 reassortant virus vaccines derived from two different avian influenza virus donors.

We compared the abilities of the six internal RNA segments of two avian influenza viruses, A/Mallard/Alberta/88/76 (H3N8) and A/Mallard/NY/6750/78 (H2N2), to confer attenuation on wild-type human influenza A/Bethesda/1/85 (H3N2) virus in seronegative adult volunteers. Live avian-human influenza A reassortant virus vaccines derived from either avian virus parent were comparable in the following properties: safety, infectivity, immunogenicity, and genetic stability. Since the avian influenza A/Mallard/Alberta/76 virus offered no clear advantage as a donor virus, we will conduct our future evaluations on live influenza A virus reassortants derived from the more extensively characterized avian influenza A/Mallard/NY/78 virus.     

Avian influenza A virus H7N9 in China,a role reversal from reassortment receptor to the donator

Reassortment can introduce one or more gene segments of influenza A viruses (IAVs) into another, resulting in novel subtypes. Since 2013, a new outbreak of human highly pathogenic avian influenza has emerged in the Yangtze River Delta (YRD) and South-Central regions of China. In this study, using Anhui province as an example, we discuss the possible impact of H7N9 IAVs on future influenza epidemics through a series of gene reassortment events. Sixty-one human H7N9 isolates were obtained from five outbreaks in Anhui province from 2013 to 2019. Bioinformatics analyses revealed that all of them were characterized by low pathogenicity and high human or mammalian tropism and had introduced novel avian influenza A virus (AIV) subtypes such as H7N2, H7N6, H9N9, H5N6, H6N6, and H10N6 through gene reassortment. In reassortment events, Anhui isolates may donate one or more segments of HA, NA, and the six internal protein-coding genes for the novel subtype AIVs. Our study revealed that H7N9, H9N2, and H5N1 can serve as stable and persistent gene pools for AIVs in the YRD and South-Central regions of China. Novel AIV subtypes might be generated continuously by reassortment. These AIVs may have obtained human-type receptor-binding abilities from their donors and prefer binding to them, which can cause human epidemics through accidental spillover infections. Facing the continual threat of emerging avian influenza, constant monitoring of AIVs should be conducted closely for agricultural and public health.     

Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health

Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.     

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus

Virus Genes - H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007...