H1N1亚型流感病毒鸡胚增殖参数的优化

为了研究最佳的H1N1亚型流感病毒鸡胚增殖参数,本试验进行了孵化前种蛋的选择与保存、鸡胚孵化中各参数设定等因素对H1N1亚型流感病毒产毒量影响的研究。其中种蛋的选择与保存,主要考察了蛋重、蛋形指数、保存期、消毒时间及方法等因素,结果显示,蛋重为55~65 g,蛋形指数为1.30~1.35,种蛋保存期为1~4 d,保存温度为16~18 ℃,保存湿度为70%~80%,保存期种蛋的甲醛熏蒸消毒时间为30 min时,可以为H1N1亚型流感疫苗生产提供最佳的种蛋。孵化过程中孵化参数对H1N1亚型流感病毒产毒量的影响,本试验主要将孵化温度、湿度、翻蛋、通风等参数作为研究对象,结果显示在生产H1N1亚型流感疫苗时,最佳孵化参数设定为:温度1~7 d为38.2 ℃、8~9 d为38.0 ℃、10 d为37.8 ℃,湿度1~10 d为65%~70%,翻蛋频率为1次/2 h,前后倾角各为45°,通风风门设定为1~5 d为4、6~10 d为5。本试验结果为H1N1亚型流感疫苗生产提供优质的鸡胚孵化技术,确保鸡胚尿囊液的质量和收获量。     

干扰素对鸡胚内H9N2亚型禽流感病毒滴度的影响

为探讨干扰素在动物上的运用,笔者利用H9N2亚型禽流感病毒与不同剂量干扰素（IFN）一起接种鸡胚,运用HA方法测定病毒效价,研究了干扰素干扰H9N2亚型禽流感病毒在鸡胚内增殖的情况,试验结果表明,高中低剂量干扰素都有抑制病毒增殖的作用,而3个试验组之间没有明显差异（P>0.05）。但是在一定的时间内,高剂量组干扰素与中低剂量组相比能够明显的抑制病毒增殖,表明高剂量组干扰素在一定时间内抑制H9N2禽流感病毒的作用比较明显,这在生产中有一定的应用价值。     

鸡胚中H9N2亚型禽流感病毒的分离鉴定

将来自有产蛋下降的肉用型父母代种鸡群的种蛋孵化,每日观察鸡胚死亡情况。在孵化的50个鸡胚中9日龄时开始死亡1个,尿囊液无血凝性,盲传一代后,鸡胚尿囊液有血凝性。在HI试验中,对H9亚型禽流感病毒(AIV)单因子血清在1∶26稀释度时呈现血凝抑制活性,对新城疫病毒(NDV)和H5亚型AIV单因子血清不呈血凝抑制活性,由此确定为低致病性禽流感病毒,命名为ZKH90901。对该毒株进行HA和NA基因扩增克隆测序,把获得的HA和NA基因与已经发表的H9N2毒株的相应序列比较,结果表明该毒株确实为H9N2亚型禽流感病毒,HA基因的裂解位点序列为KSGR↓GLF,符合低致病性禽流感病毒H9N2蛋白裂解位点的分子特征,仍属于低致病力毒株。从鸡胚中分离到H9N2亚型禽流感病毒在国内尚未见报道。     

H5N1亚型禽流感病毒NP基因的克隆与表达

根据已知H5N1亚型禽流感病毒NP基因序列设计、合成PCR克隆引物。自H5N1阳性病料中提取总RNA,反转录后采用高可信度DNA聚合酶,经PCR扩增NP基因,采用Invitrogen定向表达系统进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NP蛋白,分子质量约66.0ku。采用单克隆抗体和阳性血清经ELISA、免疫印迹分析重组蛋白的免疫反应性。结果表明:所获得的重组NP蛋白在ELISA分析中均能与阳性血清和单克隆抗体发生特异性结合,但在免疫印迹分析中仅与阳性血清发生特异性结合。     

Establishment of a Duplex Real-time RT-PCR for Detection of H6N1 Subtype Avian Influenza Virus

According to the sequences of HA and NA genes of H6 and N1 subtype avian influenza virus (AIV),two pairs of specific primers and two TaqMan probes with different fluorescence were designed.The duplex Real-time RT-PCR assay was developed and optimized to simultaneously detect H6 and N1 subtypes AIV in one reaction.The result showed that the specificity of this assay was high and only amplified H6 and N1 subtypes AIV,and was not cross-reactive with other H and N subtypes AIV,newcastle disease virus and infectious bronchitis virus.The detection limit of this assay was 100 copies/μL of H6N1 subtype AIV.This newly developed duplex Real-time RT-PCR assay was a rapid,specific and sensitive method for the detection of H6N1 subtype AIV,and it could provid a technical support to prevent and control H6N1 subtype AIV.     

H6N1亚型禽流感病毒二重荧光RT-PCR检测方法的建立

根据GenBank中H6、N1亚型禽流感病毒(AIV)的HA、NA基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.经反应条件优化,本试验建立了检测H6N1亚型AIV的二重荧光RT-PCR方法.该法特异性强,只对H6亚型和N1亚型AIV进行特异性扩增,对其他H亚型AIV、N亚型AIV及新城疫病毒、传染性支气管炎病毒等病原体的检测均为阴性;该法敏感性好,对H6N1亚型AIV的检测限为100拷贝/μL.本试验建立的H6N1亚型AIV的二重荧光RT-PCR方法,具有快速、敏感、特异的优点,为H6N1亚型AIV的防控提供技术支撑.     

H5N1亚型禽流感病毒疫苗的研究现状及应用前景

H5N1亚型禽流感病毒是目前发现的禽流感病毒中感染性最强、致死率最高、流行最广的一类病毒,该病毒已在世界上多个国家发生流行,给禽类养殖业带来了巨大的经济损失。当前针对该病尚无高效特异的治疗方法,进行疫苗的接种是目前最为有效的预防措施和关键环节。因此,研制安全、高效、廉价的新型疫苗成为当前禽流感防制工作的热点之一,且取得了大量的研究成果。作者从禽流感病毒灭活疫苗、减毒活疫苗、基因工程疫苗和RNAi技术应用等方面对H5N1亚型禽流感病毒疫苗的研究现状作一综述,归纳出该领域中存在的问题和不足,并对禽流感疫苗的应用前景作一展望,以期为深入进行禽流感的防制研究提供参考。     

猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立

根据GenBank中H1N1和H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)、神经氨酸酶(neuraminidase,NA)和M基因保守序列,分别设计合成了5对特异性引物,利用RT-PCR技术对SIV的型和亚型进行鉴定。结果表明,该方法的型RT-PCR可以检测出10⁴ EID₅₀病毒量所提取的RNA;H1、H3、N1和N2的亚型RT-PCR均可以检测出10⁴ EID₅₀病毒量所提取的RNA。除每对特异性引物所对应的亚型外,对其他亚型及猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测均为阴性,应用该方法对临床样品进行检测,其结果与病毒分离结果符合率为100%。结果表明,该方法特异性好、敏感性高,有望成为SIV的一种特异、敏感、快速的分型检测方法,为猪流感分子流行病学的调查奠定了良好的基础。     

一株鸭源H1N6亚型禽流感病毒的分离鉴定及HA和NA基因序列分析

为了解禽流感病毒（AIV）在广西中越边境地区的流行情况,本研究在该地区活禽市场开展禽流感病原监测。监测过程中分离鉴定出1株H1N6亚型禽流感病毒,命名为A/Duck/Guangxi/F01/2016（H1N6）,对其HA和NA基因进行序列测定,并与GenBank中下载的相关参考序列进行比对和遗传进化分析。结果显示,分离株HA基因与A/sparrow/Guangxi/GXs-1/2012（H1N2）的核苷酸同源性最高（96.9%）,NA基因与A/Pavo cristatus/Jiangxi/JA1/2016（H5N6）的核苷酸同源性最高（98.2%）。HA基因裂解位点氨基酸序列为PSIQSR↓GLF,符合低致病性禽流感病毒分子特征;与部分N6亚型禽流感病毒一样,分离株NA基因有11个氨基酸缺失。此外,本研究还对分离毒株的受体亲和性进行了测定,结果显示该病毒优先结合唾液酸α-2,3-Gal受体。本研究结果表明A/Duck/Guangxi/F01/2016（H1N6）是一株重组低致病性禽流感病毒。     

1株H1N1亚型猪流感病毒的全基因组特征及其对小鼠的致病性

旨在了解河南省猪流感病毒的流行情况及其遗传进化和基因组特征。2018年4月,从河南省某一出现疑似流感症状猪群中采集鼻拭子样品150份用于分离病毒,对分离病毒的全基因组进行序列测定和分析。同时感染6周龄BALB/c小鼠,研究其对小鼠的致病性。结果显示,获得1株H1N1亚型病毒[命名为A/swine/Henan/NY20/2018（H1N1）]。遗传进化表明,其HA和NA基因属于欧亚类禽H1N1分支,PB2、PB1、PA、NP和M基因属于2009甲型H1N1分支,NS基因属于经典H1N1分支。HA蛋白的裂解位点序列为PSIQSR↓GL,具有低致病性流感病毒的分子特征,在小鼠肺和鼻甲有效复制并能引起肺组织病理学变化。本研究分离到1株3源重排H1N1亚型病毒,对小鼠呈现一定致病力,提示应进一步加强对SIV的监测。     

一株鹅源H5N1亚型禽流感病毒人工感染鸡的病理学研究

鸡肌肉接种鹅源禽流感病毒A/goose/Guangdong/2/1996(H5N1)后,引起约70%发病率和60%死亡率,并表现出一些显微和超微病理学变化,主要为全身性充血、淤血、出血和血栓形成,多种器官的细胞变性、坏死、炎症,以及胰腺、肾脏、肝脏、心脏、肺脏、脑、肠、胸腺、脾脏、法氏囊等的细胞凋亡。     

一株H5N6亚型禽流感病毒对鸭和小鼠的致病性研究

为了研究一株从鹭中分离到的禽流感病毒(AIV)A/Heron/Guangdong/C1/2013(H5N6)对鸭和小鼠的致病力,本研究通过对鸭和小鼠滴鼻点眼和鸡的颈静脉注射进行攻毒试验,观察其致病力和组织病理学等变化,对其生物学特性进行初步研究。结果显示,该毒株的鸡胚半数感染量(EID₅₀)为10^-8.16/0.1 mL,静脉接种致病指数(IVPI)为2.76。对鸭的半数致死量(LD₅₀)为10^-4.0/0.2 mL,对小鼠的LD₅₀为10^-4.67/0.05 mL。以10⁶ EID₅₀/只滴鼻点眼感染鸭,主要表现为食欲下降、精神萎靡、肿头流泪等症状,大多数鸭在感染后4~7 d死亡,感染后第7 天肝脏、肺脏、肾脏仍在排毒,解剖可见心包积液、肺脏淤血、肾脏肿大等症状,病理切片可见心脏、肝脏、脾脏、肾脏炎性细胞浸润,脑细胞核固缩等病变。以5×10⁵ EID₅₀/只滴鼻感染小鼠,主要表现为食欲下降、精神萎靡、被毛粗乱、聚堆等症状,大部分小鼠在感染后5~7 d死亡,第7天时只有肺脏仍在排毒,各脏器解剖学病变不明显,病理切片可见心脏、肾脏、肺脏炎性细胞浸润,脑细胞核固缩等病变。研究结果表明,该H5N6亚型AIV毒株对鸭和小鼠具有很强的致病力,IVPI大于1.2,为高致病性AIV,本研究为H5N6亚型AIV研究和防控提供了理论基础。     

Pathogenicity of a Strain of H5N6 Subtype Avian Influenza Virus in Ducks and Mice

To investigate the pathogenicity of A/Hero/Guangdong/C1/2013(H5N6), an AIV strain isolated from Heron on ducks and mice, pathogenicity of this new virus strain and changing of histopathology as well as a preliminary study on its biological characteristics were studied by virus challenges test via intranasal and eye-drop and in chicken via jugular vein injection.Results showed that the EID₅₀ of this strain of virus was 10^-8.16/0.1 mL in embryonated chicken eggs and the intravenous inoculation of pathogenic index (IVPI) was 2.76.In ducks and mice, the 50% lethal doses (LD₅₀) of it were determined to be 10^-4.0/0.2 mL and 10^-4.67/0.05 mL, respectively.Symptoms of infection included loss of appetite, depression, swollen head and tears after being infected with 10⁶ EID₅₀ per duck by intranasal and eye-drop administration.Most of ducks died 4 to 7 days post-infection, liver, lung and kidney still eliminated toxicants at day 7 post-infection.Anatomy showed symptoms of pericardial effusion, pulmonary congestion and kidney enlargement, while pathological section showed pathological change like karyopycnosis and inflammatory cell infiltration in heart, liver, kidney and spleen.Mice developed symptoms of infection like loss of appetite, depression, shaggy coat and ruffled coat after being infected with 5×10⁵ EID₅₀ per mouse by intranasal and eye-drop administration.Most of the infected mice died 5 to 7 days post-infection and only liver still eliminated toxicants at day 7 post-infection.Although organ anatomy showed no obvious pathological changes, pathological section showed pathological changes like karyopycnosis and inflammatory cell infiltration in heart, kidney and lung.Our research demonstrated that this H5N6 subtype AIV had a strong pathogenicity and could be defined as a highly pathogenic AIV strain as its IVPI was greater than 1.2.Our work laid a theory foundation for study, prevention and control of H5N6 subtype AIV.     

H7N9亚型禽流感病毒三重PCR检测方法的建立

In order to develop a rapid and simultaneous assay for H7N9 subtype avian influenza virus （AIV）, three pairs of specific primers were designed according to the conserved sequences of the hemagglutinin (HA) gene of H7 subtype AIV, the neuramidinase (NA) gene of N9 subtype AIV, and the matix (M) gene of all subtypes AIV. The reaction conditions were optimized, and the specificity and sensitivity of this method were evaluated to develop a triplex PCR assay. It was shown that H7N9 subtype AIV could be amplified into three specific bands by this triplex PCR, the lengths of these bands were 330 (H7 AIV), 207 (N9 AIV) and 632 bp (all AIV), respectively. Samples containing H7 or N9 subtype AIV could be amplified into two specific bands, which were 330 and 632, 207 and 632 bp, respectively. Samples containing other subtypes AIV could be amplified into a 632 bp specific band. No specific band was amplified from other avian pathogenic virus. Sensitivity test results showed that as low as 10³ copies/μL H7N9 subtype AIV could be detected. This triplex PCR could simultaneously diagnose H7N9 subtype AIV, single H7 subtype AIV, single N9 subtype AIV and other subtype AIV in one tube. This assay was a rapid, specific and sensitive method for the detection of H7N9 subtype AIV. It could be applied in rapid diagnosis for clinical samples, and also provided a technical support to prevent and control H7N9 subtype AIV.     

甲型H1N1流感病毒双抗体夹心ELISA检测方法的初步建立

建立甲型H1N1流感病毒双抗体夹心ELISA检测方法,通过优化IPTG浓度和诱导时间确定HA融合蛋白的最佳表达条件,并进行Western bloc和血凝试验鉴定.用纯化蛋白制备单克隆抗体,建立检测甲型H1N1流感病毒的双抗体夹心ELISA检测方法,对其交叉反应、符合率进行验证.结果表明,HA蛋白在BL21 (DE3)中...     

H9亚型禽流感病毒对MDCK细胞增殖及其培养基过氧化氢含量的影响

为了探讨H9亚型禽流感病毒对MDCK细胞增殖及其培养基过氧化氢含量的影响,将不同剂量的H9亚型禽流感病毒接种MDCK细胞单层,吸附1.5h后弃去再加入新的DMEM维持液,每隔12h取上清液测定HA滴度、乳酸脱氢酶(LDH)活力以及过氧化氢(H2O2)含量。同时,本试验采用MTT法测定不同剂量病毒接种对MDCK细胞增殖的影响。结果表明,以10-3EID50、10-4EID50病毒剂量接种细胞后,病毒HA滴度分别在36h和48h达到滴度最大值(9log2)以及LHD活力的最大值,上清液中H2O2含量在48h时显著(P<0.05)高于空白对照组和低剂量病毒接种组;接种病毒后36h以内对MDCK细胞生长具有显著地(P<0.05)促进作用,之后会显著(P<0.05)抑制细胞的增殖。综上所述,H9亚型禽流感病毒能在前期促进MDCK细胞的增殖,随着病毒增殖对细胞损伤加大,LDH漏出量增加,促凋亡因子H2O2参与介导了这一过程。     

巢式PCR检测H1亚型禽流感病毒方法的建立

为建立快速、准确检测H1亚型禽流感病毒(avian influenza virus,AIV)的方法,本研究针对H1亚型AIV血凝素(HA)基因保守区设计合成了2对特异性引物并优化反应条件进行巢式PCR反应。特异性和敏感性试验结果表明,该PCR方法能特异性检测到H1亚型AIV,其敏感性最低能检测到13.2 fg/μL的H1亚型AIV RNA模板。本研究建立的巢式PCR方法为H1亚型AIV的早期诊断及有效防制提供了快速、特异且敏感的检测方法。     

Pathogenesis and Subsequent Cross‐Protection of Influenza Virus Infection in Pigs Sustained by an H1N2 Strain

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.     

H5N1禽流感病毒感染小鼠后内参基因的筛选

本研究旨在评定H5N1禽流感病毒(H5N1 avian influenza virus,H5N1 AIV)感染小鼠后,脾脏组织中6个候选内参基因酪氨酸3/色氨酸5-单加氧酶激活蛋白(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,Ywhaz)、次黄嘌呤磷酸核糖转移酶1(hypoxanthine-guanine phosphoribosyltransferase 1,Hprt1)、琥珀酸脱氢酶A亚基(succinate dehydrogenase flavoprotein subunit,Sdha)、β-肌动蛋白(β-actin,Actb)、肽基脯氨酰异构酶A(peptidyl prolyl isomerase A,Ppia)和18S核糖体RNA(18S ribosomal RNA,18S rRNA)的稳定性。通过H5N1 AIV强毒株A/DK/GD/378/08感染小鼠,采用Real-time PCR方法分别于12、24、48、72、96、120和144 h检测感染组与对照组中6个候选内参基因的表达水平,然后用Genorm软件对内参的稳定性进行评价。结果表明,正常小鼠脾脏组织中6个内参基因稳定度由高到低分别为Ywhaz/Hprt、Sdha、Actb、Ppia、18S rRNA;H5N1 AIV感染的小鼠脾脏组织中6个内参基因稳定度由高到低分别为Ppia/Sdha、18S rRNA、Hprt、Ywhaz、Actb;综合评价正常和H5N1 AIV感染后小鼠脾脏组织中6个内参基因的表达稳定度由高到低分别为Sdha/Hprt1、Ywhaz、Ppia、Actb、18S rRNA。因此,小鼠脾脏组织中Ppia和Sdha是研究H5N1 AIV在机体中复制时最理想的2个内参基因;Sdha和Hprt1则是在研究H5N1 AIV与机体相互作用时最理想的2个内参基因。