Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

抗H3N2亚型猪流感病毒单克隆抗体的制备与鉴定

采用纯化的H3N2亚型猪流感病毒(SIV)尿囊液作为免疫原免疫6～8周龄Balb/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,用间接ELISA方法筛选分泌抗SIV-H3N2的阳性细胞株,经克隆获得7株亲和力较高的杂交瘤细胞株,分别命名为1C9、2C5、2F10、3D3、4E8、5C7、5D12,用其制备的腹水ELISA效价可达1×106。通过抗体亚型测定,间接免疫荧光试验及免疫印迹试验分析鉴定,该7株单抗均为抗H3N2亚型SIV的特异性单克隆抗体,而且与其他亚型猪流感病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒和猪瘟病毒等均无交叉反应,为H3N2亚型SIV的鉴别诊断奠定了基础。     

重组欧洲禽源H1N1亚型猪流感病毒疫苗株的构建及免疫保护效力

以国内欧洲禽源H1N1亚型猪流感病毒流行株A/swine/Shanghai/1/2014(H1N1)(SH1)为材料,RT-PCR扩增HA和NA基因并将其克隆至PBD载体中,构建HA和NA基因的重组质粒。将HA和NA基因重组质粒及来源于流感病毒高产株PR8的6个内部基因(PB2、PB1、PA、NP、M和NS)重组质粒共转染293T细胞,从而成功构建了重组欧洲禽源H1N1亚型猪流感病毒疫苗株SH/PR8。以相同病毒剂量接种MDCK细胞,检测不同时间点的血凝效价,绘制病毒生长曲线,结果表明,重组病毒SH/PR8相对于原始野生株SH1在MDCK细胞上具有更高的病毒滴度。重组病毒SH/PR8制备的灭活疫苗经过一免和二免小鼠后,检测血清中血凝抑制抗体、中和抗体和IgG抗体,结果显示首免后抗体效价持续升高,4周后抗体效价达到峰值。小鼠攻毒试验结果表明,免疫组体质量下降不明显且未出现死亡,而非免疫组小鼠体质量持续下降并且于攻毒后6d小鼠出现100%死亡。肺脏组织病毒含量滴定以及组织病理学观察结果显示,免疫组能够有效抑制SH1病毒在肺脏中的复制,减轻肺脏病理变化。总之,本研究表明重组SH/PR8疫苗候选株相对于原始毒株SH1在MDCK细胞上具有更高的复制能力,制备的重组灭活疫苗能够诱导机体产生高水平的抗体,能够针对欧洲禽源H1N1亚型猪流感病毒的攻击提供很好的免疫保护。     

Novel swine influenza virus subtype H3N1 in Italy

To date, three subtypes of swine influenza viruses, H1N1, H1N2, and H3N2 have been isolated in Italy. In 2006, a novel swine influenza virus subtype (H3N1) was isolated from coughing pigs. RT-PCR performed on lung tissues, experimental infection in pigs with the novel isolate, and cloning the virus by plaque assay confirmed this unique H and N combination. The novel isolate was also antigenically and genetically characterized. Genetic and phylogenetic analysis showed that the complete HA gene of the H3N1 strain has the highest nucleotide identity to three Italian H3N2 strains, one isolated in 2001 and two in 2004, whereas the full length NA sequence is closely related to three H1N1 subtype viruses isolated in Italy in 2004. The remaining genes are also closely related to respective genes found in H1N1 and H3N2 SIVs currently circulating in Italy. This suggests that the novel SIV could be a reassortant between the H3N2 and H1N1 SIVs circulating in Italy.     

Efficacy of swine influenza A virus vaccines against an H3N2 virus variant

We compared the efficacy of 3 commercial vaccines against swine influenza A virus (SIV) and an experimental homologous vaccine in young pigs that were subsequently challenged with a variant H3N2 SIV, A/Swine/Colorado/00294/2004, selected from a repository of serologically and genetically characterized H3N2 SIV isolates obtained from recent cases of swine respiratory disease. The experimental vaccine was prepared from the challenge virus. Four groups of 8 pigs each were vaccinated intramuscularly at both 4 and 6 wk of age with commercial or homologous vaccine. Two weeks after the 2nd vaccination, those 32 pigs and 8 nonvaccinated pigs were inoculated with the challenge virus by the deep intranasal route. Another 4 pigs served as nonvaccinated, nonchallenged controls. The serum antibody responses differed markedly between groups. After the 1st vaccination, the recipients of the homologous vaccine had hemagglutination inhibition (HI) titers of 1:640 to 1:2560 against the challenge (homologous) virus. In contrast, even after 2nd vaccination, the commercial-vaccine recipients had low titers or no detectable antibody against the challenge (heterologous) virus. After the 2nd vaccination, all the groups had high titers of antibody to the reference H3N2 virus A/Swine/Texas/4199-2/98. Vaccination reduced clinical signs and lung lesion scores; however, virus was isolated 1 to 5 d after challenge from the nasal swabs of most of the pigs vaccinated with a commercial product but from none of the pigs vaccinated with the experimental product. The efficacy of the commercial vaccines may need to be improved to provide sufficient protection against emerging H3N2 variants.     

具有HI活性的H1亚型流感病毒特异性单克隆抗体的制备与应用

为了制备具有HI活性的HI亚型流感病毒特异性单克隆抗体(MAb),本研究以H1N1亚型猪流感病毒(SIV)株A/Swine/Guangdong/718/01(H1N1)为免疫原,免疫BALB/c小鼠,经常规细胞融合后,血凝抑制(HI)方法进行检测,融合细胞经稀释克隆纯化后,获得11株能稳定分泌抗血凝素特异性HI MAb的杂交瘤细胞株。鉴定表明,所获MAb与其他具有血凝活性的病毒以及其他14个HA亚型的流感病毒均不具有HI交叉反应,表明这11株MAb均具有良好的流感病毒亚型特异性。其中A6F、2BBF和2BB与其他H1亚型流感病毒分离株的HI试验证实我国不同地区分离株之间的抗原性存在一定差异。11株MAb对H1SIV抗原的HI试验结果显示其HI效价有明显差异。叠加实验表明这些MAb分别识别HA抗原的不同表位。间接免疫荧光试验表明,2BBF、8HB、1DH、7FC和2BB均可与2009年流行H1N1病毒A/California/04/2009HA抗原发生特异性反应。这些MAb特异性的研制为H1亚型流感病毒的疫情病原学快速诊断以及病毒抗原性变异的相关研究提供了物质基础。     

猪流感病毒H1N1亚型HA蛋白单克隆抗体的制备与鉴定

为获得H1N1亚型猪流感病毒(SIV)血凝素(HA)蛋白的单克隆抗体(McAb),选取A/swine/NanChang/H1N1/2009毒株,病毒经增殖、超速离心后,收集病毒粒子作为免疫原,将SIV粒子包被建立了间接ELISA方法。将免疫4次的BALB/c小鼠,经间接ELISA测定小鼠血清效价,将效价高的小鼠脾细胞与骨髓瘤细胞(sp2/0)进行融合。采用间接ELISA的方法筛选效价高的阳性细胞孔,采用有限稀释法进行3轮亚克隆,得到了5株杂交瘤细胞株,分别命名为2C6、5G5、1D5、3G3、4F11。随后对5株杂交瘤细胞的亚型进行鉴定,显示2C6、1D5、4F11为IgM亚型,5G5、3G3为IgG1亚型,5株杂交瘤的L链均为k链。间接免疫荧光(IFA)和Western blot检测表明,5G5能够与HA蛋白发生特异性的结合。本研究为进一步建立H1亚型SIV的相关检测方法及对HA蛋白的功能研究奠定基础。     

H3N2亚型猪流感病毒NS2基因表达产物特性分析

采用RT-PCR方法克隆猪流感病毒H3N2亚型NS2全长基因,构建NS2基因原核表达质粒pET-28a-NS2和真核表达质粒p3xFLAG-CMVNS2,在大肠杆菌和真核细胞内表达NS2基因,并制备抗NS2多克隆抗体。用所制备的抗体分析p3xFLAG-CMV-NS2转染表达NS2蛋白和病毒感染细胞内的NS2蛋白。结果表明:经终浓度为1 mmol/L的IPTG诱导后,重组蛋白NS2在大肠杆菌中得到表达。表达蛋白纯化后免疫Wistar大鼠制备抗NS2蛋白多克隆抗体,Western-blot分析表明抗NS2多克隆抗体可以识别大肠杆菌表达的NS2蛋白、转染Vero细胞表达的NS2蛋白和病毒感染细胞内的NS2蛋白。间接免疫荧光发现NS2蛋白主要定位于细胞质。本试验为进一步研究NS2蛋白在病毒复制过程中的生物学功能和猪流感病毒的复制机理奠定基础。     

猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立

对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

类人源猪H3N2亚型流感病毒HA,NA遗传进化分析

2007-2008年从吉林省某猪场采集疑似流感发病猪的鼻咽拭子,经病毒分离鉴定获得3株H3N2亚型流感病毒,分别命名为A/swine/Jilin/5/2007（Sw/Jilin/5/07）、A/swine/Jilin/19/2007（Sw/Jilin/19/07）、A/swine/Jilin/37/2008（Sw/Jilin/37/08）。HA进化树分析结果表明：3株H3N2亚型流感病毒属于近代人源病毒谱系;但是在NA进化树中,Sw/Jilin/37/08株与早期人源和近代人源关系密切,暗示它可能是早期人源和近代人源H3N2亚型猪流感病毒之间的过渡毒株,进一步证实猪在流感病毒种间传播过程中充当＂中间宿主＂作用。     

Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype

Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in na?ve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease.     

一株H9N2亚型猪流感病毒的遗传进化和致病性分析

本研究从有流感症状的病猪中分离到一株H9N2亚型猪流感病毒(SIV),命名为A/swine/Jiangsu/1/2015(SW/JS/1/15)。为探究其遗传特征和生物学特性,本研究采用RT-PCR技术扩增其全部基因节段后测序并进行遗传分析,并研究了其对鸡和豚鼠的致病特性。遗传进化分析显示,分离病毒SW/JS/1/15株是由BJ/94系、DK1系、G1系和F/98系4个分支病毒重组而成,8个基因节段均属于G57基因型。分离株HA蛋白裂解位点为PSRSSR*GL,符合低致病性流感病毒的特征。HA蛋白有9个潜在糖基化位点,其中218位糖基化位点缺失,145位与313位各新增一个糖基化位点。与疫苗株SH/F/98、SD/6/96、GD/SS/94相比,分离病毒HA抗原位点发生了G^90E、S^127R、S^145N、D^153G、N^167S、A^168N、A^198T、T^200R、N^201D、和Q^235M(H9numbering)突变;NA蛋白发生6个氨基酸突变:K^367R、K/E^368N、D^369N、D^401E、K^143N和T^434P。同时NA蛋白颈部缺失aa63~aa65。分离病毒的8个基因节段与2株禽源H9N2病毒的相应基因高度同源,其6个内部基因与两株人源H7N9病毒的内部基因高度同源。致病性试验结果显示分离病毒可以感染鸡和豚鼠,但不能在豚鼠群内水平传播,且可能作为H7N9等新型流感病毒内部基因供体,同时表明猪可以感染禽流感病毒(AIV),且可能是AIV获得感染哺乳动物能力的过渡宿主。本研究为H9N2亚型SIV的致病性以及遗传特征的研究提供科学依据。     

H3N2亚型猪流感病毒NS1基因的克隆表达及间接ELISA检测方法的建立

采用RT-PCR方法成功扩增了H3N2亚型猪流感病毒(Swine influenza virus,SIV)四川分离株(A/Swine/Sichuan/01/2006)的NS1基因,将其克隆于原核表达载体pET-32a(+)中,构建了重组质粒pET32-NS1.将该质粒转化进大肠杆菌RosettaTM,经终浓度为0.5 mmol/L的IPTG诱导表达后,通过SDS-PAGE电泳表明融合的NS1蛋白得到了大量的表达,该融合蛋白的相对分子质量约为43 500.Western-blotting结果显示该蛋白能与阳性血清发生特异性反应具有很好的免疫反应原性.结果表明,所建立的ELISA方法与几种常见猪群传染病病原无交叉反应,具有较好的特异性、敏感性和重复性,可进一步优化应用于临床SIV的检测.     

猪流感(H_1N_1H_3N_2亚型)二价灭活疫苗试制报告

猪流行性感冒(swine influenza,SI)是由猪流行性感冒病毒(SIV)引起的猪的一种急性、热性和高度接触性的呼吸道传染病,其临床上以突发高热、咳嗽、呼吸困难、衰竭和死亡为特征[1].     

Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs

OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.     

基因重排H1N2亚型猪流感病毒的分离鉴定和生物学特性的研究

本研究对2005年～2006年广西和海南省疑似猪流感(SD)病猪的组织病料进行了病毒分离,并对分离毒株进行了亚型鉴定和生物学特性的研究.结果显示:分离的3株流感病毒均为H1N2亚型猪流感病毒(SIV).能凝集多种动物的红细胞,但凝集谱与以往报道略有不同;为热不稳定型病毒;在电镜下可观察到典型流感病毒粒子.动物试验显示:小白鼠、大白鼠和家兔对分离毒株不敏感,而豚鼠较敏感.能较好地复制出流感症状和病理变化;本体试验动物仅有轻微的临床症状,但能检测到抗体升高的变化,并能从鼻腔和上呼吸道检测和分离到SIV.核苷酸同源性分析显示:分离毒株Sw/GX/17/05、Sw/GX/13/06和Sw/HN/1/05的血凝素(HA)基因分别与基因重排H1N2 亚型SIV A/Swine/lndiana 9K035/99、A/SW/MN/23124-T/01和A/swine/Zhejiang/1/04的核苷酸同源性最高,分别达97.4%、97.0%和95.7%;神经氨酸酶(NA)基因均与基因重排H1N2 亚型流感病毒A/Trurkey/MO/24093/99 的核苷酸同源性最高,达97.2%～98.0%.核苷酸同源性分析进一步证实了分离毒株为基因重排H1N2亚型SIV.     

H9N2亚型猪流感病毒对小鼠的致病性及其分子特性研究

本实验从河北地区疑似流感发病猪体内分离到一株病毒,经鉴定为H9N2亚型猪流感(SIV)病毒.将该分离株经滴鼻、点眼途径感染小鼠,观察临床症状和病理变化,同时对血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)和基质蛋白基因(M)进行克隆和序列测定,与GenBank中登录的相关序列进行比对并绘制系统发育进化树.致病性结果显示:感染小鼠出现精神不振,体重下降,并引起以弥漫性肺泡损伤为主的临床症状和病理变化.序列分析结果显示:该分离株与禽流感病毒(AW) A/chicken/Hebei/4/2008 (H9N2)(简称CK/HB/4/08)参考株的HA、NA、NP和M基因的核苷酸序列和推导的氨基酸序列的同源性最高.HA蛋白的裂解位点序列为PARSSR↓GLF,属于低致病性流感病毒的裂解位点.HA、NP、NA和M基因的遗传进化分析均显示该分离株与AIV的CK/HB/4/08株位于同一分支,具有较近的亲缘关系;由此推测该分离株可能是由CK/HB/4/08演化而来,并在跨物种传播的过程中发生了部分变异.     

H3N2亚型犬流感病毒的分离鉴定

为了解广州地区犬流感的流行情况,本研究采集107份犬鼻咽拭子样品和58份血清样品,SPF鸡胚分离病毒并进行抗体检测。结果显示:分离获得3株H3N2亚型犬流感病毒。分离株的EID50为10-2.42/0.1 mL～10-3.5/0.1 mL;MDT为177.6 h～192 h;对乙醚、氯仿、酸和温度均敏感;对血凝素为热不稳定型;能够凝集公鸡、豚鼠、猪和牛的红细胞。     

Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice

The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 10⁸ or 10⁷. Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals.