登革病毒1、2型单、双价病毒样颗粒的免疫原性研究

目的初步研究登革病毒1、2型单、双价病毒样颗粒（VLP）的免疫原性。方法表达纯化各型VLP。用纯化好的VLP免疫BALB／c小鼠,将BALB／c小鼠随机分PBS组、灭活DENV1、灭活DENV2、纯化VLP1、纯化VLP2、纯化VLP1＋2组,以灭活病毒为阳性对照,PBS为阴性对照。ELISA法检测小鼠血清抗vLP抗体效价以及血清IFN-1、TNF-α细胞因子水平,流式细胞术检测小鼠脾细胞CD4＋细胞和CD8＋细胞数。结果登革病毒1、2型单、双价VLP均能刺激免疫小鼠产生一定程度的抗血清效价,VLP1的抗体水平较VLP2的低,双价VLP组针对DENV2抗原的抗体水平上升明显,是单价VLP2的2-3倍;攻毒后。灭活DENV1和VLP2可以产生高水平的IFN-1,分别为60和35pg／ml,双价VLP组的较低;VLV2组的TNF-α一直保持较高水平,双价vLP组中攻DENV2组的TNF咀水平较DENVl组高;三次免疫后,各实验组较之PBS组CD4＋细胞的比例都有下降,而CD8＋细胞变化不大,除灭活DENV1组有增加,其余各组都有减少。结论无论单、双价VLP均能刺激小鼠产生一定程度的体液免疫和细胞免疫反应,联合免疫有一定程度的协同性。     

流感/禽流感病毒及其致病力鉴别的基因芯片技术研究

目的 建立流感/禽流感病毒及其致病力鉴别的基因芯片检测技术.方法 以血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)基冈作为靶片段,设计病毒检测和致病力特异性鉴别探针,建立基因芯片鉴别检测技术,采用单引物扩增法(SPA)处理样本核酸,分别对此芯片进行特异性、敏感性和符合率评价.结果 此芯片能够特异性的检测H1N1、H3N2、B型流感病毒及H5N1、H9N2禽流感病毒,敏感性分别为8HAU、16HAU、32HAU及8HAU、8HAU.致病力鉴别探针敏感性为32HAU.同RT-PCR方法比较,检测灵敏度为83.9%.结论 建立的常见流感病毒检测基因芯片特异性高、敏感性高、灵敏度高,更能够对致病力进行有效甄别,可作为临床诊断、传染病防控等方面的有益补充.     

Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications

The goal of the study was to establish if there was a relationship between molecular patterns and virus evolution. Therefore the complete genome sequence of two distinct apathogenic Newcastle disease virus (NDV) strains was determined and a third genome size category, containing 15,198 nucleotides, was recognized. Phylogenetic analysis revealed that two major separations resulting in three genome size categories occurred during the history of NDV. An ancient division in the primordial reservoir (wild waterbird species) led to two basal sister clades, class I and II, with genome sizes 15,198 (due to a 12 nucleotide insert in the phosphoprotein gene) and 15,186 nucleotides, respectively. Ancestors of only class II viruses colonized chicken populations and subsequently converted to virulent forms. These took place more than once and resulted in an early lineage [including genotypes I–IV and H33(W)] with genome size of 15,186 nucleotides. A second division occurred in the 20th century in the secondary (chicken) host. This gave rise to the branching-off of a clade (including recent genotypes V–VIII consisting of only pathogenic viruses) with the concomitant insertion of six nucleotides into the 5′ non-coding region of the nucleoprotein gene thereby increasing the genome size to 15,192 nucleotides.     

Biological characteristics and immunological properties in Muscovy ducks of H5N6 virus-like particles composed of HA-TM/HA-TMH3 and M1

Highly pathogenic avian influenza viruses (HPAIVs), including H5N6 strains, pose threats to the health of humans and poultry. Waterfowl play a crucial role as a reservoir of HPAIVs. Since current influenza vaccines induce poor antibody titres in waterfowl, there is an urgent need to develop an efficient vaccine against H5N6 infection. In this study, we constructed two H5N6 virus-like particles (VLPs) composed of matrix-1 (M1) and haemagglutinin of wildtype (HA-TM) or haemagglutinin with transmembrane domain replacement (HA-TM_H3) (designated as H5N6 VLPs-TM and H5N6 VLPs-TM_H3). Biological characteristics of the composed H5N6 VLPs were compared including localization, expression, contents of HA trimers, thermal stability, morphology and immunogenicity in Muscovy ducks. Our results indicate that the H5N6 VLPs-TM_H3 contained more HA trimers and presented better thermal stability. Moreover, Muscovy ducks immunized with H5N6 VLPs-TM_H3 produced higher titres of HI antibody and IFN-γ compared with those immunized with the same dose of H5N6 VLP-TM, thus providing a promising approach for the development of influenza virus vaccines for waterfowl.

RESEARCH HIGHLIGHTS

• H5N6 VLPs-TM_H3 had more HA trimers and resisted higher temperature than H5N6 VLPs-TM

• H5N6 VLPs-TM_H3 induced higher titre of HI than H5N6 VLPs-TM in Muscovy ducks

     

Artificially designed hepatitis B virus core particles composed of multiple epitopes of type A and O foot-and-mouth disease virus as a bivalent vaccine candidate

Recently, many countries, including China, have experienced a series of type A and O foot-and-mouth disease virus (FMDV) epidemics, causing serious economic losses. Although concerns about the safety of inactivated FMD vaccines have been raised, the development of a safe and effective subunit vaccine is necessary. We constructed two chimeric virus-like particles (VLPs; rHBc/AO and rHBc/AOT VLPs) displaying tandem repeats of B cell epitopes (VP1 residue 134-161 and 200-213) derived from type A and O FMDV and one T cell epitope (3 A residue 21-35) using the truncated hepatitis B virus core (HBc) carrier. Our results indicate that the chimeric HBc can self-assemble into VLPs with these FMDV epitopes displayed on the surface. Immunization with the chimeric VLPs induced specific IgG and neutralization antibodies against type A and O FMDV in mice. Compared with the commercial type A/O FMDV bivalent inactivated vaccine, rHBc/AO and rHBc/AOT VLPs significantly stimulated the production of Th1 type cytokines (IFN-γ and IL-2), whereas Th2 cytokine production (IL-4 and IL-10) was decreased. Compared with rHBc/AO, rHBc/AOT induced increased Th2 cytokine and specific IgG production. These results demonstrate that the VLPs constructed in the current study induced both humoral and cellular immune responses and may represent potential bivalent VLP vaccines targeting both FMDV type A and O strains.     

A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus

A latex agglutination test (LAT) was developed for detecting antibodies against avian influenza virus. The recombinant avian influenza virus nucleoprotein expressed in Escherichia coli was purified, coupled with latex beads, and used as an antigen for the LAT. The LAT was capable of detecting anti-avian influenza virus antibodies irrespective of the avian-influenza subtype, and in most cases, the results correlated with the results of an agar gel precipitation test (AGPT). However, in comparison with the AGPT, the LAT could detect the anti-avian influenza virus antibodies for a longer period of time after the infection. The nonspecific agglutination observed in uninfected chicken sera was resolved by pretreating the sera with dried chicken-liver powder for 1 h. The LAT is easy to perform, and even after considering the time required for pretreatment of the serum, the total time required for obtaining the results is reduced in comparison to the time required in the case of the AGPT. This easy and rapid LAT is considered to be useful for monitoring avian influenza virus infection in the field.     

基于iTRAQ技术的人感染H7N9禽流感病例血浆蛋白质组学特征分析

目的 分析人感染H7N9禽流感病例与正常人之间血浆蛋白质组学差异.方法 收集华北地区首例人感染H7N9禽流感病例发病期和恢复期血浆标本,以及同时期病例父亲(未感染病毒)血浆标本,分离提取血浆蛋白,通过同位素标记相对与绝对定量(isotopically labeled relative and absolute quantification,iTRAQ)技术联合液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)技术对样本进行蛋白质质谱检测.利用PANTHER平台对差异蛋白质组进行Gene Ontology(GO)注释以及基因路径(pathway)分析.结果 本研究鉴定得到与人感染H7N9禽流感病毒感染相关的差异表达蛋白共250个,其中表达上调蛋白合计159个,表达下调蛋白合计91个.这些差异蛋白共参与了11个生物学过程、共参与了7个分子通路.结论 入感染H7N9禽流感病毒可能导致7个分子通路发生改变.     

新城疫病毒血凝素-神经氨酸酶蛋白的可溶性超表达与免疫活性检测

目的检测新城疫病毒(NDV)血凝素-神经氨酸酶(HN)蛋白在大肠杆菌中的可溶性表达量,及其免疫活性。方法采用融合表达的方法将HN蛋白基因分别与Grifin、谷胱甘肽S移酶(GST)、麦芽糖结合蛋白(MBP)、Nus A、小泛素样相关修饰物(SUMO)、硫氧还蛋白(thioredoxin)、蛋白G(protein G)、γ-晶状体蛋白(γ-crystallin)、Ars C、Ppi B 10种融合标签基因采用柔性接头进行连接,构建10个重组表达质粒,经酶切及测序鉴定正确后,将这10个重组质粒分别转入大肠杆菌BL21中,用不同条件进行诱导表达,经SDS-PAGE检测并选出最佳诱导蛋白表达的条件,筛选出能够显著促进HN蛋白可溶性表达的标签,并用Western blotting对所表达蛋白的含量进行定性分析,同时纯化的重组蛋白用Western blotting和间接ELISA验证其免疫原性。结果成功构建了10个不同融合标签的HN融合蛋白表达载体,筛选出融合标签麦芽糖结合蛋白(MBP)能够显著促进HN蛋白可溶性表达,且Western blotting显示所表达重组蛋白的表达量是最多的。经Western blotting和间接ELISA验证重组HN蛋白具有良好的免疫原性。结论融合标签MBP可以显著提高HN蛋白在大肠杆菌中的可溶性表达量。     

Vaccination of gallinaceous poultry for H5N1 highly pathogenic avian influenza: Current questions and new technology

Vaccination of poultry for avian influenza virus (AIV) is a complex topic as there are numerous technical, logistic and regulatory aspects which must be considered. Historically, control of high pathogenicity (HP) AIV infection in poultry has been accomplished by eradication and stamping out when outbreaks occur locally. Since the H5N1 HPAIV from Asia has spread and become enzootic, vaccination has been used on a long-term basis by some countries to control the virus, other countries have used it temporarily to aid eradication efforts, while others have not used it at all. Currently, H5N1 HPAIV is considered enzootic in China, Egypt, Viet Nam, India, Bangladesh and Indonesia. All but Bangladesh and India have instituted vaccination programs for poultry. Importantly, the specifics of these programs differ to accommodate different situations, resources, and industry structure in each country. The current vaccines most commonly used are inactivated whole virus vaccines, but vectored vaccine use is increasing. Numerous technical improvements to these platforms and novel vaccine platforms for H5N1 vaccines have been reported, but most are not ready to be implemented in the field.     

Identification of a natural multi-recombinant of Newcastle disease virus

Newcastle disease (ND), caused by ND virus (NDV), is one of the most serious illnesses of birds, particularly chickens, and has been one of the major causes of economic losses in the poultry industry. Live vaccines are widely used to prevent chicken from NDV all over the world. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in NDV evolution. In this study, we performed the phylogenetic and recombination analysis on complete NDV genomes. A natural multi-recombinant cockatoo/Indonesia/14698/90 (AY562985) was identified. Its two minor parental-like strains might be from the NDV vaccine lineage and anhinga/U.S.(Fl)/44083/93 lineage, respectively. Our study suggests that recombination plays a role in NDV evolution. Especially, the study also suggests that live vaccines have capacity to play roles in shaping NDV evolution by homologous recombination with circulating virus.     

Detection of amantadine-resistant variants among avian influenza viruses isolated in North America and Asia

Here, we report the diversity of amantadine-resistant mutants among avian influenza A viruses with pandemic potential (H5, H6, H7, and H9 hemagglutinin subtypes). Drug-resistant variants were not detected among 1979--83 isolates, whereas 31.1% of H5 and 10.6% of H9 strains from Southeast Asia isolated in 2000-04 carried mutations in M2 protein. In North America, resistant variants occurred among H7 viruses only (16.4% of those tested). H6 viruses were amantadine-sensitive. These findings prompt concern regarding the control of pandemic influenza, the possibility that the next pandemic virus will be amantadine-resistant and the need to monitor the use of the drug in poultry.     

Co-circulation of genetically distinct groups of avian paramyxovirus type 1 in pigeon Newcastle disease in Iran

Pigeons are considered as one of the major natural reservoirs in the epidemiology of Newcastle disease (ND). In this study, the partial sequence of fusion protein gene of 17 pigeon-origin ND viruses (NDVs) isolated during 2012–2013 in Iran was analysed. Since the studied isolates showed F0 protein cleavage sites compatible with velogenic NDVs, all were considered as virulent NDVs. Two isolates carried 112RRQKRF117 as the cleavage site motif, whereas the rest demonstrated 112KRQKRF117 motif which just recently has been reported among Iranian virulent NDVs. Phylogenetic analysis divided all these diverse isolates in two distinct clusters within class II genotype VI. Based on the partial fusion protein gene sequence, 15 out of 17 isolates showed the highest genetic identity to subgenotype VIb/2 and the other two isolates were placed in a distinct genetic group of genotype VI. Based on recent findings, at least two different sublineages of genotype VI are causing the ND outbreaks in the pigeon population and are circulating simultaneously along with virulent NDVs of genotype VII in various species in Iran. The continuing circulation of a diverse group of virulent NDVs as an enzootic in widespread species such as pigeon can cause outbreaks in commercial poultry flocks and also failure in controlling programmes. Therefore, the constant monitoring and awareness of the virus characteristics should be considered in controlling programmes against ND in Iran.     

Monoclonal antibodies identify a strain-specific epitope on the HN glycoprotein of Newcastle disease virus strain Australia-Victoria

A panel of monoclonal antibodies raised against the hemagglutinin-neuraminidase glycoprotein (HN) of the Australia-Victoria strain of Newcastle disease virus has been used to compare that strain and eight other strains of the virus. The ability of the antibodies to neutralize infectivity, inhibit hemagglutination and neuraminidase, and bind to purified virions in solid-phase radioimmunoassays was determined for each strain. Of the four antigenic sites delineated by these antibodies on the HN of the homologous strain, site 1 (that with the greatest neutralizing susceptibility), is apparently conserved in all the strains tested as revealed by neutralization assays. The least neutralizing site, number 4, is also conserved in most of the strains tested. Site 2, which lies at or near the neuraminidase site, appears to be conserved in the avirulent strains but not in the virulent strains. An antibody to site 3 is unable to bind to a significant extent to any of the heterologous strains tested, and thus recognizes a strain-specific epitope. Inhibition of hemagglutination and neuraminidase by antibodies to each site were also examined and the results suggest that antibodies to sites 1 and 2 may distinguish virulent and avirulent strains at least with respect to these functions.     

一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性

目的以非包涵体形式表达含戊型肝炎病毒(HEV)中和抗原表位的新型HEV重组蛋白,并对其进行鉴定和分析。方法将HEV开放阅读框架2(ORF2)编码452~617位氨基酸的基因片段连接到载体pET28a( ),转化大肠杆菌,获取表达克隆。以Ni-NTA层析柱纯化表达的蛋白,并用SDS-PAGE、Westernblot和直接电镜负染等方法分析鉴定,最后免疫小鼠检测特异性抗体的产生水平。结果表达的重组蛋白天然可溶,相对分子质量(Mr)约为22000,并可形成直径为20nm左右的病毒样颗粒,能与戊型肝炎患者血清发生Westernblot阳性反应,免疫小鼠后可诱导产生高滴度的特异性抗体。体外中和试验显示产生的抗体具有中和HEV的活性。结论原核表达的、含HEV中和抗原表位的、长度仅166个氨基酸的HEVORF2近3′端编码蛋白能够形成病毒样颗粒,而且该新型病毒样颗粒具有良好的抗原性和免疫原性。     

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.     

Virus-like particle (VLP)-based vaccines for pandemic influenza: Performance of a VLP vaccine during the 2009 influenza pandemic

The influenza pandemic of 2009 demonstrated the inability of the established global capacity for egg-based vaccine production technology to provide sufficient vaccine for the population in a timely fashion. Several alternative technologies for developing influenza vaccines have been proposed, among which non-replicating virus-like particles (VLPs) represent an attractive option because of their safety and immunogenic characteristics. VLP vaccines against pandemic influenza have been developed in tobacco plant cells and in Sf9 insect cells infected with baculovirus that expresses protein genes from pandemic influenza strains. These technologies allow rapid and large-scale production of vaccines (3-12 weeks). The 2009 influenza outbreak provided an opportunity for clinical testing of a pandemic influenza VLP vaccine in the midst of the outbreak at its epicenter in Mexico. An influenza A(H1N1)2009 VLP pandemic vaccine (produced in insect cells) was tested in a phase II clinical trial involving 4,563 healthy adults. Results showed that the vaccine is safe and immunogenic despite high preexisting anti-A(H1N1)2009 antibody titers present in the population. The safety and immunogenicity profile presented by this pandemic VLP vaccine during the outbreak in Mexico suggests that VLP technology is a suitable alternative to current influenza vaccine technologies for producing pandemic and seasonal vaccines.     

Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the detection of antibodies to influenza A virus in domestic and wild avian and mammalian species

An epitope-blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of antibodies to influenza A virus in taxonomically diverse domestic and wild vertebrate species. In contrast to the bELISAs published previously that require reagent production, manipulation by the end-user, or have not been evaluated for use with both mammalian and avian species, this assay is performed using commercially available recombinant nucleoprotein antigen and corresponding nucleoprotein-specific monoclonal antibody and has been shown to work with multiple avian and mammalian species. The efficacy of the bELISA as a serum screening assay was compared to the agar gel immunodiffusion (AGID) assay using 251 serum samples obtained from experimentally infected mallards (Anas platyrhynchos) and raccoons (Procyon lotor). The concordance between the AGID assay and bELISA was 94.1% (95% CI = 89.9, 98.3) for raccoons, and 71.2% (95% CI = 63.5, 78.9) for mallards and 82.8% (95% CI = 78.2, 87.3) overall. The bELISA was more sensitive than the AGID assay as demonstrated by the detection of antibodies to influenza A virus at earlier time points in experimental infection studies and at higher serial dilutions. The efficacy of the bELISA to monitor natural influenza A virus exposure was also compared to the AGID assay using an additional 745 serum samples from six avian species and six mammalian species. This bELISA provides a rapid, reliable, and inexpensive technique for large-scale surveillance of influenza A virus exposure in taxonomically diverse vertebrate species.     

Molecular characterization,isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.     

A multiplexed immunoassay for detection of antibodies against avian influenza virus

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.