应用HA、NA、M1和M2蛋白制备H5N1高致病性禽流感病毒样颗粒

目的:应用重组杆状病毒表达系统制备由HA、NA、M1和M2蛋白组成的H5N1高致病性禽流感病毒样颗粒,为研究H5N1高致病性禽流感疫苗奠定基础。方法:构建能共表达A/chicken/Jilin/2003(H5N1)禽流感病毒血凝素(HA)和神经氨酸酶(NA)、A/PR/8/34(H1N1)流感病毒基质蛋白(M1)和离子通道蛋白(M2)的2个二元重组杆状病毒,共同感染HighFive细胞,同时表达HA、NA、M1和M2蛋白,使这4种蛋白在感染的细胞内自主组装成病毒样颗粒。经差速离心和蔗糖密度梯度超速离心收获病毒样颗粒,通过Western印迹鉴定病毒样颗粒的组成,透射电镜观察病毒样颗粒形态,血凝试验测定病毒样颗粒的活性。结果:HA、NA、M1、M2蛋白在昆虫细胞中共表达,并组装成病毒样颗粒;电镜观察到病毒样颗粒的形态与流感病毒一致,直径约80 nm;血凝试验显示该病毒样颗粒具有凝集鸡红细胞的活性。结论:应用该方法可以制备流感病毒样颗粒,为H5N1流感疫苗研究提供了可行方案。     

NA Proteins of Influenza A Viruses H1N1/2009, H5N1, and H9N2 Show Differential Effects on Infection Initiation,Virus Release,and Cell-Cell Fusion

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins’ different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

Influenza A virus neuraminidase limits viral superinfection

Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of alpha-2,3- and alpha-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.     

Healthy human subjects have CD4+ T cells directed against H5N1 influenza virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.     

Proposed lead molecules against Hemagglutinin of avian influenza virus (H5N1)

Human infection with avian influenza H5N1 is an emerging infectious disease characterized by respiratory symptoms and a high fatality rate. Hemagglutinin and neuraminidase are the two surface proteins responsible for infection by influenza virus. Till date, neuraminidase has been the major target for antiviral drugs. In the present study we chose hemagglutinin protein as it mediates the binding of the virus to target cells through sialic acid residues on the host cell-surface. Hemagglutinin of H5 avian influenza (PDB ID: 1JSN) was used as the receptor protein. Ligands were generated by structure-based de novo approach and virtual screening of ZINC database. A total of 11,104 conformers were generated and docked into the receptor binding site using 'High Throughput Virtual Screening'. We proposed potential lead molecules against the receptor binding site of hemagglutinin based on the results obtained from in silico docking and hydrogen bond interaction between the ligand and the 1JSN protein molecule. We found sialic acid derivative 1 to be the lead molecules amongst the ligands generated by structure based de novo approach. However the molecules obtained from ZINC database were showing better docking scores as well as conserved hydrogen bond interactions. Thus we proposed ZINC00487720 and ZINC00046810 as potential lead molecules that could be used as an inhibitor to the receptor binding site of hemagglutinin. They could now be studied in vivo to validate the in silico results.     

Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background

Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.

Methodology/Principal Findings

Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.

Conclusions/Significance

These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.     

Molecular docking of selected phytocompounds with H1N1 Proteins

The H1N1 influenza virus is a serious threat to human population. Oseltamivir and Zanamivir are known antiviral drugs for swine flu with observed side effects. These drugs are viral neuraminidase and hemagglutinin inhibitor prevents early virus multiplication by blocking sialic acid cleavage on host cells. Therefore, it is of interest to identify naturally occurring novel compounds to control viral growth. Thus, H1N1 proteins (neuraminidase and hemagglutinin) were screened with phytocompounds isolated from Tulsi plant (Ocimum sanctum L.) using molecular docking tools. This identified Apigenin as an alternative to Oseltamivir and Zanamivir with improved predicted binding properties. Hence, it is of interest to consider this compound for further in vitro and in vivo evaluation.     

Immunization with a Live Attenuated H7N9 Influenza Vaccine Protects Mice against Lethal Challenge

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.     

A novel pathogenic mechanism of highly pathogenic avian influenza H5N1 viruses involves hemagglutinin mediated resistance to serum innate inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立

为了对致病性强、危害性大的H5、H7、H9亚型禽流感病毒进行同时集成化快速检测,通过对GenBank已报道的禽流感病毒的HA基因进行序列分析比较,设计了H5、H7、H9 3个亚型的特异性引物和分别用3个荧光基团标记的Taqman MGB核酸探针。将各个亚型引物与探针优化组合,筛选出能够同时检测禽流感病毒H5、H7、H9 3个亚型、且对Ct值和扩增效率影响不大的3组引物和探针,建立了三重实时荧光RT-PCR方法。该方法特异性好,在我们检测的样品中,没有发现假阳性和假阴性现象。同时敏感性高,检测禽流感病毒H5、H7、H9亚型的敏感性分别达到1 0001、000、500个模板拷贝数;此外抗干扰能力强,对禽流感H5、H7、H9 3个亚型的不同模板浓度进行组合,仍可有效地同时检测3个病毒亚型。所建立的方法对保存的89个禽流感病毒样品进行检测,结果与经典检测方法(病毒分离鉴定、HA、HI)的符合率达100%。用上述建立的方法与鸡胚分离法同时对新鲜采集的4 000多份临床样品进行检测,两种方法的检测结果符合率为100%。     

高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

Microarray for diagnostics of human pathogenic influenza A viruses subtypes

An oligonucleotide microchip was developed for diagnostics of human pathogenic Influenza A viruses subtypes. It contains discriminating probes for H1-, H2-, H3-, H5-, H7- and H9-subtypes of hemagglutinin and for N1-, N2-, and N7-subtypes of neuraminidase. The additional set of probes was used for M-gene of Influenza A viruses definition. Microchip was tested on samples pathogenic H5N1 avian influenza viruses, pandemic H1N1 swine influenza viruses and seasonal H1N1 and H3N2 influenza viruses. The microchip can be used for the analysis of both cultured strains and clinical specimens.     

Construction and comparison of different source neuraminidase candidate vaccine strains for human infection with Eurasian avian-like influenza H1N1 virus

Human infections with Eurasian avian-like swine influenza H1N1 viruses have been reported in China in past years. One case resulted in death and others were mild case. In 2016, the World Health Organization recommended the use of A/Hunan/42443/2015(H1N1) virus to construct the first candidate vaccine strain for Eurasian avian-like swine influenza H1N1 viruses. Previous reports showed that the neuraminidase of A/Puerto Rico/8/34(H1N1) might improve the viral yield of reassortant viruses. Therefore, we constructed two reassortant candidate vaccine viruses of A/Hunan/42443/2015(H1N1) by reverse genetic technology, with (6+2) and (7+1) gene constitution, respectively. The (6+2) virus had hemagglutinin and neuraminidase from A/Hunan/42443/2015, and the (7+1) one had hemagglutinin from A/Hunan/42443/2015, while all the other genes were from A/Puerto Rico/8/34. Our data revealed that although the neuraminidase of the (7+1) virus was from high yield A/Puerto Rico/8/34, the hemagglutination titer and the hemagglutinin protein content of the (7+1) virus was not higher than that of the (6+2) virus. Both of the (7+1) and (6+2) viruses reached a similar level to that of A/Puerto Rico/8/34 at the usual harvest time in vitro. Therefore, both reassortant viruses are potential candidate vaccine viruses, which could contribute to pandemic preparedness.     

Adaptation of influenza A viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity

Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by alpha(2-3) or alpha(2-6) linkages. One of these cell lines had less than 1/10 as much N-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.