Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

Structural basis for dsRNA recognition by NS1 protein of influenza A virus

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain （RBD） bound to a double-stranded RNA （dsRNA） at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel a-helices, dsRNA is anchored by a pair of invariable arginines （Arg38） from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide： H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3＇ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation （S31N）, a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.

Antiviral activity of five Asian medicinal pant crude extracts against highly pathogenic H5N1 avian influenza virus

Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Novel H1N1 influenza A virus infection in a patient with acute rejection after liver transplantation

BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

Establishing an ELISA,Based on the HA1 Protein,for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.

Adaptation in polymerase basic protein 1 family from influenza A virus to climate change

Global climate changes affect the functioning of ecosystems, in particular host-pathogen interactions, with major consequences in health ecology, however, it is less addressed how the change in global temperature affects the protein family of influenza virus. In this study, we studied the adaptation of polymerase basic protein 1 (PB1) family from influenza A virus to temperature change. 3841 PB1 proteins sampled from 1956-2011 were quantified by the amino-acid pair predictability and then compared their general changes with the temperature changes (Had-CRUT3v and CRUTEM4v data sets) of corresponding years on a 5? by 5? grid-box basis. Also, point-to-point comparisons were conducted from 1956 to 1998 in all and different species. The results showed that both changes in the temperature and unpredictable portion of PB1 proteins had similar trends from 1956 to 2011, which provides the evidence of virus adaptation at protein level to climate change.

In silico modification of oseltamivir as neuraminidase inhibitor of influenza A virus subtype H1N1

This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures.The docking result showed that AD3BF2 D ligand（N-[（1S,6R）-5-amino-5-{[（2R,3S,4S）-3,4-dihydroxy-4-（hydroxymethyl） tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-（1-ethylpropoxy）-1-cyclohexene-1-carboxylate） had better binding energy values than standard oseltamivir.AD3BF2 D had several interactions,including hydrogen bonds,with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation.The results showed that AD3BF2 D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS 1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction （RT-PCR） and cloned into a prokaryotic expression vector, pET-28a（＋）, and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay （ELISA） was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line

The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We engineered an NS1-expressing 293 （293-NS1） cell line with no response to IFN stimulation. Compared with the parental 293 cells,

The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in <i>Escherichia coli</i>DH5<i>α</i>strain

Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.

Therapeutic Trial of an Endothelin Receptor Agonist for the Highly Pathogenic Avian Influenza A/H5N1 Virus Infection in Chicks

The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people since 1997, and has shown a mortality rate of over 50%. The high mortality in human cases is thought to be enhanced by the excessive secretion of various endogenous factors, including cytokines and interleukins, stimulated by viral infections. Chickens infected with A/H5N1 viruses experience sudden death without showing severe clinical symptoms or inflammation. However, severe hemorrhage and congestion are seen in various tissues in sporadic chicken cases of A/H5N1-infections, especially in the pulmonary tissues, thus indicating that there is ischemia due to vascular abnormalities. Our previous studies have focused on the expression pattern of endothelin-1, which modulates the vascular tone via endothelin receptors. An Indonesian sporadic strain of A/H5N1 virus was intranasally administered to 10-day-old chicks, and the expression of endothelin was examined in the infected birds. All birds died within five days of inoculation, and had moderate inflammation accompanied by severe hemorrhage and congestion in the lungs. Immunohistochemical studies showed enhanced expression of endothelin-1 in the infected lungs. In addition, the real-time PCR analyses revealed that endothelin-1 and endothelin receptor A mRNA were significantly elevated in the birds with A/H5N1 infections. Subsequently, H5N1-infected birds were inoculated with bosentan hydrate, a competitive antagonist of endothelin receptors. Interestingly, the mortality rate of the infected birds was dramatically decreased in a dose-dependent manner by the administration of bosentan hydrate. The pathological lesions, including congestion and hemorrhage in the pulmonary tissues, were clearly inhibited. These findings are promising, and suggest that endothelin receptor antagonists are a potential treatment for the highly pathogenic avian flu.