Phylogenetic analysis of surface proteins of novel H1N1 virus isolated from 2009 pandemic

Swine Influenza Virus (H1N1) is a known causative agent of swine flu. Transmission of Swine Influenza Virus form pig to human is not a common event and may not always cause human influenza. The 2009 outbreak by subtype H1N1 in humans is due to transfer of Swine Influenza Virus from pig to human. Thus to analyze the origin of this novel virus we compared two surface proteins (HA and NA) with influenza viruses of swine, avian and humans isolates recovered from 1918 to 2008 outbreaks. Phylogenetic analyses of hemagglutinin gene from 2009 pandemic found to be clustered with swine influenza virus (H1N2) circulated in U.S.A during the 1999-2004 outbreaks. Whereas, neuraminidase gene was clustered with H1N1 strains isolated from Europe and Asia during 1992-2007 outbreaks. This study concludes that the new H1N1 strain appeared in 2009 outbreak with high pathogenicity to human was originated as result of re-assortment (exchange of gene). Moreover, our data also suggest that the virus will remain sensitive to the pre-existing therapeutic strategies.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Insights from investigating the interaction of oseltamivir (Tamiflu) with neuraminidase of the 2009 H1N1 swine flu virus

The neuraminidase (NA) of influenza virus is the target of anti-flu drugs oseltamivir and zanamivir. Clinical practices showed that oseltamivir was effective to treat the 2009-H1N1 influenza but failed to the 2006-H5N1 avian influenza. To perform an in-depth analysis on such a drug-resistance problem, the 2009-H1N1-NA structure was developed. To compare it with the crystal 2006-H5N1-NA structure as well as the 1918 influenza virus H1N1-NA structure, the multiple sequential and structural alignments were performed. It has been revealed that the hydrophobic residue Try347 in H5N1-NA does not match with the hydrophilic carboxyl group of oseltamivir as in the case of H1N1-NA. This may be the reason why H5N1 avian influenza virus is drug-resistant to oseltamivir. The finding provides useful insights for how to modify the existing drugs, such as oseltamivir and zanamivir, making them not only become more effective against H1N1 virus but also effective against H5N1 virus.     

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.     

Molecular character of influenza A/H1N1 2009: Implications for spread and control

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior “seasonal” H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 “swine flu” vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.     

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009

In 2013, three reassortant swine influenza viruses (SIVs)—two H1N2 and one H3N2—were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human‐like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human‐like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human‐lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human‐lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

Analysis of synonymous codon usage bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended, and the intra-genomic codon usage bias of 09H1N1 is quite low. Base composition constraint, dinucleotide biases and translational selection are the main factors influencing the codon usage bias of 09H1N1. At the genome level, we find that the codon usage bias of 09H1N1 is similar to H1N1 (A/swine/Kansas/77778/2007H1N1), H9N2 from Asia, H1N2 from Asia and North America and H3N2 from North America. Our results provide insight for understanding the processes governing evolution, regulation of gene expression, and revealing the evolution of 09H1N1.     

中国“人-猪-禽”基因重排H1N2亚型猪流感病毒全基因克隆及遗传进化的研究

[目的]为了研究2006年从广西病猪肺组织中分离的H1N2亚型猪流感病毒(SIV)A/Swine/Guangxi/13/2006(H1N2)(Sw/Gx/13/06)的遗传学特性和8个基因的来源.[方法]运用RT PCR方法对其全基因进行了克隆并运用分子生物学软件对其基因序列进行了遗传进化分析.[结果]血凝素(HA)、核蛋白(NP)、基质蛋白(M)和非结构蛋白(NS)基因来源于猪古典H1N1亚型流感病毒;神经氨酸酶(NA)和聚合酶蛋白(PB1)基因来源于人的H3N2亚型流感病毒;聚合酶蛋白(PA)和聚合酶蛋白(PB2)基因来自于禽流感病毒.[结论]可见Sw/GX/13/06是一株"人-猪-禽"三源基因重排H1N2亚型SIV且与美国(1999-2001年)和韩国(2002年)分离到该型病毒的有明显的亲缘关系.据我们所知,这是中国首次报道含有禽流感病毒基因片段的重排H1N2 SIV,该病毒是否对养猪业和人类公共卫生健康具有潜在的威胁,有待于进一步研究.     

High genetic and antigenic similarity between a swine H3N2 influenza A virus and a prior human influenza vaccine virus: A possible immune pressure-driven cross-species transmission

In late April of 2009, a global outbreak of human influenza was reported. The causative agent is a highly unusual reassortant H1N1 influenza virus carrying genetic segments derived from swine, human and avian influenza viruses. In this study, we compared the HA, NA and other gene segments of a swine H3N2 influenza A virus, A/Swine/Guangdong/z5/2003, which was isolated from pigs in 2003 in Guangdong Province, China, to the predominant human and swine H3N2 viruses. We found that the similarity of gene segments of A/Swine/Guangdong/z5/2003 was closer to Moscow/99-like human H3N2 virus than Europe swine H3N2 viruses during 1999-2002. These results suggest that A/Swine/Guangdong/z5/2003 may be porcine in origin, possibly being driven by human immune pressure induced by either natural H3N2 virus infection or use of A/Moscow/10/99 (H3N2)-based human influenza vaccine. The results further confirm that swine may play a dual role as a “shelter” for hosting influenza virus from humans or birds and as a “mixing vessel” for generating reassortant influenza viruses, such as the one causing current influenza pandemic.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

A/H1N1流感—世界关注的焦点

2009年4月,A/H1N1流感在墨西哥和美国暴发。随后,疫情迅速蔓延到美洲、欧洲、亚洲多个国家。A/H1N1流感病毒是一种以前在人或动物身上从未观测到的新病毒。遗传进化和抗原特性分析表明该病毒和猪流感病毒密切相关,与人类的季节性流感病毒有明显区别。但是流行病学信息表明A/H1N1流感病毒只攻击人类,并在人与人之间传播,尚未发现动物向人类传播的情况。本文从A/H1N1流感病毒的生物学特性、临床特征、公共卫生意义等方面全面阐述了A/H1N1流感的最新研究进展,为正确认识和科学防控A/H1N1流感提供参考。     

In silico genome analysis and drug efficacy test of influenza A virus (H1N1) 2009

The H1N1 2009 virus is pandemic in many countries. The genome of this virus contains eight segments. Among the eight segments maximum numbers of mutation occur at the segment 1 and segment 4 which codes for PB2 subunit and hemagglutinin (HA) and less number of mutations occur in segment 6 which codes for neuraminidase (NA) protein. Neuraminidase (NA) inhibitors (Oseltamivir and Zanamivir) are presently used as an anti-flu drugs. In the present study, the in silico efficacy of different drugs was tested against the swine flu virus. 3D structures of neuraminidase (NA) proteins of H1N1 2009 were generated using Geno3D. The 3D structure of H1N1 1918 was downloaded from PDB. Interaction study was done using Arguslab 4 and PyMol view. Oseltamivir and Zanamivir have good number of interactions with H1N1 2009 virus and the scoring function also support to this result. When compared with the 1918 H1N1 viral protein, 2009 H1N1 NA protein shows more number of interaction and good scoring function. The RMSD value of before and after docking are found to be same at 0.04A° for both the drugs. The force field energy of NA protein 2009 was found to be −15603.529 KJ/mol before docking. The force field energy was found to be decreased after docking at −17620.740 KJ/mol with Tamiflu and −17652.242 KJ/mol with Zanamivir. The number of interaction and scoring function shows that Oseltamivir and Zanamivir will be able to effectively control the present pandemic H1N1 virus 2009.     

人H5N1亚型禽流感病毒安徽株NS1基因的克隆及在原核系统的表达

利用RT-PCR方法,从人H5N1亚型禽流感病毒安徽株扩增到了NS1基因,对其进行了克隆、序列测定和分析,并在原核系统高效表达和纯化了NS1蛋白。进化分析表明,A/Anhui/01/2005毒株与近些年国内分离的水禽H5N1病毒进化关系更为接近。NS1与福建、湖南分离的禽流感病毒同源性最高,分别达到99.1%和98.2%。序列分析表明,与病毒的致病性相关的92位氨基酸为Asp,与病毒的细胞因子抗性相关的80~84位氨基酸发生缺失,与断裂/多聚腺苷酸化特异性因子结合的基序改变为GFEWN,和病毒致死性相关的PL基序为ESEV。随后在大肠杆菌高效表达并纯化了NS1蛋白。NS1基因及其编码产物的特性分析以及在原核系统的表达,为进一步研究NS1的致病机制和抗病毒药物研制奠定了基础。     

Analysis of cellular proteome alterations in porcine alveolar macrophage cells infected with 2009 (H1N1) and classical swine H1N1 influenza viruses

The H1N1/2009 influenza virus has the potential to cause a human pandemic, and sporadic cases of human-to-pig transmission have been reported. In this study, two influenza viruses were isolated from pigs. A phylogenetic analysis showed that the A/swine/NanChang/F9/2010(H1N1) (F9/10) strain shared a high degree of homology with the pandemic H1N1/2009 virus, and A/swine/GuangDong/34/2006 (H1N1) (34/06) strains was a classical swine influenza virus. A proteomic analysis was performed to investigate possible alterations of protein expression in porcine alveolar macrophage (PAM) cells infected by the F9/10 and 34/06 viruses over different time courses. Using 2-DE in association with MALDI-TOF MS/MS, we identified 13 up-regulated and 21 down-regulated protein spots, including cytoskeleton proteins, cellular signal transduction proteins, molecular biosynthesis proteins and heat shock proteins. The most significant changes in the infected cells were associated with molecular biosynthesis proteins and heat shock proteins. We analysed the biological characteristics of the F9/10 and 34/06 viruses in vivo and in vitro. The F9/10 virus showed greater pathogenicity than the 34/06 virus in PAM cells and mice. This study provides insights into the biologic characteristics, potential virulence alteration and cross-species transmission mechanisms of the pandemic H1N1/2009.     

江苏首例甲型H1N1(2009)流感病毒分离及其血凝素分子遗传特征分析

2009年6月12日,江苏确诊首例甲型H1N1(2009)病例。通过细胞和鸡胚分离系统,我们分离到一株具有较高血凝活性的病毒,命名为A/Jiangsu/1/2009。为了跟踪病毒的变异情况,我们开展了病毒的全基因组测序工作,在此基础上对其血凝素基因(Haemagglutinin,HA)的遗传特性进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Jiangsu/1/2009HA蛋白的有4个氨基酸发生了突变,但都不在已知的抗原位点上。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1的特点。与禽流感H1病毒相比,分离株HA蛋白受体结合位点上的E190D和G225D发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究首次对早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.     

Analysis of Synonymous Codon Usage Bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended...