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.     

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.     

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses

Summary The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.     

Epidemiological update on swine influenza (H1N1) in pigs

The 2009 H1N1 pandemic has slowed down its spread after initial speed of transmission. The conventional swine influenza H1N1 virus (SIV) in pig populations worldwide needs to be differentiated from pandemic H1N1 influenza virus, however it is also essential to know about the exact role of pigs in the spread and mutations taking place in pig-to-pig transmission. The present paper reviews epidemiological features of classical SIV and its differentiation with pandemic influenza.     

一株2011年出现的季节性甲型H1N1流行性感冒病毒血凝素基因特性的研究

本文通过比较2011年分离培养的1株季节性甲型H1N1流行性感冒(简称流感)病毒(A/Shanghai/1167/2011(H1N1))与历年季节性甲型H1N1流感病毒的血凝素(HA)基因,追溯该病毒的基因变异与来源,探讨该毒株的出现对流感防控工作的意义.采用反转录-聚合酶链反应(RT-PCR)方法扩增病毒的HA和神经氨酸酶(NA)片段,并进行测序;应用分子生物学软件对获得的序列进行分析,绘制基因进化树;同时,通过血凝抑制试验检测2011年下半年健康人群中该流感病毒的抗体水平.结果显示,A/Shanghai/1167/2011(H1N1)的HA基因序列与世界卫生组织(WHO)2007～2008年季节性甲型H1N1流感病毒疫苗株A/Brisbane/59/2007(H1N1)最接近,同源性达99.2%,与新型甲型H1N1流感病毒A/California/07/2009疫苗株同源性仅为72.4%.其HA基因裂解位点为PSIQSR↓GLF,尚未出现高致病性的分子特征.HA片段共编码557个氨基酸,有9个潜在的糖基化位点,序列与2009年前WHO疫苗株A/NewCaledonia/20/1999(H1N1)、A/SolomonIslands/3/2006(H1N1)和/Brisbane/59/2007(H1N1)相比,分别有15、12和4处不同,这些差异分布在Sa、Sb、Ca1、Ca2、Cb 5个抗原决定簇的氨基酸差异分别有5、5和2处.该毒株在健康人群血清的抗体阳性率为34.33%,几何平均效价(GMT)为10.38.A/Shanghai/1167/2011(H1N1)是2011年出现在上海地区的一个季节性甲型H1N1流感病毒毒株,其抗原变异与既往季节性甲型H1N1流感病毒相比不大,但在以A(H1N1)pdm09为主要流行株的年份检测到散在发生的既往季节性甲型H1N1流感病毒毒株应当引起重视,其在人群中的抗体水平较低,易引起流行,需要提高对类流感人群中此种毒株的持续监测.     

Genetic analysis and rescue of a triple-reassortant H3N2 influenza A virus isolated from swine in eastern China

One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like HIN1, NS from classical swine H1NI, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.     

新甲型H1N1(2009)病毒的早期分子特征

摘要：【目的】本世纪首次流感大流行的病原属于甲型H1N1流感病毒,在遗传特性和抗原等方面都有别于人群中流行多年的季节性H1N1流感病毒。为了深入了解病毒的遗传特性,跟踪病毒的演化趋势,及时发现具有流行病学意义的变异株,本研究对早期分离的甲型H1N1(2009)病毒的分子特性进行了详细分析。【方法】通过GenBank的流感资源中心下载相关毒株的基因组信息, 序列分析采用DNAStar软件包的EditSeq和MegAlign比较与病毒致病性和宿主特异性相关的氨基酸变化情况。以A/California/07/2009（H1N1）作为新甲型H1N1(2009)的代表株进行详细的分子特征分析。【结果】A/California/07/2009不具备高致病性流感病毒的分子特征;病毒编码的11个蛋白大部分保留有猪流感病毒的分子特征,同时也具有一些禽和人流感病毒的特征;PB1-F2在11aa,57aa和87aa后发生断裂,具有古典猪H1N1和人H1N1双重特点,这是甲型H1N1（2009）病毒一个特有的分子特征。【结论】首次详细分析了新甲型H1N1（2009）病毒的分子特征。随着病毒在人群中的进一步适应和持续存在,这些分子特征将发生变化,应该特别关注这些变化对病毒的传播力和致病性的影响。     

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.     

Pandemic swine influenza virus (H1N1): A threatening evolution

“Survival of the fittest” is an old axiom laid down by the great evolutionist Charles Darwin and microorganisms seem to have exploited this statement to a great extent. The ability of viruses to adapt themselves to the changing environment has made it possible to inhabit itself in this vast world for the past millions of years. Experts are well versed with the fact that influenza viruses have the capability to trade genetic components from one to the other within animal and human population. In mid April 2009, the Centers for Disease Control and Prevention and the World Health Organization had recognized a dramatic increase in number of influenza cases. These current 2009 infections were found to be caused by a new strain of influenza type A H1N1 virus which is a re-assortment of several strains of influenza viruses commonly infecting human, avian, and swine population. This evolution is quite dependent on swine population which acts as a main reservoir for the reassortment event in virus. With the current rate of progress and the efforts of heath authorities worldwide, we have still not lost the race against fighting this virus. This article gives an insight to the probable source of origin and the evolutionary progress it has gone through that makes it a potential threat in the future, the current scenario and the possible measures that may be explored to further strengthen the war against pandemic.     

Genetic characterization of swine influenza viruses isolated in Japan between 2009 and 2012

Eleven swine influenza viruses (SIVs) isolated from pigs in Japanese institutions between 2009 and 2012 were genetically characterized. Seven H1N1 were shown to have originated from A(H1N1)pdm09 viruses. Two H1N2 viruses contained H1 and N2 genes of Japanese H1N2 SIV origin together with internal genes of A(H1N1)pdm09 viruses. Two H3N2 viruses isolated during animal quarantine were identified as triple reassortant H3N2 viruses maintained among pigs in North America. This study shows that A(H1N1)pdm09 viruses and their reassortant strains are already present in domestic pigs in Japan and that novel SIVs are possibly being imported from abroad.     

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.     

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。     

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。     

Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.     

一例混合感染中H3N2和H7N9流感病毒的分子遗传特性

【目的】揭示一例混合感染中H3N2和N7N9流感病毒的分子遗传特性。【方法】通过荧光定量PCR法对标本进行流感病毒分型检测。通过二代测序技术对病毒分离物进行全基因组测序分析。【结果】2013年4月在南京市检测到一例人季节性H3N2流感病毒和禽流感H7N9病毒混合感染,混合病毒分别命名为A/Nanjing/M1/2013 (H3N2) (M1-H3N2)和A/Nanjing/M2/2013 (H7N9) (M2-H7N9)。分离株M2-H7N9 HA蛋白的Q226L位点和PB2蛋白E627K位点发生突变,增强了病毒对人体的感染能力。【结论】报道了一起人混合感染H3N2和N7N9流感病毒病例,提示人可能成为流感病毒基因“混合器”,应高度关注H7N9病毒与人季节性流感病毒的基因重配现象。     

引发2009年流感暴发的H1N1新型流感病毒的研究进展

引起流感世界性大流行的主要原因与流感病毒表面抗原血凝素(HA)和神经氨酸酶(NA)频发的变异有很大关系,抗原的变异使得流感病毒可以逃逸机体的免疫防御,而且使许多应用中的疫苗失去防御效果。综述2009年世界暴发的H1N1新型流感病毒的结构在进化过程中发生的变异,有助于增加人们对流感病毒的了解,从而有效的治疗和预防流感大流行。     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

Genetic comparison of H5N1 influenza A viruses isolated from chickens in Japan and Korea

Outbreaks of highly pathogenic avian influenza (HPAI) caused by H5N1 virus occurred during 2003 to 2004 in Korea and Japan. The H5N1 viruses isolated in both countries were genetically similar at > 99% identity in the nucleotide sequences of all eight RNA segments, indicating that they belong to genotype V and are distinct from HPAI viruses prevalent in southeast Asia that belong to genotype Z. These findings indicate that the H5N1 viruses that caused the HPAI outbreaks in both Korea and Japan were derived from a common ancestor.     

Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China

A total of 100 HIN1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang,Hubei and Guangdong between June and November 2009,were provided by local CDC laboratories.After MDCK cell culture,57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.A total of 39 HA sequences,52 NA sequences,36 PB2 sequences,31 PB1 sequences,40 PA sequences,48 NP sequences,51 MP sequences and 36 NS sequences were obtained,including 20 whole genome sequences.Sequence comparison revealed they shared a high degree of homology (96％～99％) with known epidemic strains (A/Califomia/04/2009(H1N1).Phylogenetic analysis showed that although the sequences were highly conserved,they clustered into a small number of groups with only a few distinct strains.Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences:A/Hubei/86/2009 PKVRDQEG→PKVRDQEA,A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER,A/Hubei/75/2009PKVRDQEG→PKVRDQGG,the A/Hubei/75/2009 was isolated from an acute case,while the other two were from patients with mild symptoms.Other key sites such as 119,274,292 and 294 amino acids of NA protein,627 of PB2 protein were conserved.Meanwhile,all the M2 protein sequences possessed the Ser32Asn mutation,suggesting that these viruses were resistant to adamantanes.Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.