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.     

H3N2型猪流感病毒M2蛋白表达分析

流感病毒的M2蛋白在流感病毒复制中起着重要作用,是抗流感病毒的靶标分子。本研究以提取的病毒基因组RNA作为模板,RT-PCR扩增H3N2亚型猪流感病毒M2基因,分别构建了重组原核表达载体和重组真核表达载体,建立了M2蛋白的原核和真核表达系统。通过大肠杆菌表达系统,制备了M2重组蛋白,并免疫大鼠制备了多克隆抗体。Western blotting和间接免疫荧光方法检测表明所制备的抗体能识别真核表达的M2蛋白和病毒感染细胞后表达M2蛋白,具有良好的特异性。重组M2真核表达载体转染Vero细胞,表达的重组M2蛋白大小为20kDa,定位于细胞浆中,与病毒感染细胞中的M2蛋白定位相同。Western blotting检测表明M2蛋白在病毒感染细胞12h后才能检出,晚于NS1、NP和M1,属于病毒复制的晚期蛋白,可作为病毒复制晚期的指示分子。本研究为弄清M2蛋白在病毒复制过程中的生物学功能奠定了基础。     

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.     

Identification of host encoded microRNAs interacting with novel swine-origin influenza A (H1N1) virus and swine influenza virus

The discovery of microRNAs (miRNAs) is a remarkable breakthrough in the field of life science, and they are important actors which regulate gene expression in diverse cellular processes. Recently, several reports indicated that miRNAs can also target viruses and regulate virus replication. Here we discovered 36 pig-encoded miRNAs and 22 human-encoded miRNAs which have putative targets in swine influenza virus (SIV) and Swine-Origin 2009 A/H1N1 influenza virus (S-OIV) genes respectively. Interestingly, the putative interactions of ssc-miR-124a, ssc-miR-136 and ssc-miR-145 with their SIV target genes had been found to be maintained almost throughout all of the virus evolution. Enrichment analysis of previously reported miRNA gene expression profiles revealed that three miRNAs are expressed at higher levels in human lung or trachea tissue. The hsa-miR-145 and hsa-miR-92a putatively target the HA gene and hsa-miR-150 putatively targets the PB2 gene. Analysis results based on the location distribution from which virus was isolated and sequence conservation imply that some putative miRNA-mediated host-virus interactions may characterize the location-specificity.     

H3N2亚型猪流感病毒的拯救及HA与NA基因的替换

摘要：【目的】为研究流感病毒突破种间屏障分子机制,筛选流感基因工程疫苗株。【方法】本实验以猪流感病毒A/Swine/Henan/S4/01(H3N2)为亲本株,利用反向遗传学操作技术,采用RT-PCR技术对该病毒的8个基因片段分段进行扩增,通过与双向转录载体pHW2000连接, 重组质粒转染293T和MDCK共培养细胞,拯救出全部基因均来自于亲本株的猪流感病毒rgH3N2,并分别以人流感病毒A/ PR/8/34(H1N1)、禽流感病毒A/Duck/Nanchang /4-165/2000 (H4N6 )、马流感病毒A/Equine/Fuyun/2008/(H3N8)的HA和NA基因替换A/Swine/Henan/S4/01的相应基因,【结果】生物学实验结果表明rgH3N2在鸡胚半数感染量、组织培养半数感染量、稳定性试验等方面都与亲本株保持一致。rgH3N2经鸡胚多次传代后血凝价最高可达到1:256,接种MDCK细胞60 h后,血凝价可以达到1:64。基因替换后成功拯救出的重组病毒rgH1N1、rgH4N6和rgH3N8在鸡胚和细胞上均具有较高的增殖能力。【结论】病毒的成功拯救为流感病毒突破种间屏障分子机制,HA、NA基因在流感病毒跨种属传播中所扮演角色的研究和流感基因工程疫苗株的筛选奠定了基础。     

H3N2亚型猪流感病毒M1基因克隆及表达特性分析

从H3N2亚型猪流感病毒感染的鸡胚尿囊液中提取病毒基因组RNA,采用RT-PCR方法克隆M1全长基因,将M1基因亚克隆至pET-28a(+)表达载体中,构建重组表达质粒pET-28a-M1,将该重组质粒转化大肠杆菌BL21并经IPTG诱导表达。诱导产物经SDS-PAGE电泳分析显示重组蛋白M1获得大量表达,表达蛋白纯化后免疫Wistar大鼠制备多克隆抗体。Westernblotting检测结果表明制备的抗M1蛋白多克隆抗体可以识别大肠杆菌表达的M1蛋白和病毒感染细胞的病毒M1蛋白。构建M1基因真核重组质粒p3xFLAG-CMV-7.1-M1并转染Vero细胞,Western blotting检测表明抗M1蛋白多克隆抗体可以识别在Vero细胞中得到表达的M1蛋白,成功建立M1基因的真核表达系统。分析了病毒感染过程中M1蛋白的变化及其作为病毒复制指示分子的可能性。鼠源M1蛋白多克隆抗体的制备和M1基因真核重组表达质粒的构建,为进一步研究猪流感病毒的复制机理以及M1蛋白在病毒复制过程中的生物学功能奠定了基础。     

Studies on neuraminidase from influenza virus A(H3N2) obtained by two procedures

• 1.1. Neuraminidase was obtained by (A) bromelain solubilization or (B) by treatment with N-lauroylsarcosine.
• 2.2. 5-N-acetyl-2-O(3-methoxyphenyl)-α-d-neuraminic acid, employed as substrate, avoids the interference produced by the thiobarbituric acid method, and is not interfered by the ampholytes.
• 3.3. Only about 20% of original enzyme activity was lost after electrofocusing. The sample from procedure A showed two peaks, corresponding to pis 4.4 and 5.6. The sample from procedure B, having a higher activity, showed only one peak at pI 4.4.
• 4.4. Samples A and B showed different K_m and hydrolysis rate with N-acetylneuraminyl-lactose and glycophorin A. It was not found significantly different with other substrates: α₁-acid glycoprotein, brain gangliosides. 5-N-acetyl-2-O-(3-methoxyphenyl)-α-d-neuraminic acid and 2'-(4-methyl umbelliferyl)-α-d-N-acetylneuraminic acid.

     

Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.     

抗甲3型流感病毒卵黄抗体的研制

制备抗H3N2型流感病毒特异性鸡卵黄免疫球蛋白（IgY),防治同型流感病毒引起的流感,制备H3N2型流感病毒,通过两种途径免疫母鸡,以水稀释-硫酸铵沉淀及离子交换层析法提纯,并进行理化性质分析和动物效力试验。两种途径免疫均可使母鸡产生特异性IgY,其中以静脉为主的混合免疫法效果较好,提纯后纯度可达90%以上,通过被动免疫可使小鼠对同型流感病毒的攻击产生保护作用。结果表明,已成功地制备出高效价的H3N2型流感病毒特异性IgY。     

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.     

Evolution of swine H3N2 influenza viruses in the United States

During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851-8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4, 382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.     

H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用

为建立H1亚型猪流感病毒抗体检测方法,扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分,构建原核表达载体pET30a-HA1,并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原,建立间接ELISA检测方法。利用该检测方法检测了2008?2009年采集的猪血清785份,阳性率为15.54％,不同省份的阳性率存在差异 (8％~47％)。以IDEXX相关试剂盒检测结果作为参照,该方法的诊断特异性达到91％,诊断敏感性达到95％。     

H9N2亚型猪流感病毒感染BALB/c小鼠动物模型的建立

目的建立H9N2亚型猪流感病毒感染BALB/c小鼠动物模型,为研究病毒致病机制提供模型动物。方法通过滴鼻的方法将H9N2亚型猪流感病毒感染BALB/c小鼠,观察小鼠的症状和组织病理变化。结果 BALB/c小鼠的临床症状明显,病理变化典型。结论 H9N2亚型猪流感病毒感染BALB/c小鼠的疾病模型成功建立。     

An influenza A (H1N1) virus, closely related to swine influenza virus, responsible for a fatal case of human influenza.

In July 1991, an influenza A virus, designated A/Maryland/12/91 (A/MD), was isolated from the bronchial secretions of a 27-year-old animal caretaker. He had been admitted to the hospital with bilateral pneumonia and died of acute respiratory distress syndrome 13 days later. Antigenic analyses with postinfection ferret antisera and monoclonal antibodies to recent H1 swine hemagglutinins indicated that the hemagglutinin of this virus was antigenically related to, but distinguishable from, those of other influenza A (H1N1) viruses currently circulating in swine. Oligonucleotide mapping of total viral RNAs revealed differences between A/MD and other contemporary swine viruses. However, partial sequencing of each RNA segment of A/MD demonstrated that all segments were related to those of currently circulating swine viruses. Sequence analysis of the entire hemagglutinin, nucleoprotein, and matrix genes of A/MD revealed a high level of identity with other contemporary swine viruses. Our studies on A/MD emphasize that H1N1 viruses in pigs obviously continue to cross species barriers and infect humans.     

Seasonality of influenza A(H3N2) virus: a Hong Kong perspective (1997-2006)

Background

The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia.

Methodology/Principal Findings

281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997–2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations.

Conclusions/Significance

The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population.     

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。     

Explaining the geographical origins of seasonal influenza A (H3N2)

Most antigenically novel and evolutionarily successful strains of seasonal influenza A (H3N2) originate in East, South and Southeast Asia. To understand this pattern, we simulated the ecological and evolutionary dynamics of influenza in a host metapopulation representing the temperate north, tropics and temperate south. Although seasonality and air traffic are frequently used to explain global migratory patterns of influenza, we find that other factors may have a comparable or greater impact. Notably, a region''s basic reproductive number (R₀) strongly affects the antigenic evolution of its viral population and the probability that its strains will spread and fix globally: a 17–28% higher R₀ in one region can explain the observed patterns. Seasonality, in contrast, increases the probability that a tropical (less seasonal) population will export evolutionarily successful strains but alone does not predict that these strains will be antigenically advanced. The relative sizes of different host populations, their birth and death rates, and the region in which H3N2 first appears affect influenza''s phylogeography in different but relatively minor ways. These results suggest general principles that dictate the spatial dynamics of antigenically evolving pathogens and offer predictions for how changes in human ecology might affect influenza evolution.     

High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2)

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-microM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-microM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI(50) = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI(50) > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity.