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 .

Genome-wide comparison inferred the origin and evolution of B-cell epitopes on the proteins of human influenza A virus

The novel strain H1N1 caused the outbreak of first pandemic influenza in 21 century. Now it is a common component of current seasonal influenza viruses. The recent transmission and plentiful genome sequences available provided a good opportunity to study the origin and evolution of epitopes on the proteins of human influenza virus. In the present study, the B-cell epitope compositions in the pandemic strains, circulating traditional seasonal strains, swine strains as well as highly virulent avian strain H5N1 were identified with the aid of the Immune Epitope DataBase (IEDB) and were compared at genomic level. A total of 14210 distinct sequences down-loaded from NCBI database were used for analysis. Some epitopes on proteins HA or NA, not conserved in recent seasonal strains, were found in 2009 pandemic strains but existed in the early human strains (1919-1935). The pandemic strain shared higher conserved epitopes with “bird flu” virus H5N1than classic human seasonal strains. The epitopes that could exist at common antigenic regions of HA protein are needed to further identify. The genetic exchanges between human and swine population by transmission was very active but the princepal side of the transmission could be from swine to human. These results provided valuable information on influenza A virus evolution and transmission by means of epitope analysis at genomic level.

Avian influenza virus ecology in wild birds of Western Siberia

Background:The aim of the study was to explore the ecological diversity of wild birds in Siberia, which are the natural reservoir of avian influenza virus(AIV).Methods:Cloacal swabs and intestinal fragments were collected from wild migratory birds from 2007-2014. Isolated viruses were grown in the allantoic cavity of embryonated chicken eggs. The presence of virus was determined using hemagglutination assays. Primary identification and subtyping of influenza viruses was confirmed by RT-PCR.Results:A total of 2300 samples obtained from wild migratory birds of 8 orders were collected and tested. Influenza was detected in 185 birds of 3 orders. Species of family Anatidae(order Anseriformes) such as European Teal(Anas crecca), Garganey Teal(A. querquedula), and Shoveler(A. clypeata) play the main role in AIV circulation in the south of Western Siberia. The proportion of viral carriers among waterfowl ranged from 5.6 to 20% in 2007-2014. The order Charadriiformes had lower virus isolation rates of not more than 1.4%.Conclusions:Wild migratory waterfowl of orders Anseriformes and Charadriiformes are the main reservoir of AIV in the south of Western Siberia. This area plays a key role in persistence, evolution, and geographical distribution of avian influenza.

Swine-origin influenza-virus-induced acute lung injury:Novel or classical pathogenesis?

Influenza viruses are common respiratory pathogens in humans and can cause serious infection that leads to the development of pneumonia.Due to their hostrange diversity,genetic and antigenic diversity,and potential to reassort genetically in vivo,influenza A viruses are continual sources of novel influenza strains that lead to the emergence of periodic epidemics and outbreaks in humans.Thus,newly emerging viral diseases are always major threats to public health.In March 2009,a novel influenza virus suddenly emerged and caused a worldwide pandemic.The novel pandemic influenza virus was genetically and antigenically distinct from previous seasonal human influenza A/H1N1 viruses;it was identified to have originated from pigs,and further genetic analysis revealed it as a subtype of A/H1N1,thus later called a swine-origin influenza virus A/H1N1.Since the novel virus emerged,epidemiological surveys and research on experimental animal models have been conducted,and characteristics of the novel influenza virus have been determined but the exact mechanisms of pulmonary pathogenesis remain to be elucidated.In this editorial,we summa-rize and discuss the recent pandemic caused by the novel swine-origin influenza virus A/H1N1 with a focus on the mechanism of pathogenesis to obtain an insight into potential therapeutic strategies.

The nature of proteins in influenza

Mutation can alter the structure of viral proteins to form different structure. Carbon distribution is responsible for these changes in structure. The carbon distribution in proteins of human Influenza A virus is analyzed here. Results reveal that the carbon contents are high in surface proteins, optimum in polymerase proteins and less in nuclear proteins. Polymerase proteins have better carbon distribution pattern than the other proteins. Thymine distribution in different frames of mRNAs are checked as it has link with carbon distribution pattern in the corresponding proteins. Results show that frame 4 is violating from thymine distribution. This is responsible for production of protein with different carbon distribution. Unusual thymine distribution in frame 3 are observed. The thymine distributions are different in viral mRNA compared to normal one. Minimizing the excess thymine in H1N1 mRNAs might improve the protein performance. Mutational study based on carbon distribution should be better exploited for further improving the protein stability, activity and ultimately for gene therapy.

The emergence of pandemic influenza viruses

Pandemic influenza has posed an increasing threat to public health worldwide in the last decade.In the 20th century,three human pandemic influenza outbreaks occurred in 1918,1957 and 1968,causing significant mortality.A number of hypotheses have been proposed for the emergence and development of pandemic viruses,including direct introduction into humans from an avian origin and reassortment between avian and previously circulating human viruses,either directly in humans or via an intermediate mammalian host.However,the evolutionary history of the pandemic viruses has been controversial,largely due to the lack of background genetic information and rigorous phylogenetic analyses.The pandemic that emerged in early April 2009 in North America provides a unique opportunity to investigate its emergence and development both in human and animal aspects.Recent genetic analyses of data accumulated through long-term influenza surveillance provided insights into the emergence of this novel pandemic virus.In this review,we summarise the recent literature that describes the evolutionary pathway of the pandemic viruses.We also discuss the implications of these findings on the early detection and control of future pandemics.

Crystal structure of the swine-origin A (H1N1)- 2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus

Influenza virus is the causative agent of the seasonal and occasional pandemic flu.The current H1N1 influenza pandemic,announced by the WHO in June 2009,is highly contagious and responsible for global economic losses and fatalities.Although the H1N1 gene segments have three origins in terms of host species,the virus has been named swine-origin influenza virus(S-OIV)due to a predominant swine origin.2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects.Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection.Primary sequence analysis of the 2009 S-OIV hemagglutinin(HA)reveals its closest relationship to that of the 1918 pandemic influenza virus,however,analysis at the structural level is necessary to critically assess the functional significance.In this report,we report the crystal structure of soluble hemagglutinin H1(09H1)at 2.9Å,illustrating that the 09H1 is very similar to the 1918 pandemic HA(18H1)in overall structure and the structural modules,including the five defined antiboby(Ab)-binding epitopes.Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV,and people born around the 1918 are resistant to the current pandemic,yet younger generations are more susceptible to the 2009 pandemic.

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen （HA） from the strains A/New Caledonia/20/99 （HIN1） （pV1AS） and A/Califorrtia/04/2009 （H1N1） （pVEH1）, respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD50 of mouse-adapted A/New Caledonia/20/99 （H1N1）. Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses （P〈0.05） than the pVAX1 control group. Additionally, the pV1A5 vaccine effec- tively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group （P〈0.05）. Our study indicates that the seasonal influenza DNA vac- cine completely protects against the homologous A/New Caledonia/20/99 virus （H1N1）, while the pandemic influenza DNA vaccine only partially protects against this virus.

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.

Detection and pathogenesis of a novel swine H3N2 influenza virus containing three genes from the 2009 pandemic H1N1 influenza viruses in Korea in 2015

Dear Editor,Influenza A viruses cause pandemics at an interval of approximately 10-40 years,and pigs are regarded as a＂mixing vessel＂because they are easily infected with avian and human influenza viruses（Ito et al.,1998）.According to previous studies,H3N2,H1N2,and H1N1 subtypes o（swine influenza viruses have been detected in Korean pigs （Pascua et al., 2013; Kim et al., 2014; Song et al., 2007）. Moreover, a novel H3N2 influenza virus containing the matrix （34） gene from a 2009 pandemic influenza virus was detected in Korean pigs in 2013 （Pascua et al., 2013）, an H1N2 influenza virus con- taining the internal genes from a 2009 pandemic influ- enza virus was found in Korean pigs in 2014 （Kim et al., 2014）, and an H1N1 influenza virus containing all genes from the classical swine influenza viruses was isolated from Korean pigs in 2007 （Song et al., 2007）.

Seasonal influenza vaccination may mitigate the potential impact of an H5N1 pandemic

Seasonal influenza is a highly contagious, acute respiratory illness that affects people of all ages. The major pathogens, influenza A viruses, are classified into serologically defined antigenic subtypes of the hemagglutinin （HA） and neuraminidase （NA）. Of 16 identified HA and 9 NA subtypes, only H1N1 and H3N2 subtypes are now circulating among humans. Influenza vaccines have been available for over 60 years and well proven to be an effective public health intervention to control seasonal influenza epidemics. Seasonal influenza vaccines presently available, inactivated or split, contain the circulating strains of influenza A virus H3N2, H1N1 and influenza B virus. The composition of the vaccine is renewed semi-annually, as necessary, based on surveillance data.

Special features of the 2009 pandemic swine-origin influenza AH1N1 hemagglutinin and neuraminidase

Since the 2009 pandemic H1N1 swine-origin influenza A virus (09 S-OIV) has reminded the world about the global threat of the ever changing influenza virus,many questions regarding the detailed re-assortment of influenza viruses yet remain unanswered.Influenza A virus is the causative agent of the pandemic flu and contains 2 major antigenic glycoproteins on its surface:(i) hemagglutinin (HA);and (ii) neuraminidase (NA).The structures of the 09 S-OIV HA and NA proteins (09H1 and 09N1) have recently been resolved in our laboratory and provide some clues as to why the 09 S-OIV re-assortment virus is highly infectious with severe consequences in humans.For example,the 09H1 is highly similar to the HA of the 1918 influenza A pandemic virus in overall structure and especially in regards to its 5 defined antibody binding epitopes.For 09N1,its most distinctive feature is the lack of a 150-loop active site cavity,which was previously predicted to be present in all N1 NAs,and we hypothesize that the 150-loop may play a important role in the substrate specificity (α2,3 or α2,6 linked sialic acid receptors) and enzymatic mechanism of influenza NA.Combination of the HA and NA with special characteristics for the 09 S-OIV might contribute to its high increased transmissibility in humans.

Stigmatization and Scientific Inquiry in the Tracing of the Origin of the 1918 Influenza Pandemic in the United States

The United States was hard hit by the great influenza pandemic of 1918.The national policy of putting the war first,the unprecedented scale of military training,and the worldwide troop movements and engagement created the conditions for the spread of the pandemic and at the same time seriously weakened US preparedness.The unprecedented pandemic threw American society into extreme panic and spawned all kinds of hypotheses about the pandemic’s geographic origin.Some of the press turned scientifically“tracing the flu”into a succession of pejorative geopolitical exonyms,stigmatizing it as“Spanish flu,”“Russian flu,”“German poisoning,”“Chinese plague,”etc.The groundless ascription of a geographic origin to the influenza pandemic was questioned at the time by insightful American medical professionals and even by Chinese medical experts.In the aftermath of the pandemic,tracking its source became a professional issue of pure medical science,with the search for the pathogen of the pandemic becoming a priority.The discovery and genetic sequencing of the 1918 influenza virus by scientists in the US and other countries led to landmark advances in the discovery of the pathogen,so the importance of tracing it back to its place of origin has taken a back seat.Although evidence of the geographic origin of the 1918 influenza pandemic is not conclusive,medical science has developed enough to disprove the ridiculous“geographic tracking”in the US during the pandemic.

华南地区猪场猪流感和甲型H1N1/2009流感病毒的血清学调查

为了解华南地区猪场猪流感病毒（SIV）和甲型H1N1／2009流感病毒（H1N1pdm09）的流行情况，本研究采用血凝抑制试验（HI），对2013年～2014年从5个省份采集的2208份血清样品进行SIV和H1N1pdm09流感病毒血清学检测，结果显示阳性血清共1269份（57．42％），其中广东省和广西省的感染率最高（p〈0．01）。H1N1SIV、H3N2SIV和H1N1pdm09流感病毒抗体阳性率分别为22．51％、32．97％和26．49％。H1N1pdm09流感病毒的抗体几何平均滴度（GMT）最高（91．93＋1．04，p〈0．05）。H1N1SIV和H3N2SIV在冬季血清阳性率最高，然而在冬末（2月）阳性率却最低（p〈0．01）。H1N1pdm09流感病毒抗体阳性率在5月和9月出现高峰（p〈0．01）。母猪的H1N1SIV和H1N1pdm09流感病毒抗体阳性率高于仔猪和育肥猪，但仔猪的H3N2SIV抗体阳性率却比较高。本研究为华南地区猪场SIV和H1N1pdm09流感病毒的预防提供参考依据。

不同年龄组甲型H1N1流感患者临床特征及预后分析

目的对不同年龄组确诊甲型H1N1流感患者的临床特征及预后进行分析,为临床该疾病的社区诊治提供帮助。方法从2009-09-01—11-01连续监测以发热、咳嗽、咳痰等呼吸道症状为主诉就诊的病例,体温≥37.5℃。抽取满足上述定义的病例。对检测患者行咽拭子H1N1的RT-PCR检测,将确诊甲型H1N1流感患者分为青少年组(年龄<18岁)和成人组(年龄≥18岁),并对他们的临床症状、血常规、C反应蛋白、胸部X线平片及用药情况、预后等进行观察及随访。结果 104例确诊为甲型H1N1流感的患者,青少年组患者29例,成人组患者75例,临床症状多以呼吸道症状为主,两组患者发热程度,咳嗽、咳痰,恶心、呕吐或腹泻,头痛,全身酸痛,全身乏力,关节痛等症状间差异无统计学意义(P>0.05)。血常规各项目、C反应蛋白水平及胸部X线平片表现两组差异亦无统计学意义(P>0.05)。成人组咳嗽、咳痰持续时间较青少年组显著延长,差异有统计学意义(P=0.006),青少年组的住院率高于成人组,差异有统计学意义(P=0.036)。随访104例甲型H1N1流感确诊患者均痊愈。使用达菲为成人患者11例(11/104,10.58%),104例患者均使用清热解毒中药如清开灵等。结论 H1H1感染青少年与成人后,临床症状、血常规、CRP及胸部X线平片无差异,主要以呼吸道症状为主,但咳嗽、咳痰持续时间在成人较长。实验室检查两组患者白细胞以正常为主,淋巴细胞表现为正常或降低,未见增高,中药可能在抗甲型H1N1流感中具有一定作用。

新世纪流感大流行的思考

2009年从墨西哥开始暴发了一场席卷全世界的流感疫情.此次大流行的毒株,甲型H1N1病毒,包含了猪源、禽源和人源流感病毒的基因片段.研究该毒株的基因重配、进化历程及其生物学特性,将对防控此次流行具有重要意义.目前,该毒株的遗传进化关系已明确,通过遗传性状分析可获知该毒株可能的生物学性状,但流感大流行动向、毒株遗传变化、毒力及致病性变化仍在密切监控中.流感病毒生态系统具有复杂性,其基因组易突变、易重配、易在自然宿主保存,使得流感大流行存在一定的必然性.正视流感大流行的威胁,积极提高流感病毒在生态系统中的监控,加强流行病学调查,发展疫苗与药物,建立有效公共卫生保障体系,才能降低流感大流行的破坏性.

近年来华东地区家鸭中禽流感病毒的亚型分布

【目的】为了研究近年来华东地区家鸭中禽流感病毒的亚型分布情况。【方法】对2002-2006年分离自华东地区家鸭的180株禽流感病毒的HA亚型和其中88株禽流感病毒的NA亚型分别进行了测定。【结果】近年来华东地区家鸭中至少存在9种HA亚型和6种NA亚型组成的H1N1,H3N1,H3N2,H3N8,H4N6,H5N1,H5N2,H6N2,H6N8,H8N4,H9N2,H10N3,H11N2共13种亚型的禽流感病毒。【结论】华东地区家鸭中有多种亚型的禽流感病毒分布,应加强家鸭禽流感的监测和防制工作。

猪流感病毒SYBR GreenⅠ定量RT-PCR检测方法的建立及应用

为研究猪流感病毒(SIV)感染后SIV在体内的分布规律,本研究设计针对SIV NP基因保守区引物,建立了SIV SYBR GreenⅠ荧光定量PCR方法。该方法对SIV核酸检测灵敏度为30TCID50,对健康猪肺组织cDNA、猪瘟病毒(CSFV)、猪蓝耳病病毒(PRRSV)核酸呈阴性反应。采用建立的荧光定量RT-PCR方法对人工感染A/swine/Shanghai/1/2007/H1N2(Sw/SH/1/2007)猪进行检测,结果显示:病毒在猪鼻腔粘膜持续存在至感染后第8d;肛门拭子在感染后2d～8d可持续检测到病毒核酸;喉头、气管在感染后第3d可检到病毒核酸,并持续至第10d;肺部淋巴结、脾脏在感染后5d～7d检测到病毒核酸阳性;其它脏器均未检测到病毒核酸,从而确定呼吸道系统及脾脏是SIV定殖的主要场所,SIV感染猪后向外排毒周期约为1周。

2006--2008年华东地区家禽不同HA亚型低致病性禽流感的病原学监测

目的采用血清学试验和RT-PCR方法在2006-2008年5月期间从华东地区家禽中分离到473株禽流感病毒(AIVs),其中家鸭AIVs的分离率最高(14.7%),鸡AIVs分离率最低(2.92%)。共分离到H1、H3、H4、H6、H9、H10、H11七种HA亚型低致病性禽流感病毒(LPAIVs),其中H3亚型LPAIVs在家禽中的分布占优势。禽流感病毒的分离呈现一定的季节性,一般春秋冬三个季节分离率相对较高。

浙江省甲型H1N1流感病毒血凝素基因序列分析

目的分析浙江省2009-2011年甲型H1N1流感病毒血凝素基因(HA)序列进化特征。方法提取浙江省2009-2011年8株甲型H1N1流感病毒基因组RNA,RT-PCR扩增血凝素基因,测序并拼接出ORF;从GenBank数据库下载2009-2012年国内外甲型H1N1流感病毒HA基因序列,选择20株作为代表进行分析。采用MEGA软件对其进行种系发生树的构建。结果扩增并测序获得8株甲型H1N1流感病毒1 701bp的HA基因ORF,与国内外甲型H1N1流感病毒HA序列比对后发现所有序列的同源性均较高,2009年和2010年浙江省3株病毒HA与北美早期毒株A/California/04/2009(H1N1)同源性达到99.4%～99.5%,2011年浙江省5株病毒HA与A/California/04/2009(H1N1)同源性为98.5%～99.1%。在1株来自重症患者的毒株中发现了D222G突变。结论与早期毒株相比,虽然浙江省2011年的甲型H1N1流感病毒血凝素基因累积了更多的变异,但所有毒株的同源性达到98.5%以上。血凝素中D222G突变可能与重症甲型H1N1流感病毒感染相关。