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.     

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.     

21世纪的首次大流行：2009甲型（H1N1）流感

2009年4月初,在墨西哥和美国出现一种新型甲型（H1N1）流感病毒。该病毒通过人-人传播迅速在全球范围蔓延。该病毒拥有来自人流感病毒、禽流感病毒和猪流感病毒的基因片段,其HA基因与引发1918年大流行的流感病毒株的HA基因同源性很高。该病毒倾向于感染儿童、青少年、孕妇,以及具有心肺疾病的人。据观察,它在人群中的传播能力高于季节性流感。部分感染患者具有在季节性流感中罕见的呕吐和腹泻症状。先前的流感病毒大流行和2009年爆发的甲型H1N1流感病毒大流行表明,由于流感病毒变异速度快、容易发生基因重排,新产生的变异毒株很可能造成新的大流行,威胁人类健康。由于禽流感病毒和人流感病毒都能感染猪,猪被认为是通过基因重排生成新的大流行病毒的＂混合容器＂。     

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．     

Cytokine and chemokine responses in pediatric patients with severe pneumonia associated with pandemic A/H1N1/2009 influenza virus

Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty‐seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)‐10 (P = 0.027) and IL‐5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon‐γ (P = 0.009), tumor necrosis factor‐α (P = 0.01), IL‐4 (P = 0.024), and IL‐2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL‐8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection.     

Effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic H1N1 2009 vaccine: a randomized controlled trial

Vaccination with the non-adjuvanted split-virion A/California/7/2009 influenza vaccine (pandemic H1N1 2009 vaccine) began in October 2009 in Japan. The present study was designed to assess the effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the pandemic H1N1 2009 vaccine in healthy adult volunteers. One hundred and seventeen participants aged 22 to 62 were randomly assigned to two study groups. In Group 1 (the priming group), participants were first vaccinated with the seasonal trivalent influenza vaccine followed by two separate one-dose vaccinations of the pandemic H1N1 2009 vaccine, whereas in Group 2 (the non-priming group), the participants were first vaccinated with one dose of the pandemic H1N1 2009 vaccine, followed by simultaneous vaccination of the seasonal trivalent vaccine and the second dose of the pandemic H1N1 2009 vaccine. The participants in Group 2 had a seroprotection rate (SPR) of 79.7% and a seroconversion rate (SCR) of 79.7% in the hemagglutination-inhibition test after the first dose of the pandemic H1N1 2009 vaccine, indicating that the pandemic H1N1 2009 vaccine is sufficiently immunogenic. On the other hand, the participants of Group 1 had a significantly weaker antibody response, with a SPR of 60.8% and a SCR of 58.5%. These results indicate that prior vaccination with the seasonal trivalent influenza vaccine inhibits the antibody response to the pandemic H1N1 2009 vaccine. Therefore, the pandemic H1N1 2009 vaccine should be administered prior to vaccination with the seasonal trivalent influenza vaccine.     

Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza.     

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.     

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.     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

Real-time characterization of the molecular epidemiology of an influenza pandemic

Early characterization of the epidemiology and evolution of a pandemic is essential for determining the most appropriate interventions. During the 2009 H1N1 influenza A pandemic, public databases facilitated widespread sharing of genetic sequence data from the outset. We use Bayesian phylogenetics to simulate real-time estimates of the evolutionary rate, date of emergence and intrinsic growth rate (r₀) of the pandemic from whole-genome sequences. We investigate the effects of temporal range of sampling and dataset size on the precision and accuracy of parameter estimation. Parameters can be accurately estimated as early as two months after the first reported case, from 100 genomes and the choice of growth model is important for accurate estimation of r₀. This demonstrates the utility of simple coalescent models to rapidly inform intervention strategies during a pandemic.     

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。     

Phylogenetic analyses of pandemic influenza A (H1N1) virus in university students at Tobetsu, Hokkaido, Japan

Pandemic influenza H1N1 virus (A[H1N1]pdm09) emerged in 2009. To determine the phylogeography of A(H1N1)pdm09 in a single population, 70 strains of the virus were isolated from university students or trainee doctors at Tobetsu, Hokkaido, Japan, between September and December 2009. The nucleotide sequences of the HA1 region of the HA genes and described phylogenetic relationships of the strains circulating among them were analyzed. It was found that the 70 isolates could be phylogenetically separated into three groups and that two epidemics were caused by different groups of the virus. The three groups were also distinguishable from each other by three amino acid changes: A197T, S203T and Q293H. The substitution of S203T, which is located in the antigenic site, suggests antigenic drift of the virus.     

Development and diagnostic application/evaluation of pandemic (H1N1) 2009 influenza virus-specific monoclonal antibodies

We prepared mAb specific to the H1N1 2009 virus (H1N1 2009) to facilitate development of an RDT with enhanced sensitivity and specificity. Among these antibodies, we identified two clones--hybridomas 1H7E1 and 3A3H7-that specifically bound to H1N1 2009 (non-seasonal) and were very suitable for application to a diagnostic kit. The affinity constants (K(a)) of 1H7E1 and 3A3H7 were 1.10 × 10(10) and 2.35 × 10(10), respectively. To identify the antibodies, we performed ELISA and immunoblot analyses and found that 1H7E1 recognized a conformational epitope of HA while 3A3H7 recognized a linear epitope. In clinical evaluations using specimens from 215 patients, a lateral flow rapid testing kit comprising these mAb showed a sensitivity of 81.5% (75/92) and a specificity of 96.7% (119/123). Results using the RDT kit were well correlated with conventional RT-PCR methods as commonly and commercially used. Based on our findings, we believe that use of these mAb with a rapid evaluation kit could serve as a good diagnostic tool for H1N1 2009.     

B型流感病毒B/Guangxi-Jiangzhou/1352/2018毒株的遗传进化及其对小鼠的致病性分析北大核心CSCD

B型流感病毒(influenza B virus,IBV)比A型流感病毒(influenza A virus,IAV)更易引发并发症,在一定季节内造成的疾病负担甚至超过IAV,但目前人们对IBV的关注较少。为了分析IBV临床毒株B/Guangxi-Jiangzhou/1352/2018的遗传进化特点,本研究构建了系统进化树,并以世界卫生组织推荐的疫苗株为参考,对其血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)进行了氨基酸序列同源性及突变位点分析。分析结果发现B/Guangxi-Jiangzhou/1352/2018毒株无谱系间重配现象,与同年疫苗株B/Colorado/06/2017匹配性较差。另外,测定了B/Guangxi-Jiangzhou/1352/2018毒株感染小鼠的半数致死量(median lethal dose,LD_(50))及其对小鼠的致病性。结果表明,B/Guangxi-Jiangzhou/1352/2018毒株感染小鼠的LD_(50)为10^(5.9) TCID_(50)(median tissue culture infective dose),小鼠肺脏中病毒滴度在感染后1 d达到高峰,炎性细胞因子的mRNA水平在感染后12 h达到高峰,且感染后肺脏中的肺泡损伤严重,有大量炎性细胞浸润。本研究证明了IBV临床毒株B/Guangxi-Jiangzhou/1352/2018可以感染小鼠并诱发典型的肺脏炎症,为研究IBV致病及传播机制奠定了基础,为评价新型流感疫苗、抗病毒和抗炎症药物提供了理想的动物模型。     

Proteomics search of influenza A viruses for adaptive mutations to human hosts

Evaluation of: Miotto O, Heiny AT, Albrecht R et al. Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains. PLoS ONE 5(2), e9025 (2010).

The emergence of an influenza pandemic is of great concern globally. It is, therefore, necessary to have a better understanding of the adaptation of influenza A viruses to humans. The mutation patterns affecting host tropism may provide information on the mechanisms and determinants of the host barrier. The work by Miotto et al. describes a catalog of mutations observed specifically in human influenza A viruses by analyzing almost 100,000 influenza A virus protein sequences. These sites may be important for host tropism and characteristic mutations of human influenza viruses may be required for efficient human-to-human transmission. The catalog can be used for genetic surveillance of zoonotic strains of the influenza A virus to determine their pandemic potential, as well as for basic research on the influenza A virus.     

Interspecies transmission and host restriction of avian H5N1 influenza virus

Long-term endemicity of avian H5N1 influenza virus in poultry and continuous sporadic human infections in several countries has raised the concern of another potential pandemic influenza. Suspicion of the avian origin of the previous pandemics results in the close investigation of the mechanism of interspecies transmission. Entry and fusion is the first step for the H5N1 influenza virus to get into the host cells affecting the host ranges. Therefore receptor usage study has been a major focus for the last few years. We now know the difference of the sialic acid structures and distributions in different species, even in the different parts of the same host. Many host factors interacting with the influenza virus component proteins have been identified and their role in the host range expansion and interspecies transmission is under detailed scrutiny. Here we review current progress in the receptor usage and host factors. Supported by the National Basic Research Program of China (Grant Nos. 2005CB523001, 2005CB523002), National Key Technologies Research & Development Program (Grant 2006BAD06A01/2006BAD06A04); US National Institutes of Health (NIH) (Grant 3 U19 AI051915-05S1), the National Natural Science Foundation of China (Grant 30599434). GAO FG is a distinguished young investigator of the NSFC (Grant No. 30525010).