Dynamic patterns of circulating seasonal and pandemic A(H1N1)pdm09 influenza viruses from 2007-2010 in and around Delhi, India

Influenza surveillance was carried out in a subset of patients with influenza-like illness (ILI) presenting at an Employee Health Clinic (EHS) at All India Institute of Medical Sciences (AIIMS), New Delhi (urban) and pediatric out patients department of civil hospital at Ballabhgarh (peri-urban), under the Comprehensive Rural Health Services Project (CRHSP) of AIIMS, in Delhi region from January 2007 to December 2010. Of the 3264 samples tested, 541 (17%) were positive for influenza viruses, of which 221 (41%) were pandemic Influenza A(H1N1)pdm09, 168 (31%) were seasonal influenza A, and 152 (28%) were influenza B. While the Influenza viruses were detected year-round, their types/subtypes varied remarkably. While there was an equal distribution of seasonal A(H1N1) and influenza B in 2007, predominance of influenza B was observed in 2008. At the beginning of 2009, circulation of influenza A(H3N2) viruses was observed, followed later by emergence of Influenza A(H1N1)pdm09 with co-circulation of influenza B viruses. Influenza B was dominant subtype in early 2010, with second wave of Influenza A(H1N1)pdm09 in August-September, 2010. With the exception of pandemic H1N1 emergence in 2009, the peaks of influenza activity coincided primarily with monsoon season, followed by minor peak in winter at both urban and rural sites. Age group analysis of influenza positivity revealed that the percent positivity of Influenza A(H1N1)pdm09 influenza virus was highest in >5–18 years age groups (OR 2.5; CI = 1.2–5.0; p = 0.009) when compared to seasonal influenza. Phylogenetic analysis of Influenza A(H1N1)pdm09 from urban and rural sites did not reveal any major divergence from other Indian strains or viruses circulating worldwide. Continued surveillance globally will help define regional differences in influenza seasonality, as well as, to determine optimal periods to implement influenza vaccination programs among priority populations.     

No Serological Evidence of Influenza A H1N1pdm09 Virus Infection as a Contributing Factor in Childhood Narcolepsy after Pandemrix Vaccination Campaign in Finland

Background

Narcolepsy cataplexy syndrome, characterised by excessive daytime sleepiness and cataplexy, is strongly associated with a genetic marker, human leukocyte antigen (HLA) DQB1*06:02. A sudden increase in the incidence of childhood narcolepsy was observed after vaccination with AS03-adjuvanted Pandemrix influenza vaccine in Finland at the beginning of 2010. Here, we analysed whether the coinciding influenza A H1N1pdm pandemic contributed, together with the Pandemrix vaccination, to the increased incidence of childhood narcolepsy in 2010. The analysis was based on the presence or absence of antibody response against non-structural protein 1 (NS1) from H1N1pdm09 virus, which was not a component of Pandemrix vaccine.

Methods

Non-structural (NS) 1 proteins from recombinant influenza A/Udorn/72 (H3N2) and influenza A/Finland/554/09 (H1N1pdm09) viruses were purified and used in Western blot analysis to determine specific antibody responses in human sera. The sera were obtained from 45 patients who fell ill with narcolepsy after vaccination with AS03-adjuvanted Pandemrix at the end of 2009, and from controls.

Findings

Based on quantitative Western blot analysis, only two of the 45 (4.4%) Pandemrix-vaccinated narcoleptic patients showed specific antibody response against the NS1 protein from the H1N1pdm09 virus, indicating past infection with the H1N1pdm09 virus. Instead, paired serum samples from patients, who suffered from a laboratory confirmed H1N1pdm09 infection, showed high levels or diagnostic rises (96%) in H1N1pdm virus NS1-specific antibodies and very high cross-reactivity to H3N2 subtype influenza A virus NS1 protein.

Conclusion

Based on our findings, it is unlikely that H1N1pdm09 virus infection contributed to a sudden increase in the incidence of childhood narcolepsy observed in Finland in 2010 after AS03-adjuvanted Pandemrix vaccination.     

Epidemiological and Virological Characteristics of Influenza Viruses Circulating in Cambodia from 2009 to 2011

Background

The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011.

Methodology/Principal Findings

Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009–2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade.

Conclusions/Significance

In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective.     

Highly Predictive Model for a Protective Immune Response to the A(H1N1)pdm2009 Influenza Strain after Seasonal Vaccination

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young (<31 years, N = 17) versus elderly (≥50 years, N = 20) donors sero-negative to the newly emerged A(H1N1)pdm09 influenza virus strain and correlated it with the serological response to that specific strain after seasonal influenza vaccination. Extensive multi-chromatic FACS analysis (36 lymphocyte sub-populations measured) was used to quantitatively assess the cellular immune status before vaccination. We identified CD4⁺ T cells, and amongst them particularly naive CD4⁺ T cells, as the best correlates for a successful A(H1N1)pdm09 immune response. Moreover, the number of influenza strains a donor was sero-negative to at baseline (NSSN) in addition to age, as expected, were important predictive factors. Age, NSSN and CD4⁺ T cell count at baseline together predicted sero-protection (HAI≥40) to A(H1N1)pdm09 with a high accuracy of 89% (p-value = 0.00002). An additional validation study (N = 43 vaccinees sero-negative to A(H1N1)pdm09) has confirmed the predictive value of age, NSSN and baseline CD4⁺ counts (accuracy = 85%, p-value = 0.0000004). Furthermore, the inclusion of donors at ages 31–50 had shown that the age predictive function is not linear with age but rather a sigmoid with a midpoint at about 50 years. Using these results we suggest a clinically relevant prediction model that gives the probability for non-protection to A(H1N1)pdm09 influenza strain after seasonal multi-valent vaccination as a continuous function of age, NSSN and baseline CD4 count.     

Evolution of the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus in Morocco During Two Influenza Seasons 2009–2011

To study genetic evolution of Moroccan influenza A(H1N1)pdm09 virus strains, we conducted a molecular characterization of the hemagglutinin gene subunit 1 (HA1) of 36 influenza A(H1N1)pdm09 virus strains. The stains were collected from patients in Rabat and Casablanca during two influenza seasons 2009–2010 and 2010–2011. Nucleotide and amino acid sequences of 14 influenza A(H1N1)pdm09 virus strains from 2009 to 2010 were ~97 and 99 %, respectively, similar to the reference strain A/California/07/2009 (H1N1). Phylogenetic analysis of 22 influenza A(H1N1)pdm09 virus strains from 2010 to 2011 revealed a co-circulation of three well-described different genetic groups. Most important, none of the identified groups showed significant changes at the antigenic site of the virus HA1 subunit which may alter the efficacy of California/07/2009 (H1N1) vaccine.     

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the p _epitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012–2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.     

Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.     

海南省2016-2018年A(H1N1)pdm09亚型流感病毒基因特性分析

2009年A(H1N1)pdm09亚型流感病毒在墨西哥暴发,之后在全世界流行。为了解海南省2016-2018年A(H1N1)pdm09亚型流感病毒流行态势,分析血凝素(HA)与神经氨酸酶(NA)基因遗传进化特征与变异情况,本研究从中国流感监测信息系统获取海南省2016-2018年流感病毒病原学监测数据,选取5家流感监测网络实验室分离鉴定的37株A(H1N1)pdm09亚型流感毒株进行HA与NA基因测序,利用MEGA 10.1.8构建HA与NA基因种系进化树,并分析其氨基酸变异情况。结果显示,2016-2018年共出现3次A(H1N1)pdm09亚型流感病毒活动高峰。2017年10月份以后的分离株(4/8)与2018年大部分分离株(21/22)独立于疫苗株A/Michigan/45/2015聚为一个小支,发生20余处HA与NA氨基酸位点变异。与疫苗株A/California/7/2009(2010-2016)相比,2016-2018年流感病毒分离株在HA基因抗原决定簇上发生7处氨基酸变异并有一个潜在糖基化位点,未发现HA基因受体结合位点变异与NA基因耐药性变异。本研究提示,2016-2018年,A(H1N1)pdm09亚型流感病毒逐步发生规律性进化,氨基酸变异频率有增加趋势,今后应持续加强流感病毒病原学监测,密切追踪A(H1N1)pdm09亚型流感病毒基因变异情况,为科学防控提供理论依据。     

Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection

Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010–2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.     

Detection of the Influenza A(H1N1)pdm09 Virus Carrying the K-15E,P83S and Q293H Mutations in Patients Who Have Undergone Bone Marrow Transplant

The 2009 pandemic influenza A(H1N1)pdm09 virus emerged and caused considerable morbidity and mortality in the third world, especially in Brazil. Although circulating strains of A(H1N1)pdm09 are A/California/04/2009-like (CA-04-like) viruses, various studies have suggested that some mutations in the viral hemagglutinin (HA) may be associated with enhanced severity and fatality. This phenomenon is particularly challenging for immunocompromised individuals, such as those who have undergone bone marrow transplant (BMT), because they are more likely to display worse clinical outcomes to influenza infection than non-immunocompromised individuals. We studied the clinical and viral aspects of post-BMT patients with confirmed A(H1N1)pdm09 diagnosis in the largest cancer hospital in Brazil. We found a viral strain with K-15E, P83S and Q293H polymorphisms in the HA, which is presumably more virulent, in these individuals. Despite that, these patients showed only mild symptoms of infection. Our findings complement the discovery of mild cases of infection with the A(H1N1)pdm09 virus with the K-15E, P83S and Q293H mutations in Brazil and oppose other studies that have linked these changes with increased disease severity. These results could be important for a better comprehension of the impact of the pandemic influenza in the context of BMT.     

Comparative analyses of pandemic H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets

Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.     

AID Activity in B Cells Strongly Correlates with Polyclonal Antibody Affinity Maturation in-vivo Following Pandemic 2009-H1N1 Vaccination in Humans

The role of Activation-Induced Cytidine Deaminase (AID) in somatic hypermutation and polyclonal antibody affinity maturation has not been shown for polyclonal responses in humans. We investigated whether AID induction in human B cells following H1N1pdm09 vaccination correlated with in-vivo antibody affinity maturation against hemagglutinin domains in plasma of young and elderly individuals. AID was measured by qPCR in B cells from individuals of different ages immunized with the H1N1pdm09 influenza vaccine. Polyclonal antibody affinity in human plasma for the HA1 and HA2 domains of the H1N1pdm09 hemagglutinin was measured by antibody-antigen complex dissociation rates using real time kinetics in Surface Plasmon Resonance. Results show an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination. Levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlated with increase in polyclonal antibody affinity to the HA1 globular domain (but not to the conserved HA2 stalk). In the younger population, significant affinity maturation to the HA1 globular domain was observed, which associated with initial levels of AID and fold-increase in AID after vaccination. In some older individuals (>65 yr), higher affinity to the HA1 domain was observed before vaccination and H1N1pdm09 vaccination resulted in minimal change in antibody affinity, which correlated with low AID induction in this age group. These findings demonstrate for the first time a strong correlation between AID induction and in-vivo antibody affinity maturation in humans. The ability to generate high affinity antibodies could have significant impact on the elucidation of age-specific antibody responses following vaccination and eventual clinical efficacy and disease outcome.     

High Rate of A(H1N1)pdm09 Infections among Rural Thai Villagers, 2009–2010

Background

Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.

Methods

A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.

Results

The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003.

Conclusion

Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.     

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.     

Incidence and Epidemiology of Hospitalized Influenza Cases in Rural Thailand during the Influenza A (H1N1)pdm09 Pandemic, 2009–2010

Background

Data on the burden of the 2009 influenza pandemic in Asia are limited. Influenza A(H1N1)pdm09 was first reported in Thailand in May 2009. We assessed incidence and epidemiology of influenza-associated hospitalizations during 2009–2010.

Methods

We conducted active, population-based surveillance for hospitalized cases of acute lower respiratory infection (ALRI) in all 20 hospitals in two rural provinces. ALRI patients were sampled 1∶2 for participation in an etiology study in which nasopharyngeal swabs were collected for influenza virus testing by PCR.

Results

Of 7,207 patients tested, 902 (12.5%) were influenza-positive, including 190 (7.8%) of 2,436 children aged <5 years; 86% were influenza A virus (46% A(H1N1)pdm09, 30% H3N2, 6.5% H1N1, 3.5% not subtyped) and 13% were influenza B virus. Cases of influenza A(H1N1)pdm09 first peaked in August 2009 when 17% of tested patients were positive. Subsequent peaks during 2009 and 2010 represented a mix of influenza A(H1N1)pdm09, H3N2, and influenza B viruses. The estimated annual incidence of hospitalized influenza cases was 136 per 100,000, highest in ages <5 years (477 per 100,000) and >75 years (407 per 100,000). The incidence of influenza A(H1N1)pdm09 was 62 per 100,000 (214 per 100,000 in children <5 years). Eleven influenza-infected patients required mechanical ventilation, and four patients died, all adults with influenza A(H1N1)pdm09 (1) or H3N2 (3).

Conclusions

Influenza-associated hospitalization rates in Thailand during 2009–10 were substantial and exceeded rates described in western countries. Influenza A(H1N1)pdm09 predominated, but H3N2 also caused notable morbidity. Expanded influenza vaccination coverage could have considerable public health impact, especially in young children.     

2009−2015年中国甲型H1N1流感病毒血凝素基因进化分析

【目的】为了解中国地区2009?2015年甲型H1N1流感病毒流行态势,分析血凝素(Hemagglutinin,HA)基因的变异情况及其遗传进化特征。【方法】汇集国家流感中心2009?2015年流感周报的流感流行数据,分析甲型H1N1流感的流行病学特征;从全球共享禽流感数据倡议组织数据库及美国国家生物技术中心数据库下载甲型H1N1流感病毒HA基因序列,采用生物学软件进行系统进化和遗传特性的分析。【结果】2009?2015年全国共发生4次甲型H1N1流感的流行高峰。2009?2015年毒株与参考毒株A/California/07/2009(H1N1)的HA基因同源性逐年降低。遗传进化分析显示同一年份的毒株在系统进化树上基本呈现集中分布,2011年的毒株独立形成2个分支。分子特征表现为HA基因的4个抗原决定簇氨基酸位点均有变异,其中Ca区的203位、Sa区的163位和Sb区的185位氨基酸位点逐渐替换为新的氨基酸。除2010年与2012年,其他年份的毒株通过不同模型均得到正向压力选择HA氨基酸位点240。【结论】甲型H1N1流感在中国地区成为主要流行的亚型之一。HA基因与其编码的氨基酸逐年变异,未来进一步的流感监测能力还需加强。     

Guillain-Barré Syndrome and Adjuvanted Pandemic Influenza A (H1N1) 2009 Vaccines: A Multinational Self-Controlled Case Series in Europe

Background

The risk of Guillain-Barré syndrome (GBS) following the United States'' 1976 swine flu vaccination campaign in the USA led to enhanced active surveillance during the pandemic influenza (A(H1N1)pdm09) immunization campaign. This study aimed to estimate the risk of GBS following influenza A(H1N1)pdm09 vaccination.

Methods

A self-controlled case series (SCCS) analysis was performed in Denmark, Finland, France, Netherlands, Norway, Sweden, and the United Kingdom. Information was collected according to a common protocol and standardised procedures. Cases classified at levels 1–4a of the Brighton Collaboration case definition were included. The risk window was 42 days starting the day after vaccination. Conditional Poisson regression and pooled random effects models estimated adjusted relative incidences (RI). Pseudo likelihood and vaccinated-only methods addressed the potential contraindication for vaccination following GBS.

Results

Three hundred and three (303) GBS and Miller Fisher syndrome cases were included. Ninety-nine (99) were exposed to A(H1N1)pdm09 vaccination, which was most frequently adjuvanted (Pandemrix and Focetria). The unadjusted pooled RI for A(H1N1)pdm09 vaccination and GBS was 3.5 (95% Confidence Interval (CI): 2.2–5.5), based on all countries. This lowered to 2.0 (95% CI: 1.2–3.1) after adjustment for calendartime and to 1.9 (95% CI: 1.1–3.2) when we accounted for contra-indications. In a subset (Netherlands, Norway, and United Kingdom) we further adjusted for other confounders and there the RI decreased from 1.7 (adjusted for calendar month) to 1.4 (95% CI: 0.7–2.8), which is the main finding.

Conclusion

This study illustrates the potential of conducting European collaborative vaccine safety studies. The main, fully adjusted analysis, showed that the RI of GBS was not significantly elevated after influenza A(H1N1)pdm09 vaccination (RI = 1.4 (95% CI: 0.7–2.8). Based on the upper limits of the pooled estimate we can rule out with 95% certainty that the number of excess GBS cases after influenza A(H1N1)pdm09 vaccination would be more than 3 per million vaccinated.     

Effectiveness of A(H1N1)pdm09 Influenza Vaccine in Adults Recommended for Annual Influenza Vaccination

Introduction

Because of variability in published A(H1N1)pdm09 influenza vaccine effectiveness estimates, we conducted a study in the adults belonging to the risk groups to assess the A(H1N1)pdm09 MF59-adjuvanted influenza vaccine effectiveness.

Methods

VE against influenza and/or pneumonia was assessed in the cohort study (n>25000), and vaccine effectiveness against laboratory-confirmed A(H1N1)pdm09 influenza was assessed in a matched case-control study (16 pairs). Odds ratios (OR) and their 95% confidence intervals (95% CI) were calculated by using multivariate logistic regression; vaccine effectiveness was estimated as (1-odds ratio)*100%.

Results

Vaccine effectiveness against laboratory-confirmed A(H1N1)pdm09 influenza and influenza and/or pneumonia was 98% (84–100%) and 33% (2–54%) respectively. The vaccine did not prevent influenza and/or pneumonia in 18–59 years old subjects, and was 49% (16–69%) effective in 60 years and older subjects.

Conclusions

Even though we cannot entirely rule out that selection bias, residual confounding and/or cross-protection has played a role, the present results indicate that the MF59-adjuvanted A(H1N1)pdm09 influenza vaccine has been effective in preventing laboratory-confirmed A(H1N1)pdm09 influenza and influenza and/or pneumonia, the latter notably in 60 years and older subjects.     

Polymerase Discordance in Novel Swine Influenza H3N2v Constellations Is Tolerated in Swine but Not Human Respiratory Epithelial Cells

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection.