Genetic characterization of Thai swine influenza viruses after the introduction of pandemic H1N1 2009

Pandemic H1N1 2009 (pH1N1), influenza virus containing triple reassortant internal genes (TRIG) from avian, human, and swine influenza viruses emerged in 2009 as a highly infectious virus that was able to be transmitted from humans to pigs. During June 2010–May 2012, influenza virus surveillance was conducted in Thai pig population. Twenty-three samples (1.75 %) were successfully isolated from total of 1,335 samples. Interestingly, pH1N1 (7 isolates, 30.34 %), reassortant pH1N1 (rH1N1) (1 isolate, 4.35 %), Thai endemic H1N1 (enH1N1) (3 isolates, 13.04 %), reassortant H3N2 with pH1N1 internal genes (rH3N2) (9 isolates, 39.13 %), and reassortant H1N2 with pH1N1 internal genes (rH1N2) (3 isolates, 13.04 %) were found. It should be noted that rH1N1, rH1N2, and rH3N2 viruses contained the internal genes of pH1N1 virus having a TRIG cassette descendant from the North American swine lineage. Although all isolates in this study were obtained from mild clinically sick pigs, the viruses were still highly infective and possibly may play an important role in human–animal interfacing transmission. In addition, the TRIG cassette may have an influence on antigenic shift resulting in emergence of novel viruses, as seen in this study. Continuing surveillance of influenza A natural hosts, particularly in pigs is necessary.     

A novel reassortant H1N2 virus related to the pandemic H1N1 2009 influenza virus isolated from Korean pigs

Since the discovery of the pandemic H1N1 (pH1N1) virus in 2009, a novel reassortant H1N2 virus (A/Swine/Korea/VDS1/2010) containing the pH1N1 segments has been detected in Korean pig populations. The hemagglutinin and neuraminidase genes of this virus are derived from reassortant H1N1- and H1N2-group viruses, respectively, identified in Korean pigs, while other genes originate from contemporary circulating pH1N1 viruses. The antigenic and biological properties of this novel virus, as determined by clinical, pathological, serological, and genetic analyses, are similar to those of pH1N1 viruses, which infect swine easily (Weingartl et al. J Virol 84:2245–2256, 2010; Brookes et al. PLoS one 5:e9068, 2010; Lange et al. J Gen Virol 90:2119–2123, 2009). Determining whether this virus will become established and pose a threat to mammalian populations requires further investigation.     

2009 pandemic H1N1-associated myocarditis in a previously healthy adult

Influenza infection most commonly affects the upper and lower respiratory tracts, but can involve extrapulmonary sites, including the myocardium. We report on a case of myocarditis caused by documented 2009 pandemic H1N1 influenza in a previously healthy adult, and review the literature on influenza myocarditis.     

Molecular characterization and comparative analysis of pandemic H1N1/2009 strains with co-circulating seasonal H1N1/2009 strains from eastern India

During the peak outbreak (July-September 2009), a total 1886 patients were screened in eastern India, of which 139 (7.37%) and 52 (2.76%) were positive for pH1N1 and seasonal H1N1, respectively. Full-length HA1, NA, NS1 and PB1-F2 genes of representative strains were sequenced. Phylogenetic analysis of deduced amino acid sequences of pH1N1 strains revealed HA1 and NS1 to be of North American swine lineage, and the NA gene of Eurasian swine lineage. Consistent with previous reports, the PB1-F2 gene of pH1N1 strains was unique due to a mutation resulting in a truncated protein of 11 aa. The HA, NA and NS1 genes of H1N1/2009 strains clustered with H1N1 strains of 2000-2009, whereas a subset of strains contained a pH1N1-like truncated PB1-F2. The truncated PB1-F2 may confer the advantage of lower pathogenicity but higher replication and infectivity to the human H1N1 strains. This is the first report of seasonal H1N1/2009 strains with a pH1N1/2009-like gene segment.     

Modelling during an emergency: the 2009 H1N1 influenza pandemic

During the 2009 H1N1 pandemic, decision-makers had access to mathematical and computational models that were not available in previous pandemics in 1918, 1957, and 1968. How did models contribute to policy and action during the 2009 H1N1 pandemic? Modelling encountered six primary challenges: (i) expectations of modelling were not clearly defined; (ii) appropriate real-time data were not readily available; (iii) modelling results were not generated, shared, or disseminated in time; (iv) decision-makers could not always decipher the structure and assumptions of the models; (v) modelling studies varied in intervention representations and reported results; and (vi) modelling studies did not always present the results or outcomes that are useful to decision-makers. However, there were also seven general successes: (i) modelling characterized the role of social distancing measures such as school closure; (ii) modelling helped to guide data collection; (iii) modelling helped to justify the value of the vaccination programme; (iv) modelling helped to prioritize target populations for vaccination; (v) modelling addressed the use of antiviral medications; (vi) modelling helped with health system preparedness planning; and (vii) modellers and decision-makers gained a better understanding of how to work with each other. In many ways, the 2009 pandemic served as practice and a learning opportunity for both modellers and decision-makers. Modellers can continue working with decision-makers and other stakeholders to help overcome these challenges, to be better prepared when the next emergency inevitably arrives.     

Increased incidence of narcolepsy following the 2009 H1N1 pandemic

Narcolepsy is an autoimmune sleep disease characterized by excessive daytime sleepiness, cataplexy, and intrusive rapid eye movement sleep. Deficit in endogenous orexin is a major pathogenic component of the disease. This disorder is also associated with the gene marker HLADQB1*0602. After the 2009, H1N1 pandemic, a remarkable increased incidence of narcolepsy, especially in children, was noticed in different countries. In Finland and Sweden, this was attributed to the H1N1 vaccine, especially the adjuvanted pandemic vaccines. In China, there was a 3-fold increase in narcolepsy onset but was due to H1N1 virus infection per se. This review will first summarize the epidemiological data of post H1N1 narcolepsy, then possible mechanisms of the H1N1-related immune process to link the HLA (human leukocyte antigen) genetic background and hypocretin deficiency in narcolepsy–cataplexy will be discussed.     

Emergence of novel reassortant H3N2 swine influenza viruses with the 2009 pandemic H1N1 genes in the United States

Reassortant H1 swine influenza viruses (SIVs) carrying 2009 pandemic H1N1 virus (pH1N1) genes have been isolated from pigs worldwide. Seven novel reassortant H3N2 SIVs were identified from diseased pigs in the USA from winter 2010 to spring 2011. These novel viruses contain three or five internal genes from pH1N1 and continue to circulate in swine herds. The emergence of novel reassortant H3N2 SIVs demonstrates reassortment between pH1N1 and endemic SIVs in pigs and justifies continuous surveillance.     

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009)

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.     

Analysis of antigen epitopes and molecular pathogenic characteristics of the 2009 H1N1 pandemic influenza A virus in China

In order to further predict the epidemic trend and develop vaccines for 2009 H1N1 virus, we monitored its epitopes and molecular pathogenic characteristics during the epidemic process. We also analyzed the similarity of antigenic and genetic characteristics among the novel 2009 H1N1, representative seasonal H1N1 strains, and vaccine strains. 2009 H1N1 isolates had high similarity of hemagglutinin (HA) antigenic sites with H1N1 viruses isolated before 1940 and up to 80.0% similarity with 1918 H1N1. The elderly people born before 1940 have relatively low 2009 H1N1 infection rate, which might be responsible for their previous infection with either 1918 H1N1 virus or an early progeny. Compared to seasonal H1N1 vaccine strains from 1999 to 2010, the HA, neuraminidase (NA), and nucleoprotein (NP) proteins of the isolates had highly conserved CTL epitopes (60.5-65.8%, 69.6-82.6%, and 76.7%, respectively). The seriousness and mortality rate of 2009 H1N1 infections were similar to seasonal influenza, which may be related to the molecular characteristics of low toxicity of 2009 H1N1 and cross-T-cell immunity, due to vaccination or exposure to seasonal H1N1 virus. Some strains of 2009 H1N1 acquired mutations at antigenic and glycosylation sites. It is of particular interest that Haishu/SWL110/10 and Beijing/SE2649/09, isolated after November 2009, gained a new glycosylation site at the position 179 of HA protein, near the RBD. Thus, in the future, vaccination with glycosylated 2009 H1N1 virus may prevent the seasonal epidemic caused by strains with glycosylation site mutation near the receptor binding domain (RBD).     

Positive rates of 2009 novel influenza A (H1N1) was high in school-aged individuals: significance in pandemic control

In this study, data from a pandemic H1N1 outbreak in Korea were analyzed according to time, geography (districts), and age. A total of 252,271 samples collected nationwide were referred to the Greencross Reference Laboratory from June 2009 to February 2010 for H1N1 confirmation testing. Of these samples, 105,300 (41.7%) were H1N1-positive. With time, positivity was highest (57.0%) from October 26 - November 1 (4 weeks after Chuseok). The positive rates among districts show the highest value in Ulsan City (63.1%) and the lowest in Gyeongnam Province (32.8%). The positive rates for ages 0-2, 3-5, 6-11, 12-17, 18-20, 21-30, 31-40, 41-50, 51-60, and > 60 yr were 17.0%, 33.1%, 56.2%, 55.5%, 55.3%, 41.5%, 28.2%, 30.5%, 31.1%, and 16.8%, respectively, indirectly indicating propagation of H1N1 through schools. Pandemic control should involve school-targeted strategies.     

Interim report on the A/H1N1 influenza virus pandemic in Marseille,France, April-November 2009

We report here the results of a 7-month survey of the influenza A/H1N1 pandemic in the Virology laboratory of the public hospitals of Marseille (April–November 2009). In total, 8 587 samples were analysed during this period, of which 1 974 (23%) were positive for the novel influenza variant. The analysis of results obtained using rapid influenza diagnostic tests (RIDTs) revealed a global sensitivity of 49.4% (vs. molecular qRT-PCR detection), strongly correlated with age groups (varying from 30% to 58% for patients >40 age and <10, respectively), indicating that RIDTs can be helpful in accelerating the management of suspected cases. Epidemiological analysis showed that the winter influenza wave began in October in Marseille (i.e. 2 to 3 months earlier than usual seasonal influenza outbreaks) and that the majority of autochthonous cases were observed in patients younger than 20 years old, with a low number of cases in patients over 60 years old. In November 2009, 22.2% (167/754) of patients with a laboratory diagnosis of influenza A/H1N1 infection were hospitalized, of which 9% (15/167) were admitted to an intensive care unit (ICU). Patients in the extreme age groups (>40 years old and <1) were significantly more often hospitalized than others, and 2.4% of hospitalized patients died. During the last 3 weeks of the period, the average number of bed-days attributable to H1N1sw-positive patients was 31.4, of which 5.9 were in ICUs.     

中国甲型H1N1流感疫苗质量分析

目的 通过对甲型H1N1流感疫苗批签发中的检测数据进行分析和比较,了解我国甲型H1N1流感疫苗的总体质量状况.方法 按照各企业注册标准对甲型H1N1流感疫苗进行资料审查和全项检定,对关键项目检测结果进行分析和比较.结果 甲型H1N1流感疫苗批签发总体合格率为99.8%,有效成分血凝素含量在标示量的90%～103%范围内,甲醛、卵清蛋白和内毒素含量等安全性指标均符合规定.结论 我国甲型H1N1流感疫苗各项检测总体情况良好,能充分保证疫苗的安全性和有效性,中国食品药品检定研究院的独立检验和批签发对保证上市疫苗的质量发挥了重要作用.

Abstract：

Objective To analyze the laboratory testing data of 2009 pandemic influenza A (H1N1) vaccines during lot release procedure, thus to know the overall quality status of this vaccines.Methods National Institutes for Food and Drug Control(NIFDC) carried out the laboratory test according to the specifications of each manufacture, and the results was analyzed and compared between manufacturer and NIFDC. Results 99.8% of vaccines batches were released by NIFDC, haemagglutinin contents were between 90% to 103 % of labeled values, and testing results slightly differ between manufactures and NIFDC,other items related to safety were all meet specifications. Conclusion The quality of H1N1 vaccines in China were satisfying, the lot release and independent test by NIFDC play important roles to ensure the vaccines' quality.     

Fatal cases of 2009 pandemic influenza A (H1N1) in Korea

The aim of this study was to describe the features of deaths associated with the 2009 pandemic influenza A (H1N1) by 26 November 2009 in Korea. We collected standardized case reports on 115 confirmed deaths through a nationwide enhanced influenza surveillance system. The median age was 61 yr (interquartile range [IQR], 0.2-97 yr) and 58 (50.4%) were females. The case fatality rate was estimated as 16 per 100,000 cases. The age-related mortality rate had a J-shaped curve. Eighty-three patients (72.2%) had at least 1 underlying medical disease. Bacterial co-infections were detected in the blood or sputum specimens from 34 patients. Of the 63 patients who were hospitalized in the intensive care unit (ICU), the median time from symptom onset to hospital admission was 2 days (IQR, 0-22 days), and the median time from hospitalization to ICU admission was 1 day (IQR, 0-17 days). Neuraminidase inhibitors were administered to 100 patients (87.0%), 36% of whom began treatment within 2 days. In conclusion, fatal cases from the 2009 influenza A (H1N1) infection in Korea are mainly aged individuals with underlying disease, and associated with pneumonia, bacterial co-infections, and multi-organ failure.     

Lymphocytopenia as a marker for pandemic influenza A/H1N1 2009 virus infection in children

Lymphocytopenia has been reported in adults with pandemic influenza A/H1N1 2009 infection, but data in children are inconclusive. Data from 76 children presented with flu‐like symptoms between July and November 2009 and tested for pandemic influenza A/H1N1 2009 virus and white blood cell (WBC) counts were analyzed. Samples from 37 (48.7%) children resulted in a positive PCR assay for pandemic influenza A/H1N1 2009 virus. When comparing data from these children with data from 39 (51.3%) children with uncomplicated flu‐like illness and negative PCR assay for pandemic influenza A/H1N1 2009 virus, no difference in disease duration, median age, red blood cell count, hemoglobin concentration, C reactive protein concentration, and absolute neutrophil count was observed, whereas significant differences were apparent when considering WBC count, relative and absolute lymphocyte count, absolute lymphocyte count z‐score, and platelet count. Receiver operating characteristic curve analysis revealed that the best absolute lymphocyte count and absolute lymphocyte count z‐score cut‐points that simultaneously maximized sensitivity and specificity were 2,256 cells/µl and ?0.89, respectively, sensitivity being 0.81 (95% CI: 0.68–0.94), specificity 0.87 (95% CI: 0.77–0.98), positive predictive value 0.85 (95% CI: 0.74–0.97), and negative predictive value 0.83 (95% CI: 0.71–0.94). In conclusion, lymphocytopenia is a marker for influenza A/H1N1 2009 virus infection in children. Absolute lymphocyte count <2,556 cells/µl or absolute lymphocyte count z‐score < ?0.89 may be useful cut‐offs to discriminate against children at higher risk of infection during epidemics. Considering that the pandemic virus is highly likely to continue to circulate in the coming winter season, these findings provide direct and practical implications for the near future. J. Med. Virol. 83:1–4, 2011. © 2010 Wiley‐Liss, Inc.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.