Enhanced mammalian transmissibility of seasonal influenza A/H1N1 viruses encoding an oseltamivir-resistant neuraminidase

Between 2007 and 2009, oseltamivir resistance developed among seasonal influenza A/H1N1 (sH1N1) virus isolates at an exponential rate, without a corresponding increase in oseltamivir usage. We hypothesized that the oseltamivir-resistant neuraminidase (NA), in addition to being relatively insusceptible to the antiviral effect of oseltamivir, might confer an additional fitness advantage on these viruses by enhancing their transmission efficiency among humans. Here we demonstrate that an oseltamivir-resistant clinical isolate, an A/Brisbane/59/2007(H1N1)-like virus isolated in New York State in 2008, transmits more efficiently among guinea pigs than does a highly similar, contemporaneous oseltamivir-sensitive isolate. With reverse genetics reassortants and point mutants of the two clinical isolates, we further show that expression of the oseltamivir-resistant NA in the context of viral proteins from the oseltamivir-sensitive virus (a 7:1 reassortant) is sufficient to enhance transmissibility. In the guinea pig model, the NA is the critical determinant of transmission efficiency between oseltamivir-sensitive and -resistant Brisbane/59-like sH1N1 viruses, independent of concurrent drift mutations that occurred in other gene products. Our data suggest that the oseltamivir-resistant NA (specifically, one or both of the companion mutations, H275Y and D354G) may have allowed resistant Brisbane/59-like viruses to outtransmit sensitive isolates. These data provide in vivo evidence of an evolutionary mechanism that would explain the rapidity with which oseltamivir resistance achieved fixation among sH1N1 isolates in the human reservoir.     

Reassortment between seasonal H1N1 and pandemic (H1N1) 2009 influenza viruses is restricted by limited compatibility among polymerase subunits

Reassortment is important for influenza virus evolution and the generation of novel viruses with pandemic potential; however, the factors influencing reassortment are still poorly understood. Here, using reverse genetics and a replicon assay, we demonstrated that a mixed polymerase complex containing a pandemic (H1N1) 2009 influenza virus PB2 on a seasonal H1N1 virus background has reduced polymerase activity, leading to impaired virus viability. Adaptation of viruses containing the mixed polymerase complex resulted in compensatory mutations in PB1. Taken together, our results identify the cooperation between PB2 and PB1 as an important restricting factor for reassortment of influenza viruses.     

Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.     

Assessing the in vitro fitness of an oseltamivir-resistant seasonal A/H1N1 influenza strain using a mathematical model

In 2007, the A/Brisbane/59/2007 (H1N1) seasonal influenza virus strain acquired the oseltamivir-resistance mutation H275Y in its neuraminidase (NA) gene. Although previous studies had demonstrated that this mutation impaired the replication capacity of the influenza virus in vitro and in vivo, the A/Brisbane/59/2007 H275Y oseltamivir-resistant mutant completely out-competed the wild-type (WT) strain and was, in the 2008-2009 influenza season, the primary A/H1N1 circulating strain. Using a combination of plaque and viral yield assays, and a simple mathematical model, approximate values were extracted for two basic viral kinetics parameters of the in vitro infection. In the ST6GalI-MDCK cell line, the latent infection period (i.e., the time for a newly infected cell to start releasing virions) was found to be 1-3 h for the WT strain and more than 7 h for the H275Y mutant. The infecting time (i.e., the time for a single infectious cell to cause the infection of another one) was between 30 and 80 min for the WT, and less than 5 min for the H275Y mutant. Single-cycle viral yield experiments have provided qualitative confirmation of these findings. These results, though preliminary, suggest that the increased fitness success of the A/Brisbane/59/2007 H275Y mutant may be due to increased infectivity compensating for an impaired or delayed viral release, and are consistent with recent evidence for the mechanistic origins of fitness reduction and recovery in NA expression. The method applied here can reconcile seemingly contradictory results from the plaque and yield assays as two complementary views of replication kinetics, with both required to fully capture a strain's fitness.     

Amino acid changes in hemagglutinin contribute to the replication of oseltamivir-resistant H1N1 influenza viruses

Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008.     

Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses

The highly pathogenic avian influenza (HPAI) H5N1 virus lineage has undergone extensive genetic reassortment with viruses from different sources to produce numerous H5N1 genotypes, and also developed into multiple genetically distinct sublineages in China. From there, the virus has spread to over 60 countries. The ecological success of this virus in diverse species of both poultry and wild birds with frequent introduction to humans suggests that it is a likely source of the next human pandemic. Therefore, the evolutionary and ecological characteristics of its emergence from wild birds into poultry are of considerable interest. Here, we apply the latest analytical techniques to infer the early evolutionary dynamics of H5N1 virus in the population from which it emerged (wild birds and domestic poultry). By estimating the time of most recent common ancestors of each gene segment, we show that the H5N1 prototype virus was likely introduced from wild birds into poultry as a non-reassortant low pathogenic avian influenza H5N1 virus and was not generated by reassortment in poultry. In contrast, more recent H5N1 genotypes were generated locally in aquatic poultry after the prototype virus (A/goose/Guangdong/1/96) introduction occurred, i.e., they were not a result of additional emergence from wild birds. We show that the H5N1 virus was introduced into Indonesia and Vietnam 3-6 months prior to detection of the first outbreaks in those countries. Population dynamics analyses revealed a rapid increase in the genetic diversity of A/goose/Guangdong/1/96 lineage viruses from mid-1999 to early 2000. Our results suggest that the transmission of reassortant viruses through the mixed poultry population in farms and markets in China has selected HPAI H5N1 viruses that are well adapted to multiple hosts and reduced the interspecies transmission barrier of those viruses.     

Molecular character of influenza A/H1N1 2009: Implications for spread and control

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior “seasonal” H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 “swine flu” vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

甲型H1N1流感病毒在季流病毒、禽流感病毒共感染时变异可能性的验证

目的 甲型H1N1流感病毒A/California/7/2009分别与A/Brisbane/10/07和A/ShenZhen/406H/06共感染小型香猪,预测甲流病毒在与季流H3N2病毒/甲流病毒与禽流感病毒共感染时是否会发生变异.方法 分别将A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)对5～6月龄小型猪共感染,小型猪经复方氯胺酮0.1 mL/kg麻醉后进行滴鼻感染,感染后第5天安乐死动物,取动物肺组织作病毒测序分析.结果 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2)共感染后,A/California/7/2009病毒PB1基因993位G→A突变,PA基因1659位G→A突变,没有氨基酸的变异.A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后A/California/7/2009病毒PB2基因1711位T→C突变.碱基的突变未引起氨基酸的变异.结论 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后在猪的体内没有发生病毒重组、变异.     

Sero-prevalence and incidence of A/H1N1 2009 influenza infection in Scotland in winter 2009-2010

Background

Sero-prevalence is a valuable indicator of prevalence and incidence of A/H1N1 2009 infection. However, raw sero-prevalence data must be corrected for background levels of cross-reactivity (i.e. imperfect test specificity) and the effects of immunisation programmes.

Methods and Findings

We obtained serum samples from a representative sample of 1563 adults resident in Scotland between late October 2009 and April 2010. Based on a microneutralisation assay, we estimate that 44% (95% confidence intervals (CIs): 40–47%) of the adult population of Scotland were sero-positive for A/H1N1 2009 influenza by 1 March 2010. Correcting for background cross-reactivity and for recorded vaccination rates by time and age group, we estimated that 34% (27–42%) were naturally infected with A/H1N1 2009 by 1 March 2010. The central estimate increases to >40% if we allow for imperfect test sensitivity. Over half of these infections are estimated to have occurred during the study period and the incidence of infection in late October 2009 was estimated at 4.3 new infections per 1000 people per day (1.2 to 7.2), falling close to zero by April 2010. The central estimate increases to over 5.0 per 1000 if we allow for imperfect test specificity. The rate of infection was higher for younger adults than older adults. Raw sero-prevalences were significantly higher in more deprived areas (likelihood ratio trend statistic = 4.92,1 df, P = 0.03) but there was no evidence of any difference in vaccination rates.

Conclusions

We estimate that almost half the adult population of Scotland were sero-positive for A/H1N1 2009 influenza by early 2010 and that the majority of these individuals (except in the oldest age classes) sero-converted as a result of natural infection with A/H1N1 2009. Public health planning should consider the possibility of higher rates of infection with A/H1N1 2009 influenza in more deprived areas.     

Age distribution of cases of 2009 (H1N1) pandemic influenza in comparison with seasonal influenza

Introduction

Several aspects of the epidemiology of 2009 (H1N1) pandemic influenza have not been accurately determined. We sought to study whether the age distribution of cases differs in comparison with seasonal influenza.

Methods

We searched for official, publicly available data through the internet from different countries worldwide on the age distribution of cases of influenza during the 2009 (H1N1) pandemic influenza period and most recent seasonal influenza periods. Data had to be recorded through the same surveillance system for both compared periods.

Results

For 2009 pandemic influenza versus recent influenza seasons, in USA, visits for influenza-like illness to sentinel providers were more likely to involve the age groups of 5–24, 25–64 and 0–4 years compared with the reference group of >64 years [odds ratio (OR) (95% confidence interval (CI)): 2.43 (2.39–2.47), 1.66 (1.64–1.69), and 1.51 (1.48–1.54), respectively]. Pediatric deaths were less likely in the age groups of 2–4 and <2 years than the reference group of 5–17 years [OR (95% CI): 0.46 (0.25–0.85) and 0.49 (0.30–0.81), respectively]. In Australia, notifications for laboratory-confirmed influenza were more likely in the age groups of 10–19, 5–9, 20–44, 45–64 and 0–4 years than the reference group of >65 years [OR (95% CI): 7.19 (6.67–7.75), 5.33 (4.90–5.79), 5.04 (4.70–5.41), 3.12 (2.89–3.36) and 1.89 (1.75–2.05), respectively]. In New Zealand, consultations for influenza-like illness by sentinel providers were more likely in the age groups of <1, 1–4, 35–49, 5–19, 20–34 and 50–64 years than the reference group of >65 years [OR (95% CI): 2.38 (1.74–3.26), 1.99 (1.62–2.45), 1.57 (1.30–1.89), 1.57 (1.30–1.88), 1.40 (1.17–1.69) and 1.39 (1.14–1.70), respectively].

Conclusions

The greatest increase in influenza cases during 2009 (H1N1) pandemic influenza period, in comparison with most recent seasonal influenza periods, was seen for school-aged children, adolescents, and younger adults.     

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.     

Exhaled aerosol transmission of pandemic and seasonal H1N1 influenza viruses in the ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected 'donor' ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.     

Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 μM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 μM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.     

Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009

The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American αH1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the γH1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.     

Age-matched comparison of children hospitalized for 2009 pandemic H1N1 influenza with those hospitalized for seasonal H1N1 and H3N2

BACKGROUND: A wide spectrum of clinical manifestation ranging from deaths to a mild course of disease has been reported in children infected with the 2009 pandemic H1N1 (pH1N1) influenza. METHODOLOGY/MAJOR FINDINGS: We conducted an age-matched control study comparing children hospitalized for pH1N1 with historic controls infected with seasonal H1N1 and H3N2 influenza to correct for the effect of age on disease susceptibility and clinical manifestations. We also compared children with pH1N1 to children concurrently admitted for seasonal influenza during the pandemic period to adjust for differences in health-seeking behavior during the pandemic or other potential bias associated with historic controls. There was no death or intensive care admission. Children with pH1N1 were more likely to have at least one risk condition for influenza, an underlying chronic pulmonary condition, more likely to have asthma exacerbation and to be treated with oseltamivir. There was no difference in other aspects of the clinical course or outcome. CONCLUSION: Disease manifestation of children hospitalized for pH1N1 infection was mild in our patient population.     

Reassortment Networks and the evolution of pandemic H1N1 swine-origin influenza

Prior research developed Reassortment Networks to reconstruct the evolution of segmented viruses under both reassortment and mutation. We report their application to the swine-origin pandemic H1N1 virus (S-OIV). A database of all influenza A viruses, for which complete genome sequences were available in Genbank by October 2009, was created and dynamic programming was used to compute distances between all corresponding segments. A reassortment network was created to obtain the minimum cost evolutionary paths from all viruses to the exemplar S-OIV A/California/04/2009. This analysis took 35 hours on the Cray Extreme Multithreading (XMT) supercomputer, which has special hardware to permit efficient parallelization. Six specific H1N1/H1N2 bottleneck viruses were identified that almost always lie on minimum cost paths to S-OIV. We conjecture that these viruses are crucial to S-OIV evolution and worthy of careful study from a molecular biology viewpoint. In phylogenetics, ancestors are typically medians that have no functional constraints. In our method, ancestors are not inferred, but rather chosen from previously observed viruses along a path of mutation and reassortment leading to the target virus. This specificity and functional constraint render our results actionable for further experiments in vitro and in vivo.