Asparagine Substitution at PB2 Residue 701 Enhances the Replication,Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus

The 2009/2010 pandemic influenza virus (H1N1pdm) contains an avian-lineage PB2 gene that lacks E627K and D701N substitutions important in the pathogenesis and transmission of avian-origin viruses in humans or other mammals. Previous studies have shown that PB2-627K is not necessary because of a compensatory Q591R substitution. The role that PB2-701N plays in the H1N1pdm phenotype is not well understood. Therefore, PB2-D701N was introduced into an H1N1pdm virus (A/New York/1682/2009 (NY1682)) and analyzed in vitro and in vivo. Mini-genome replication assay, in vitro replication characteristics in cell lines, and analysis in the mouse and ferret models demonstrated that PB2-D701N increased virus replication rates and resulted in more severe pathogenicity in mice and more efficient transmission in ferrets. In addition, compared to the NY1682-WT virus, the NY1682-D701N mutant virus induced less IFN-λ and replicated to a higher titer in primary human alveolar epithelial cells. These findings suggest that the acquisition of the PB2-701N substitution by H1N1pdm viruses may result in more severe disease or increase transmission in humans.     

Predicting the Antigenic Structure of the Pandemic (H1N1) 2009 Influenza Virus Hemagglutinin

The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.     

利用靶定基质蛋白基因m的小干扰RNA抑制2009甲型H1N1流感病毒的复制

目的:设计、构建并筛选针对流感病毒基质蛋白基因m的小干扰RNA(siRNA),检测其对2009甲型H1N1流感病毒复制的抑制效果。方法:设计3条针对流感病毒m基因的siRNA,并克隆到短发夹型(shRNA)siRNA表达载体pSilencer2.1-U6-hygro上;经测序证明构建成功后,将表达3种siRNA的质粒和阴性对照质粒psiRNA-control分别转染MDCK细胞,用潮霉素筛选稳定表达细胞株,用H1N1流感病毒感染细胞,通过Real-time PCR和Western印迹检测干扰效果。结果:构建了3个针对流感病毒m基因的siRNA表达质粒,3种siRNA均使m基因的mRNA水平降低,其中M1-306的抑制效率达60%;3种siRNA均使流感病毒NA蛋白的表达量降低,M2-25、M1-105的抑制效率明显,M1-306略低。结论:针对m基因的siRNA可以有效抑制流感病毒在MDCK细胞中的复制。     

Genomic Signature and Mutation Trend Analysis of Pandemic (H1N1) 2009 Influenza A Virus

A novel swine-origin pandemic influenza A(H1N1) virus (H1N1pdm, also referred to as S-OIV) was identified as the causative agent of the 21^st century''s first influenza pandemic, but molecular features conferring its ability of human-to-human transmission has not been identified. Here we compared the protein sequences of 2009 H1N1pdm strains with those causing other pandemics and the viruses isolated from humans, swines and avians, and then analyzed the mutation trend of the residues at the signature and non-signature positions, which are species- and non-species-associated, respectively, in the proteins of H1N1pdm during the pandemic of 2009. We confirmed that the host-specific genomic signatures of 2009 H1N1pdm, which are mainly swine-like, were highly identical to those of the 1918 H1N1pdm. During the short period of time when the pandemic alert level was raised from phase 4 to phase 6, one signature residue at the position of NP-100 mutated from valine to isoleucine. Four non-signature residues, at positions NA-91, NA-233, HA-206, and NS1-123, also changed during the epidemic in 2009. All these mutant residues, except that at NA-91, are located in the viral functional domains, suggesting that they may play roles in the human adaption and virulence of 2009 H1N1pdm.     

Transmission of a 2009 H1N1 Pandemic Influenza Virus Occurs before Fever Is Detected, in the Ferret Model

During the early phase of the 2009 influenza pandemic, attempts were made to contain the spread of the virus. Success of reactive control measures may be compromised if the proportion of transmission that occurs before overt clinical symptoms develop is high. In this study we investigated the timing of transmission of an early prototypic strain of pandemic H1N1 2009 influenza virus in the ferret model. Ferrets are the only animal model in which this can be assessed because they display typical influenza-like clinical signs including fever and sneezing after infection. We assessed transmission from infected animals to sentinels that were placed either in direct contact or in adjacent cages, the latter reflecting the respiratory droplet (RD) transmission route. We found that pre-symptomatic influenza transmission occurred via both contact and respiratory droplet exposure before the earliest clinical sign, fever, developed. Three of 3 animals exposed in direct contact between day 1 and 2 after infection of the donor animals became infected, and 2/3 of the animals exposed at this time period by the RD route acquired the infection, with the third animal becoming seropositive indicating either a low level infection or significant exposure. Moreover, this efficient transmission did not temporally correlate with respiratory symptoms, such as coughs and sneezes, but rather with the peak viral titre in the nose. Indeed respiratory droplet transmission did not occur late in infection, even though this was when sneezing and coughing were most apparent. None of the 3 animals exposed at this time by the RD route became infected and these animals remained seronegative at the end of the experiment. These data have important implications for pandemic planning strategies and suggest that successful containment is highly unlikely for a human-adapted influenza virus that transmits efficiently within a population.     

Absence of 2009 Pandemic H1N1 Influenza A Virus in Fresh Pork

The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.     

Genetic Characterization of the Influenza A Pandemic (H1N1) 2009 Virus Isolates from India

Background

The Influenza A pandemic H1N1 2009 (H1N1pdm) virus appeared in India in May 2009 and thereafter outbreaks with considerable morbidity and mortality have been reported from many parts of the country. Continuous monitoring of the genetic makeup of the virus is essential to understand its evolution within the country in relation to global diversification and to track the mutations that may affect the behavior of the virus.

Methods

H1N1pdm viruses were isolated from both recovered and fatal cases representing major cities and sequenced. Phylogenetic analyses of six concatenated whole genomes and the hemagglutinin (HA) gene of seven more isolates from May-September 2009 was performed with reference to 685 whole genomes of global isolates available as of November 24, 2009. Molecular characterization of all the 8 segments was carried out for known pathogenic markers.

Results

The first isolate of May 2009 belonged to clade 5. Although clade 7 was the dominant H1N1pdm lineage in India, both clades 6 and 7 were found to be co-circulating. The neuraminidase of all the Indian isolates possessed H275, the marker for sensitivity to the neuraminidase inhibitor Oseltamivir. Some of the mutations in HA are at or in the vicinity of antigenic sites and may therefore be of possible antigenic significance. Among these a D222G mutation in the HA receptor binding domain was found in two of the eight Indian isolates obtained from fatal cases.

Conclusions

The majority of the 13 Indian isolates grouped in the globally most widely circulating H1N1pdm clade 7. Further, correlations of the mutations specific to clade 7 Indian isolates to viral fitness and adaptability in the country remains to be understood. The D222G mutation in HA from isolates of fatal cases needs to be studied for pathogenicity.     

Factors Associated with Household Transmission of Pandemic (H1N1) 2009 among Self-Quarantined Patients in Beijing,China

As the pandemic (H1N1) 2009 progressed, the Ministry of Health of China advised cases with mild symptoms to remain home for isolation and observation, which may have increased the risk for infection among other household members. Describing the transmission characteristics of this novel virus is indispensable to effectively controlling the spread of disease; thus, the aim of this study was to assess risk factors associated with household transmission of pandemic H1N1 from self-quarantined patients in Beijing, the capital city of China. A 1:2 case-control study with 54 case households and 108 control households was conducted between August 1 and September 30, 2009 in Beijing. Cases were households with a self-quarantined index patient and a secondary case, while controls were households with a self-quarantined index patient and a close contact. Controls were also matched to cases for sex and age of index case-patient. A structured interview guide was used to collect the data. Conditional logistical models were employed to estimate Odds Ratios (OR) with 95% confidence intervals (95% CI). Results indicated that higher education level (OR 0.42; 95% CI 0.22-0.83), sharing room with an index case-patient (OR 3.29; 95%CI 1.23-8.78), daily room ventilation (OR 0.28; 95%CI 0.08-0.93), and hand washing ≥3/d (OR 0.71; 95%CI 0.48-0.94) were related to the household transmission of pandemic H1N1 from self-quarantined patients. These results highlight that health education, as well as the quarantine of the index case-patient immediately after infection, frequent hand hygiene, and ventilation are critical to mitigating household spread of pandemic H1N1 virus and minimizing its impact. Household contacts should be educated to promote these in-home practices to contain transmission, particularly when household members are quarantined at home.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic

The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.The recent emergence of swine-origin H1N1 influenza A virus (pandemic H1N1/09) in humans has heightened awareness of how the burden of morbidity and mortality due to influenza is associated with the appearance of new genetic variants (5) and of the genetic and epidemiological determinants of viral transmission (8). The emergence of pandemic H1N1/09 is also unprecedented in recorded history as it means that three antigenically distinct lineages of influenza A virus—pandemic H1N1/09 and the seasonal H1N1 and H3N2 viruses— currently cocirculate within human populations.Although the presence of multiple subtypes of influenza A virus may place an additional burden on public health resources, it also provides a unique opportunity to compare the patterns and dynamics of evolution in these viruses on a similar time scale. Indeed, one of the most interesting secondary effects of the current H1N1/09 pandemic has been an increased vigilance for cases of influenza-like illness and hence an intensified sampling of seasonal H1N1 and H3N2 viruses during the typical influenza “off-season” (i.e., spring-summer) in the northern hemisphere. Because the influenza season in the northern hemisphere generally runs from November through March, with a usual peak in January or February, influenza viruses sampled outside of this period are of special interest.The current model for the global spatiotemporal dynamics of influenza A virus is that the northern and southern hemispheres represent ecological “sinks” for this virus, with little ongoing viral transmission during the summer months (9). In contrast, more continual viral transmission occurs within the tropical “source” population (13) that is most likely centered on an intense transmission network in east and southeast Asia (10). However, the precise epidemiological and evolutionary reasons for this major geographic division, and for the seasonality of influenza A virus in general, remain uncertain (1, 4). Evidence for this “sink-source” ecological model is that viruses sampled from successive seasons in localities such as New York State do not usually form linked clusters on phylogenetic trees, indicating that they are not connected by direct transmission through the summer months (7). Similar conclusions can be drawn for the United States as a whole and point to multiple introductions of phylogenetically distinct lineages during the winter (6), followed by complex patterns of spatial diffusion (14). However, despite the growing epidemiological and phylogenetic data supporting this model, it is also evident that there is relatively little sequence data from seasonal influenza viruses that are sampled from April to October in the northern hemisphere. Hence, it is uncertain whether extended chains of transmission can occur during this time period, even though this may have an important bearing on our understanding of influenza seasonality.To address these issues, we examined the evolutionary behavior of seasonal H1N1 and H3N2 viruses as they cocirculated during a single time period—(late) April to June 2009—within a single locality (New York State). Not only are levels of influenza virus transmission in the northern hemisphere usually very low during this time period, but in this particular season the human host population was also experiencing the emerging epidemic of pandemic H1N1/09.     

Pandemic (H1N1) 2009 Influenza Community Transmission Was Established in One Australian State When the Virus Was First Identified in North America

Background

In mid-June 2009 the State of Victoria in Australia appeared to have the highest notification rate of pandemic (H1N1) 2009 influenza in the world. We hypothesise that this was because community transmission of pandemic influenza was already well established in Victoria at the time testing for the novel virus commenced. In contrast, this was not true for the pandemic in other parts of Australia, including Western Australia (WA).

Methods

We used data from detailed case follow-up of patients with confirmed infection in Victoria and WA to demonstrate the difference in the pandemic curve in two Australian states on opposite sides of the continent. We modelled the pandemic in both states, using a susceptible-infected-removed model with Bayesian inference accounting for imported cases.

Results

Epidemic transmission occurred earlier in Victoria and later in WA. Only 5% of the first 100 Victorian cases were not locally acquired and three of these were brothers in one family. By contrast, 53% of the first 102 cases in WA were associated with importation from Victoria. Using plausible model input data, estimation of the effective reproductive number for the Victorian epidemic required us to invoke an earlier date for commencement of transmission to explain the observed data. This was not required in modelling the epidemic in WA.

Conclusion

Strong circumstantial evidence, supported by modelling, suggests community transmission of pandemic influenza was well established in Victoria, but not in WA, at the time testing for the novel virus commenced in Australia. The virus is likely to have entered Victoria and already become established around the time it was first identified in the US and Mexico.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.     

Estimating the Disease Burden of Pandemic (H1N1) 2009 Virus Infection in Hunter New England,Northern New South Wales,Australia, 2009

Introduction

On May 26, 2009, the first confirmed case of Pandemic (H1N1) 2009 virus (pH1N1) infection in Hunter New England (HNE), New South Wales (NSW), Australia (population 866,000) was identified. We used local surveillance data to estimate pH1N1-associated disease burden during the first wave of pH1N1 circulation in HNE.

Methods

Surveillance was established during June 1-August 30, 2009, for: 1) laboratory detection of pH1N1 at HNE and NSW laboratories, 2) pH1N1 community influenza-like illness (ILI) using an internet survey of HNE residents, and 3) pH1N1-associated hospitalizations and deaths using respiratory illness International Classification of Diseases 10 codes at 35 HNE hospitals and mandatory reporting of confirmed pH1N1-associated hospitalizations and deaths to the public health service. The proportion of pH1N1 positive specimens was applied to estimates of ILI, hospitalizations, and deaths to estimate disease burden.

Results

Of 34,177 specimens tested at NSW laboratories, 4,094 (12%) were pH1N1 positive. Of 1,881 specimens from patients evaluated in emergency departments and/or hospitalized, 524 (26%) were pH1N1 positive. The estimated number of persons with pH1N1-associated ILI in the HNE region was 53,383 (range 37,828–70,597) suggesting a 6.2% attack rate (range 4.4–8.2%). An estimated 509 pH1N1-associated hospitalizations (range 388–630) occurred (reported: 184), and up to 10 pH1N1-associated deaths (range 8–13) occurred (reported: 5). The estimated case hospitalization ratio was 1% and case fatality ratio was 0.02%.

Discussion

The first wave of pH1N1 activity in HNE resulted in symptomatic infection in a small proportion of the population, and the number of HNE pH1N1-associated hospitalizations and deaths is likely higher than officially reported.