Effect of D222G mutation in the hemagglutinin protein on receptor binding, pathogenesis and transmissibility of the 2009 pandemic H1N1 influenza virus

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like (α2-6 sialylated glycan) receptors and an increase in the binding to avian-like (α2-3 sialylated glycan) receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Sialic acid recognition of the pandemic influenza 2009 H1N1 virus: binding mechanism between human receptor and influenza hemagglutinin

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.     

The seroprevalence of pandemic influenza H1N1 (2009) virus in China

Background

Mainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.

Methodology/Principal Findings

Stored serum samples (n = 2,379) collected during 2006-2008 were used to estimate baseline serum reactogenicity to pH1N1. In January 2010, we used a multistage-stratified random sampling method to select 50,111 subjects who met eligibility criteria and collected serum samples and administered a standardized questionnaire. Antibody response to pH1N1 was measured using haemagglutination inhibition (HI) assay and the weighted seroprevalence was calculated using the Taylor series linearization method. Multivariable logistic regression analyses were used to examine risk factors for pH1N1 seropositivity. Baseline seroprevalence of pH1N1 antibody (HI titer ≥40) was 1.2%. The weighted seroprevalence of pH1N1 among the Chinese population was 21.5%(vaccinated: 62.0%; unvaccinated: 17.1%). Among unvaccinated participants, those aged 6-15 years (32.9%) and 16-24 years (30.3%) had higher seroprevalence compared with participants aged 25–59 years (10.7%) and ≥60 years (9.9%, P<0.0001). Children in kindergarten and students had higher odds of seropositivity than children in family care (OR: 1.36 and 2.05, respectively). We estimated that 207.7 million individuals (15.9%) experienced pH1N1 infection in China.

Conclusions/Significance

The Chinese population had low pre-existing immunity to pH1N1 and experienced a relatively high attack rate in 2009 of this virus. We recommend routine control measures such as vaccination to reduce transmission and spread of seasonal and pandemic influenza viruses.     

Molecular mechanism of the enhanced virulence of 2009 pandemic Influenza A (H1N1) virus from D222G mutation in the hemagglutinin: a molecular modeling study

D222G mutation of the hemagglutinin (HA) is of special interest because of its close association with the enhanced virulence of 2009 pandemic influenza A (H1N1) virus through the increased binding affinity to α2,3-linked sialylated glycan receptors. However, there is still a lack of detailed understanding about the molecular mechanism of this enhanced virulence. Here, molecular dynamics simulation and binding free energy calculation were performed to explore the altered glycan receptor binding mechanism of HA upon the D222G mutation by studying the interaction of one α2,3-linked sialylglycan (sequence: SIA-GAL-NAG) with the wild type and D222G mutated HA. The binding free energy calculation based on the molecular mechanics generalized Born surface area (MM-GBSA) method indicates that the D222G mutated HA has a much stronger binding affinity with the studied α2,3-linked glycan than the wild type. This is consistent with the experimental result. The increased binding free energy of D222G mutant mainly comes from the increased energy contribution of Gln223. The structural analysis proves that the altered electrostatic potential of receptor binding domain (RBD) and the increased flexibility of 220-loop are the essential reasons leading to the increased affinity of HA to α2,3-linked sialic acid glycans. The obtained results of this study have allowed a deeper understanding of the receptor recognition mechanism and the pathogenicity of influenza virus, which will be valuable to the structure-based inhibitors design targeting influenza virus entry process.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Comparative analysis of hemagglutinin of 2009 H1N1 influenza A pandemic indicates its evolution to 1918 H1N1 pandemic

To gain insight into the possible origin of the hemagglutinin of 2009 outbreak, we performed its comparative analysis with hemagglutinin of influenza viral strains from 2005 to 2008 and the past pandemics of 1977, 1968, 1957 and 1918. This insilico analysis showed a maximum sequence similarity between 2009 and 1918 pandemics. Primary structure analysis, antigenic and glycosylation site analyses revealed that this protein has evolved from 1918 pandemic. Phylogenetic analysis of HA amino acid sequence of 2009 influenza A(H1N1) viruses indicated that this virus possesses a distinctive evolutionary trait with 1918 influenza A virus. Although the disordered sequences are different among all the isolates, the disordered positions and sequences between 2009 and 1918 isolates show a greater similarity. Thus these analyses contribute to the evidence of the evolution of 2009 pandemic from 1918 influenza pandemic. This is the first computational evolutionary analysis of HA protein of 2009 H1N1 pandemic.     

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.     

Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.     

Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.     

Prevalence of 2009 pandemic influenza A (H1N1) virus antibodies, Tampa Bay Florida--November-December, 2009

Background

In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.

Methods

During November-December 2009, we collected leftover serum from a blood bank, a pediatric children''s hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.

Results

During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.

Conclusions

After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults.     

Genetical features of influenza virus A (H1N1) strain that caused the 2009 pandemic

Genetical features of the A(H1N1) influenza virus strain that caused the 2009 pandemic are analyzed in the review. Mutations typical for this strain, unique and similar to influenza viruses of swine, avian and seasonal types, and phenotypic (pathologic) features associated with them, that are experimentally confirmed, are described. A possibility of reassortation of avian and swine influenza viruses and possible epidemiologic consequences are discussed.     

Persistence of the 2009 pandemic influenza A (H1N1) virus in water and on non-porous surface

Knowledge of influenza A virus survival in different environmental conditions is a key element for the implementation of hygiene and personal protection measures by health authorities. As it is dependent on virus isolates even within the same subtype, we studied the survival of the 2009 H1N1 pandemic (H1N1pdm) virus in water and on non-porous surface. The H1N1pdm virus was subjected to various environmental parameters over time and tested for infectivity. In water, at low and medium salinity levels and 4°C, virus survived at least 200 days. Increasing temperature and salinity had a strong negative effect on the survival of the virus which remained infectious no more than 1 day at 35°C and 270 parts per thousand (ppt) of salt. Based on modeled data, the H1N1pdm virus retained its infectivity on smooth non-porous surface for at least 7 days at 35°C and up to 66 days at 4°C. The H1N1pdm virus has thus the ability to persist in water and on glass surface for extended periods of time, even at 35°C. Additional experiments suggest that external viral structures in direct contact with the environment are mostly involved in loss of virus infectivity.     

Extension and verification of the SEIR model on the 2009 influenza A (H1N1) pandemic in Japan

In order to understand the evolution of the 2009 influenza A (H1N1) pandemic within local regions of Japan, we studied the significance of regional migration between these regions. For this purpose, we have employed an extended SEIR model to describe the immigration of infected people and the stochastic variation of the infectious efficiency. We then applied a data assimilation technique in order to study how the agreement of the simulation results with the observed data depends on the presence/absence of immigration and the degree of variation of the infectious efficiency. Reproducibility is evaluated by log-likelihood values. The log-likelihood does not indicate the significance of immigration. Although there are multiple waves in the time course of the number of reported infected individuals, these waves could be explained by the stochastic nature of infectious events.     

Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic

Background

Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges.

Methodology

A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations.

Results and Conclusions

Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.