Pdm09 Hemagglutinin Research Articles

Comparing the transmission potential from sequence and surveillance data of 2009 North American influenza pandemic waves.

Technological advancements in phylodynamic modeling coupled with the accessibility of real-time pathogen genetic data are increasingly important for understanding the infectious disease transmission dynamics. In this study, we compare the transmission potentials of North American influenza A(H1N1)pdm09 derived from sequence data to that derived from surveillance data. The impact of the choice of tree-priors, informative epidemiological priors, and evolutionary parameters on the transmission potential estimation is evaluated. North American Influenza A(H1N1)pdm09 hemagglutinin (HA) gene sequences are analyzed using the coalescent and birth-death tree prior models to estimate the basic reproduction number (R 0 ). Epidemiological priors gathered from published literature are used to simulate the birth-death skyline models. Path-sampling marginal likelihood estimation is conducted to assess model fit. A bibliographic search to gather surveillance-based R 0 values were consistently lower (mean≤1.2) when estimated by coalescent models than by the birth-death models with informative priors on the duration of infectiousness (mean≥1.3 to ≤2.88 days). The user-defined informative priors for use in the birth-death model shift the directionality of epidemiological and evolutionary parameters compared to non-informative estimates. While there was no certain impact of clock rate and tree height on the R 0 estimation, an opposite relationship was observed between coalescent and birth-death tree priors. There was no significant difference (p=0.46) between the birth-death model and surveillance R 0 estimates. This study concludes that tree-prior methodological differences may have a substantial impact on the transmission potential estimation as well as the evolutionary parameters. The study also reports a consensus between the sequence-based R 0 estimation and surveillance-based R 0 estimates. Altogether, these outcomes shed light on the potential role of phylodynamic modeling to augment existing surveillance and epidemiological activities to better assess and respond to emerging infectious diseases.

A Comprehensive Molecular and Epidemiological Characterization of Influenza Viruses Circulating 2016–2020 in North Macedonia

Influenza viruses know no boundaries, representing an example of rapid virus evolution combined with pressure exerted by the host’s immune system. Seasonal influenza causes 4–50 million symptomatic cases in the EU/EEA each year, with a global death toll reaching 650,000 deaths. That being the case, in 2014 North Macedonia introduced the sentinel surveillance in addition to the existing influenza surveillance in order to obtain more precise data on the burden of disease, circulating viruses and to implement timely preventive measures. The aims of this study were to give a comprehensive virological and epidemiological overview of four influenza seasons (2016–2020), assess the frequency and distribution of influenza circulating in North Macedonia and to carry out molecular and phylogenetic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09, A(H3N2) from ILI and SARI patients. Our results showed that out of 1,632 tested samples, 46.4% were influenza positive, with influenza A(H1N1)pdm09 accounting for the majority of cases (44%), followed by influenza B (32%) and A(H3N2) (17%). By comparing the sentinel surveillance system to the routine surveillance system, we showed that the newly applied system works efficiently and gives great results in the selection of cases. Statistically significant differences (p = < 0.0000001) were observed when comparing the number of reported ILI cases among patients aged 0–4, 5–14, 15–29, and 30–64 years to the reference age group. The phylogenetic analysis of the HA sequences unveiled the resemblance of mutations circulating seasonally worldwide, with a vast majority of circulating viruses belonging to subclade 6B.1A. The PROVEAN analysis showed that the D187A substitution in the receptor binding site (RBS) of the A(H1N1)pdm09 HA has a deleterious effect on the its function. The A(H3N2) viruses fell into the 3C.2a and 3C.3a throughout the analyzed seasons. Molecular characterization revealed that various substitutions in the A(H3N2) viruses gradually replaced the parental variant in subsequent seasons before becoming the dominant variant. With the introduction of sentinel surveillance, accompanied by the advances made in whole-genome sequencing and vaccine therapeutics, public health officials can now modify their approach in disease management and intervene effectively and in a timely manner to prevent major morbidity and mortality from influenza.

Structure-Guided Creation of an Anti-HA Stalk Antibody F11 Derivative That Neutralizes Both F11-Sensitive and -Resistant Influenza A(H1N1)pdm09 Viruses.

The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational rearrangements of HA for virus infection. Here we characterized molecular interactions between the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand the structural basis of the actions and modifications of this antibody. In silico structural analyses using a model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structure-guided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for the F11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivative that can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered conformational rearrangements and suggest that in silico guiding experiments can be useful to create anti-HA stalk antibodies with new phenotypes.

Comparison of the Protective Efficacy of Neutralizing Epitopes of 2009 Pandemic H1N1 Influenza Hemagglutinin.

The 2009 H1N1 influenza (Pdm09) pandemic has been referred to as the first influenza pandemic of the twenty-first century. There is a marked difference in antigenicity between the pandemic H1N1 virus and past seasonal H1N1 viruses, which allowed the pandemic virus to spread rapidly in humans. Antibodies (Abs) against hemagglutinin (HA), especially neutralizing Abs against epitopes in the head of HA, play critical roles in defending the host against the virus. Some preexisting neutralizing Abs that recognize neutralizing epitopes of Pdm09 HA, thereby affording cross-protection, have been reported. To better understand the protective effects of epitopes in Pdm09 HA, we constructed a series of plasmid DNAs (DNA vaccines) by cloning various combinations of Pdm09 neutralizing epitopes into the HA backbone derived from A/PR/8/1934 (H1N1). We subsequently compared the protective immune responses induced by these various forms of HA in a mouse model. We found that the plasmid DNAs with epitope substitutions provided better protection against lethal virus challenge and induced higher strain-specific antibody titers, with epitope Sa being the most effective. Moreover, the combination of epitopes Sa and Sb provided almost complete protection in mice. These findings provide new insights into the protective efficacy of neutralizing epitopes of influenza HA.

Evaluation of potential immunogenicity differences between Pandemrix™ and Arepanrix™

ABSTRACTIn retrospective observational studies, an increased relative risk of incident narcolepsy was observed in some European countries among recipients of the AS03-adjuvanted, A(H1N1)pdm09, inactivated, detergent-split virion vaccine Pandemrix™ manufactured in Dresden, Germany (D-Pan H1N1). A similar increased risk was not observed in a retrospective population-based study in individuals in Quebec province, Canada, who received Aprepanrix™, a Quebec-manufactured AS03-adjuvanted A(H1N1)pdm09 inactivated, detergent-split virion vaccine (Q-Pan H1N1). Antibody responses in D-Pan versus Q-Pan vaccinees (adults/children) measured as hemagglutination inhibition (HI) titers 21 d post-vaccination were found to be equivalent (NCT01161160). The current post-hoc analysis was conducted to determine whether antibody avidity differed following immunization with the 2 vaccines. Using surface plasmon resonance, we evaluated the capacity of serum specimens (drawn from the comparative immunogenicity trial) from a subset of subjects aged 3–9 y who received either D-Pan or Q-Pan (N = 28/group), to bind to recombinant A(H1N1)pdm09 hemagglutinin. IgG antibodies were purified from Day 21 sera. Binding was assessed by end association level; dissociation by retention of antigen-antibody complexes at the end of the dissociation phase, and kd. Inter-run variability for the control monoclonal antibody, association levels and dissociation levels was low (CVs 1.3%, 7.8% and 1.4%, respectively); non-specific binding was negligible. High avidity and slow dissociation was observed for both groups (kd ≤ 10−4/s; geometric mean [IQR] association and dissociation levels for D-Pan/Q-Pan: 15.4 RU [13.4–17.7]/12.4 RU [10.8–14.3] and 94.5% [92.5–96.5]/95.5% [93.5–97.6], respectively). Association, but not dissociation levels correlated with HI titers. No significant differences in avidity parameters were observed between D-Pan and Q-Pan sera.

Phylogenetic analyses of influenza A (H1N1)pdm09 hemagglutinin gene during and after the pandemic event in Brazil

Pandemic influenza A H1N1 [A(H1N1)pdm09] was first detected in Brazil in May 2009, and spread extensively throughout the country causing a peak of infection during June to August 2009. Since then, it has continued to circulate with a seasonal pattern, causing high rates of morbidity and mortality. Over this period, the virus has continually evolved with the accumulation of new mutations. In this study we analyze the phylogenetic relationship in a collection of 220 A(H1N1)pdm09 hemagglutinin (HA) gene sequences collected during and after the pandemic period (2009 to 2014) in Brazil. In addition, we have looked for evidence of viral polymorphisms associated with severe disease and compared the range of viral variants with the vaccine strain (A/California/7/2009) used throughout this period.The phylogenetic analyses in this study revealed the circulation of at least eight genetic groups in Brazil. Two (G6-pdm and G7-pdm) co-circulated during the pandemic period, showing an early pattern of viral diversification with a low genetic distance from vaccine strain. Other phylogenetic groups, G5, G6 (including 6B, 6C and 6D subgroups), and G7 were found in the subsequent epidemic seasons from 2011 to 2014. These viruses exhibited more amino acid differences from the vaccine strain with several substitutions at the antigenic sites. This is associated with a theoretical decrease in the vaccine efficacy. Furthermore, we observed that the presence of any polymorphism at residue 222 of the HA gene was significantly associated with severe/fatal cases, reinforcing previous reports that described this residue as a potential virulence marker.This study provides new information about the circulation of some viral variants in Brazil, follows up potential genetic markers associated with virulence and allows infer if the efficacy of the current vaccine against more recent A(H1N1)pdm09 strains may be reduced.

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity.

Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.

Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface.

The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. Influenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.

3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus.

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4β→8)-epicatechin-3′-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.

Functional balance between the hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 HA D222 variants.

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/106) was higher than that of G222 (1.7 nmol/h/106 viruses) and E/N222 variants (4.4 nmol/h/106 viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity in intravenous immunoglobulin as a potential therapeutic against emerging influenza viruses.

Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations. We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC. ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG. IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines.

Genetic analysis of post-pandemic 2010-2011 influenza A(H1N1)pdm09 hemagglutinin virus variants that caused mild, severe, and fatal infections in Northern Greece.

Since its appearance, influenza A(H1N1)pdm09 caused considerable morbidity and mortality in Northern Greece. Genetic analysis of post-pandemic circulating strains scoped to investigate any correlation between genetic variations that emerged during viral evolution and severity of infection. Pharyngeal swabs/aspirates (n = 1,870) were examined with real-time reverse transcription-polymerase chain reaction. Hemagglutinin sequences were analyzed on 110 strains (37 fatal/73 non-fatal cases), followed by statistical and phylogenetic analysis. Influenza A(H1N1)pdm09 was detected in 848 samples. Coexistence of clusters 3, 4, 5, 6, and 7 indicated co-circulation of lineages in Northern Greece. Genetic analysis showed that HA sequences had 96-99% sequence similarity with the vaccine strain and that there was no association between any co-circulating lineage and severity. Several viruses accumulated variations in HA antigenic sites. D222G was significantly associated with fatal infections, supporting its association with increased viral pathogenesis. On the other hand, four variations were associated with milder disease outcomes. Certain signature amino acid changes persisted during and/or after the pandemic, indicating their offer of selective advantages to the virus. Negative selection was observed in 70% of pandemic variations as they probably did not contribute to the virus fitness. It is of interest that persistent variations were highly identified in the vicinity of antigenic or receptor-binding sites. Of those, K171R was associated only with fatal infections. Also of interest, only strains that were isolated from fatal infections had variations that altered both their acid-base and polarity properties. Genetic changes that may alter the antigenicity, pathogenicity and transmissibility of circulating virus variants need to be determined and closely monitored.

IgM, IgG, and IgA antibody responses to influenza A(H1N1)pdm09 hemagglutinin in infected persons during the first wave of the 2009 pandemic in the United States.

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.

A(H1N1)pdm09 hemagglutinin D222G and D222N variants are frequently harbored by patients requiring extracorporeal membrane oxygenation and advanced respiratory assistance for severe A(H1N1)pdm09 infection

In patients with A(H1N1)pdm09 infection, severe lung involvement requiring admission to intensive care units (ICU) has been reported. Mutations at the hemagglutinin (HA) receptor binding site (RBS) have been associated with increased virulence and disease severity, representing a potential marker of critical illness. To assess the contribution of HA-RBS variability in critically ill patients, A(H1N1)pdm09 virus from adult patients with severe infection admitted to ICU for extracorporeal membrane oxygenation support (ECMO) during influenza season 2009-2011 in Piemonte (4·2 million inhabitants), northwestern Italy, was studied. We retrospectively analyzed HA-RBS polymorphisms in ICU patients and compared with those from randomly selected inpatients with mild A(H1N1)pdm09 disease and outpatients with influenza from the local surveillance program. By HA-RBS direct sequencing of respiratory specimens, D222G and D222N viral variants were identified in a higher proportion in ICU patients (n=8/24, 33·3%) than in patients with mild disease (n=2/34, 6%) or in outpatients (n=0/44) (Fisher's exact test P<0·0001; OR 38·5; CI 95% 4·494-329·9). Eleven ICU patients died (42%), three of them carrying the D222G variant, which was associated with RBS mutation S183P in two. D222G and D222N mutants were identified in upper and lower respiratory samples. A(H1N1)pdm09 HA substitutions D222G and D222N were harbored in a significantly higher proportion by patients with acute respiratory distress for A(H1N1)pdm09 severe infection requiring ICU admission and ECMO. These data emphasize the importance of monitoring viral evolution for understanding virus-host adaptation aimed at the surveillance of strain circulation and the study of viral correlates of disease severity.

Age-Associated Cross-reactive Antibody-Dependent Cellular Cytotoxicity Toward 2009 Pandemic Influenza A Virus Subtype H1N1

During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed. We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein. A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals. ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Influenza virus A(H1N1)pdm09 hemagglutinin polymorphism and associated disease in southern Germany during the 2010/11 influenza season

A novel influenza A virus emerged in early 2009 to cause the first influenza pandemic of the 21st century. Understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. In this study, we investigated the molecular evolution of influenza virus A(H1N1)pdm09 in the 2010/11 influenza season in southern Germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. Phylogenetic analysis revealed co-circulation of different genetic groups. The D222G mutation, which had previously been observed in severe cases, was not detected. Immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. Interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. The H275Y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient.

A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico

BackgroundPandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model.MethodsThe nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model.ResultsViruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p = 0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p = 0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222.ConclusionsWe confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.

Reconstruction of the Evolutionary Dynamics of the A(H1N1)pdm09 Influenza Virus in Italy during the Pandemic and Post-Pandemic Phases

The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.

In vivo evaluation of pathogenicity and transmissibility of influenza A(H1N1)pdm09 hemagglutinin receptor binding domain 222 intrahost variants isolated from a single immunocompromised patient

The influenza A(H1N1)pdm09 virus has circulated worldwide and continued to cause complicated infections and deaths. Reports have identified an increased prevalence of the hemagglutinin receptor binding domain D222G mutation in viruses isolated from individuals who have suffered such severe infections, but this association is still unclear. Virus isolated from a nasopharyngeal wash of a severely ill immunocompromised patient at the time of diagnosis contained the D222, but isolates collected later in his course from a bronchoalveolar lavage contained primarily the G222 mutation and was mixed with a minor population of D222. These clinical isolates were compared to a G222 plaque purified virus in the ferret model. The G222 predominant clinical isolate was the most pathogenic in ferrets and developed the most diversity at the 222 amino acid position during infection, suggesting that increased diversity and not a specific polymorphism at HA 222 may be important in predicting pathogenic potential.