Comparable Fitness and Transmissibility between Oseltamivir-Resistant Pandemic 2009 and Seasonal H1N1 Influenza Viruses with the H275Y Neuraminidase Mutation

Limited antiviral compounds are available for the control of influenza, and the emergence of resistant variants would further narrow the options for defense. The H275Y neuraminidase (NA) mutation, which confers resistance to oseltamivir carboxylate, has been identified among the seasonal H1N1 and 2009 pandemic influenza viruses; however, those H275Y resistant variants demonstrated distinct epidemiological outcomes in humans. Specifically, dominance of the H275Y variant over the oseltamivir-sensitive viruses was only reported for a seasonal H1N1 variant during 2008-2009. Here, we systematically analyze the effect of the H275Y NA mutation on viral fitness and transmissibility of A(H1N1)pdm09 and seasonal H1N1 influenza viruses. The NA genes from A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal), and A/Brisbane/59/2007 (Brisbane) were individually introduced into the genetic background of CA04. The H275Y mutation led to reduced NA enzyme activity, an increased K_m for 3′-sialylactose or 6′-sialylactose, and decreased infectivity in mucin-secreting human airway epithelial cells compared to the oseltamivir-sensitive wild-type counterparts. Attenuated pathogenicity in both RG-CA04^NA-H275Y and RG-CA04 × Brisbane^NA-H275Y viruses was observed in ferrets compared to RG-CA04 virus, although the transmissibility was minimally affected. In parallel experiments using recombinant Brisbane viruses differing by hemagglutinin and NA, comparable direct contact and respiratory droplet transmissibilities were observed among RG-NewCal^HA,NA, RG-NewCal^HA,NA-H275Y, RG-Brisbane^HA,NA-H275Y, and RG-NewCal^HA × Brisbane^NA-H275Y viruses. Our results demonstrate that, despite the H275Y mutation leading to a minor reduction in viral fitness, the transmission potentials of three different antigenic strains carrying this mutation were comparable in the naïve ferret model.     

Influenza virus H1N1pdm09 infections in the young and old: evidence of greater antibody diversity and affinity for the hemagglutinin globular head domain (HA1 Domain) in the elderly than in young adults and children

The H1N1 2009 influenza virus (H1N1pdm09) pandemic had several unexpected features, including low morbidity and mortality in older populations. We performed in-depth evaluation of antibody responses generated following H1N1pdm09 infection of naïve ferrets and of 130 humans ranging from the very young (0 to 9 years old) to the very old (70 to 89 years old). In addition to hemagglutination inhibition (HI) titers, we used H1N1pdm09 whole-genome-fragment phage display libraries (GFPDL) to evaluate the antibody repertoires against internal genes, hemagglutinin (HA), and neuraminidase (NA) and also measured antibody affinity for antigenic domains within HA. GFPDL analyses of H1N1pdm09-infected ferrets demonstrated gradual development of antibody repertoires with a focus on M1 and HA1 by day 21 postinfection. In humans, H1N1pdm09 infection in the elderly (>70 years old) induced antibodies with broader epitope recognition in both the internal genes and the HA1 receptor binding domain (RBD) than for the younger age groups (0 to 69 years). Importantly, post-H1N1 infection serum antibodies from the elderly demonstrated substantially higher avidity for recombinant HA1 (rHA1) (but not HA2) than those from younger subjects (50% versus <22% 7 M urea resistance, respectively) and lower antibody dissociation rates using surface plasmon resonance. This is the first study in humans that provides evidence for a qualitatively superior antibody response in the elderly following H1N1pdm09 infection, indicative of recall of long-term memory B cells or long-lived plasma cells. These findings may help explain the age-related morbidity and mortality pattern observed during the H1N1pdm09 pandemic.     

Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum

The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein‐based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.     

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.     

Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.     

Emergence and dissemination of a swine H3N2 reassortant influenza virus with 2009 pandemic H1N1 genes in pigs in China

The 2009 pandemic influenza virus (pdm/09) has been frequently introduced to pigs and has reassorted with other swine viruses. Recently, H3N2 reassortants with pdm/09-like internal genes were isolated in Guangxi and Hong Kong, China. Genetic and epidemiological analyses suggest that these viruses have circulated in swine for some time. This is the first evidence that swine reassortant viruses with pdm/09-like genes may have become established in the field, altering the landscape of human and swine influenza.     

Evolution of the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus in Morocco During Two Influenza Seasons 2009–2011

To study genetic evolution of Moroccan influenza A(H1N1)pdm09 virus strains, we conducted a molecular characterization of the hemagglutinin gene subunit 1 (HA1) of 36 influenza A(H1N1)pdm09 virus strains. The stains were collected from patients in Rabat and Casablanca during two influenza seasons 2009–2010 and 2010–2011. Nucleotide and amino acid sequences of 14 influenza A(H1N1)pdm09 virus strains from 2009 to 2010 were ~97 and 99 %, respectively, similar to the reference strain A/California/07/2009 (H1N1). Phylogenetic analysis of 22 influenza A(H1N1)pdm09 virus strains from 2010 to 2011 revealed a co-circulation of three well-described different genetic groups. Most important, none of the identified groups showed significant changes at the antigenic site of the virus HA1 subunit which may alter the efficacy of California/07/2009 (H1N1) vaccine.     

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.     

Hemagglutinin 222D/G Polymorphism Facilitates Fast Intra-Host Evolution of Pandemic (H1N1) 2009 Influenza A Viruses

The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA) in position 222 (HA-D222G) as well as HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09 (mpJena/5258), developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i.) symptoms (body weight loss and clinical score) continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid α-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1)pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.     

A Large Proportion of the Mexican Population Remained Susceptible to A(H1N1)pdm09 Infection One Year after the Emergence of 2009 Influenza Pandemic

Background

The 2009 H1N1 influenza pandemic initially affected Mexico from April 2009 to July 2010. By August 2010, a fourth of the population had received the monovalent vaccine against the pandemic virus (A(H1N1)pdm09). To assess the proportion of the Mexican population who remained potentially susceptible to infection throughout the summer of 2010, we estimated the population seroprevalence to A(H1N1)pdm09 in a serosurvey of blood donors.

Methods

We evaluated baseline cross-reactivity to the pandemic strain and set the threshold for seropositivity using pre-pandemic (2005–2008) stored serum samples and sera from confirmed A(H1N1)pdm09 infected individuals. Between June and September 2010, a convenience sample serosurvey of adult blood donors, children, and adolescents was conducted in six states of Mexico. Sera were tested by the microneutralization (MN) and hemagglutination inhibition (HI) assays, and regarded seropositive if antibody titers were equal or exceeded 1:40 for MN and 1:20 for HI. Age-standardized seroprevalence were calculated using the 2010 National Census population.

Results

Sera from 1,484 individuals were analyzed; 1,363 (92%) were blood donors, and 121 (8%) children or adolescents aged ≤19 years. Mean age (standard deviation) was 31.4 (11.5) years, and 276 (19%) were women. A total of 516 (35%) participants declared history of influenza vaccination after April 2009. The age-standardized seroprevalence to A(H1N1)pdm09 was 48% by the MN and 41% by the HI assays, respectively. The youngest quintile, aged 1 to 22 years, had the highest the seroprevalence; 61% (95% confidence interval [CI]: 56, 66%) for MN, and 56% (95% CI: 51, 62%) for HI.

Conclusions

Despite high transmission of A(H1N1)pdm09 observed immediately after its emergence and extensive vaccination, over a half of the Mexican population remained potentially susceptible to A(H1N1)pdm09 infection. Subsequent influenza seasons with high transmission of A(H1N1)pdm09, as 2011–2012 and 2013–2014, are compatible with these findings.     

海南省2016-2018年A(H1N1)pdm09亚型流感病毒基因特性分析

2009年A(H1N1)pdm09亚型流感病毒在墨西哥暴发,之后在全世界流行。为了解海南省2016-2018年A(H1N1)pdm09亚型流感病毒流行态势,分析血凝素(HA)与神经氨酸酶(NA)基因遗传进化特征与变异情况,本研究从中国流感监测信息系统获取海南省2016-2018年流感病毒病原学监测数据,选取5家流感监测网络实验室分离鉴定的37株A(H1N1)pdm09亚型流感毒株进行HA与NA基因测序,利用MEGA 10.1.8构建HA与NA基因种系进化树,并分析其氨基酸变异情况。结果显示,2016-2018年共出现3次A(H1N1)pdm09亚型流感病毒活动高峰。2017年10月份以后的分离株(4/8)与2018年大部分分离株(21/22)独立于疫苗株A/Michigan/45/2015聚为一个小支,发生20余处HA与NA氨基酸位点变异。与疫苗株A/California/7/2009(2010-2016)相比,2016-2018年流感病毒分离株在HA基因抗原决定簇上发生7处氨基酸变异并有一个潜在糖基化位点,未发现HA基因受体结合位点变异与NA基因耐药性变异。本研究提示,2016-2018年,A(H1N1)pdm09亚型流感病毒逐步发生规律性进化,氨基酸变异频率有增加趋势,今后应持续加强流感病毒病原学监测,密切追踪A(H1N1)pdm09亚型流感病毒基因变异情况,为科学防控提供理论依据。     

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.     

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the p _epitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012–2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.     

Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009

Background

In April 2009, a novel triple-reassortant swine influenza A H1N1 virus (“A/H1N1pdm”; also known as SOIV) was detected and spread globally as the first influenza pandemic of the 21^st century. Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.

Methodology/Principal Findings

By Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades (2, 3, and 7) were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47×10⁻³ amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493–757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.

Conclusions/Significance

These findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics.     

H1N1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis

Background

The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil.

Methods

We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir.

Results

A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples.

Conclusions

Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.     

Variations in the Hemagglutinin of the 2009 H1N1 Pandemic Virus: Potential for Strains with Altered Virulence Phenotype?

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice--herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations--which are also found in the 1918 and seasonal H1N1 viruses-in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies.     

Design of Escherichia coli-Expressed Stalk Domain Immunogens of H1N1 Hemagglutinin That Protect Mice from Lethal Challenge

The hemagglutinin protein (HA) on the surface of influenza virus is essential for viral entry into the host cells. The HA1 subunit of HA is also the primary target for neutralizing antibodies. The HA2 subunit is less exposed on the virion surface and more conserved than HA1. We have previously designed an HA2-based immunogen derived from the sequence of the H3N2 A/HK/68 virus. In the present study, we report the design of an HA2-based immunogen from the H1N1 subtype (PR/8/34). This immunogen (H1HA0HA6) and its circular permutant (H1HA6) were well folded and provided complete protection against homologous viral challenge. Antisera of immunized mice showed cross-reactivity with HA proteins of different strains and subtypes. Although no neutralization was observable in a conventional neutralization assay, sera of immunized guinea pigs competed with a broadly neutralizing antibody, CR6261, for binding to recombinant Viet/04 HA protein, suggesting that CR6261-like antibodies were elicited by the immunogens. Stem domain immunogens from a seasonal H1N1 strain (A/NC/20/99) and a recent pandemic strain (A/Cal/07/09) provided cross-protection against A/PR/8/34 viral challenge. HA2-containing stem domain immunogens therefore have the potential to provide subtype-specific protection.     

Polymerase Discordance in Novel Swine Influenza H3N2v Constellations Is Tolerated in Swine but Not Human Respiratory Epithelial Cells

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection.