Human Seasonal H3N2 Research Articles

2018-2019 human seasonal H3N2 influenza A virus spillovers into swine with demonstrated virus transmission in pigs were not sustained in the pig population.

Human seasonal H3 clade 3C3a influenza A viruses (IAV) were detected four times in U.S. pigs from commercial swine farms in Michigan, Illinois, and Virginia in 2019. To evaluate the relative risk of this spillover to the pig population, whole genome sequencing and phylogenetic characterization were conducted, and the results revealed that all eight viral gene segments were closely related to 2018-2019 H3N2 human seasonal IAV. Next, a series of in vitro viral kinetics, receptor binding, and antigenic characterization studies were performed using a representative A/swine/Virginia/A02478738/2018(H3N2) (SW/VA/19) isolate. Viral replication kinetic studies of SW/VA/19 demonstrated less efficient replication curves than all 10 swine H3N2 viruses tested but higher than three human H3N2 strains. Serial passaging experiments of SW/VA/19 in swine cells did not increase virus replication, but changes at HA amino acid positions 9 and 159 occurred. In swine transmission studies, wild-type SW/VA/19 was shed in nasal secretions and transmitted to all indirect contact pigs, whereas the human seasonal strain A/Switzerland/9715293/2013(H3N2) from the same 3C3a clade failed to transmit. SW/VA/19 induced minimal macroscopic and microscopic lung lesions. Collectively, these findings demonstrate that these human seasonal H3N2 3C3a-like viruses did not require reassortment with endemic swine IAV gene segments for virus shedding and transmission in pigs. Limited detections in the U.S. pig population in the subsequent period of time suggest a yet-unknown restriction factor likely limiting the spread of these viruses in the U.S. pig population.IMPORTANCEInterspecies human-to-swine IAV transmission occurs globally and contributes to increased IAV diversity in pig populations. We present data that a swine isolate from a 2018-2019 human-to-swine transmission event was shed for multiple days in challenged and contact pigs. By characterizing this introduction through bioinformatic, molecular, and animal experimental approaches, these findings better inform animal health practices and vaccine decision-making. Since wholly human seasonal H3N2 viruses in the United States were not previously identified as being transmissible in pigs (i.e., reverse zoonosis), these findings reveal that the interspecies barriers for transmission to pigs may not require significant changes to all human seasonal H3N2, although additional changes may be required for sustained transmission in swine populations.

Reverse zoonosis of the 2022–2023 human seasonal H3N2 detected in swine

The Iowa State University Veterinary Diagnostic Laboratory detected nineteen human-to-swine reverse zoonoses of the 2022–2023 human seasonal H3N2 between November 2022 and November 2023. Cases from seven U.S. locations were detected: 3 Colorado, 1 Illinois, 1 Indiana, 2 Missouri, 7 North Carolina, 1 Ohio, and 1 Pennsylvania. One additional case was detected in Mexico and two cases were identified from Chile. Case samples were comprised of 4 nasal swabs and 15 oral fluids. Virus was successfully isolated from two of four nasal swab samples, but isolation from oral fluids was unsuccessful. The swine detections of H3 human viruses were classified to one of two human-seasonal H3 clades, 3C.2a1b.2a.2b and 3C.2a1b.2a.2a.1. Phylogenetic inference indicated at minimum 7 reverse zoonotic events occurred, with possible swine-to-swine transmission following the initial spillover. Twelve neuraminidase genes were sequenced, and nine were classified as human-seasonal H3N2 lineage: the remaining were endemic swine IAV NA genes from the N2.2002B, N2.1998, or the N1.Classical lineage, suggesting reassortment. The two viral isolates obtained from nasal swab samples were sequenced and were entirely human-lineage viruses. Seven swine samples with human seasonal H3 were sequenced and revealed co-detections with H1 1A.3.3.3 (gamma), with internal gene segments from both the triple reassortant internal gene (TRIG) and pandemic 2009 lineages. Serologic investigation of samples from swine production systems provided evidence for infection with human seasonal H3N2. One farm in the United States and four farms in Mexico had concurrent virologic evidence. The swine-isolated 3C.2a1b.2a.2b H3N2 was antigenically distinct from endemic 1990.4.A, 2010.1, and 2010.2 swine H3N2 lineages, but retained antigenic similarity to a recent human seasonal H3N2 (A/Darwin/6/2021). Pigs experimentally inoculated with a representative isolate demonstrated replication in the nose and lungs and minimal to mild macroscopic and microscopic lung lesions, but primary pigs did not transmit the virus to indirect contacts. If sustained in the pig population, this human seasonal H3 would represent the first new lineage detected in pigs the 2020 decade and present an emerging threat to swine health and production.

Large-Scale Serological Survey of Influenza A Virus in South Korean Wild Boar (Sus scrofa).

In this comprehensive large-scale study, conducted from 2015 to 2019, 7,209 wild boars across South Korea were sampled to assess their exposure to influenza A viruses (IAVs). Of these, 250 (3.5%) were found to be IAV-positive by ELISA, and 150 (2.1%) by the hemagglutination inhibition test. Detected subtypes included 23 cases of pandemic 2009 H1N1, six of human seasonal H3N2, three of classical swine H1N1, 13 of triple-reassortant swine H1N2, seven of triple-reassortant swine H3N2, and seven of swine-origin H3N2 variant. Notably, none of the serum samples tested positive for avian IAV subtypes H3N8, H5N3, H7N7, and H9N2 or canine IAV subtype H3N2. This serologic analysis confirmed the exposure of Korean wild boars to various subtypes of swine and human influenza viruses, with some serum samples cross-reacting between swine and human strains, indicating potential infections with multiple IAVs. The results highlight the potential of wild boar as a novel mixing vessel, facilitating the adaptation of IAVs and their spillover to other hosts, including humans. In light of these findings, we recommend regular and frequent surveillance of circulating influenza viruses in the wild boar population as a proactive measure to prevent potential human influenza pandemics and wild boar influenza epizootics.

Mutual information networks reveal evolutionary relationships within the influenza A virus polymerase.

The influenza A virus (IAV) RNA polymerase is an essential driver of IAV evolution. Mutations that the polymerase introduces into viral genome segments during replication are the ultimate source of genetic variation, including within the three subunits of the IAV polymerase (polymerase basic protein 2, polymerase basic protein 1, and polymerase acidic protein). Evolutionary analysis of the IAV polymerase is complicated, because changes in mutation rate, replication speed, and drug resistance involve epistatic interactions among its subunits. In order to study the evolution of the human seasonal H3N2 polymerase since the 1968 pandemic, we identified pairwise evolutionary relationships among ∼7000 H3N2 polymerase sequences using mutual information (MI), which measures the information gained about the identity of one residue when a second residue is known. To account for uneven sampling of viral sequences over time, we developed a weighted MI (wMI) metric and demonstrate that wMI outperforms raw MI through simulations using a well-sampled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dataset. We then constructed wMI networks of the H3N2 polymerase to extend the inherently pairwise wMI statistic to encompass relationships among larger groups of residues. We included hemagglutinin (HA) in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitch-hiking on antigenic changes in HA. The wMI networks reveal coevolutionary relationships among residues with roles in replication and encapsidation. Inclusion of HA highlighted polymerase-only subgraphs containing residues with roles in the enzymatic functions of the polymerase and host adaptability. This work provides insight into the factors that drive and constrain the rapid evolution of influenza viruses.

Introductions of Human-Origin Seasonal H3N2, H1N2 and Pre-2009 H1N1 Influenza Viruses to Swine in Brazil.

In South America, the evolutionary history of influenza A virus (IAV) in swine has been obscured by historically low levels of surveillance, and this has hampered the assessment of the zoonotic risk of emerging viruses. The extensive genetic diversity of IAV in swine observed globally has been attributed mainly to bidirectional transmission between humans and pigs. We conducted surveillance in swine in Brazil during 2011-2020 and characterized 107 H1N1, H1N2, and H3N2 IAVs. Phylogenetic analysis based on HA and NA segments revealed that human seasonal IAVs were introduced at least eight times into swine in Brazil since the mid-late 1980s. Our analyses revealed three genetic clades of H1 within the 1B lineage originated from three distinct spillover events, and an H3 lineage that has diversified into three genetic clades. The N2 segment from human seasonal H1N2 and H3N2 viruses was introduced into swine six times and a single introduction of an N1 segment from the human H1N1 virus was identified. Additional analysis revealed further reassortment with H1N1pdm09 viruses. All these introductions resulted in IAVs that apparently circulate only in Brazilian herds. These results reinforce the significant contributions of human IAVs to the genetic diversity of IAV in swine and reiterate the importance of surveillance of IAV in pigs.

Interspecies Transmission from Pigs to Ferrets of Antigenically Distinct Swine H1 Influenza A Viruses with Reduced Reactivity to Candidate Vaccine Virus Antisera as Measures of Relative Zoonotic Risk.

During the last decade, endemic swine H1 influenza A viruses (IAV) from six different genetic clades of the hemagglutinin gene caused zoonotic infections in humans. The majority of zoonotic events with swine IAV were restricted to a single case with no subsequent transmission. However, repeated introduction of human-seasonal H1N1, continual reassortment between endemic swine IAV, and subsequent drift in the swine host resulted in highly diverse swine IAV with human-origin genes that may become a risk to the human population. To prepare for the potential of a future swine-origin IAV pandemic in humans, public health laboratories selected candidate vaccine viruses (CVV) for use as vaccine seed strains. To assess the pandemic risk of contemporary US swine H1N1 or H1N2 strains, we quantified the genetic diversity of swine H1 HA genes, and identified representative strains from each circulating clade. We then characterized the representative swine IAV against human seasonal vaccine and CVV strains using ferret antisera in hemagglutination inhibition assays (HI). HI assays revealed that 1A.3.3.2 (pdm09) and 1B.2.1 (delta-2) demonstrated strong cross reactivity to human seasonal vaccines or CVVs. However, swine IAV from three clades that represent more than 50% of the detected swine IAVs in the USA showed significant reduction in cross-reactivity compared to the closest CVV virus: 1A.1.1.3 (alpha-deletion), 1A.3.3.3-clade 3 (gamma), and 1B.2.2.1 (delta-1a). Representative viruses from these three clades were further characterized in a pig-to-ferret transmission model and shown to exhibit variable transmission efficiency. Our data prioritize specific genotypes of swine H1N1 and H1N2 to further investigate in the risk they pose to the human population.

Naïve Human Macrophages Are Refractory to SARS-CoV-2 Infection and Exhibit a Modest Inflammatory Response Early in Infection.

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 exposed MDM with notable absence of IFN-β induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.

Characterization of a 2016-2017 Human Seasonal H3 Influenza A Virus Spillover Now Endemic to U.S. Swine.

ABSTRACTIn 2017, the Iowa State University Veterinary Diagnostic Laboratory detected a reverse-zoonotic transmission of a human seasonal H3 influenza A virus into swine (IAV-S) in Oklahoma. Pairwise comparison between the recently characterized human seasonal H3 IAV-S (H3.2010.2) hemagglutinin (HA) sequences detected in swine and the most similar 2016–2017 human seasonal H3 revealed 99.9% nucleotide identity. To elucidate the origin of H3.2010.2 IAV-S, 45 HA and 27 neuraminidase (NA) sequences from 2017 to 2020 as well as 11 whole-genome sequences (WGS) were genetically characterized. Time to most recent common human ancestor was estimated between August and September 2016. The N2 NA was of human origin in all but one strain from diagnostic submissions with NA sequences, and the internal gene segments from WGS consisted of matrix genes originating from the 2009 pandemic H1N1 and another 5 internal genes of triple reassortant swine origin (TTTTPT). Pigs experimentally infected with H3.2010.2 demonstrated efficient nasal shedding and replication in the lungs, mild pneumonia, and minimal microscopic lung lesions and transmitted the virus to indirect contact swine. Antigenically, H3.2010.2 viruses were closer to a human seasonal vaccine strain, A/Hong Kong/4801/2014, than to the H3.2010.1 human seasonal H3 viruses detected in swine in 2012. This was the second sustained transmission of a human seasonal IAV into swine from the 2010 decade after H3.2010.1. Monitoring the spillover and detection of novel IAV from humans to swine may help vaccine antigen selection and could impact pandemic preparedness.IMPORTANCE H3.2010.2 is a new phylogenetic clade of H3N2 circulating in swine that became established after the spillover of a human seasonal H3N2 from the 2016–2017 influenza season. The novel H3.2010.2 transmitted and adapted to the swine host and demonstrated reassortment with internal genes from strains endemic to pigs, but it maintained human-like HA and NA. It is genetically and antigenically distinct from the H3.2010.1 H3N2 introduced earlier in the 2010 decade. Human seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection to improve the ability to prevent respiratory disease in swine as well as the risk of zoonotic transmission.

Antigenic Distance between North American Swine and Human Seasonal H3N2 Influenza A Viruses as an Indication of Zoonotic Risk to Humans.

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 strains were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained the closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and the most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold reductions compared to the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of postinfection and postvaccination human serum cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before the 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection. IMPORTANCE Human H3N2 influenza A viruses spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation and increased H3N2 diversity in pig populations. The evolution of H3N2 in swine led to a reduced similarity to human seasonal H3N2 and the vaccine strains used to protect human populations. We quantified the antigenic phenotypes and found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the decade of incursion but had substantial differences compared to recent human vaccine strains. Additionally, pandemic preparedness vaccine strains demonstrated a loss of similarity to contemporary swine strains. Finally, human sera revealed that although these adults had antibodies against human H3N2 strains, many had limited immunity to swine H3N2, especially older adults born before 1970. Antigenic assessment of swine H3N2 provides critical information for pandemic preparedness and candidate vaccine development.

Ubiquitous influenza A virus in Chilean swine before the H1N1pdm09 introduction.

Influenza A virus (IAV) was a neglected swine pathogen in South America before the 2009 H1N1 pandemic (A(H1N1)pdm2009). The A(H1N1)pdm2009 strain has widely spread among the Chilean swine population and co-circulates with endemic H1N2 and H3N2 viruses. The presence of IAV as a swine pathogen in Chilean swine before the 2009 pandemic is unknown. To understand the IAV in swine prior to 2009, aY retrospective study of samples from pigs affected with respiratory diseases was conducted. Ninety formalin-fixed and paraffin-embedded lung tissues belonging to 21 intensive pig production companies located in five different administrative regions of Chile, collected between 2005 and 2008, were evaluated. The tissues were tested by immunohistochemistry (IHC), identifying that 9 out of 21 farms (42.8%) and 31 out of 90 (34.4%) samples were IAV positive. Only three out of the 31 IHC-positive samples were positive upon RNA extraction and rtRT-PCR analysis. Partial nucleotide sequences were obtained from one sample and characterized as an H3N2 subtype closely related to a human seasonal H3N2 IAVs that circulated globally in the mid-90s. These results indicate that IAV was circulating in swine before 2009 and highlight the value of conducting retrospective studies through genomic strategies to analyse historical samples.

Infection and risk factors of human and avian influenza in pigs in south China

The coinfection of swine influenza (SI) strains and avian/human-source influenza strains in piggeries can contribute to the evolution of new influenza viruses with pandemic potential. This study analyzed surveillance data on SI in south China and explored the spatial predictor variables associated with different influenza infection scenarios in counties within the study area. Blood samples were collected from 7670 pigs from 534 pig farms from 2015 to 2017 and tested for evidence of infection with influenza strains from swine, human and avian sources. The herd prevalences for EA H1N1, H1N1pdm09, classic H1N1, HS-like H3N2, seasonal human H1N1 and avian influenza H9N2 were 88.5, 64.5, 60.3, 57.8, 12.9 and 10.3 %, respectively. Anthropogenic factors including detection frequency, chicken density, duck density, pig density and human population density were found to be better predictor variables for three influenza infection scenarios (infection with human strains, infection with avian strains, and coinfection with H9N2 avian strain and at least one swine strain) than were meteorological and geographical factors. Predictive risk maps generated for the four provinces in south China highlighted that the areas with a higher risk of the three infection scenarios were predominantly clustered in the delta area of the Pearl River in Guangdong province and counties surrounding Poyang Lake in Jiangxi province. Identification of higher risk areas can inform targeted surveillance for influenza in humans and pigs, helping public health authorities in designing risk-based SI control strategies to address the pandemic influenza threat in south China.

Characterization of contemporary 2010.1 H3N2 swine influenza A viruses circulating in United States pigs

In 2012, swine influenza surveillance detected a novel reassorted influenza A virus (IAV) strain containing human-seasonal hemagglutinin (HA) and neuraminidase (NA). Subsequently, these viruses reassorted, maintaining only the human-origin H3, which resulted in a new lineage of viruses that became the most frequently detected H3 clade in US swine (2010.1 HA clade). Here, we assessed the antigenic phenotype, virulence, and transmission characteristics of this virus lineage following its introduction to swine. Relative to 2010.1 viruses from 2012 and 2014, recent 2010.1 contemporary strains from 2015 to 2017 resulted in equivalent macroscopic lung lesions and transmission in pigs. A single mutation at amino acid residue 145 within the previously defined HA antigenic motif was associated with a change of antigenic phenotype, potentially impairing vaccine efficacy. Contemporary 2010.1 viruses circulating in swine since 2012 were significantly different from both pre-2012H3N2 in swine and human-seasonal H3N2 viruses and demonstrated continued evolution within the lineage.

Immune-engineered H7N9 influenza hemagglutinin improves protection against viral influenza virus challenge

ABSTRACT The influenza hemagglutinin (HA) isolated from avian H7N9 influenza virus strains elicit weak immune responses. This low immunogenicity may be due to a regulatory T cell (Treg)–stimulating epitopes in HA from the H7N9 isolate A/Anhui/1/2013 (Anh/13). In this report, this Treg stimulating sequence was removed from the wild-type (WT) H7 HA amino acid sequence and replaced with a conserved CD4 + T cell stimulating sequences from human seasonal H3N2 strains and designed OPT1 H7 HA. The effectiveness of this optimized H7 HA protein was determined using a humanized mouse (HLA-DR3) expressing the human leukocyte antigen (HLA) DR3 allele. HLA-DR3 mice were pre-immunized by infecting with H3N2 influenza virus, A/Hong Kong/4108/2014 and then vaccinated intramuscularly with either the WT H7 HA from Anh/13 or the OPT1 H7 HA antigen without adjuvant. The OPT1 H7 HA vaccination group elicited higher H7 HA-specific IgG titers that resulted in a lower mortality, weight loss, and lung viral titer following lethal challenge with the H7N9 Anh/13 influenza virus compared to WT-vaccinated mice. Overall, T-cell epitope-engineered vaccines can improve the immunogenicity of H7 HA antigens resulting in enhanced survival and lower morbidity against H7N9 influenza virus challenge.

Serosurvey of Coyotes (Canis latrans), Foxes (Vulpes vulpes, Urocyon cinereoargenteus), and Raccoons (Procyon lotor) for Exposure to Influenza a Viruses in the USA.

We tested coyote (Canis latrans), fox (Urocyon cinereoargenteus, Vulpes vulpes), and raccoon (Procyon lotor) sera for influenza A virus (IAV) exposure. We found 2/139 samples (1 coyote, 1 raccoon) had IAV antibodies and hemagglutination inhibition assays revealed the antibodies to the 2009/2010 H1N1 human pandemic virus or to the 2007 human seasonal H1N1 virus.

Attachment Patterns of Human and Avian Influenza Viruses to Trachea and Colon of 26 Bird Species - Support for the Community Concept.

Avian influenza A viruses (AIVs) have a broad host range, but are most intimately associated with waterfowl (Anseriformes) and, in the case of the H13 and H16 subtypes, gulls (Charadriiformes). Host associations are multifactorial, but a key factor is the ability of the virus to bind host cell receptors and thereby initiate infection. The current study aims at investigating the tissue attachment pattern of a panel of AIVs, comprising H3N2, H6N1, H12N5, and H16N3, to avian trachea and colon tissue samples obtained from host species of different orders. Virus attachment was not restricted to the bird species or order from which the virus was isolated. Instead, extensive virus attachment was observed to several distantly related avian species. In general, more virus attachment and receptor expression were observed in trachea than in colon samples. Additionally, a human seasonal H3N2 virus was studied. Unlike the studied AIVs, this virus mainly attached to tracheae from Charadriiformes and a very limited set of avian cola. In conclusion, the reported results highlight the importance of AIV attachment to trachea in many avian species. Finally, the importance of chickens and mallards in AIVs dynamics was illustrated by the abundant AIV attachment observed.

H5N8 and H7N9 packaging signals constrain HA reassortment with a seasonal H3N2 influenza A virus

Influenza A virus (IAV) has a segmented genome, which (i) allows for exchange of gene segments in coinfected cells, termed reassortment, and (ii) necessitates a selective packaging mechanism to ensure incorporation of a complete set of segments into virus particles. Packaging signals serve as segment identifiers and enable segment-specific packaging. We have previously shown that packaging signals limit reassortment between heterologous IAV strains in a segment-dependent manner. Here, we evaluated the extent to which packaging signals prevent reassortment events that would raise concern for pandemic emergence. Specifically, we tested the compatibility of hemagglutinin (HA) packaging signals from H5N8 and H7N9 avian IAVs with a human seasonal H3N2 IAV. By evaluating reassortment outcomes, we demonstrate that HA segments carrying H5 or H7 packaging signals are significantly disfavored for incorporation into a human H3N2 virus in both cell culture and a guinea pig model. However, incorporation of the heterologous HAs was not excluded fully, and variants with heterologous HA packaging signals were detected at low levels in vivo, including in naïve contact animals. This work indicates that the likelihood of reassortment between human seasonal IAV and avian IAV is reduced by divergence in the RNA packaging signals of the HA segment. These findings offer important insight into the molecular mechanisms governing IAV emergence and inform efforts to estimate the risks posed by H7N9 and H5N8 subtype avian IAVs.

Endogenous Cellular MicroRNAs Mediate Antiviral Defense against Influenza A Virus.

The reciprocal interaction between influenza virus and host microRNAs (miRNAs) has been implicated in the regulation of viral replication and host tropism. However, the global roles of the cellular miRNA repertoire and the mechanisms of miRNA-mediated antiviral defense await further elucidation. In this study, we systematically screened 297 cellular miRNAs from human and mouse epithelial cells and identified five inhibitory miRNAs that efficiently inhibited influenza virus replication in vitro and in vivo. Among these miRNAs, hsa-mir-127-3p, hsa-mir-486-5p, hsa-mir-593-5p, and mmu-mir-487b-5p were found to target at least one viral gene segment of both the human seasonal influenza H3N2 and the attenuated PR8 (H1N1) virus, whereas hsa-miR-1-3p inhibited viral replication by targeting the supportive host factor ATP6V1A. Moreover, the number of miRNA binding sites in viral RNA segments was positively associated with the activity of host miRNA-induced antiviral defense. Treatment with a combination of the five miRNAs through agomir delivery pronouncedly suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice. These data suggest that the highly expressed miRNAs in respiratory epithelial cells elicit effective antiviral defenses against influenza A viruses and will be useful for designing miRNA-based therapies against viral infection.

Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants.

Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines.IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015–2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015–2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2–4 fold lower for the 2015–2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Influenza A(H3N2) Virus in Swine at Agricultural Fairs and Transmission to Humans, Michigan and Ohio, USA, 2016.

In 2016, a total of 18 human infections with influenza A(H3N2) virus occurred after exposure to influenza-infected swine at 7 agricultural fairs. Sixteen of these cases were the result of infection by a reassorted virus with increasing prevalence among US swine containing a hemagglutinin gene from 2010–11 human seasonal H3N2 strains.