Potential pandemic risk of circulating swine H1N2 influenza viruses.

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans due to a potential lack of population level immunity. Here we describe a pipeline to characterize and triage influenza viruses for their pandemic risk and examine the pandemic potential of two widespread swine origin viruses. Our analysis reveals that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against a α-H1 clade strain (α-swH1N2) but do against a γ-H1 clade strain. The α-swH1N2 virus replicates efficiently in human airway cultures and exhibits phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, α-swH1N2 is capable of efficient airborne transmission to both naïve ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity show reduced α-swH1N2 viral shedding and less severe disease signs. Despite this, H1N1pdm09-immune ferrets that became infected via the air can still onward transmit α-swH1N2 with an efficiency of 50%. These results indicate that this α-swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness and pathology in animals with prior immunity.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple non-human mammalian species1,2. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera3-6. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here we use pseudovirus deep mutational scanning7 to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine strains. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.

Biomarkers associated with vaccine-associated enhanced respiratory disease following influenza A virus infection in swine.

Influenza A virus (IAV) is a major pathogen in the swine industry. Whole-inactivated virus (WIV) vaccines in swine are highly effective against homologous viruses but provide limited protection to antigenically divergent viruses and may lead to vaccine-associated enhanced respiratory disease (VAERD) after heterologous infection. Although VAERD is reproducible in laboratory studies, clinical diagnosis is challenging, as it would require both knowledge of prior vaccine history and evidence of severe disease by assessment of pathologic lesions at necropsy following infection with a heterologous virus. The objective of this study was to identify potential biomarkers for VAERD for antemortem clinical diagnosis. Naïve pigs were split into two groups, and one group was vaccinated with IAV WIV vaccine. All pigs were then challenged with a heterologous virus to induce VAERD in the vaccinated group and necropsied at 5 days post infection (dpi). Blood was collected on 0, 1, 3, and 5 dpi, and assessed by hematology, plasma chemistry, acute phase proteins, and citrullinated H3 histone (CitH3) assays. Additionally, cytokine and CitH3 levels were assessed in bronchoalveolar lavage fluid (BALF) collected at necropsy. Compared to nonvaccinated challenged pigs, blood collected from vaccinated and challenged (V/C) pigs with VAERD had elevated white blood cells and neutrophils, elevated C-reactive protein and haptoglobin acute phase proteins, and elevated CitH3. In BALF, the proinflammatory cytokine IL-8 and CitH3 were elevated in V/C pigs. In conclusion, a profile of elevated white blood cells and neutrophils, elevated C-reactive protein and haptoglobin, and elevated CitH3 may be relevant for a clinical antemortem IAV VAERD diagnosis.

Detection and Characterization of Influenza A Virus Endemic Circulation in Suckling and Nursery Pigs Originating from Vaccinated Farms in the Same Production System.

Inactivated influenza A virus (IAV) vaccines help reduce clinical disease in suckling piglets, although endemic infections still exist. The objective of this study was to evaluate the detection of IAV in suckling and nursery piglets from IAV-vaccinated sows from farms with endemic IAV infections. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. IAV RNA was detected in 1.64% and 31.01% of individual nasal swabs and oral fluids, respectively. H1N2 was detected most often, with sporadic detection of H1N1 and H3N2. Whole-genome sequences of IAV isolated from suckling piglets revealed an H1 hemagglutinin (HA) from the 1B.2.2.2 clade and N2 neuraminidase (NA) from the 2002A clade. The internal gene constellation of the endemic H1N2 was TTTTPT with a pandemic lineage matrix. The HA gene had 97.59% and 97.52% nucleotide and amino acid identities, respectively, to the H1 1B.2.2.2 used in the farm-specific vaccine. A similar H1 1B.2.2.2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nurseries from farms with endemic infections in spite of using farm-specific IAV vaccines in sows.

Divergent Pathogenesis and Transmission of Highly Pathogenic Avian Influenza A(H5N1) in Swine.

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), ßIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.

Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Human-to-swine introductions and onward transmission of 2009 H1N1 pandemic influenza viruses in Brazil.

Introduction: Once established in the human population, the 2009 H1N1 pandemic virus (H1N1pdm09) was repeatedly introduced into swine populations globally with subsequent onward transmission among pigs. Methods: To identify and characterize human-to-swine H1N1pdm09 introductions in Brazil, we conducted a large-scale phylogenetic analysis of 4,141 H1pdm09 hemagglutinin (HA) and 3,227 N1pdm09 neuraminidase (NA) gene sequences isolated globally from humans and swine between 2009 and 2022. Results: Phylodynamic analysis revealed that during the period between 2009 and 2011, there was a rapid transmission of the H1N1pdm09 virus from humans to swine in Brazil. Multiple introductions of the virus were observed, but most of them resulted in self-limited infections in swine, with limited onward transmission. Only a few sustained transmission clusters were identified during this period. After 2012, there was a reduction in the number of human-to-swine H1N1pdm09 transmissions in Brazil. Discussion: The virus underwent continuous antigenic drift, and a balance was established between swine-to-swine transmission and extinction, with minimal sustained onward transmission from humans to swine. These results emphasize the dynamic interplay between human-to-swine transmission, antigenic drift, and the establishment of swine-to-swine transmission in shaping the evolution and persistence of H1N1pdm09 in swine populations.

Reverse-zoonoses of 2009 H1N1 pandemic influenza A viruses and evolution in United States swine results in viruses with zoonotic potential.

The 2009 H1N1 pandemic (pdm09) lineage of influenza A virus (IAV) crosses interspecies barriers with frequent human-to-swine spillovers each year. These spillovers reassort and drift within swine populations, leading to genetically and antigenically novel IAV that represent a zoonotic threat. We quantified interspecies transmission of the pdm09 lineage, persistence in swine, and identified how evolution in swine impacted zoonotic risk. Human and swine pdm09 case counts between 2010 and 2020 were correlated and human pdm09 burden and circulation directly impacted the detection of pdm09 in pigs. However, there was a relative absence of pdm09 circulation in humans during the 2020-21 season that was not reflected in swine. During the 2020-21 season, most swine pdm09 detections originated from human-to-swine spillovers from the 2018-19 and 2019-20 seasons that persisted in swine. We identified contemporary swine pdm09 representatives of each persistent spillover and quantified cross-reactivity between human seasonal H1 vaccine strains and the swine strains using a panel of monovalent ferret antisera in hemagglutination inhibition (HI) assays. The swine pdm09s had variable antigenic reactivity to vaccine antisera, but each swine pdm09 clade exhibited significant reduction in cross-reactivity to one or more of the human seasonal vaccine strains. Further supporting zoonotic risk, we showed phylogenetic evidence for 17 swine-to-human transmission events of pdm09 from 2010 to 2021, 11 of which were not previously classified as variants, with each of the zoonotic cases associated with persistent circulation of pdm09 in pigs. These data demonstrate that reverse-zoonoses and evolution of pdm09 in swine results in viruses that are capable of zoonotic transmission and represent a potential pandemic threat.

Mucin 4 is a cellular biomarker of necrotizing bronchiolitis in influenza A virus infection.

Influenza A virus (IAV) in the human and swine host infects epithelial cells lining the respiratory tract causing a necrotizing bronchitis and bronchiolitis. These epithelial surfaces are protected by large glycoproteins called mucins. Mucin 4 (MUC4) is a transmembrane mucin that consists of an alpha subunit responsible for surface protection and intracellular beta subunit involved in signal transduction which repress apoptosis and stimulate epithelial proliferation. This study was designed to determine the expression and potential role of MUC4 during IAV infection. We used immunohistochemistry in combination with machine learning image analysis to quantify differential protein expression of MUC4 subunits in IAV-infected and uninfected lung in a porcine model. MUC4 protein basal expression in control animals varied significantly by litter. MUC4 protein expression was significantly increased in bronchioles with necrotizing bronchiolitis compared to histologically normal bronchioles, likely representing a regenerative response to restore mucosal integrity of conducting airways. Understanding the impact of differential MUC4 expression among healthy individuals and during IAV infection will facilitate control strategies by elucidating mechanisms associated with susceptibility to IAV that can be therapeutically or genetically regulated and may be extended to other respiratory diseases.

PARNAS: Objectively Selecting the Most Representative Taxa on a Phylogeny.

The use of next-generation sequencing technology has enabled phylogenetic studies with hundreds of thousands of taxa. Such large-scale phylogenies have become a critical component in genomic epidemiology in pathogens such as SARS-CoV-2 and influenza A virus. However, detailed phenotypic characterization of pathogens or generating a computationally tractable dataset for detailed phylogenetic analyses requires objective subsampling of taxa. To address this need, we propose parnas, an objective and flexible algorithm to sample and select taxa that best represent observed diversity by solving a generalized k-medoids problem on a phylogenetic tree. parnas solves this problem efficiently and exactly by novel optimizations and adapting algorithms from operations research. For more nuanced selections, taxa can be weighted with metadata or genetic sequence parameters, and the pool of potential representatives can be user-constrained. Motivated by influenza A virus genomic surveillance and vaccine design, parnas can be applied to identify representative taxa that optimally cover the diversity in a phylogeny within a specified distance radius. We demonstrated that parnas is more efficient and flexible than existing approaches. To demonstrate its utility, we applied parnas to 1) quantify SARS-CoV-2 genetic diversity over time, 2) select representative influenza A virus in swine genes derived from over 5 years of genomic surveillance data, and 3) identify gaps in H3N2 human influenza A virus vaccine coverage. We suggest that our method, through the objective selection of representatives in a phylogeny, provides criteria for quantifying genetic diversity that has application in the the rational design of multivalent vaccines and genomic epidemiology. PARNAS is available at https://github.com/flu-crew/parnas.

Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in influenza A virus from North American swine.

Influenza A viruses (IAVs) of the H1N1 classical swine lineage became endemic in North American swine following the 1918 pandemic. Additional human-to-swine transmission events after 1918, and a spillover of H1 viruses from wild birds in Europe, potentiated a rapid increase in genomic diversity via reassortment between introductions and the endemic classical swine lineage. To determine mechanisms affecting reassortment and evolution, we conducted a phylogenetic analysis of N1 and paired HA swine IAV genes in North America between 1930 and 2020. We described fourteen N1 clades within the N1 Eurasian avian lineage (including the N1 pandemic clade), the N1 classical swine lineage, and the N1 human seasonal lineage. Seven N1 genetic clades had evidence for contemporary circulation. To assess antigenic drift associated with N1 genetic diversity, we generated a panel of representative swine N1 antisera and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assays and antigenic cartography. Within the N1 genes, the antigenic similarity was variable and reflected shared evolutionary history. Sustained circulation and evolution of N1 genes in swine had resulted in a significant antigenic distance between the N1 pandemic clade and the classical swine lineage. Between 2010 and 2020, N1 clades and N1-HA pairings fluctuated in detection frequency across North America, with hotspots of diversity generally appearing and disappearing within 2 years. We also identified frequent N1-HA reassortment events (n = 36), which were rarely sustained (n = 6) and sometimes also concomitant with the emergence of new N1 genetic clades (n = 3). These data form a baseline from which we can identify N1 clades that expand in range or genetic diversity that may impact viral phenotypes or vaccine immunity and subsequently the health of North American swine.

Swine-to-Ferret Transmission of Antigenically Drifted Contemporary Swine H3N2 Influenza A Virus Is an Indicator of Zoonotic Risk to Humans.

Human-to-swine transmission of influenza A (H3N2) virus occurs repeatedly and plays a critical role in swine influenza A virus (IAV) evolution and diversity. Human seasonal H3 IAVs were introduced from human-to-swine in the 1990s in the United States and classified as 1990.1 and 1990.4 lineages; the 1990.4 lineage diversified into 1990.4.A-F clades. Additional introductions occurred in the 2010s, establishing the 2010.1 and 2010.2 lineages. Human zoonotic cases with swine IAV, known as variant viruses, have occurred from the 1990.4 and 2010.1 lineages, highlighting a public health concern. If a variant virus is antigenically drifted from current human seasonal vaccine (HuVac) strains, it may be chosen as a candidate virus vaccine (CVV) for pandemic preparedness purposes. We assessed the zoonotic risk of US swine H3N2 strains by performing phylogenetic analyses of recent swine H3 strains to identify the major contemporary circulating genetic clades. Representatives were tested in hemagglutination inhibition assays with ferret post-infection antisera raised against existing CVVs or HuVac viruses. The 1990.1, 1990.4.A, and 1990.4.B.2 clade viruses displayed significant loss in cross-reactivity to CVV and HuVac antisera, and interspecies transmission potential was subsequently investigated in a pig-to-ferret transmission study. Strains from the three lineages were transmitted from pigs to ferrets via respiratory droplets, but there were differential shedding profiles. These data suggest that existing CVVs may offer limited protection against swine H3N2 infection, and that contemporary 1990.4.A viruses represent a specific concern given their widespread circulation among swine in the United States and association with multiple zoonotic cases.

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.

Pigs lacking TMPRSS2 displayed fewer lung lesions and reduced inflammatory response when infected with influenza A virus.

Influenza A virus (IAV) infection is initiated by hemagglutinin (HA), a glycoprotein exposed on the virion's lipid envelope that undergoes cleavage by host cell proteases to ensure membrane fusion, entry into the host cells, and completion of the viral cycle. Transmembrane protease serine S1 member 2 (TMPRSS2) is a host transmembrane protease expressed throughout the porcine airway epithelium and is purported to play a major role in the HA cleavage process, thereby influencing viral pathogenicity and tissue tropism. Pigs are natural hosts of IAV and IAV disease causes substantial economic impact on the pork industry worldwide. Previous studies in mice demonstrated that knocking out expression of TMPRSS2 gene was safe and inhibited the spread of IAV after experimental challenge. Therefore, we hypothesized that knockout of TMPRSS2 will prevent IAV infectivity in the swine model. We investigated this hypothesis by comparing pathogenesis of an H1N1pdm09 virus challenge in wildtype (WT) control and in TMPRSS2 knockout (TMPRSS2 -/-) pigs. We demonstrated that TMPRSS2 was expressed in the respiratory tract in WT pigs with and without IAV infection. No differences in nasal viral shedding and lung lavage viral titers were observed between WT and TMPRSS2 -/- pigs. However, the TMPRSS2 -/- pig group had significantly less lung lesions and significant reductions in antiviral and proinflammatory cytokines in the lung. The virus titer results in our direct challenge model contradict prior studies in the murine animal model, but the reduced lung lesions and cytokine profile suggest a possible role for TMPRSS2 in the proinflammatory antiviral response. Further research is warranted to investigate the role of TMPRSS2 in swine IAV infection and disease.

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.

Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene.

Influenza A viruses (FLUAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of FLUAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin FLUAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin FLUAV to transmit and become established in pigs. We generated a reassortant FLUAV (VIC11pTRIG) containing surface gene segments from a human FLUAV strain and internal gene segments from the 2009 pandemic and TRIG FLUAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant analysis identified three mutants that emerged during replication in pigs, which were mapped near the receptor binding site of the hemagglutinin (HA). The variants replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA, and one of them was present in all contact pigs. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission. IMPORTANCE Influenza A viruses cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of FLUAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human FLUAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and neuraminidase (NA) on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid substitution that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.

Antigenic Characterization and Pandemic Risk Assessment of North American H1 Influenza A Viruses Circulating in Swine.

The first pandemic of the 21st century was caused by an H1N1 influenza A virus (IAV) introduced from pigs into humans, highlighting the importance of swine as reservoirs for pandemic viruses. Two major lineages of swine H1 circulate in North America: the 1A classical swine lineage (including that of the 2009 H1N1 pandemic) and the 1B human seasonal-like lineage. Here, we investigated the evolution of these H1 IAV lineages in North American swine and their potential pandemic risk. We assessed the antigenic distance between the HA of representative swine H1 and human seasonal vaccine strains (1978 to 2015) in hemagglutination inhibition (HI) assays using a panel of monovalent antisera raised in pigs. Antigenic cross-reactivity varied by strain but was associated with genetic distance. Generally, the swine 1A lineage viruses that seeded the 2009 H1 pandemic were antigenically most similar to the H1 pandemic vaccine strains, with the exception of viruses in the genetic clade 1A.1.1.3, which had a two-amino acid deletion mutation near the receptor-binding site, which dramatically reduced antibody recognition. The swine 1B lineage strains, which arose from previously circulating (pre-2009 pandemic) human seasonal viruses, were more antigenically similar to pre-2009 human seasonal H1 vaccine viruses than post-2009 strains. Human population immunity was measured by cross-reactivity in HI assays to representative swine H1 strains. There was a broad range of titers against each swine strain that was not associated with age, sex, or location. However, there was almost no cross-reactivity in human sera to the 1A.1.1.3 and 1B.2.1 genetic clades of swine viruses, and the 1A.1.1.3 and 1B.2.1 clades were also the most antigenically distant to the human vaccine strains. Our data demonstrate that the antigenic distances of representative swine strains from human vaccine strains represent an important part of the rational assessment of swine IAV for zoonotic risk research and pandemic preparedness prioritization. IMPORTANCE Human H1 influenza A viruses (IAV) spread to pigs in North America, resulting in a sustained circulation of two major groups of H1 viruses in swine. We quantified the genetic diversity of H1 in swine and measured antigenic phenotypes. We demonstrated that the swine H1 lineages were significantly different from the human vaccine strains and that this antigenic dissimilarity increased over time as the viruses evolved in swine. Pandemic preparedness vaccine strains for human vaccines also demonstrated a loss in similarity with contemporary swine strains. Human sera revealed a range of responses to swine IAV, including two groups of viruses with little to no immunity. The surveillance and risk assessment of IAV diversity in pig populations are essential to detect strains with reduced immunity in humans and provide critical information for pandemic preparedness.

Changes in the Hemagglutinin and Internal Gene Segments Were Needed for Human Seasonal H3 Influenza A Virus to Efficiently Infect and Replicate in Swine.

The current diversity of influenza A viruses (IAV) circulating in swine is largely a consequence of human-to-swine transmission events and consequent evolution in pigs. However, little is known about the requirements for human IAVs to transmit to and subsequently adapt in pigs. Novel human-like H3 viruses were detected in swine herds in the U.S. in 2012 and have continued to circulate and evolve in swine. We evaluated the contributions of gene segments on the ability of these viruses to infect pigs by using a series of in vitro models. For this purpose, reassortant viruses were generated by reverse genetics (rg) swapping the surface genes (hemagglutinin-HA and neuraminidase-NA) and internal gene segment backbones between a human-like H3N1 isolated from swine and a seasonal human H3N2 virus with common HA ancestry. Virus growth kinetics in porcine intestinal epithelial cells (SD-PJEC) and in ex-vivo porcine trachea explants were significantly reduced by replacing the swine-adapted HA with the human seasonal HA. Unlike the human HA, the swine-adapted HA demonstrated more abundant attachment to epithelial cells throughout the swine respiratory tract by virus histochemistry and increased entry into SD-PJEC swine cells. The human seasonal internal gene segments improved replication of the swine-adapted HA at 33 °C, but decreased replication at 40 °C. Although the HA was crucial for the infectivity in pigs and swine tissues, these results suggest that the adaptation of human seasonal H3 viruses to swine is multigenic and that the swine-adapted HA alone was not sufficient to confer the full phenotype of the wild-type swine-adapted virus.