Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

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.

Pulmonary MicroRNA expression after heterologous challenge with swine influenza A virus (H1N2) in immunized and non-immunized pigs.

MicroRNAs (miRNAs) contribute to post-transcriptional modulation of the host response during influenza A virus (IAV) infection and may be involved in shaping disease severity. Differential disease severity was achieved in two groups of pigs by immunization of one group with a commercial swine IAV vaccine prior to heterologous IAV (H1N2) challenge of both groups. Lung tissue was harvested 1, 3, and 14 days after challenge and miRNA expression was quantified. Gene Ontology term enrichment analysis was employed to examine the functional relevance of genes potentially regulated by differentially expressed miRNAs in pigs with varying degrees of disease severity following IAV infection. Results suggested that the miRNA response associated with less severe disease may modulate host mechanisms essential for viral life cycle, e.g. transcription, translation, and protein trafficking. During more severe disease, miRNA-mediated regulation may focus on dampening virus-specific processes e.g. virion assembly and viral protein processing, and controlling host metabolism.

Pathogenicity and escape to pre-existing immunity of a new genotype of swine influenza H1N2 virus that emerged in France in 2020.

In 2020, a new genotype of swine H1N2 influenza virus (H1avN2-HA 1C.2.4) was identified in France. It rapidly spread within the pig population and supplanted the previously predominant H1avN1-HA 1C.2.1 virus. To characterize this new genotype which is genetically and antigenically distant from the other H1avNx viruses detected in France, an experimental study was conducted to compare the outcomes of H1avN2 and H1avN1 infections in pigs and evaluate the protection conferred by the only inactivated vaccine currently licensed in Europe containing an HA 1C (clade 1C.2.2) antigen. Infection with H1avN2 induced stronger clinical signs and earlier shedding than H1avN1. The neutralizing antibodies produced following H1avN2 infection were unable to neutralize H1avN1, and vice versa, whereas the cellular-mediated immunity cross-reacted. Vaccination slightly altered the impact of H1avN2 infection at the clinical level, but did not prevent shedding of infectious virus particles. It induced a cellular-mediated immune response towards H1avN2, but did not produce neutralizing antibodies against this virus. As in vaccinated animals, animals previously infected by H1avN1 developed a cross-reacting cellular immune response but no neutralizing antibodies against H1avN2. However, H1avN1 pre-infection induced a better protection against the H1avN2 infection than vaccination, probably due to higher levels of non-neutralizing antibodies and a mucosal immunity. Altogether, these results showed that the new H1avN2 genotype induced a severe respiratory infection and that the actual vaccine was less effective against this H1avN2-HA 1C.2.4 than against H1avN1-HA 1C.2.1, which may have contributed to the H1avN2 epizootic and dissemination in pig farms in France.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

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.

The evolution, pathogenicity and transmissibility of quadruple reassortant H1N2 swine influenza virus in China: A potential threat to public health.

Swine are regarded as "intermediate hosts" or "mixing vessels" of influenza viruses, capable of generating strains with pandemic potential. From 2020 to 2021, we conducted surveillance on swine H1N2 influenza (swH1N2) viruses in swine farms located in Guangdong, Yunnan, and Guizhou provinces in southern China, as well as Henan and Shandong provinces in northern China. We systematically analyzed the evolution and pathogenicity of swH1N2 isolates, and characterized their replication and transmission abilities. The isolated viruses are quadruple reassortant H1N2 viruses containing genes from pdm/09 H1N1 (PB2, PB1, PA and NP genes), triple-reassortant swine (NS gene), Eurasian Avian-like (HA and M genes), and recent human H3N2 (NA gene) lineages. The NA, PB2, and NP of SW/188/20 and SW/198/20 show high gene similarities to A/Guangdong/Yue Fang277/2017 (H3N2). The HA gene of swH1N2 exhibits a high evolutionary rate. The five swH1N2 isolates replicate efficiently in human, canine, and swine cells, as well as in the turbinate, trachea, and lungs of mice. A/swine/Shandong/198/2020 strain efficiently replicates in the respiratory tract of pigs and effectively transmitted among them. Collectively, these current swH1N2 viruses possess zoonotic potential, highlighting the need for strengthened surveillance of swH1N2 viruses.

Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals.

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian "avian-like" (EA) swine H1N1 and "human-like" H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals.

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.

Human infection with a reassortant swine-origin influenza A(H1N2)v virus in Taiwan, 2021.

BACKGROUND: Influenza A virus infections occur in different species, causing mild-to-severe symptoms that lead to a heavy disease burden. H1N1, H1N2 and H3N2 are major subtypes of swine influenza A viruses in pigs and occasionally infect humans. METHODS: A case infected by novel influenza virus was found through laboratory surveillance system for influenza viruses. Clinical specimens were tested by virus culture and/or real-time RT-PCR. The virus was identified and characterized by gene sequencing and phylogenetic analysis. RESULTS: In 2021, for the first time in Taiwan, an influenza A(H1N2)v virus was isolated from a 5-year old girl who was suffering from fever, runny nose and cough. The isolated virus was designated A/Taiwan/1/2021(H1N2)v. Full-genome sequencing and phylogenetic analyses revealed that A/Taiwan/1/2021(H1N2)v is a novel reassortant virus containing hemagglutinin (HA) and neuraminidase (NA) gene segments derived from swine influenza A(H1N2) viruses that may have been circulating in Taiwan for decades, and the other 6 internal genes (PB2, PB2, PA, NP, M and NS) are from human A(H1N1)pdm09 viruses. CONCLUSION: Notably, the HA and NA genes of A/Taiwan/1/2021(H1N2)v separately belong to specific clades that are unique for Taiwanese swine and were proposed to be introduced from humans in different time periods. Bidirectional transmission between humans and swine contributes to influenza virus diversity and poses the next pandemic threat.

Prevalence, Genetics and Evolutionary Properties of Eurasian Avian-like H1N1 Swine Influenza Viruses in Liaoning.

Swine influenza virus (SIV) is an important zoonosis pathogen. The 2009 pandemic of H1N1 influenza A virus (2009/H1N1) highlighted the importance of the role of pigs as intermediate hosts. Liaoning province, located in northeastern China, has become one of the largest pig-farming areas since 2016. However, the epidemiology and evolutionary properties of SIVs in Liaoning are largely unknown. We performed systematic epidemiological and genetic dynamics surveillance of SIVs in Liaoning province during 2020. In total, 33,195 pig nasal swabs were collected, with an SIV detection rate of 2%. Our analysis revealed that multiple subtypes of SIVs are co-circulating in the pig population in Liaoning, including H1N1, H1N2 and H3N2 SIVs. Furthermore, 24 H1N1 SIVs were confirmed to belong to the EA H1N1 lineage and divided into two genotypes. The two genotypes were both triple reassortant, and the predominant one with polymerase, nucleoprotein (NP), and matrix protein (M) genes originating from 2009/H1N1; hemagglutinin (HA) and neuraminidase (NA) genes originating from EA H1N1; and the nonstructural protein (NS) gene originating from triple reassortant H1N2 (TR H1N2) was detected in Liaoning for the first time. According to our evolutionary analysis, the EA H1N1 virus in Liaoning will undergo further genome variation.

Predicting Cross-Species Infection of Swine Influenza Virus with Representation Learning of Amino Acid Features.

Swine influenza viruses (SIVs) can unforeseeably cross the species barriers and directly infect humans, which pose huge challenges for public health and trigger pandemic risk at irregular intervals. Computational tools are needed to predict infection phenotype and early pandemic risk of SIVs. For this purpose, we propose a feature representation algorithm to predict cross-species infection of SIVs. We built a high-quality dataset of 1902 viruses. A feature representation learning scheme was applied to learn feature representations from 64 well-trained random forest models with multiple feature descriptors of mutant amino acid in the viral proteins, including compositional information, position-specific information, and physicochemical properties. Class and probabilistic information were integrated into the feature representations, and redundant features were removed by feature space optimization. High performance was achieved using 20 informative features and 22 probabilistic information. The proposed method will facilitate SIV characterization of transmission phenotype.

Co-circulation of multiple influenza A reassortants in swine harboring genes from seasonal human and swine influenza viruses.

Since the influenza pandemic in 2009, there has been an increased focus on swine influenza A virus (swIAV) surveillance. This paper describes the results of the surveillance of swIAV in Danish swine from 2011 to 2018. In total, 3800 submissions were received with a steady increase in swIAV-positive submissions, reaching 56% in 2018. Full-genome sequences were obtained from 129 swIAV-positive samples. Altogether, 17 different circulating genotypes were identified including six novel reassortants harboring human seasonal IAV gene segments. The phylogenetic analysis revealed substantial genetic drift and also evidence of positive selection occurring mainly in antigenic sites of the hemagglutinin protein and confirmed the presence of a swine divergent cluster among the H1pdm09Nx (clade 1A.3.3.2) viruses. The results provide essential data for the control of swIAV in pigs and emphasize the importance of contemporary surveillance for discovering novel swIAV strains posing a potential threat to the human population.

A bivalent live attenuated influenza virus vaccine protects against H1N2 and H3N2 viral infection in swine.

Swine Influenza A virus (swIAV) poses a substantial burden to the swine industry due to its highly contagious nature, acute viral disease, and ability to cause up to 100 % morbidity. Currently, North American swine are predominately infected with three subtypes of swIAV: H1N1, H1N2, and H3N2. The ability of influenza viruses to cross both directions between humans and swine means that both human and swine-origin viruses as well as new reassortant viruses can pose a substantial public health or pandemic threat. Since the primary method of protection and control against influenza is through vaccination, more effective, new vaccine platforms need to be developed. This study uses two Canadian swIAV isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69] to design a bivalent live attenuated influenza virus vaccine (LAIV) through reverse genetics. The hemagglutinin (HA) cleavage site from both SD191-WT and SD69-WT were engineered from a trypsin-sensitive to an elastase-sensitive motif, to generate SD191-R342V and SD69-K345V, respectively. The elastase dependent SD191-R342V virus possesses a mutation from arginine to valine at amino acid (aa) 342 on HA, whereas the elastase dependent SD69-K345V virus possesses a mutation from lysine to valine at aa 345 on HA. Both elastase dependent swIAVs are completely dependent on elastase, display comparable growth properties to the wild type (WT) viruses, are genetically stable in vitro, and entirely non-virulent in pigs. Moreover, when these elastase dependent swIAVs were administered together in pigs, they were found to stimulate antibody responses and IFN-γ secreting cells, as well as prevent viral replication and lung pathology associated with WT H1N2 and H3N2 swIAV challenge. Therefore, this bivalent LAIV demonstrates the strong candidacy to protect swine against the predominant influenza subtypes in North America.

Ser-Leu substitution at P2 position of the hemagglutinin cleavage site attenuates replication and pathogenicity of Eurasian avian-like H1N2 swine influenza viruses.

Swine influenza viruses not only constitute a potential economic problem for livestock, but also pose a substantial threat to human health. Mutation in the proteolytic cleavage site of hemagglutinin (HA) is recognized as an essential factor of tissue tropism and viral pathogenicity. However, the molecular properties of the cleavage site of Eurasian avian-like swine (EA) H1N2 virus remain largely unknown. In this study, we found a serine-leucine (Ser-Leu) substitution at the P2 position of the HA cleavage site (S328 L) in naturally occurring EA H1N2 virus. To study the effect of this substitution, we used reverse genetics to generate recombinant wild-type and mutant viruses containing a single amino acid mutation at the P2 position in A/swine/Guangdong/YJ28/2014 (YJ28) or A/swine/Guangdong/DG2/2015 (DG2) background. In vitro experiments showed that the Ser-Leu substitution at the P2 position attenuated the viral replication and HA cleavage efficiency. In vivo analyses revealed that, while all mice inoculated with r/DG2-S328 L or r/YJ28 viruses survived, the survival rates of r/DG2- and r/YJ28-L328S-inoculated animals were 20 % and 40 %, respectively. Furthermore, the Ser-Leu substitution at the P2 position attenuated the replication in nasal turbinate and lungs. In summary, this amino acid change may be useful to understand the molecular properties of the cleavage site and be valuable for vaccine development.

Swine Influenza A Viruses and the Tangled Relationship with Humans.

Influenza A viruses (IAVs) are the causative agents of one of the most important viral respiratory diseases in pigs and humans. Human and swine IAV are prone to interspecies transmission, leading to regular incursions from human to pig and vice versa. This bidirectional transmission of IAV has heavily influenced the evolutionary history of IAV in both species. Transmission of distinct human seasonal lineages to pigs, followed by sustained within-host transmission and rapid adaptation and evolution, represent a considerable challenge for pig health and production. Consequently, although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine around the world, extensive diversity can be found in the hemagglutinin (HA) and neuraminidase (NA) genes, as well as the remaining six genes. We review the complicated global epidemiology of IAV in swine and the inextricably entangled implications for public health and influenza pandemic planning.

Genetic Variability among Swine Influenza Viruses in Italy: Data Analysis of the Period 2017-2020.

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs' evolution could generate strains with zoonotic potential.

Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus.

The surveillance of swine influenza A viruses in France revealed the emergence of an antigenic variant following deletions and mutations that are fixed in the HA-encoding gene of the European human-like reassortant swine H1N2 lineage. In this study, we compared the outcomes of the parental (H1huN2) and variant (H1huN2Δ146-147) virus infections in experimentally-inoculated piglets. Moreover, we assessed and compared the protection that was conferred by an inactivated vaccine currently licensed in Europe. Three groups of five unvaccinated or vaccinated piglets were inoculated with H1huN2 or H1huN2Δ146-147 or mock-inoculated, respectively. In unvaccinated piglets, the variant strain induced greater clinical signs than the parental virus, in relation to a higher inflammatory response that involves TNF-α production and a huge afflux of granulocytes into the lung. However, both infections led to similar levels of virus excretion and adaptive (humoral and cellular) immune responses in blood. The vaccinated animals were clinically protected from both infectious challenges and did not exhibit any inflammatory responses, regardless the inoculated virus. However, whereas vaccination prevented virus shedding in H1huN2-infected animals, it did not completely inhibit the multiplication of the variant strain, since live virus particles were detected in nasal secretions that were taken from H1huN2Δ146-147-inoculated vaccinated piglets. This difference in the level of vaccine protection was probably related to the poorer ability of the post-vaccine antibodies to neutralize the variant virus than the parental virus, even though post-vaccine cellular immunity appeared to be equally effective against both viruses. These results suggest that vaccine antigens would potentially need to be updated if this variant becomes established in Europe.