Molecular epidemiology and vaccine compatibility analysis of seasonal influenza A viruses in the context of COVID-19 epidemic in Wuhan, China.

The COVID-19 pandemic had a significant impact on the global influenza vaccination and the epidemics of seasonal influenza. To further explore the molecular epidemiology of influenza viruses and assess vaccine effectiveness, we collected influenza cases in Wuhan during the 2022-2023 influenza season. Among 1312 clinical samples, 312 samples tested positive for influenza viruses using reverse transcription polymerase chain reaction. These positive samples included 146A/H1N1 subtypes (46.8%), 164A/H3N2 subtypes (52.6%) and 2 influenza B virus types (0.6%). Based on the whole genome sequence information of hemagglutinin (HA) and neuraminidase (NA) from 27A/H1N1 influenza virus strains and 26A/H3N2 influenza virus strains obtained in this study, a phylogenetic analysis was conducted. The analysis revealed that all A/H1N1 strains belonged to the evolutionary branch 6B.1A.5a.2a, and they exhibited specific substitutions at positions K71Q, Q206E, E241A, and R276K. Similarly, all A/H3N2 strains were classified into the 3C.2a1b.2a.1a subclade and displayed amino acid substitutions at positions S172H, N175Y, I176T, K187N, and S214P. Notably, the A/H3N2 strains also acquired a new potential glycosylation site at position N174. Using an epitope model, the predicted vaccine effectiveness was assessed for the A/H1N1 and A/H3N2 strains. The predicted vaccine effectiveness against the Wuhan influenza epidemic strain was over 85% for the A/H1N1 vaccine strain. However, the effectiveness against the A/H3N2 vaccine strain was only 48.7%. To further verify the protection of influenza vaccine against circulating influenza viruses in the region, we conducted in vivo and in vitro animal studies. The results of in vitro neutralization experiment showed that rabbit serum antibodies inoculated with quadrivalent isolated influenza vaccine had neutralization ability against all 24 isolated influenza viruses. In vivo experiments showed that vaccinated mice had fewer lung lesions when infected with the influenza strain circulating in Wuhan, suggesting that vaccination can effectively reduce the occurrence of severe lung damage. These findings emphasize the importance of accurately predicting seasonal influenza strains for effective influenza prevention and control, especially during the co-circulation of SARS-CoV-2 and influenza viruses. This study provides valuable information on the seasonal influenza virus in Wuhan during the COVID-19 pandemic and serves as a basis for vaccine prediction and updates.

First detection of influenza A virus subtypes H1N1 and H3N8 in the Antarctic region: King George Island, 2023.

RELEVANCE: Influenza A virus is characterized by a segmented single-stranded RNA genome. Such organization of the virus genome determines the possibility of reassortment, which can lead to the emergence of new virus variants. The main natural reservoir of most influenza A virus subtypes are wild waterfowl. Seasonal migrations gather waterfowl from all major migration routes to nesting areas near the northern and southern polar circles. This makes intercontinental spread of influenza A viruses possible. Objective ‒ to conduct molecular genetic monitoring and study the phylogenetic relationships of influenza A virus variants circulating in Antarctica in 2023. MATERIALS AND METHODS: We studied 84 samples of biological material obtained from birds and marine mammals in April‒May 2023 in coastal areas of Antarctica. For 3 samples, sequencing was performed on the Miseq, Illumina platform and phylogenetic analysis of the obtained nucleotide sequences of the influenza A virus genomes was performed. RESULTS: The circulation of avian influenza virus in the Antarctic region was confirmed. Heterogeneity of the pool of circulating variants of the influenza A virus (H3N8, H1N1) was revealed. Full-length genomes of the avian influenza virus were sequenced and posted in the GISAID database (EPI_ISL_19032103, 19174530, 19174467). CONCLUSION: The study of the genetic diversity of influenza A viruses circulating in the polar regions of the Earth and the identification of the conditions for the emergence of new genetic variants is a relevant task for the development of measures to prevent biological threats.

Whole-Genome Analysis of the Influenza A(H1N1)pdm09 Viruses Isolated from Influenza-like Illness Outpatients in Myanmar and Community-Acquired Oseltamivir-Resistant Strains Present from 2015 to 2019.

In this study, we describe the genetic characteristics of influenza A(H1N1)pdm09 strains detected in Myanmar from 2015 to 2019. Whole genomes from 60 A(H1N1)pdm09 virus isolates were amplified using real-time polymerase chain reaction and successfully sequenced using the Illumina iSeq100 platforms. Eight individual phylogenetic trees were retrieved for each segment along with those of the World Health Organization (WHO)-recommended Southern Hemisphere vaccine strains for the respective years. A(H1N1)pdm09 viruses from 2015 were found to belong to clade 6B, those from 2016 to 6B.1, 2017 to 6B.1A, and 2019 to 6B.1A.5a, and were genetically distinct from the Southern Hemisphere vaccine strains for the respective seasons, A/California/7/2009 and A/Michigan/45/2015. We observed one virus with intra-subtype reassortment, collected in the 2015 season. Importantly, three viruses possessed the H275Y substitution in the neuraminidase protein, appearing to be community-acquired without the prior administration of neuraminidase inhibitors. These viruses exhibited highly reduced susceptibility to oseltamivir and peramivir. This study demonstrates the importance of monitoring genetic variations in influenza viruses that will contribute to the selection of global influenza vaccines.

Genomic evolution of influenza during the 2023-2024 season, the johns hopkins health system.

Influenza, a human disease caused by viruses in the Orthomyxoviridae family, is estimated to infect 5% -10 % of adults and 20% -30 % of children annually. Influenza A (IAV) and Influenza B (IBV) viruses accumulate amino acid substitutions (AAS) in the hemagglutinin (HA) and neuraminidase (NA) proteins seasonally. These changes, as well as the dominating viral subtypes, vary depending on geographical location, which may impact disease prevalence and the severity of the season. Genomic surveillance is crucial for capturing circulation patterns and characterizing AAS that may affect disease outcomes, vaccine efficacy, or antiviral drug activities. In this study, whole-genome sequencing of IAV and IBV was attempted on positive remnant clinical samples (587) collected from 580 patients between June 2023 and February 2024 in the Johns Hopkins Health System (JHHS). Full-length HA segments were obtained from 424 (72.2 %) samples. H1N1pdm09 (71.7 %) was the predominant IAV subtype, followed by H3N2 (16.7 %) and IBV-Victoria clade V1A.3a.2 (11.6 %). Within H1N1pdm09 HA sequences, the 6B1A.5a.2a.1 (60.5 %) clade was the most represented. Full-length NA segments were obtained from 421 (71.7 %) samples. Within H1N1pdm09 and IBV, AAS previously proposed to change susceptibility to NA inhibitors were infrequently detected. Phylogeny of HA and NA demonstrated heterogeneous HA and NA H1N1pdm09 and IBV subclades. No significant differences were observed in admission rates or use of supplemental oxygen between different subtypes or clades. Influenza virus genomic surveillance is essential for understanding the seasonal evolution of influenza viruses and their association with disease prevalence and outcomes.

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.

Resurgence of influenza with increased genetic diversity of circulating viruses during the 2022-2023 season.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.

The evolution, complexity, and diversity of swine influenza viruses in China: A hidden public health threat.

Swine influenza viruses (SIVs), including H1N1, H1N2, and H3N2, have spread throughout the global pig population. Potential pandemics are a concern with the recent sporadic cross-species transmission of SIVs to humans. We collected 1421 samples from Guangdong, Fujian, Henan, Yunnan and Jiangxi provinces during 2017-2018 and isolated 29 viruses. These included 21H1N1, 5H1N2, and 3H3N2 strains. Genome analysis showed that the domestic epidemic genotypes of H1N1 were mainly G4 and G5 reassortant EA swine H1N1. These genotypes have a clear epidemic advantage. Two strains were Clade 6B.1 pdm/09H1N1, suggesting a possible pig-to-human transmission route. Notably, three new H1N2 genotypes were identified using the genomic backbones of G4 or G5 viruses for recombination. The identification of various subtypes and genotypes highlight the complexity and diversity of SIVs in China and need for continuous monitoring of SIV evolution to assess the risks and prepare for potential influenza pandemics.

Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) virus in a child in Shandong Province, China.

BACKGROUND: Influenza A virus infections can occur in multiple species. Eurasian avian-like swine influenza A (H1N1) viruses (EAS-H1N1) are predominant in swine and occasionally infect humans. A Eurasian avian-like swine influenza A (H1N1) virus was isolated from a boy who was suffering from fever; this strain was designated A/Shandong-binzhou/01/2021 (H1N1). The aims of this study were to investigate the characteristics of this virus and to draw attention to the need for surveillance of influenza virus infection in swine and humans. METHODS: Throat-swab specimens were collected and subjected to real-time fluorescent quantitative polymerase chain reaction (RT‒PCR). Positive clinical specimens were inoculated onto Madin-Darby canine kidney (MDCK) cells to isolate the virus, which was confirmed by a haemagglutination assay. Then, whole-genome sequencing was carried out using an Illumina MiSeq platform, and phylogenetic analysis was performed with MEGA X software. RESULTS: RT‒PCR revealed that the throat-swab specimens were positive for EAS-H1N1, and the virus was subsequently successfully isolated from MDCK cells; this strain was named A/Shandong-binzhou/01/2021 (H1N1). Whole-genome sequencing and phylogenetic analysis revealed that A/Shandong-binzhou/01/2021 (H1N1) is a novel triple-reassortant EAS-H1N1 lineage that contains gene segments from EAS-H1N1 (HA and NA), triple-reassortant swine influenza H1N2 virus (NS) and A(H1N1) pdm09 viruses (PB2, PB1, PA, NP and MP). CONCLUSIONS: The isolation and analysis of the A/Shandong-binzhou/01/2021 (H1N1) virus provide further evidence that EAS-H1N1 poses a threat to human health, and greater attention should be given to the surveillance of influenza virus infections in swine and humans.

Genotypic Variants of Pandemic H1N1 Influenza A Viruses Isolated from Severe Acute Respiratory Infections in Ukraine during the 2015/16 Influenza Season.

Human type A influenza viruses A(H1N1)pdm09 have caused seasonal epidemics of influenza since the 2009-2010 pandemic. A(H1N1)pdm09 viruses had a leading role in the severe epidemic season of 2015/16 in the Northern Hemisphere and caused a high incidence of acute respiratory infection (ARI) in Ukraine. Serious complications of influenza-associated severe ARI (SARI) were observed in the very young and individuals at increased risk, and 391 fatal cases occurred in the 2015/16 epidemic season. We analyzed the genetic changes in the genomes of A(H1N1)pdm09 influenza viruses isolated from SARI cases in Ukraine during the 2015/16 season. The viral hemagglutinin (HA) fell in H1 group 6B.1 for all but four isolates, with known mutations affecting glycosylation, the Sa antigenic site (S162N in all 6B.1 isolates), or virulence (D222G/N in two isolates). Other mutations occurred in antigenic site Ca (A141P and S236P), and a subgroup of four strains were in group 6B.2, with potential alterations to antigenicity in A(H1N1)pdm09 viruses circulating in 2015/16 in Ukraine. A cluster of Ukrainian isolates exhibited novel D2E and N48S mutations in the RNA binding domain, and E125D in the effector domain, of immune evasion nonstructural protein 1 (NS1). The diverse spectrum of amino-acid substitutions in HA, NS1, and other viral proteins including nucleoprotein (NP) and the polymerase complex suggested the concurrent circulation of multiple lineages of A(H1N1)pdm09 influenza viruses in the human population in Ukraine, a country with low vaccination coverage, complicating public health measures against influenza.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Antibody Responsiveness to Influenza: What Drives It?

The induction of a specific antibody response has long been accepted as a serological hallmark of recent infection or antigen exposure. Much of our understanding of the influenza antibody response has been derived from studying antibodies that target the hemagglutinin (HA) protein. However, growing evidence points to limitations associated with this approach. In this review, we aim to highlight the issue of antibody non-responsiveness after influenza virus infection and vaccination. We will then provide an overview of the major factors known to influence antibody responsiveness to influenza after infection and vaccination. We discuss the biological factors such as age, sex, influence of prior immunity, genetics, and some chronic infections that may affect the induction of influenza antibody responses. We also discuss the technical factors, such as assay choices, strain variations, and viral properties that may influence the sensitivity of the assays used to measure influenza antibodies. Understanding these factors will hopefully provide a more comprehensive picture of what influenza immunogenicity and protection means, which will be important in our effort to improve influenza vaccines.

Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay.

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/µL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.

Analysis of the Evolution of Pandemic Influenza A(H1N1) Virus Neuraminidase Reveals Entanglement of Different Phenotypic Characteristics.

The influenza A virus (IAV) neuraminidase (NA) is essential for virion release from cells and decoy receptors and an important target of antiviral drugs and antibodies. Adaptation to a new host sialome and escape from the host immune system are forces driving the selection of mutations in the NA gene. Phylogenetic analysis shows that until 2015, 16 amino acid substitutions in NA became fixed in the virus population after introduction in the human population of the pandemic IAV H1N1 (H1N1pdm09) in 2009. The accumulative effect of these substitutions, in the order in which they appeared, was analyzed using recombinant proteins and viruses in combination with different functional assays. The results indicate that NA activity did not evolve to a single optimum but rather fluctuated within a certain bandwidth. Furthermore, antigenic and enzymatic properties of NA were intertwined, with several residues affecting multiple properties. For example, the substitution K432E in the second sialic acid binding site, next to the catalytic site, was shown to affect catalytic activity, substrate specificity, and the pH optimum for maximum activity. This substitution also altered antigenicity of NA, which may explain its selection. We propose that the entanglement of NA phenotypes may be an important determining factor in the evolution of NA.IMPORTANCE Since its emergence in 2009, the pandemic H1N1 influenza A virus (IAV) has caused significant disease and mortality in humans. IAVs contain two envelope glycoproteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA). NA is essential for virion release from cells and decoy receptors, is an important target of antiviral drugs, and is increasingly being recognized as an important vaccine antigen. Not much is known, however, about the evolution of this protein upon the emergence of the novel pandemic H1N1 virus, with respect to its enzymatic activity and antigenicity. By reconstructing the evolutionary path of NA, we show that antigenic and enzymatic properties of NA are intertwined, with several residues affecting multiple properties. Understanding the entanglement of NA phenotypes will lead to better comprehension of IAV evolution and may help the development of NA-based vaccines.

Next-Generation Sequencing Analysis of the Within-Host Genetic Diversity of Influenza A(H1N1)pdm09 Viruses in the Upper and Lower Respiratory Tracts of Patients with Severe Influenza.

The influenza A(H1N1)pdm09 virus emerged in April 2009 with an unusual incidence of severe disease and mortality, and currently circulates as a seasonal influenza virus. Previous studies using consensus viral genome sequencing data have overlooked the viral genomic and phenotypic diversity. Next-generation sequencing (NGS) may instead be used to characterize viral populations in an unbiased manner and to measure within-host genetic diversity. In this study, we used NGS analysis to investigate the within-host genetic diversity of influenza A(H1N1)pdm09 virus in the upper and lower respiratory samples from nine patients who were admitted to the intensive care unit (ICU). A total of 47 amino acid substitution positions were found to differ between the upper and lower respiratory tract samples from all patients. However, the D222G/N substitution in hemagglutinin (HA) protein was the only amino acid substitution common to multiple patients. Furthermore, the substitution was detected only in the six samples from the lower respiratory tract. Therefore, it is important to investigate influenza A(H1N1)pdm09 virus populations using multiple paired samples from the upper and lower respiratory tract to avoid overlooking potentially important substitutions, especially in patients with severe disease.IMPORTANCE The D222G/N substitution in the hemagglutinin (HA) protein of influenza A(H1N1)pdm09 virus has been reported to be associated with disease severity and mortality in numerous previous studies. In the present study, 75% of lower respiratory samples contained heterogeneous influenza populations that carried different amino acids at position 222 of the HA protein, whereas all upper respiratory samples only contained the wild-type 222D. These results suggest the influenza A(H1N1)pdm09 virus has diversified inside the host owing to differences in tissue specificity. In this study, the within-host genetic diversity of influenza A(H1N1)pdm09 virus was investigated for the first time using next-generation sequencing analysis of the viral whole-genome in samples extracted from the upper and lower respiratory tracts of patients with severe disease.

Distribution and evolution of H1N1 influenza A viruses with adamantanes-resistant mutations worldwide from 1918 to 2019.

H1N1 influenza is a kind of acute respiratory infectious disease that has a high socioeconomic and medical burden each year around the world. In the past decades, H1N1 influenza viruses have exhibited high resistance to adamantanes, which has become a serious issue. To understand the up-to-date distribution and evolution of H1N1 influenza viruses with adamantanes-resistant mutations, we conducted a deep analysis of 15875 M2 protein and 8351 MP nucleotides sequences. Results of the distribution analyses showed that 77.32% of H1N1 influenza viruses harbored-resistance mutations of which 73.52% were S31N, And the mutant variants mainly appeared in North America and Europe and H1N1 influenza viruses with S31N mutation became the circulating strains since 2009 all over the world. In addition, 80.65% of human H1N1 influenza viruses and 74.61% of swine H1N1 influenza viruses exhibited adamantanes resistance, while the frequency was only 1.86% in avian H1N1 influenza viruses. Studies from evolutionary analyses indicated that the avian-origin swine H1N1 influenza viruses replaced the classical human H1N1 influenza viruses and became the circulating strains after 2009; The interspecies transmission among avian, swine, and human strains over the past 20 years contributed to the 2009 swine influenza pandemic. Results of our study clearly clarify the historical drug resistance level of H1N1 influenza viruses around the world and demonstrated the evolution of adamantanes-resistant mutations in H1N1 influenza viruses. Our findings emphasize the necessity for monitoring the adamantanes susceptibility of H1N1 influenza viruses and draw attention to analyses of the evolution of drug-resistant H1N1 influenza variants.

Emergence of Novel Reassortant H1N1 Avian Influenza Viruses in Korean Wild Ducks in 2018 and 2019.

Influenza A virus subtype H1N1 has caused global pandemics like the "Spanish flu" in 1918 and the 2009 H1N1 pandemic several times. H1N1 remains in circulation and survives in multiple animal sources, including wild birds. Surveillance during the winter of 2018-2019 in Korea revealed two H1N1 isolates in samples collected from wild bird feces: KNU18-64 (A/Greater white-fronted goose/South Korea/KNU18-64/2018(H1N1) and WKU19-4 (A/wild bird/South Korea/WKU19-4/2019(H1N1). Phylogenetic analysis indicated that M gene of KNU18-64(H1N1) isolate resembles that of the Alaskan avian influenza virus, whereas WKU19-4(H1N1) appears to be closer to the Mongolian virus. Molecular characterization revealed that they harbor the amino acid sequence PSIQRSGLF and are low-pathogenicity influenza viruses. In particular, the two isolates harbored three different mutation sites, indicating that they have different virulence characteristics. The mutations in the PB1-F2 and PA protein of WKU19-4(H1N1) indicate increasing polymerase activity. These results corroborate the kinetic growth data for WKU19-4 in MDCK cells: a dramatic increase in the viral titer after 12 h post-inoculation compared with that in the control group H1N1 (CA/04/09(pdm09)). The KNU18-64(H1N1) isolate carries mutations indicating an increase in mammal adaptation; this characterization was confirmed by the animal study in mice. The KNU18-64(H1N1) group showed the presence of viruses in the lungs at days 3 and 6 post-infection, with titers of 2.71 ± 0.16 and 3.71 ± 0.25 log10(TCID50/mL), respectively, whereas the virus was only detected in the WKU19-4(H1N1) group at day 6 post-infection, with a lower titer of 2.75 ± 0.51 log10(TCID50/mL). The present study supports the theory that there is a relationship between Korea and America with regard to reassortment to produce novel viral strains. Therefore, there is a need for increased surveillance of influenza virus circulation in free-flying and wild land-based birds in Korea, particularly with regard to Alaskan and Asian strains.

Persistence of pdm2009-H1N1 internal genes of swine influenza in pigs, Thailand.

Swine influenza is one of the important zoonotic diseases of pigs. We conducted a longitudinal survey of swine influenza A viruses (S-IAV) circulating in a pig farm with history of endemic S-IAV infection from 2017 to 2018. The samples were collected from 436 pigs including nasal swab samples (n = 436) and blood samples (n = 436). Our result showed that 18.81% (82/436) were positive for influenza A virus and subsequently 57 S-IAV could be isolated. Then 24 out of 57 S-IAVs were selected for whole genome sequencing and could be subtyped as S-IAV-H1N1 (n = 18) and S-IAV-H3N2 (n = 6). Of 24 S-IAVs, we observed 3 genotypes of S-IAVs including rH1N1 (pdm + 1), rH1N1 (pdm + 2), and rH3N2 (pdm + 2). Since all genotypes of S-IAVs in this study contained internal genes from pdmH1N1-2009, it could be speculated that pdmH1N1-2009 was introduced in a pig farm and then multiple reassorted with endemic S-IAVs to generate diversify S-IAV genotypes. Our study supported and added the evidences that pdmH1N1-2009 and it reassortant have predominately persisted in pig population in Thailand. Thus, monitoring of S-IAVs in pigs, farm workers and veterinarians in pig farms is important and should be routinely conducted.

Zoonotic infection with swine A/H1avN1 influenza virus in a child, Germany, June 2020.

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.

Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection.

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.