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.

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.

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.

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.

Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico.

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

Influenza A(H1N1)pdm09 infection and viral load analysis in patients with different clinical presentations.

BACKGROUND Influenza viral load (VL) can be a decisive factor in determining the antiviral efficacy in viral clearance. OBJECTIVE This study aimed to evaluate the rate of infection and the role of influenza VL on the clinical spectrum of illnesses among different patient groups attended at a tertiary hospital in Brazil. METHODS Samples were collected from patients presenting acute respiratory infection from 2009 to 2013. Overall, 2262 samples were analysed and distributed into three groups: (i) asymptomatic (AS); (ii) symptomatic outpatients (OP); and (iii) hospitalised patients (HP). VL (expressed in Log10 RNA copies/mL) was calculated through a quantitative real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) assay aimed at the M gene, with human RNAseP target as internal control and normalising gene of threshold cycle values. FINDINGS A total of 162 (7.16%) H1N1pdm09 positive samples were analysed. Patients aged from 0.08 to 77 years old [median ± standard deviation (SD): 12.5 ± 20.54]. Children with 5 to 11 years old presented the highest detection (p < 0.0001). AS patients had the lowest VL, with a significant difference when compared with symptomatic patients (p = 0.0003). A higher VL was observed within two days of disease onset. Ten patients (HP group) received antiviral treatment and were followed up and presented a mean initial VL of 6.64 ± 1.82. A complete viral clearance for 50% of these patients was reached after 12 days of treatment. MAIN CONCLUSIONS It is important to evaluate AS patients as potential spreaders, as viral shedding was still present, even at lower VL. Our results suggest that patients with underlying diseases and severe clinical symptoms may be considered for prolonged viral treatment.

Antigenic characterization of novel H1 influenza A viruses in swine.

Novel H1N2 influenza A viruses (IAVs) in swine have been identified in Chile co-circulating with pandemic H1N1 2009-like (A(H1N1)pdm09-like) viruses. The objective of this study was to characterize antigenically the swine H1 IAVs circulating in Chile. Genetic analysis based on the HA1 domain and antigenic analysis by hemagglutination inhibition assay were carried out. Three antigenic clusters were identified, named Chilean H1 A (ChH1A), Chilean H1 B (ChH1B), and A(H1N1)pdm09-like. The antigenic sites of ChH1A and ChH1B strains were 10-60% distant from those of commercial vaccine strains at the amino acid sequence level. Antigenic variants were identified within the clusters ChH1A and A(H1N1)pdm09-like. Substitutions in the main antigenic sites (E153G in Sa, Q193H in Sb, D168N in Ca1, P137S in Ca2, and F71L in Cb) were detected in variants from the ChH1A cluster, whereas only a single substitution in antigenic site Sa (G155E) was detected in variants from A(H1N1)pdm09-like cluster, which confirms the importance to carrying out antigenic analyses in addition to genetic analyses to evaluate control measures such as vaccination. These results highlight the need to update vaccines for swine in Chile and the importance of continued surveillance to determine the onward transmission of antigenic variants in Chilean pig populations.

Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and the first case of oseltamivir-resistant strain in Myanmar 2017.

A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.

Influenza A Virus Field Surveillance at a Swine-Human Interface.

While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV.IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.

Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene.

Influenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015-2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% - 41.8% and 32.4% - 42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutination-inhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2-8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.

A Single Amino Acid at Position 431 of the PB2 Protein Determines the Virulence of H1N1 Swine Influenza Viruses in Mice.

Genetic reassortments occurred continuously among multiple subtypes or genotypes of influenza viruses prevalent in pigs. Of note, some reassortant viruses bearing the internal genes of the 2009 pandemic H1N1 (2009/H1N1) virus sporadically caused human infection, which highlights their potential threats to human public health. In this study, we performed phylogenetic analysis on swine influenza viruses (SIVs) circulating in Liaoning Province, China. A total of 22 viruses, including 18 H1N1 and 4 H1N2 viruses, were isolated from 5,750 nasal swabs collected from pigs in slaughterhouses from 2014 to 2016. H1N1 viruses formed four genotypes, which included Eurasian avian-like H1N1 (EA H1N1) and double/triple reassortant H1N1 derived from EA H1N1, 2009/H1N1, and triple reassortant H1N2 (TR H1N2) viruses. H1N1 SIVs with different genotypes and even those within the same genotypes represented different pathogenicities in mice. We further characterized two naturally isolated H1N1 SIVs that had similar viral genomes but differed substantially in their virulence in mice and found that a single amino acid at position 431 in the basic polymerase 2 (PB2) protein significantly affected the viral replication capacity and virulence of these two viruses. Taken together, our findings revealed the diverse genomic origins and virulence of the SIVs prevalent in Liaoning Province during 2014 to 2016, which highlights that continuous surveillance is essential to monitor the evolution of SIVs. We identified a naturally occurring amino acid mutation in the PB2 protein of H1N1 SIVs that impacts the viral replication and virulence in mice by altering the viral polymerase activity.IMPORTANCE The frequent reassortment among different influenza viruses in pigs adds complexity to the epidemiology of swine influenza. The diverse viral virulence phenotypes underline the need to investigate the possible genetic determinants for evaluating the pandemic potential to human public health. Here, we found that multiple genotypes of influenza viruses cocirculate in the swine population in Liaoning Province, China. Furthermore, we pinpointed a single amino acid at position 431 in the PB2 protein which plays a critical role in the virulence of H1N1 viruses in mice and found that the alteration of viral polymerase activities is the cause of the different virulence. Our study further indicated that the virulence of influenza virus is a polygenic trait, and the newly identified virulence-related residue in the PB2 provides important information for broadening knowledge on the genetic basis of viral virulence of influenza viruses.

Identification, Genetic Analysis, and Pathogenicity of Classical Swine H1N1 and Human-Swine Reassortant H1N1 Influenza Viruses from Pigs in China.

Swine influenza virus causes a substantial disease burden to swine populations worldwide and poses an imminent threat to the swine industry and humans. Given its importance, we characterized two swine influenza viruses isolated from Shandong, China. The homology and phylogenetic analyses showed that all eight gene segments of A/swine/Shandong/AV1522/2011(H1N1) were closely related to A/Maryland/12/1991(H1N1) circulating in North America. The HA, NA, M, and NS genes of the isolate were also confirmed to have a high homology to A/swine/Hubei/02/2008(H1N1) which appeared in China in 2008, and the virus was clustered into the classical swine lineage. The gene segments of A/swine/Shandong/AV1523/2011(H1N1) were highly homologous to the early human H1N1 and H2N2 influenza viruses, except for the HA gene, and the virus was a reassortant H1N1 virus containing genes from the classical swine (HA) and human (NA, PB2, PB1, PA, NP, M, and NS) lineages. Both the viruses could cause lethal infection and replicate efficiently in the lungs, brains, spleens, and kidneys of mice. Histopathological examinations showed that AV1522 and AV1523 viruses caused a spectrum of marked pneumonia and meningoencephalitis according to the duration of infection, demonstrating a progression of respiratory disease and neurological disease over the course of infection that ultimately resulted in lethality for the infected mice. The changes in the pathogenicity of swine influenza viruses to mammals, accompanied with the continuous reassortment and evolution of the viruses, highlights the importance of ongoing epidemiological investigation.