Pandemic H1N1 Influenza Research Articles

Pre-existing H1N1 immunity reduces severe disease with bovine H5N1 influenza virus.

The emergence of highly pathogenic H5N1 avian influenza in dairy cattle herds across the United States has caused multiple mild human infections. There is an urgent need to understand the risk of spillover into humans. Here, we show that pre-existing immunity from the 2009 H1N1 pandemic influenza virus provided protection from mortality and severe clinical disease to ferrets intranasally infected with bovine H5N1. H1N1 immune ferrets exhibited a differential tissue tropism with little bovine H5N1 viral dissemination to organs outside the respiratory tract and significantly less H5N1 virus found in nasal secretions and the respiratory tract. Additionally, ferrets with H1N1 prior immunity produced antibodies that cross-reacted with H5N1 neuraminidase protein. Taken together, these results suggest that mild disease in humans may be linked to prior immunity to human seasonal influenza viruses.

‘It’s really embarrassing […] to ask for help:’ navigating invisible and intersecting inequities in barriers to getting vaccinated

The arrival of COVID-19 vaccines in Canada marked the first time since the H1N1 influenza pandemic that adult vaccination became central in public health discourse, thus evoking feelings and discourse about individuals’ responsibility to get vaccinated. Public health institutions made efforts to ensure vaccines were accessible, and various local organizations worked to fill gaps in accessibility (e.g. vaccine clinics for equity-denied communities). However, despite knowledge of barriers to accessing healthcare, and specifically vaccines, public and health discourses emphasized individual objection as the main barrier to optimal vaccine uptake. This study explored how individuals who identified as a racialized minority or Indigenous person, perceived their personal responsibility to get vaccinated. We gathered perspectives in qualitative interviews (N = 27) when vaccines were first becoming available to adults living in Canada (May-June 2021). Theory about moral regulation, risk governance, and emotion informed our analysis. Findings suggest that individuals engaged in the governance of oneself and others towards getting vaccinated. However, participants also critiqued discourses that hold individuals responsible for accessing vaccines. They identified substantial barriers to accessing vaccines for individuals with fewer resources (e.g. travel time, missing work, childcare). When referencing individuals who do not know how to access information about vaccination, one participant stated, ‘it’s really embarrassing […] to ask for help.’ Feelings of individual responsibility may prevent some people uncertain about vaccines from asking for more information and support. We argue that the focus on vaccine hesitancy without attention to systemic barriers deflects responsibility for making vaccines accessible from centralized institutions to individuals.

Unraveling the Role of Viral Interference in Disrupting Biennial RSV Epidemics in Northern Stockholm.

Respiratory syncytial virus (RSV) primarily impacts infants and older adults, with seasonal winter outbreaks in temperate countries. Biennial cycles of RSV activity have also been identified in Northern Europe and some states in the United States. Delayed RSV activity was reported worldwide during the 2009 influenza pandemic, and a disrupted biennial pattern of RSV activity was observed in northern Stockholm following the pandemic. Biennial patterns shifted to early/large outbreaks in even-numbered years and late/small outbreaks in odd-numbered years. However, the mechanisms underpinning this change in pattern remain unknown. In this work, we constructed an age-stratified mechanistic model to explicitly test three factors that could lead to the change in RSV transmission dynamics: 1) birth rates, 2) temperatures, and 3) viral interference. By fitting the model to weekly RSV admission data over a 20-year period and comparing different models, we found that viral interference from influenza was the only mechanism that explained the shifted biennial pattern. Our work demonstrates the complex interplay between different respiratory viruses, providing evidence that supports the presence of interactions between the H1N1 pandemic influenza virus and RSV at the population level, with implications for future public health interventions.

A Self-Fulfilling Prophecy Pandemic: The 1977 "Russian Flu".

Surprisingly, the 1977 "Russian flu" H1N1 pandemic influenza virus was genetically indistinguishable from strains that had circulated decades earlier but had gone extinct in 1957. This essay puts forward the most plausible chronology to explain the reemergence of the 1977 H1N1 pandemic virus: (1) in January-February 1976, a self-limited small outbreak of a swine H1N1 influenza virus occurred among Army personnel at Fort Dix, New Jersey; (2) in March 1976, the US launched a nationwide H1N1 swine influenza vaccine program; (3) other countries then also launched their own H1N1 R&D efforts; (4) a new H1N1 outbreak, genetically unrelated to the Fort Dix swine virus but indistinguishable from previously extinct H1N1 viruses, was detected early in 1977 in China; (5) the leading Chinese influenza virologist later disclosed that the Chinese military had conducted large H1N1 vaccine R&D studies in 1976. It is likely that the resurrected H1N1 influenza viruses were laboratory-stored strains that were unfrozen and studied as part of the emergency response to a perceived epidemic threat, and that accidentally escaped. The fear of a possible H1N1 pandemic was the critical factor that gave rise to the actual H1N1 pandemic, resulting in an avoidable "self-fulfilling prophecy pandemic."

Influenza H5N1 and H1N1 viruses remain infectious in unpasteurized milk on milking machinery surfaces.

Spillover of highly pathogenic avian H5N1 into the cattle population poses a risk to humans through the close contact with farm workers. High viral loads of influenza viruses in the unpasteurized milk of infected lactating cows has the potential to contaminate equipment within milking parlors and create fomites for transmission to dairy workers. Cattle H5N1 and human 2009 H1N1 pandemic influenza viruses were found to remain infectious on surfaces commonly found in milking equipment materials for a few hours. The data presented here provide a compelling case for the risk of contaminated surfaces generated during milking to facilitate transmission of H5N1 from cattle-to-cattle and to dairy farm workers.

Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Neutrophil extracellular traps in the anti-infectious defense of human organism

Background. Neutrophil extracellular traps (NETs) are net-like structures that have been investigated in inflammatory diseases. However, the presence of NETs in infected persons without clinical symptoms has not been yet studied.Aim. To reveal NETs in healthy persons during and after the H1N1 influenza pandemic as well as to study the functional activity of NETs.Materials and methods. The study included two groups of volunteers (n = 10 in each group) aged 20–25 years. The first group of volunteers was examined in the absence of acute diseases during one month before the study and in the absence of chronic diseases in the medical history. Volunteers of the second group were in contact with patients with influenza, but did not get sick. The comparative study also included patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, abscess; 12 patients) and 9 patients with non-specific ulcerative colitis. Neutrophils were isolated from the blood by the traditional method of Ficoll density centrifugation. The number, morphology, and functional activity of NETs were determined (by capture of Klebsiella pneumoniae). SYBR Green I-based fluorescence microscopy was used to visualize and quantify NETs.Results. In healthy volunteers who were not in contact with infected patients, spontaneous NETs formation did not occur. Neutrophils of persons who were in contact with infected patients spontaneously formed NETs. In this case the number of NETs reached 8.58 ± 0.51%, and the size of NETs amounted to 39.68 ± 3.52 µm. NETs effectively captured cells of the tested microorganism, which was accompanied by retraction of network fibers and transformation of the network structure into a cloud-like one, which retained 89.38 ± 5.86 microbial cells. For comparison, the NETs in patients with acute inflammation in the abdominal cavity captured and bound 20.2 ± 1 .67 microbial cells and with non-specific ulcerative colitis – 5.53 ± 0.34 cells.Conclusion. High binding capacity of NETs is a factor contributing to effective defense of the body against the development of an infectious disease with manifested clinical symptoms.

Retraction: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

Retraction: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

Disease burden attributable to respiratory syncytial virus outbreaks in long-term care.

Respiratory syncytial virus (RSV) disease burden is significant among children; however, RSV can also cause excess morbidity and mortality among older adults. Populations in long-term care homes (LTCHs) may be at greater risk of exposure and increased infection severity. The objectives of this article are to identify evidence regarding disease burden and outcome severity attributable to RSV outbreaks among residents and staff in LTCHs; and to highlight reported population and outbreak characteristics. All types of evidence were eligible for inclusion. Data utilized by included studies was between the end of the 2010 H1N1 influenza pandemic and the beginning of the coronavirus disease 2019 (COVID-19) pandemic. Evidence from the following countries was considered: G7, the European Union, Australia and New Zealand. A total of 167 articles were identified; 58 full texts were analyzed and four sources of evidence were eligible for inclusion. Data related to population characteristics, outbreak type and resident and staff outcomes were manually charted. There is a paucity of evidence sources pertaining to RSV outbreak burden among residents and staff in LTCHs. Outbreak duration ranged from 13 to 21 days. For each outbreak, 4-7 residents had confirmed RSV infection. Attack rates ranged from 12% to 38%. A spectrum of disease attributable to RSV outbreaks in LTCHs was identified, ranging from mild cold-like symptoms to death. Integration of RSV into existing respiratory pathogen surveillance programs is important to characterize susceptibility, transmissibility and virulence of RSV in at-risk populations. There is a need for public health organizations to publish the findings from outbreak investigations to provide evidence to inform RSV outbreak prevention and response in LTCH settings.

Pandemic H1N1 influenza virus triggers a strong T helper cell response in human nasopharynx-associated lymphoid tissues

The pH1N1 belongs to influenza A family that is sometimes transmitted to humans via contact with pigs. Human tonsillar immune cells are widely used as in vitro models to study responses to influenza viruses. In the current study, human memory (M) and naïve (N) T cells responses in mononuclear cells of tonsil (TMCs) and peripheral blood (PBMCs) were stimulated by pH1N1/sH1N1, and then stained for estimation of T cells proliferation index. Individuals with an anti-pH1N1 hemagglutination (HA) inhibition (HAI) titer of forty or greater exhibited stronger HA-specific M-CD4+ T cells responses to pH1N1 in TMCs/PBMCs than those with an HAI titer of less than forty (P < 0.01). In addition, a positive correlation was observed between proliferation indices of M-CD4+ T cells induced by exposure to sH1N1/pH1N1 (p < 0.01). Moreover, a strong correlation (p < 0.001) was detected between subjects’ age and their HA-specific M-CD4+ T cells induced by pH1N1 exposure, indicating that this response was age-dependent. Finally, stimulation of TMCs with pH1N1-HA resulted in a significant M−CD8+ T cells response (p < 0.05). In conclusion, pH1N1 HA elicits a strong M-CD4+ T cells response in TMCs. Additionally, this response correlates with the response to sH1N1 suggesting cross-reactivity in T cells epitopes directed against HAs of both viral strains.

Kako se pripremiti za pandemiju ptičjeg gripa H5N1 - pouke iz pandemije influence H1N1 iz 1918

In this paper, we highlight the significant challenges encountered during the influenza H1N1 pandemic of 1918. These lessons are being utilized to prepare for potential avian influenza H5N1 pandemic outbreaks, as this virus will impact both humans and food-serving animals. Preparation for this threat is crucial because H5N1 infections are already affecting the United States and many other countries.

The CombE-IDMS Alternate Potency Method for H5N1 and H5N8 Cell-Based Vaccines.

Assaying the potency of inactivated viral influenza vaccines is performed using single radial immunodiffusion, which is the globally accepted release method for potency. Under conditions of a rapidly emerging pandemic, such as the 2009 H1N1 influenza pandemic, a recognized obstacle in the delivery of vaccines to the public is the time needed for the distribution of calibrated SRID reagents (antisera and antigen standards) to vaccine manufacturers. Previously, we first described a novel streamlined MS-based assay, CombE-IDMS, which does not rely on antisera/antibodies or reference antigens, as a potential rapidly deployable alternate potency method through a comparison with SRID on adjuvanted seasonal quadrivalent vaccine cell-based (aQIVc) materials. In this report, we further demonstrate that the CombE-IDMS method can also be applied to measure the potency of pre-pandemic H5N1 and H5N8 monovalent vaccine materials, each subtype both unadjuvanted and adjuvanted, through a forced degradation study. Overall, CombE-IDMS results align with those of the gold standard SRID method on both H5N1 and H5N8 materials under conditions of thermal, pH, oxidative and freeze/thaw stress, lending further evidence for the CombE-IDMS method's suitability as an alternate assay for potency of both seasonal and pandemic influenza vaccines.

The Mobility of Eurasian Avian-like M2 Is Determined by Residue E79 Which Is Essential for Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature (39.5 ​°C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Transplacental transfer efficiency of maternal antibodies against influenza A(H1N1)pdm09 virus and dynamics of naturally acquired antibodies in Chinese children: a longitudinal, paired mother–neonate cohort study

The 2009 pandemic H1N1 influenza A virus (A(H1N1)pdm09 virus) evolves rapidly and has continued to cause severe infections in children since its emergence in 2009. We aimed to characterise the kinetics of maternally and naturally acquired antibodies against historical A(H1N1)pdm09 strains and to assess the extent to which the response to heterologous strains following infection or vaccination affects observed A(H1N1)pdm09 strain-specific antibody titres in a Chinese paediatric population. In this retrospective study, we used residual serum samples from 528 mother-neonate pairs from a non-interventional, longitudinal cohort study in southern China conducted from Sept 20, 2013, to Aug 24, 2018, from six local hospitals in Anhua County, Hunan Province, China. Mother-neonate pairs were eligible for inclusion if the neonates were born after Sept 20, 2013, and their mothers had resided in the study sites for at least 3 months. We tested samples with a haemagglutination inhibition (HAI) assay to measure antibody levels against three historical A(H1N1)pdm09 strains that were antigenically similar to the strains that circulated during the 2009 pandemic (A/Hunan-Kaifu/SWL4204/2009 [SWL4204/09 strain], A/Hunan-Daxiang/SWL1277/2016 [SWL1277/16 strain], and A/Hunan-Yanfeng/SWL185/2018 [SWL185/18 strain]). We also determined the seroprevalence, geometric mean titres (GMTs), transfer ratio of maternal antibodies, and the dynamics of maternally and naturally acquired antibodies in children, from birth to 3 years of age. 1066 mother-neonate pairs were enrolled in the original cohort between Sept 20, 2013, and Oct 14, 2015. Of these, 528 pairs (523 mothers, 528 neonates) were selected for the present study. The median age of the mothers was 25 years (IQR 23 to 29). 291 (55%) of 528 children were boys and 237 (45%) were girls, and most children (452 [86%]) were breastfed before the age of 6 months. The GMTs and the seroprevalence for the SWL4204/09 strain were higher than those for the SWL1277/16 and SWL185/18 strains among mothers (GMTs: 10·4 [95% CI 9·8 to 11·1] vs 9·3 [8·7 to 9·8] vs 8·0 [7·5 to 8·4], p<0·0001; seroprevalence: 11·1% [95% CI 8·5 to 14·1] vs 6·9% [4·9 to 9·4] vs 4·6% [3·0 to 6·8], p=0·0003) and among neonates (GMTs: 10·7 [10·0 to 11·5] vs 9·4 [8·8 to 10·0] vs 8·1 [7·6 to 8·6], p<0·0001; seroprevalence: 13·4% [10·7 to 16·7] vs 8·7% [6·5 to 11·5] vs 6·1% [4·2 to 8·5], p=0·0002). Regardless of the A(H1N1)pdm09-specific strain, maternal antibodies could be transferred efficiently via the placenta (mean transfer ratios: 1·10 for SWL4204/09 vs 1·09 for SWL1277/16 vs 1·06 for SWL185/18; p=0·93). The A(H1N1)pdm09 strain-specific antibodies waned below the protective threshold of 1:40 within 2 months after birth. After maternal antibody waning, there were periodic increases and decreases in HAI antibody titres against three A(H1N1)pdm09 strains, and such increases were all significantly associated with a higher immune response to heterologous strains. Vaccination against the SWL4204/09 strain was associated with a poor response to the SWL185/18 strain (β-0·20, 95% CI -0·28 to -0·13; p<0·0001). Our findings suggest low pre-existing immunity against influenza A(H1N1)pdm09 virus among unvaccinated Chinese adult female and paediatric populations. This evidence, together with the rapid decay of maternal antibodies and the observed cross-reactivity among different A(H1N1)pdm09 strains, highlights the importance of accelerating maternal and paediatric influenza vaccination in China. The Key Program of the National Natural Science Foundation of China. For the Chinese translation of the abstract see Supplementary Materials section.

Detection of influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.

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

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

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

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

Spatial genetic structure of 2009 H1N1 pandemic influenza established as a result of interaction with human populations in mainland China.

Identifying the spatial patterns of genetic structure of influenza A viruses is a key factor for understanding their spread and evolutionary dynamics. In this study, we used phylogenetic and Bayesian clustering analyses of genetic sequences of the A/H1N1pdm09 virus with district-level locations in mainland China to investigate the spatial genetic structure of the A/H1N1pdm09 virus across human population landscapes. Positive correlation between geographic and genetic distances indicates high degrees of genetic similarity among viruses within small geographic regions but broad-scale genetic differentiation, implying that local viral circulation was a more important driver in the formation of the spatial genetic structure of the A/H1N1pdm09 virus than even, countrywide viral mixing and gene flow. Geographic heterogeneity in the distribution of genetic subpopulations of A/H1N1pdm09 virus in mainland China indicates both local to local transmission as well as broad-range viral migration. This combination of both local and global structure suggests that both small-scale and large-scale population circulation in China is responsible for viral genetic structure. Our study provides implications for understanding the evolution and spread of A/H1N1pdm09 virus across the population landscape of mainland China, which can inform disease control strategies for future pandemics.

The genetic diversity, replication, and transmission of 2009 pandemic H1N1 viruses in China.

The 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China. To better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009-2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared. In total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015-2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016-2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact. Our findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.