Long-term co-circulation of multiple influenza A viruses in pigs, Guangxi, China.

Influenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Immunogenicity of chimeric hemagglutinins delivered by an orf virus vector platform against swine influenza virus.

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFVΔ121cH6/1 and ORFVΔ121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine.

Novel influenza A viruses in pigs with zoonotic potential, Chile.

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) have recently been identified in Chile. The objective of this study was to evaluate their zoonotic potential. We perform phylogenetic analyses to determine the genetic origin and evolution of these viruses, and a serological analysis to determine the level of cross-protective antibodies in the human population. Eight genotypes were identified, all with pandemic H1N1 2009-like internal genes. H1N1 and H1N2 were the subtypes more commonly detected. Swine H1N2 and H3N2 IAVs had hemagglutinin and neuraminidase lineages genetically divergent from IAVs reported worldwide, including human vaccine strains. These genes originated from human seasonal viruses were introduced into the swine population since the mid-1980s. Serological data indicate that the general population is susceptible to the H3N2 virus and that elderly and young children also lack protective antibodies against the H1N2 strains, suggesting that these viruses could be potential zoonotic threats. Continuous IAV surveillance and monitoring of the swine and human populations is strongly recommended.IMPORTANCEIn the global context, where swine serve as crucial intermediate hosts for influenza A viruses (IAVs), this study addresses the pressing concern of the zoonotic potential of novel reassortant strains. Conducted on a large scale in Chile, it presents a comprehensive account of swine influenza A virus diversity, covering 93.8% of the country's industrialized swine farms. The findings reveal eight distinct swine IAV genotypes, all carrying a complete internal gene cassette of pandemic H1N1 2009 origin, emphasizing potential increased replication and transmission fitness. Genetic divergence of H1N2 and H3N2 IAVs from globally reported strains raises alarms, with evidence suggesting introductions from human seasonal viruses since the mid-1980s. A detailed serological analysis underscores the zoonotic threat, indicating susceptibility in the general population to swine H3N2 and a lack of protective antibodies in vulnerable demographics. These data highlight the importance of continuous surveillance, providing crucial insights for global health organizations.

Amino acids in the polymerase complex of shorebird-isolated H1N1 influenza virus impact replication and host-virus interactions in mammalian models.

A diverse population of avian influenza A viruses (AIVs) are maintained in wild birds and ducks yet the zoonotic potential of AIVs in these environmental reservoirs and the host-virus interactions involved in mammalian infection are not well understood. In studies of a group of subtype H1N1 AIVs isolated from migratory wild birds during surveillance in North America, we previously identified eight amino acids in the polymerase genes PB2 and PB1 that were important for the transmissibility of these AIVs in a ferret model of human influenza virus transmission. In this current study we found that PB2 containing amino acids associated with transmissibility at 67, 152, 199, 508, and 649 and PB1 at 298, 642, and 667 were associated with more rapid viral replication kinetics, greater infectivity, more active polymerase complexes and greater kinetics of viral genome replication and transcription. Pathogenicity in the mouse model was also impacted, evident as greater weight loss and lung pathology associated with greater inflammatory lung cytokine expression. Further, these AIVs all contained the avian-type amino acids of PB2-E627, D701, G590, Q591 and T271. Therefore, our study provides novel insights into the role of the AIV polymerase complex in the zoonotic transmission of AIVs in mammals.

Early-life influenza A (H1N1) infection independently programs brain connectivity, HPA AXIS and tissue-specific gene expression profiles.

Early-life adversity covers a range of physical, social and environmental stressors. Acute viral infections in early life are a major source of such adversity and have been associated with a broad spectrum of later-life effects outside the immune system or "off-target". These include an altered hypothalamus-pituitary-adrenal (HPA) axis and metabolic reactions. Here, we used a murine post-natal day 14 (PND 14) Influenza A (H1N1) infection model and applied a semi-holistic approach including phenotypic measurements, gene expression arrays and diffusion neuroimaging techniques to investigate HPA axis dysregulation, energy metabolism and brain connectivity. By PND 56 the H1N1 infection had been resolved, and there was no residual gene expression signature of immune cell infiltration into the liver, adrenal gland or brain tissues examined nor of immune-related signalling. A resolved early-life H1N1 infection had sex-specific effects. We observed retarded growth of males and altered pre-stress (baseline) blood glucose and corticosterone levels at PND42 after the infection was resolved. Cerebral MRI scans identified reduced connectivity in the cortex, midbrain and cerebellum that were accompanied by tissue-specific gene expression signatures. Gene set enrichment analysis confirmed that these were tissue-specific changes with few common pathways. Early-life infection independently affected each of the systems and this was independent of HPA axis or immune perturbations.

Molecular Determinants of Drug Resistance and Mutation Patterns in Influenza Viruses Circulating in Poland Across Multiple Epidemic Seasons: Implications for Vaccination Strategies.

BACKGROUND According to the WHO, up to 650 000 people die each year from seasonal flu-related respiratory illnesses. The most effective method of fighting the virus is seasonal vaccination. However, if an infection does occur, antiviral medications should be used as soon as possible. No studies of drug resistance in influenza viruses circulating in Poland have been systematically conducted. Therefore, the aim of the present study was to investigate the drug resistance and genetic diversity of influenza virus strains circulating in Poland by determining the presence of mutations in the neuraminidase gene. MATERIAL AND METHODS A total of 258 clinical specimens were collected during the 2016-2017, 2017-2018, and 2018-2019 epidemic seasons. The samples containing influenza A and B were analyzed by RT-PCR and Sanger sequencing. RESULTS Differences were found between the influenza virus strains detected in different epidemic seasons, demonstrating the occurrence of mutations. Influenza A virus was found to be more genetically variable than influenza B virus (P<0.001, Kruskal-Wallis test). However, there was no significant difference in the resistance prevalence between the influenza A subtypes A/H1N1/pdm09 (4.8%) and A/H3N2/ (6.1%). In contrast, more mutations of drug-resistance genes were found in the influenza B virus (P<0.001, chi-square test). In addition, resistance mutations appeared en masse in vaccine strains circulating in unvaccinated populations. CONCLUSIONS It seems important to determine whether the influenza virus strains tested for drug resistance as part of global influenza surveillance are equally representative of viruses circulating in populations with high and low vaccination rates, for all countries. Our results suggest that countries with low levels of influenza immunization may constitute reservoirs of drug-resistant influenza viruses.

Vaccine effectiveness against influenza hospitalisation in adults during the 2022/2023 mixed season of influenza A(H1N1)pdm09, A(H3N2) and B circulation, Europe: VEBIS SARI VE hospital network.

We conducted a multicentre hospital-based test-negative case-control study to measure vaccine effectiveness (VE) against PCR-confirmed influenza in adult patients with severe acute respiratory infection (SARI) during the 2022/2023 influenza season in Europe. Among 5547 SARI patients ≥18 years, 2963 (53%) were vaccinated against influenza. Overall VE against influenza A(H1N1)pdm09 was 11% (95% CI: -23-36); 20% (95% CI: -4-39) against A(H3N2) and 56% (95% CI: 22-75) against B. During the 2022/2023 season, while VE against hospitalisation with influenza B was >55%, it was ≤20% for influenza A subtypes. While influenza vaccination should be a priority for future seasons, improved vaccines against influenza are needed.

Isolation and characterization of genotype 4 Eurasian avian-like H1N1 influenza virus in pigs suffering from pneumonia.

Swine influenza viruses pose ongoing threat to pork industry throughout the world. In 2023, fattening pigs from a swine farm in Inner Mongolia of China experienced influenza-like symptoms. Co-infection of influenza A virus with Pasteurella multocida was diagnosed in lung tissues of diseased pigs and a genotype 4 (G4) Eurasian avian-like (EA) H1N1 virus was isolated, which was named as A/swine/Neimenggu/0326/2023. We demonstrated the virus preferentially bound human-like SAα2,6Gal receptor. It was noteworthy that the virus possessed multiple genetic markers for mammalian adaptation in the internal genes. Animal studies showed that compared with genotype 1 (G1) EA H1N1 virus and early prevalent G4 EA H1N1 virus, A/swine/Neimenggu/0326/2023 virus exhibited increased virus shedding, enhanced replication in lungs, and caused more severe lung lesions in pigs. These findings indicate that the G4 EA H1N1 virus poses increased threat to pork industry, controlling the prevailing viruses in pigs should be promptly implemented.

Assessment of the antigenic evolution of a clade 6B.1 human H1N1pdm influenza virus revealed differences between ferret and human convalescent sera.

BACKGROUND: Influenza viruses continually acquire mutations in the antigenic epitopes of their major viral antigen, the surface glycoprotein haemagglutinin (HA), allowing evasion from immunity in humans induced upon prior influenza virus infections or vaccinations. Consequently, the influenza strains used for vaccine production must be updated frequently. METHODS: To better understand the antigenic evolution of influenza viruses, we introduced random mutations into the HA head region (where the immunodominant epitopes are located) of a pandemic H1N1 (H1N1pdm) virus from 2015 and incubated it with various human sera collected in 2015-2016. Mutants not neutralized by the human sera were sequenced and further characterized for their haemagglutination inhibition (HI) titers with human sera and with ferret sera raised to H1N1pdm viruses from 2009 to 2015. FINDINGS: The largest antigenic changes were conferred by mutations at HA amino acid position 187; interestingly, these antigenic changes were recognized by human, but not by ferret serum. H1N1pdm viruses with amino acid changes at position 187 were very rare until the end of 2018, but have become more frequent since; in fact, the D187A amino acid change is one of the defining changes of clade 6B.1A.5a.1 viruses, which emerged in 2019. INTERPRETATION: Our findings indicate that amino acid substitutions in H1N1pdm epitopes may be recognized by human sera, but not by homologous ferret sera. FUNDING: This project was supported by funding from the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

Since the 1990s, endemic North American swine influenza A viruses (swFLUAVs) contained an internal gene segment constellation, the triple reassortment internal gene (TRIG) cassette. In 2009, the H1N1 pandemic (pdmH1N1) virus spilled back into swine but did not become endemic. However, the pdmH1N1 contributed the matrix gene (pdmM) to the swFLUAVs circulating in the pig population, which replaced the classical swine matrix gene (swM) found in the TRIG cassette, suggesting the pdmM has a fitness benefit. Others have shown that swFLUAVs containing the pdmM have greater transmission efficiency compared to viruses containing the swM gene segment. We hypothesized that the matrix (M) gene could also affect disease and utilized two infection models, resistant BALB/c and susceptible DBA/2 mice, to assess pathogenicity. We infected BALB/c and DBA/2 mice with H1 and H3 swFLUAVs containing the swM or pdmM and measured lung virus titers, morbidity, mortality, and lung histopathology. H1 influenza strains containing the pdmM gene caused greater morbidity and mortality in resistant and susceptible murine strains, while H3 swFLUAVs caused no clinical disease. However, both H1 and H3 swFLUAVs containing the pdmM replicated to higher viral titers in the lungs and pdmM containing H1 viruses induced greater histological changes compared to swM H1 viruses. While the surface glycoproteins and other gene segments may contribute to swFLUAV pathogenicity in mice, these data suggest that the origin of the matrix gene also contributes to pathogenicity of swFLUAV in mice, although we must be cautious in translating these conclusions to their natural host, swine. IMPORTANCE: The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

Simultaneous identification of DNA and RNA pathogens using metagenomic sequencing in cases of severe acute respiratory infection.

Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.

Differential lung gene expression changes in C57BL/6 and DBA/2 mice carrying an identical functional Mx1 gene reveals crucial differences in the host response.

BACKGROUND: Influenza virus infections represent a major global health problem. The dynamin-like GTPase MX1 is an interferon-dependent antiviral host protein that confers resistance to influenza virus infections. Infection models in mice are an important experimental system to understand the host response and susceptibility to developing severe disease following influenza infections. However, almost all laboratory mouse strains carry a non-functional Mx1 gene whereas humans have a functional MX1 gene. Most studies in mice have been performed with strains carrying a non-functional Mx1 gene. It is therefore very important to investigate the host response in mouse strains with a functional Mx1 gene. RESULTS: Here, we analyzed the host response to influenza virus infections in two congenic mouse strains carrying the functional Mx1 gene from the A2G strain. B6.A2G-Mx1r/r(B6-Mx1r/r) mice are highly resistant to influenza A virus (IAV) H1N1 infections. On the other hand, D2(B6).A2G-Mx1r/r(D2-Mx1r/r) mice, although carrying a functional Mx1 gene, were highly susceptible, exhibited rapid weight loss, and died. We performed gene expression analysis using RNAseq from infected lungs at days 3 and 5 post-infection (p.i.) of both mouse strains to identify genes and pathways that were differentially expressed between the two mouse strains. The susceptible D2-Mx1r/r mice showed a high viral replication already at day 3 p.i. and exhibited a much higher number of differentially expressed genes (DEGs) and many DEGs had elevated expression levels compared to B6-Mx1r/r mice. On the other hand, some DEGs were specifically up-regulated only in B6-Mx1r/r mice at day 3 p.i., many of which were related to host immune response functions. CONCLUSIONS: From these results, we conclude that at early times of infection, D2-Mx1r/r mice showed a very high and rapid replication of the virus, which resulted in lung damage and a hyperinflammatory response leading to death. We hypothesize that the activation of certain immune response genes was missing and that others, especially Mx1, were expressed at a time in D2-Mx1r/r mice when the virus had already massively spread in the lung and were thus not able anymore to protect them from severe disease. Our study represents an important addition to previously published studies in mouse models and contributes to a better understanding of the molecular pathways and genes that protect against severe influenza disease.

Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection.

The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus-influenza virus co-infection. IMPORTANCE: This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

A case of swine influenza A(H1N2)v in England, November 2023.

Under International Health Regulations from 2005, a human infection caused by a novel influenza A virus variant is considered an event that has potential for high public health impact and is immediately notifiable to the World Health Organisation. We here describe the clinical, epidemiological and virological features of a confirmed human case of swine influenza A(H1N2)v in England detected through community respiratory virus surveillance. Swabbing and contact tracing helped refine public health risk assessment, following this unusual and unexpected finding.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Characteristics of two zoonotic swine influenza A(H1N1) viruses isolated in Germany from diseased patients.

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2-6-sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV.

Influenza vaccine effectiveness in Europe: Results from the 2022-2023 VEBIS (Vaccine Effectiveness, Burden and Impact Studies) primary care multicentre study.

Background: Influenza A(H3N2) viruses dominated early in the 2022-2023 influenza season in Europe, followed by higher circulation of influenza A(H1N1)pdm09 and B viruses. The VEBIS primary care network estimated the influenza vaccine effectiveness (VE) using a multicentre test-negative study. Materials and Methods: Primary care practitioners collected information and specimens from patients consulting with acute respiratory infection. We measured VE against any influenza, influenza (sub)type and clade, by age group, by influenza vaccine target group and by time since vaccination, using logistic regression. Results: We included 38 058 patients, of which 3786 were influenza A(H3N2), 1548 influenza A(H1N1)pdm09 and 3275 influenza B cases. Against influenza A(H3N2), VE was 36% (95% CI: 25-45) among all ages and ranged between 30% and 52% by age group and target group. VE against influenza A(H3N2) clade 2b was 38% (95% CI: 25-49). Overall, VE against influenza A(H1N1)pdm09 was 46% (95% CI: 35-56) and ranged between 29% and 59% by age group and target group. VE against influenza A(H1N1)pdm09 clade 5a.2a was 56% (95% CI: 46-65) and 79% (95% CI: 64-88) against clade 5a.2a.1. VE against influenza B was 76% (95% CI: 70-81); overall, 84%, 72% and 71% were among 0-14-year-olds, 15-64-year-olds and those in the influenza vaccination target group, respectively. VE against influenza B with a position 197 mutation of the hemagglutinin (HA) gene was 79% (95% CI: 73-85) and 90% (95% CI: 85-94) without this mutation. Conclusion: The 2022-2023 end-of-season results from the VEBIS network at primary care level showed high VE among children and against influenza B, with lower VE against influenza A(H1N1)pdm09 and A(H3N2).