Multicountry Spread of Influenza A(H1N1)pdm09 Viruses with Reduced Oseltamivir Inhibition, May 2023-February 2024.

Since May 2023, a novel combination of neuraminidase mutations, I223V + S247N, has been detected in influenza A(H1N1)pdm09 viruses collected in countries spanning 5 continents, mostly in Europe (67/101). The viruses belong to 2 phylogenetically distinct groups and display ≈13-fold reduced inhibition by oseltamivir while retaining normal susceptibility to other antiviral drugs.

Avian Influenza A(H5N1) Neuraminidase Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09.

We detected high titers of cross-reactive neuraminidase inhibition antibodies to influenza A(H5N1) virus clade 2.3.4.4b in 96.8% (61/63) of serum samples from healthy adults in Hong Kong in 2020. In contrast, antibodies at low titers were detected in 42% (21/50) of serum samples collected in 2009. Influenza A(H1N1)pdm09 and A(H5N1) titers were correlated.

Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

Causal relationship between blood traits and severe influenza A(H1N1)pdm09 infection in East Asian: A Mendelian randomization study.

Although a range of blood traits have been reported to be associated with influenza A(H1N1)pdm09 (H1N1pdm09) disease severity, their underlying causal relationships and biological mechanisms have remained unclear. This study aimed to investigate the causal relationship between blood traits and H1N1pdm09 using a two-sample Mendelian randomization analysis. Based on the data from our in-house genome-wide association study (GWAS) on H1N1pdm09 disease severity (Ncase [severe] = 70, Ncontrol [mild] = 95) and GWAS summaries of 44 blood traits from Biobank Japan (N = 12 303-143 658), we identified the potential causal effect of blood traits on severe H1N1pdm09. The inverse variance weighted method analysis revealed significant causal effects of lower aspartate aminotransferase (AST, ß = -3.212, p = 0.019), low-density-lipoprotein cholesterol (LDL-C, ß = -1.372, p = 0.045), and basophil counts (Baso, ß = -1.638, p = 0.047) on severe H1N1pdm09 disease. Additionally, polygenic risk score analysis further confirmed genetic overlap between these blood traits and severe H1N1pdm09 disease. This study provided evidence linking the lower level of AST, LDL-C, and lower count of Baso with severe H1N1pdm09 disease, potentially identifying new therapeutic targets for patients with severe influenza.

Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Antigenic drift and immunity gap explain reduction in protective responses against influenza A(H1N1)pdm09 and A(H3N2) viruses during the COVID-19 pandemic: a cross-sectional study of human sera collected in 2019, 2021, 2022, and 2023.

BACKGROUND: Non-pharmaceutical interventions implemented during the COVID-19 pandemic resulted in a marked reduction in influenza infections globally. The absence of influenza has raised concerns of waning immunity, and potentially more severe influenza seasons after the pandemic. METHODS: To evaluate immunity towards influenza post-COVID-19 pandemic we have assessed influenza A epidemics in Norway from October 2016 to June 2023 and measured antibodies against circulating strains of influenza A(H1N1)pdm09 and A(H3N2) in different age groups by hemagglutination inhibition (HAI) assays in a total of 3364 serum samples collected in 2019, 2021, 2022 and 2023. RESULTS: Influenza epidemics in Norway from October 2016 until June 2023 were predominately influenza As, with a mixture of A(H1N1)pdm09 and A(H3N2) subtype predominance. We did not observe higher numbers of infections during the influenza epidemics following the COVID-19 pandemic than in pre-COVID-19 seasons. Frequencies of protective HAI titers against A(H1N1)pdm09 and A(H3N2) viruses were reduced in sera collected in 2021 and 2022, compared to sera collected in 2019. The reduction could, however, largely be explained by antigenic drift of new virus strains, as protective HAI titers remained stable against the same strain from one season to the next. However, we observed the development of an immunity gap in the youngest children during the pandemic which resulted in a prominent reduction in HAI titers against A(H1N1)pdm09 in 2021 and 2022. The immunity gap was partially closed in sera collected in 2023 following the A(H1N1)pdm09-dominated influenza seasons of 2022/2023. During the 2022/2023 epidemic, drift variants of A(H1N1)pdm09 belonging to the 5a.2a.1 clade emerged, and pre-season HAI titers were significantly lower against this clade compared to the ancestral 5a.2 clade. CONCLUSION: The observed reduction in protective antibodies against A(H1N1)pdm09 and A(H3N2) viruses post COVID-19 is best explained by antigenic drift of emerging viruses, and not waning of antibody responses in the general population. However, the absence of influenza during the pandemic resulted in an immunity gap in the youngest children. While this immunity gap was partially closed following the 2022/2023 influenza season, children with elevated risk of severe infection should be prioritized for vaccination.

Cocirculation of A(H3N2) and B/Victoria increased morbidity in hospitalized patients in the 2019-2020 A(H1N1)pdm09 predominant influenza season in Israel.

Community surveillance found the 2019-2020 A(H1N1)pdm09 predominant influenza season in Israel to be a high-intensity season with an early and steep morbidity peak. To further characterize disease severity in the 2019-2020 season, we analyzed a cohort of hospitalized patients with laboratory-confirmed influenza from this season (n = 636). Quantitative polymerase chain reaction was performed on clinical samples to detect the presence of influenza. Demographic, clinical, and laboratory data were retrieved via electronic health records and MDClone. Electronic health records were accessed to obtain data on intensive care unit patients, missing data and for data verification purposes. Univariate analysis was performed to compare demographic, comorbidity, and clinical characteristics across the three influenza strains. The A(H1N1)pdm09 predominant 2019-2020 influenza season in Israel was characterized by an early and steep morbidity peak, vaccine delays and shortages, and with the A(H3N2) and B/Victoria strains disproportionately targeting children and young adults, most probably due to reduced immunity to these strains. A greater proportion of children <5 years infected with A(H3N2) and B/Victoria developed severe influenza compared with those infected with A(H1N1)pdm09. Our study emphasizes the vulnerability of infants and young children in the face of rapidly evolving influenza strains and underscores the importance of influenza prevention measures in this population.

Detection of H275Y oseltamivir resistance gene mutation among Influenza A(H1N1)pdm09 patients by allelic discrimination real-time RT-PCR.

Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single-nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real-time RT-PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real-time RT-PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014-2021. The study highlights that the WHO-recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.

Strain development of A/H1N1pdm09 candidate vaccine viruses for the 2021-22 northern hemisphere influenza season.

The vaccine effectiveness (VE) of the A/H1N1pdm09 component of the 2017-18 quadrivalent live attenuated influenza vaccine (QLAIV) was improved by performing rational haemagglutinin (HA) mutagenesis. Introducing N125D, D127E, D222G and R223Q substitutions into the HA protein of A/Slovenia/2903/2015 (A/SLOV15) enhanced replicative fitness in primary human nasal epithelial cells (hNECs). This allowed A/SLOV15 to overcome inter-strain competition in QLAIV, resulting in improved VE.During strain development for the 2021-22 QLAIV formulation, A/H1N1pdm09 LAIV viruses containing wild-type (WT) HA and neuraminidase (NA) sequences were found to replicate poorly in embryonated eggs and hNECs. We aimed to enhance replicative fitness via the HA mutagenesis approach that was performed previously for A/SLOV15. Therefore, combinations of these four mutations were introduced into the HA protein of representative 6B.1A-5a.2 viruses, A/Victoria/2570/2019 and A/Victoria/1/2020 (A/VIC1). Replicative fitness of A/VIC1 V7 was improved ~30-fold in eggs and ~300-fold in hNECs relative to its parent, without compromising other critical LAIV characteristics.

Expression of Surfactant Protein D Distinguishes Severe Pandemic Influenza A(H1N1) from Coronavirus Disease 2019.

The differentiation between influenza and coronavirus disease 2019 (COVID-19) could constitute a diagnostic challenge during the ongoing winter owing to their clinical similitude. Thus, novel biomarkers are required to enable making this distinction. Here, we evaluated whether the surfactant protein D (SP-D), a collectin produced at the alveolar epithelium with known immune properties, was useful to differentiate pandemic influenza A(H1N1) from COVID-19 in critically ill patients. Our results revealed high serum SP-D levels in patients with severe pandemic influenza but not those with COVID-19. This finding was validated in a separate cohort of mechanically ventilated patients with COVID-19 who also showed low plasma SP-D levels. However, plasma SP-D levels did not distinguish seasonal influenza from COVID-19 in mild-to-moderate disease. Finally, we found that high serum SP-D levels were associated with death and renal failure among severe pandemic influenza cases. Thus, our studies have identified SP-D as a unique biomarker expressed during severe pandemic influenza but not COVID-19.

Reassortant Influenza A(H1N1)pdm09 Virus in Elderly Woman, Denmark, January 2021.

A case of human infection with influenza A(H1N1)pdm09 virus containing a nonstructural gene highly similar to Eurasian avian-like H1Nx swine influenza virus was detected in Denmark in January 2021. We describe the clinical case and report testing results of the genetic and antigenic characterizations of the virus.

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.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

Respiratory microbes detected in hospitalized adults with acute respiratory infections: associations between influenza A(H1N1)pdm09 virus and intensive care unit admission or fatal outcome in Vietnam (2015-2017).

BACKGROUND: Acute respiratory tract infection (ARI) is a leading cause of hospitalization, morbidity, and mortality worldwide. Respiratory microbes that were simultaneously detected in the respiratory tracts of hospitalized adult ARI patients were investigated. Associations between influenza A(H1N1)pdm09 virus (H1N1pdm) detection and intensive care unit (ICU) admission or fatal outcome were determined. METHODS: This prospective observational study was conducted between September 2015 and June 2017 at Bach Mai Hospital, Hanoi, Vietnam. Inclusion criteria were hospitalized patients aged ≥15 years; one or more of symptoms including shortness of breath, sore throat, runny nose, headache, and muscle pain/arthralgia in addition to cough and fever > 37.5 °C; and ≤ 10 days from the onset of symptoms. Twenty-two viruses, 11 bacteria, and one fungus in airway specimens were examined using a commercial multiplex real-time PCR assay. Associations between H1N1pdm detection and ICU admission or fatal outcome were investigated by univariate and multivariate logistic regression analyses. RESULTS: The total of 269 patients (57.6% male; median age, 51 years) included 69 ICU patients. One or more microbes were detected in the airways of 214 patients (79.6%). Single and multiple microbes were detected in 41.3 and 38.3% of patients, respectively. Influenza A(H3N2) virus was the most frequently detected (35 cases; 13.0%), followed by H1N1pdm (29 cases; 10.8%). Hematological disease was associated with ICU admission (p < 0.001) and fatal outcomes (p < 0.001) using the corrected significance level (p = 0.0033). Sex, age, duration from onset to sampling, or number of detected microbes were not significantly associated with ICU admission or fatal outcomes. H1N1pdm detection was associated with ICU admission (odds ratio [OR] 3.911; 95% confidence interval [CI] 1.671-9.154) and fatal outcome (OR 5.496; 95% CI 1.814-16.653) after adjusting for the confounding factors of comorbidities, bacteria/Pneumocystis jirovecii co-detection, and age. CONCLUSIONS: H1N1pdm was associated with severe morbidity and death in adult patients hospitalized with respiratory symptoms. The diagnosis of subtype of influenza virus may be epidemiologically important.

Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains.

BACKGROUND: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. METHODS: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. RESULTS: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. CONCLUSION: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

Locally Acquired Human Infection with Swine-Origin Influenza A(H3N2) Variant Virus, Australia, 2018.

In 2018, a 15-year-old female adolescent in Australia was infected with swine influenza A(H3N2) variant virus. The virus contained hemagglutinin and neuraminidase genes derived from 1990s-like human seasonal viruses and internal protein genes from influenza A(H1N1)pdm09 virus, highlighting the potential risk that swine influenza A virus poses to human health in Australia.

Illness Severity in Hospitalized Influenza Patients by Virus Type and Subtype, Spain, 2010-2017.

We conducted a retrospective cohort study to assess the effect of influenza virus type and subtype on disease severity among hospitalized influenza patients in Spain. We analyzed the cases of 8,985 laboratory-confirmed case-patients hospitalized for severe influenza by using data from a national surveillance system for the period 2010-2017. Hospitalized patients with influenza A(H1N1)pdm09 virus were significantly younger, more frequently had class III obesity, and had a higher risk for pneumonia or acute respiratory distress syndrome than patients infected with influenza A(H3N2) or B (p<0.05). Hospitalized patients with influenza A(H1N1)pdm09 also had a higher risk for intensive care unit admission, death, or both than patients with influenza A(H3N2) or B, independent of other factors. Determining the patterns of influenza-associated severity and how they might differ by virus type and subtype can help guide planning and implementation of adequate control and preventive measures during influenza epidemics.

Influenza A Virus Infections in Dromedary Camels, Nigeria and Ethiopia, 2015-2017.

We examined nasal swabs and serum samples acquired from dromedary camels in Nigeria and Ethiopia during 2015-2017 for evidence of influenza virus infection. We detected antibodies against influenza A(H1N1) and A(H3N2) viruses and isolated an influenza A(H1N1)pdm09-like virus from a camel in Nigeria. Influenza surveillance in dromedary camels is needed.

Interspecies Transmission of Reassortant Swine Influenza A Virus Containing Genes from Swine Influenza A(H1N1)pdm09 and A(H1N2) Viruses.

Influenza A(H1N1)pdm09 (pH1N1) virus has become established in swine in the United Kingdom and currently co-circulates with previously enzootic swine influenza A virus (IAV) strains, including avian-like H1N1 and human-like H1N2 viruses. During 2010, a swine influenza A reassortant virus, H1N2r, which caused mild clinical disease in pigs in the United Kingdom, was isolated. This reassortant virus has a novel gene constellation, incorporating the internal gene cassette of pH1N1-origin viruses and hemagglutinin and neuraminidase genes of swine IAV H1N2 origin. We investigated the pathogenesis and infection dynamics of the H1N2r isolate in pigs (the natural host) and in ferrets, which represent a human model of infection. Clinical and virologic parameters were mild in both species and both intraspecies and interspecies transmission was observed when initiated from either infected pigs or infected ferrets. This novel reassortant virus has zoonotic and reverse zoonotic potential, but no apparent increased virulence or transmissibility, in comparison to pH1N1 viruses.

Phylogenetic analysis and clinical characteristics of the co-occurring mutations in HA and NA genes of influenza A(H1N1)pdm09 viruses during 2015-2017 in Beijing, China.

BACKGROUND: Influenza A(H1N1)pdm09 viruses have undergone rapid evolution, and in recent years the complementary and antagonistic effects of HA and NA have gathered more attentions; however, the effects of co-occurring mutations in HA and NA on the patients' clinical characteristics are still poorly understood. In this study, we analyzed molecular epidemiology and evolution of A(H1N1) pdm09, explored co-occurring mutations of HA and NA, and investigated effect of co-occurring mutations on patients' clinical features. METHODS: A(H1N1)pdm09 was confirmed by reverse transcription-polymerase chain reaction. HA and NA genes were sequenced and phylogenetically analyzed. Clinical characteristics of the co-occurring mutations were analyzed statistically. RESULTS: By analyzing the HA and NA gene sequences of 33 A(H1N1)pdm09 viruses during the 2015-2017 influenza season, we found that all the viruses shared high similarities to each other and the HA genes of these viruses exclusively belonged to subclade 6B.1A. Several unreported substitutions in HA and NA proteins were observed, furthermore, co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). Sore throat was significantly associated with co-occurring mutations in HA and NA of A(H1N1)pdm09 (χ2, P < 0.05). CONCLUSIONS: Co-occurring mutations in HA and NA were detected in A(H1N1)pdm09 isolated during 2015-2017 in Beijing. Symptomatically, sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09. Therefore, studying the effect and mechanism of co-occurring mutations in HA and NA on patients' clinical features is of note needed.