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Abstract Avian influenza viruses undergo frequent genetic reassortment, which can coincide with phenotypic changes in transmission, pathogenicity, and host species niche . Since 2020, clade 2.3.4.4b H5 high pathogenicity avian influenza viruses (HPAIVs) have driven a global panzootic , causing mass mortality in wild birds, poultry , and, for the first time, repeated spillover infections in a variety of mammalian species . This resurgence of H5 HPAIV has coincided with a dramatic increase in the number of circulating reassortant strains ; however, the scale, impact and drivers of these reassortants remain unknown. Here, we combined statistical and phylodynamic modelling to reconstruct the global evolutionary dynamics of H5Nx viruses across four epizootic seasons (2020-2024). We identified 209 genetically distinct reassortants , stratified into three transmission categories based on their phylogenetic and epidemiological profiles. Accounting for sampling depth and HPAIV incidence, we est...

Abstract Although vaccines and treatments have strengthened our ability to combat the COVID-19 pandemic , new variants of SARS-CoV-2 continue to emerge in human populations. Because the evolution of SARS-CoV-2 is driven by mutation , a better understanding of its mutation rate and spectrum could improve our ability to forecast the trajectory of the pandemic . Here, we use circular RNA consensus sequencing (CirSeq) to determine the mutation rate of six SARS-CoV-2 variants and perform a short-term evolution experiment to determine the impact of these mutations on viral fitness. Our analyses indicate that the SARS-CoV-2 genome mutates at a rate of ∼1.5 × 10−6/base per viral passage and that the spectrum is dominated by C → U transitions. Moreover, we find that the mutation rate is significantly reduced in regions that form base-pairing interactions and that mutations that affect these secondary structures are especially harmful to viral fitness. In this work, we show that the biased mutat...

Abstract The current study aimed to investigate the genetic characterization and evolution of low pathogenic avian influenza virus H9N2 in Egypt . Ten H9N2 viruses were recently isolated from samples collected between 2019 and 2023. Phylogenetic analysis of the haemagglutinin (HA) gene segment of the H9N2 isolates showed a relatedness with G1 H9 4.2 lineage and clustered within genotype III of the Egyptian strains identified earlier in 2018. The majority of H9N2 strains had seven and eight glycosylation sites in HA and neuraminidase (NA) respectively . All strains carried H191 and L234 residues in their hemagglutinin which are markers facilitating avian-to-human cross species barrier transmission . No stalk deletions were detected in NA gene. In addition, genetic analysis of the NA and M encoding proteins revealed the absence of substitutions associated with resistance to oseltamivir and amantadine . The NA showed S372A and R403W substitutions which were previously detected in H3N2 and...

ABSTRACT Several emerging influenza viruses, including H7N9 and H5N6 viruses, trace their origins to reassortment with H9N2 viruses that contributed internal gene segments. However, the evolutionary constraints governing the reassortment of H9N2 viruses remain unknown. In seasonal human influenza A viruses, gene segments coevolve at both the nucleotide and amino acid levels. Here, we demonstrate that evolutionary relationships between gene segments, including polymerase subunits in human H3N2 viruses, differ from avian H9 viruses . Avian H9 viruses were characterized by little coevolution between gene segments or between polymerase subunits. Strikingly, protein trees built from avian H9 polymerase subunits diverge despite known functional constraints on polymerase evolution. The evolutionary divergence observed between gene segments of avian H9 viruses was consistent across isolates from different continents, suggesting that coevolution between H9 gene segments is not dependent on regi...

ABSTRACT Understanding viral evolutionary dynamics is crucial to pandemic responses, prediction of virus adaptation over time , and virus surveillance for public health strategies. Whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has enabled fine-grained studies of virus evolution in the human population . Serial passaging in vitro offers a complementary controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage . In this study, nine virus lineages , including four “ variants of concern ” and three former “ variants under investigation ,” were sampled over ≥33 serial passages (range 33–100) in Vero E6 cells . WGS was used to examine virus evolutionary dynamics and identify key mutations with implications for fitness and/or transmissibility. Viruses accumulated mutations regularly during serial passaging. Many low-frequency variants were lost , but others became fixed, suggestin...

Abstract Since 2021, the novel H10N3 has caused four cases of human infection in China, the most recent of which occurred in December 2024 , posing a potential threat to public health. Our previous studies indicated that several avian H10N3 strains are highly pathogenic in mice and can be transmitted between mammals via respiratory droplets without prior adaptation. By analyzing the genome sequence , we found that these H10N3 viruses carry the PB2-E627V mutation , which is becoming increasingly common in several subtypes of avian influenza viruses (AIV); however, its mechanism in mammalian adaptation remains unclear. Using a reverse genetics system , we investigated the role of PB2-E627V in the adaptation of H10N3 to mammals and poultry. Our findings demonstrate that the PB2-E627V mutation is critical for the high pathogenicity of novel H10N3 in mice and its ability to be transmitted through the air among mammals . Additionally, we found that the role of PB2-627 V in promoting AIV adap...

Abstract The highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b has rapidly disseminated globally , with mammalian infections reported in multiple species. Recent evidence of mammal-to-mammal transmission has heightened concerns about the virus’s potential adaptation to mammals . The polymerase basic 2 (PB2) protein E627K mutation appears to be of key importance for mammalian adaptation. We isolated an HPAI H5N1 clade 2.3.4.4b virus from wild birds in Korea with 96% E and 4% K at amino acid position 627 of PB2 . To investigate the genomic characteristics of this clade regarding mammalian adaptation, we studied the replication and transmission of the H5N1 virus in mice. Two experiments with different challenge-to-contact ratios were conducted to assess transmission dynamics and mutation development . In experiment 1, a 4:1 challenge-to-contact ratio resulted in 100% transmission among direct-contact mice , with all mice succumbing to the infection. In experiment 2, a 1:...

Abstract Nationwide surveillance of avian influenza viruses (AIVs) in live poultry markets across China has occurred since 2014, providing a resource for AIV prevalence and genetic diversity studies . Here we report that 3,237 of 18,425 samples from poultry were AIV positive (17.57%) between 2019 and 2023, with H9N2 being the dominant subtype . We developed an automated phylogeny-based nomenclature system to classify genetic clades of the dominant H9N2 lineage, the BJ94 lineage. Using this model, we found that ten haemagglutinin (HA) sub-subclades cocirculated in poultry and showed antigenic variation. In addition, 99.46% and 96.17% of H9N2 AIVs in 2021–2023 possessed human-receptor binding-related HA-L226 and human MxA-resistance-related NP-N52 mutations , respectively. H9N2 strains with these two mutations preferred human-type receptors and increased replication in human cells in vitro, regardless of the presence of PB2-V/K/E627. Moreover, H9N2 AIVs containing HA-L226, PB2-V/K627 and...

Abstract The global spread of the A/goose/Guangdong/1/96-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses is accompanied by an expanded host range and the establishment of sustained viral transmission among dairy cattle . To evaluate if the evolving H5N1 viruses have changed tissue tropism over time, we compared the binding patterns of recombinant hemagglutinin (HA) proteins derived from clade 1 (A/Vietnam/1203/04, H5VN) and circulating clade 2.3.4.4b viruses detected from a wild bird (A/Eurasian Teal/Hong Kong/AFCD-HKU-23-14009-01020/2023, H5HK) and dairy cattle (A/bovine/Ohio/B24OSU-439/2024, H5OH). The HA protein of A(H1N1)pdm09 virus was included for comparison. Using bio-layer interferometry, H1 protein preferentially bound to the 2,6-linked sialoside 6'SLNLN while H5 proteins preferentially bound to the 2,3-linked sialoside 3'SLN. H5OH showed higher binding affinity to 3'SLN than H5HK and H5VN. The attachment pattern of H1 and H5 proteins to the respirato...

ABSTRACT A panzootic of highly pathogenic avian influenza (HPAI) H5N1 viruses from clade 2.3.4.4b has triggered a multistate outbreak in US dairy cattle and an unknown number of human infections . HPAI viruses are handled in specialized biocontainment facilities. Ethical considerations limit certain evolution experiments aimed at assessing viral resistance to potential therapeutics. We have developed a replicating recombinant vesicular stomatitis virus (rVSV) where we replaced its glycoprotein with the hemagglutinin (HA) and neuraminidase (NA) genes of a 2.3.4.4b H5N1 virus (rVSV-H5N1dc2024), which enables these experiments to be performed under standard biosafety considerations. This virus grows to high titers and encodes a fluorescent reporter to track infection. We demonstrate the utility of rVSV-H5N1dc2024 in neutralization experiments, the evaluation of antibody escape, and the characterization of resistance mutations to NA inhibitors. rVSV-H5N1dc2024 or similar viruses may accele...

Highlights •  Different immune responses in mice vaccinated with influenza A(H5N1) than with other subtypes. •  Highly pathogenic avian influenza A(H5N1)-vaccinated mice had altered germinal center responses. •  A(H5N1)-vaccinated mice had fewer dLN germinal centers and more extrafollicular B cells. •  A(H5N1)-vaccinated mice had more dLN follicular helper and regulatory T cells. •  Our study represents a timely assessment of A(H5N1) risk to human health. Abstract Highly pathogenic avian influenza (HPAI) H5N1 virus vaccines typically yield lower neutralizing antibody titers in animals than influenza A virus (IAV) vaccines derived from other viral subtypes. To understand these differences, we compared the cellular immune responses in the draining lymph nodes (dLNs) of mice vaccinated with an inactivated whole H5N1 vaccine to those in mice vaccinated with seasonal H1N1pdm09, H7N9, or H9N2 IAV vaccines . H5N1-vaccinated mice exhibited reduced serum neutralizing ant...

Abstract Avian influenza H9N2 viruses cause significant economic losses to the poultry industry and pose a public health risk due to their potential to reassort with other avian influenza viruses, generating strains with zoonotic and pandemic potential . Two major H9N2 lineages dominate globally: the G1 lineage (genotype G1-B), prevalent in the Middle East, Africa and the Indian subcontinent , and the BJ/94 lineage (predominantly genotype G57), dominant in China, Vietnam, South Korea, Indonesia, and the Far East . We investigated replication, transmission, and pathogenicity of representatives of these two lineages, linking genotype to phenotype. The G57 strain A/Ck/Vietnam/H7F-14-BN4-315/2014 (Vietnam/315) was more lethal to chicken embryos than the G1-B strain A/chicken/Pakistan/UDL-01/2008 (Pakistan/UDL-01). Vietnam/315 exhibited higher replication in both directly infected and contact chickens, with increased virus shedding from the oropharynx and cloaca. In contrast, Pakistan/UDL-0...

Abstract The interspecies transmission of avian influenza viruses remains a significant public health concern. H6 viruses have gained attention following the first human infection by a chicken-origin H6N1 virus (A/Taiwan/02/2013, Hu/13), highlighting their zoonotic potential . To understand the evolutionary trajectory and mammalian adaptation of this Taiwan lineage , we compared two avian isolates (A/Chicken/Taiwan/CF19/2009, Ck/09; A/Chicken/Taiwan/2267/2012, Ck/12) and Hu/13 in vitro and in vivo. Hu/13 exhibited enhanced replication in MDCK cells , producing larger plaques and higher viral titers than Ck/09 and Ck/12. In BALB/c mice , Hu/13 demonstrated the highest pathogenicity and mortality , followed by Ck/12, while Ck/09 induced minimal morbidity. Hu/13 and Ck/12 replicated efficiently in respiratory tissues , eliciting robust cytokine responses and severe pulmonary lesions . In ferrets , Hu/13 showed relatively efficient transmission , infecting all direct physical-contact and t...

Abstract As an avian influenza virus (AIV) panzootic is underway , the threat of a human pandemic is emerging . Infections among mammalian species in frequent contact with humans should be closely monitored. One mammalian family , the Felidae , is of particular concern. Domestic cats are susceptible to AIV infection and provide a potential pathway for zoonotic spillover to humans. Here, we provide a systematic review of the scientific literature to describe the epidemiology and global distribution of AIV infections in felines reported from 2004 to 2024. We identified 607 AIV infections in felines , including 302 associated deaths , comprising 18 countries and 12 felid species . We observed a drastic flux in the number of AIV infections among domestic cats in 2023 and 2024, commensurate with the emergence of H5N1 clade 2.3.4.4b. We estimate that this phenomenon is underreported in the scientific literature and argue that increased surveillance among domestic cats is urgently needed. Sou...

Abstract Thousands of outbreaks of the highly pathogenic avian influenza A(H5N1) virus in birds and an increasing number of mammal infections are registered annually. In 2023, multiple avian influenza outbreaks were registered among wild birds, poultry and seals in Russia . The genetic characterization of seventy-seven avian viruses and three viruses from seals showed that they belonged to the 2.3.4.4b clade and represented four distinct reassortant genotypes . The majority of viruses represented genotype BB , which was widespread in Europe in 2023. Viruses from seals and four viruses from birds , isolated from outbreaks in the Far East region , belonged to the G1 (A3) genotype and had the amino acid substitution N319K in the NP protein , previously associated with an increased virulence for mammals . In addition, one virus of the G10 genotype and two viruses, representing a previously undescribed genotype (designated as Ru-23-G4) were identified. The viruses analyzed showed normal inh...

ABSTRACT Sporadic human infections of avian influenza virus (AIV) raise significant public health concerns . A critical factor limiting the transmission of AIVs is the shift in receptor-binding preference from Siaα2,3 to Siaα2,6. To reveal the adaptive selection dynamics during the host adaptation process of AIVs, this study generated a viral library with random mutations in the HA gene of the H9N2 strain . Upon passaging the viral library in chickens and mice , the predominantly selected variants exhibited a preference for Siaα2,3 receptors . Notably, the wild-type strain remained dominant in both inoculated and direct-contact chickens, while variants with the ΔL226/R229I substitutions were preferentially selected in mice. Ferrets have a predominance of Siaα2,6 in their respiratory tract. As expected, the variant harboring the N289D mutation, which prefers Siaα2,6 binding, was enriched during in vivo passaging in ferrets . The mice-adapted variant with the ΔL226/R229I mutations causes...

Abstract Surveillance of influenza A viruses in pigs (SwIAV) is critical for identification of novel genetic groups that pose a risk to pig health and might have zoonotic potential . SwIAVs circulating in pigs in England between 2014 and 2021 were characterised using whole genome sequencing (WGS). Haemagglutinin (HA) and neuraminidase (NA) sequencing data from 82 of 368 influenza A positive samples (71 submissions) were determined, identifying H1N1 and H1N2 subtypes from the 1A classical swine and 1B human-seasonal lineages respectively. The 1B lineage viruses were predominant, accounting for 68.29% of sequenced viruses, with 1A lineage viruses comprising 31.71%, primarily from the 1A.3.3.2 clade (2009 H1N1 pandemic origin). This study characterised previously undefined diversity within the 1B lineage which led to the designation of new HA clades 1B.1.1.1, 1B.1.1.2 and 1B.1.1.3. Complete genome data were obtained from 64/82 viruses thereby updating the definition of genetic diversity t...

Editor’s summary High-pathogenicity avian influenza subtype H5N1 is now present throughout the US , and possibly beyond. More cattle infections elevate the risk of the virus evolving the capacity to transmit between humans , potentially with high fatality rates . Nguyen et al. show that from a single transmission event from a wild bird to dairy cattle in December 2023, there has been cattle-to-poultry, cattle-to-peridomestic bird , and cattle-to-other mammal transmission. The movement of asymptomatic dairy cattle has facilitated the rapid dissemination of H5N1 from Texas across the US . Evolution within cattle, assessed using deep-sequencing data, has detected low-frequency sequence variants that had previously been associated with mammalian adaptation and transmission efficiency. —Caroline Ash Structured Abstract INTRODUCTION Highly pathogenic avian influenza (HPAI) viruses have critical consequences for animal health and the agricultural economy—and may have pandemic potential. HPAI ...

Abstract The avian influenza virus (AIV) primarily affects birds and poses an increasing concern due to its growing adaptability to other hosts , heightening zoonotic risks . The adaptability is a key factor in AIV to infect multiple non-avian species, including humans , companion animals , aquatic mammals , carnivores , and other mammals. The virus is evolving through genetic mutations and reassortments , leading to the emergence of AIV strains with enhanced virulence and adaptability in mammals. This highlights the critical need to understand the genetic factors of AIV, including mutations in polymerase proteins , surface antigens , and other regulatory proteins, as well as the dynamics of AIV- host interactions and environmental factors such as temperature, humidity, water salinity, and pH that govern the cross-species adaptability of the virus. This review provides comprehensive insights into the molecular/genetic changes AIV undergoes to adapt in mammalian hosts including bovines,...

Significance We explored how H5Nx influenza viruses , which can infect many different birds and mammals, could adapt to infect humans by altering the hemagglutinin (HA). HA must change to bind human-type receptors for transmission between people. We compared two strains from viruses isolated in 2016 and found that one ( 2.3.4.4e ) can switch to human receptor binding with a single mutation , while another ( 2.3.4.4b ) might require more complex changes to bind simple human-type receptors. These findings highlight the potential for specific strains to evolve and become a pandemic threat, underscoring the importance of monitoring mutations that could lead to human-type receptor adaptation. Abstract H5Nx viruses continue to wreak havoc in avian and mammalian species worldwide . The virus distinguishes itself by the ability to replicate to high titers and transmit efficiently in a wide variety of hosts in diverse climatic environments. Fortunately, transmission to and between humans is sca...