ETIDIOH - NEW

  Abstract Human influenza viruses rapidly acquire mutations in their hemagglutinin (HA) protein that erode neutralization by antibodies from prior exposures. Here, we use a sequencing-based assay to measure neutralization titers for 78 recent H3N2 HA strains against a large set of children and adult sera , measuring ~10,000 total titers . There is substantial person-to-person heterogeneity in the titers against different viral strains, both within and across age cohorts. The growth rates of H3N2 strains in the human population in 2023 are highly correlated with the fraction of sera with low titers against each strain. Notably, strain growth rates are less correlated with neutralization titers against pools of human sera, demonstrating the importance of population heterogeneity in shaping viral evolution . Overall, these results suggest that high-throughput neutralization measurements of human sera against many different viral strains can help explain the evolution of human influen...

  Abstract We detected H5N1 high pathogenicity avian influenza in captive Greater Rhea (Rhea americana). Viral genetic analysis revealed the mammalian associated PB2-E627K mutation , indicating selection of mammalian-relevant mutations in ratites . Pathologic investigation of available tissues demonstrated severe multifocal necrotising inflammation , and a strong vasculotropism. Competing Interest Statement The authors have declared no competing interest. Source: BioRxIV,  https://www.biorxiv.org/content/10.1101/2025.09.08.674895v1 ____

  Abstract The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a mixing vessel for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells , along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains , including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland , suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary c...

  Abstract Monkeypox virus (MPXV) experienced an unprecedented global outbreak in 2022, characterized by a significant departure from historical patterns: a rapid spread of the epidemic to more than 110 non-traditional endemic countries , with more than 90,000 confirmed cases; a fundamental shift in the mode of transmission , with human-to-human transmission (especially among men who have sex with men (MSM)) becoming the dominant route (95.2%); and genetic sequencing revealing a key adaptive mutation in a novel evolutionary branch ( Clade IIb ) that triggered the outbreak. These features highlight the significant evolution of MPXV in terms of host adaptation, transmission efficiency, and immune escape ability . The aim of this paper is to provide insights into the viral adaptive evolutionary mechanisms driving this global outbreak, with a particular focus on the role of immune escape (e.g., novel mechanisms of M2 proteins targeting the T cell co-stimulatory pathway) in enhancing vi...

  ABSTRACT Alterations in the PB2-627 domain of avian influenza virus (AIV) can potentially increase the risk of cross-host species infections in humans and mammals . Recently, there has been a rise in human cases of AIV infections without the presence of the known mammalian determinant PB2-E627K . Here, we identified a variant, PB2-627V , which has evolved in poultry and has contributed to the increase in human AIV infections . By screening global PB2 sequences , we discovered a new independent cluster of PB2-627V that emerged in the 2010s , prevalent in avian, mammalian, and human AIV isolates , including those of H9N2, H7N9, H3N8, 2.3.4.4b H5N1 , and other subtypes. We functionally assessed its host adaptation , fitness , and transmissibility across three subtypes of AIVs (H9N2, H7N9, and H3N8) in different host models . PB2-627V combines the viral properties of avian-like PB2-627E and human-like PB2-627K , facilitating AIVs to efficiently infect and replicate in chickens and mi...

Abstract In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA . As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans . Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammalian species including humans and pigs . We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L , which is found in all cattle sequences, and PA K497R , which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication . We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins , particularly ANP32A, and ...

  Abstract SARS-CoV and MERS-CoV are two coronaviruses that have received significant attention due to their high pathogenicity and mortality rates in human populations . In this study, we compared their evolutionary dynamics to provide a One Health perspective on their differences in terms of the results of disease control. The phylogenetic network of SARS-CoVs showed that human isolates gathered into a “super-spreader” cluster and were distinct from civet isolates . In contrast, dromedary camel- and human-isolated MERS-CoVs were clustered together. Thus, most clades of MERS-CoV can infect humans , and MERS-CoVs seem to more easily spill over the animal-to-human interface. Additionally, the civet can be easily controlled , while the intermediate host (dromedary camels) of MERS-CoV is an important livestock species , so it is impossible to eliminate all animals. This further leads to difficulties in disease control in MERS. Although MERS-CoVs are endemic to dromedary camels in both...

ABSTRACT The 2.3.4.4b clade highly pathogenic avian influenza H5N1 infected diverse non-human mammalian species , gained mammal-to-mammal transmission potential , and caused sporadic human infections . However, whether non-human mammals enable the human adaptation of 2.3.4.4b H5N1 to establish human infections is unclear. Gain-of-function research restrictions may hinder the assessment of 2.3.4.4b H5N1 human adaptations. Here, we tracked the evolution of 2.3.4.4b H5N1 that infected non-human mammals and evaluated their ability to gain human adaptations. The non-human mammal 2.3.4.4b H5N1 partly acquired classical human-adapting mutations , which are identical to the residues of H1N1pdm09 and seasonal human H3N2 viruses, while showing a few species-specific adaptations that might be potential barriers for successful human infections. The polymerase complex proteins , PA and PB2, acquired human adaptations in non-human mammals, with fox-infected viruses showing more positive selection in...

  ABSTRACT The H5N1 strain of influenza A virus (IAV) continues to cause severe infections in a range of avian and mammalian species , including sporadic but concerning cases in humans. There is growing concern that circulating H5N1 strains could lead to widespread human outbreaks . Research with highly pathogenic H5N1 viruses is restricted to Biosafety Level 3 (BSL-3) laboratories. Vesicular stomatitis virus (VSV)-based vaccine vectors expressing heterologous viral proteins from Ebola, SARS-CoV-2, Lassa virus, etc., have previously been shown to be safe and effective in animal models and human clinical trials . Here, we report the development of a recombinant VSV expressing the hemagglutinin (HA) and neuraminidase (NA) genes of H5N1 IAV (H5N1-VSV), which serves as a versatile platform to study various aspects of H5N1 IAV biology. H5N1-VSV replicated robustly to titers comparable to those of the full H5N1 virus in multiple cell lines. In mice , H5N1-VSV vaccination was safe, elicit...

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...