An #NS1-F161L #Substitution Determines #Host-Driven #Virulence Enhancement of #H5N6 Avian #Influenza Virus in #Ducks

 

Abstract

H5 subtype avian influenza virus (AIV) can infect both chickens and ducks, leading to substantial economic losses. Nevertheless, certain strains cause silent infections in ducks. In this study, a goose-origin clade 2.3.4.4h H5N6 AIV was isolated, which caused high mortality in mixed-gender white leghorn chickens but no deaths in mixed-gender mallard ducks. After independent serial in vitro passage in duck embryo fibroblasts (DEFs) and in vivo passage in specific-pathogen-free (SPF) ducks, the DEF-passage 10 (P10) virus induced markedly higher mortality rates and viral loads in SPF ducks compared to the DEF-P1 virus and the original parental virus prior to passage. Similarly, the in vivo-passaged P3 and P4 viruses exhibited significantly higher mortality rates than the P1 virus in SPF ducks, with 100% mortality and markedly increased viral titers in the organs. A whole-genome SNP analysis identified seven high-frequency mutations in the M1, NA and NS1 proteins. The NS1-F161L substitution virus exhibited significantly increased mortality rates, viral loads in multiple tissues, and a robustly induced innate immune response in ducks. Furthermore, dynamic evolutionary variations in the NS1 protein among global H5 avian influenza viruses revealed that the NS1-F161L substitution became dominant in clade 2.3.4.4b viruses in 2021 and subsequent years. Collectively, our findings demonstrate that host-driven adaptation can rapidly increase the pathogenicity of H5N6 AIVs in ducks and identify NS1-F161L as a critical virulence marker. These results offer novel insights relevant to the molecular surveillance, virulence prediction, and risk assessment of circulating H5 AIVs in waterfowl.

Source: 

Link: https://www.mdpi.com/1999-4915/18/5/488

____

Heart-nosed #bat #alphacoronaviruses use #human CEACAM6 to enter #cells

 

Abstract

Identifying viruses with zoonotic potential on the basis of their ability to enter human cells is a critical component of pandemic prediction, prevention and preparedness. Here using a computational approach that retains maximum phylogenetic diversity, we selected an optimal subset of alphacoronavirus spike proteins to screen against broad coronavirus receptor libraries. Most of the selected spike proteins did not use any of the established coronavirus receptors. However, the pseudotyped spike protein of Cardioderma cor (heart-nosed bat) coronavirus KY43 (CcCoV-KY43) could enter human cells. Using a recombinant CcCoV receptor-binding domain (RBD) and a human receptor screening platform, we identified direct interactions with the human CEACAM proteins CEACAM3, CEACAM5 and CEACAM6. Overexpression of human CEACAM6—a protein widely expressed in the human lung—conferred permissivity to otherwise refractory human cells. A crystal structure showed that the RBD binds the amino-terminal IgV-like domain of human CEACAM6. Immune surveillance studies using sera of individuals from the Taveta region of Kenya, where CcCoV-KY43 was identified, did not show significant evidence of recent spillover. Wider characterization of alphacoronaviruses related to CcCoV-KY43 showed that human CEACAM6 is used by two other CcCoVs collected in Kenya. Moreover, there was more restricted nonhuman CEACAM6 tropism for viruses isolated from Rhinolophus bats from Russia and China. Thus, alphacoronaviruses that use CEACAM6 are probably geographically widespread, and viruses from East Africa show potential for transmission to humans.

Source: 

Link: https://www.nature.com/articles/s41586-026-10394-x

____

A natural five-amino-acid insert at the S2’ #cleavage site of #MERS-CoV #spike enhances viral membrane fusion

 

Highlights

• A novel 5-aa insert, TSGVF, is present at the S2’ cleavage site of the spike protein of MERS-CoV from dromedary camels.

• Pseudovirus-based entry assays showed that the TSGVF insert increases viral entry efficiency in different human cells.

• Pseudovirus with TSGVF insert at the S2’ cleavage site showed strong resistance to TMPRSS2 inhibitor.

• The natural occurrence of TSGVF insert at the spike S2’ cleavage site enhances viral membrane fusion and syncytia formation.

Source: 

Link: https://www.sciencedirect.com/science/article/pii/S1995820X26000611?via%3Dihub

____

#Infection of the #bovine mammary #gland by avian #H5N1 subclade 2.3.4.4b #influenza viruses

 

Abstract

The emergence of the panzootic clade of highly pathogenic avian influenza H5N1 (2.3.4.4b) in 2020 marked a major expansion in the host range of influenza A viruses (IAVs), raising concerns about further cross‑species transmission events and zoonotic spillover. Introduction of 2.3.4.4b viruses into U.S. dairy herds has resulted in widespread circulation, accompanied by reduced milk yield, mastitis, and high viral loads in milk. Notably, virus circulation in dairy cattle represents a novel route for mammalian adaptation and transmission that has already led to more than 40 human cases in the U.S. since 2024. Here, we investigated whether avian clade 2.3.4.4b viruses could infect mammary tissue from Aberdeen Angus, Holstein Friesian, and Limousin cattle, three breeds commonly farmed in Europe, the Americas, and Oceania. Using mammary gland explants, we inoculated tissues with attenuated reassortant viruses expressing the haemagglutinin and neuraminidase glycoproteins of three 2.3.4.4b viruses that predated the emergence of H5N1 in US cattle: A/chicken/England/053052/2021 (AIV07), A/chicken/Scotland/054477/2021 (AIV09), and A/chicken/England/085598/2022 (AIV48). Infected epithelial cells were identified using immunohistochemistry in explants from both the teat and gland cistern for all three breeds following infection with AIV09 and AIV48, indicating that mammary tissue from each of the three tested cattle breeds cattle is permissive to H5N1 infection. Lectin staining showed expression of both α2,3‑linked and α2,6‑linked sialic acids in the mammary tissue of all donors showing that all three breeds have the potential to support infection with both avian-adapted and mammalian adapted IAVs. Together, these findings demonstrate that mammary glands from both beef and dairy cattle breeds are permissive to infection with avian‑adapted and mammalian-adapted H5N1 viruses and highlight the potential for this tissue to act as a mixing vessel for IAV reassortment, underscoring the need to include cattle in ongoing H5N1 surveillance and risk‑assessment frameworks.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1, MR/Y03368X/1, MC_UU_0034/2, MC_UU_0034/3, MC_UU_0034/1

Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/V004697/1

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.04.16.718897v1

____

Acquisition of specific #human respiratory tract binding of 2.3.4.4b #H5N1 #hemagglutinins requires multiple #mutations

 

Abstract

It has been suggested that the hemagglutinin of the human-infecting cattle-derived 2.3.4.4b virus A/Texas/34 (H5TX) requires only one mutation, namely Q226L, to switch from binding avian-type to human-type receptor preference. In this study, we examined the binding of H5TX Q226L, along with other key mutations, to sections of human trachea. We conclude that, while H5TX Q226L can bind human-type receptors, more than a single mutation is required for this protein to bind to human respiratory tract tissue. We also report changes in receptor-binding specificity of another 2.3.4.4b HA mutant, H5FR Q226L, associated with the presence of a multibasic cleavage site. This study offers insight into the determinants of evolution towards human-type receptor binding in currently circulating H5Nx viruses. It also emphasizes the importance of testing individual strains using additional methods, including tissue-based approaches, alongside synthetic glycans.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

NWO, OCENW.M20.106

Horizon, 862605

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.04.16.718875v1

____

#SARS-CoV-2 and #MERS-CoV disrupt #host #protein synthesis via nsp1 with differential effects on the integrated stress response

 

Significance

Coronaviruses cause disease across a wide range of animal species and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus nonstructural protein 1 (nsp1) promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus–host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.

Abstract

Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus nonstructural protein 1 (nsp1) has been shown to inhibit host protein synthesis and, in some coronaviruses, promote host messenger RNA (mRNA) degradation while viral mRNAs are protected. We showed previously that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induces activation of host integrated stress response (ISR) kinases protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), which promote phosphorylation of eukaryotic initiation factor 2 (eIF2α) and consequent inhibition of host protein synthesis. In contrast, eIF2α remains unphosphorylated during Middle East respiratory syndrome coronavirus (MERS-CoV) infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2α. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2α phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast, MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescence in situ hybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis.

Source: 

Link: https://www.pnas.org/doi/abs/10.1073/pnas.2536296123?af=R

____

Historical #Pandemic and Contemporary #Influenza A Viruses Reveal #PB2 M631L as a Convergent #Adaptation to #Human ANP32

 

Abstract

Understanding the genetic changes that allow avian influenza A viruses (IAVs) to switch their natural hosts and establish productive infection in humans is important for pandemic risk assessment. Adaptations in the IAV polymerase are required to overcome species-specific restrictions imposed by host ANP32 proteins. Notably, avian virus polymerase is generally only poorly supported by human ANP32 proteins due to species-specific differences. Consequently, efficient polymerase adaptation to the binding interface of human ANP32 requires distinct amino acid changes, such as PB2 E627K. A separate adaptation, PB2 M631L, has recently been reported in mammalian-adapted IAV; however, its functional role across divergent viral lineages and its relationship to host ANP32-dependent adaptation remain incompletely defined. Here, we examine PB2 M631L in the polymerases of a 1918 pandemic strain, a recombinant contemporary H1N1pdm09, and a recent clade 2.3.4.4b H5N1 virus. Using polymerase activity and protein-interaction assays, we show that PB2 M631L enhances polymerase activity and ANP32 binding in human—but not avian—contexts, and that this effect is conserved across multiple viral backgrounds. In H1N1pdm09, PB2 M631L also increased virus replication in mammalian cells. These findings indicate that PB2 M631L contributes to enhanced polymerase compatibility with human ANP32 proteins and are consistent with a role in adaptation across multiple influenza virus lineages. Our results highlight how analysis of historical pandemic strains can inform risk assessment for future emerging viruses.

Source: 

Link: https://www.mdpi.com/2076-2607/14/4/859

____

#Species - and #variant - specific #ACE2 compatibility shapes #SARS-CoV-2 #spillover potential in North American #cervids

 

Abstract

Free-ranging white-tailed deer (WTD) are established SARS-CoV-2 reservoirs, but the susceptibility of other cervid species remains unclear. Here we integrate receptor analysis, structural modeling, and field surveillance to assess SARS-CoV-2 susceptibility across North American cervids. We identify species- and variant-specific differences in ACE2–spike compatibility. Elk ACE2 exhibits weak binding to the ancestral strain (Wuhan-Hu-1) and Delta spike receptor-binding domains (RBDs), likely due to a unique K31N substitution. In contrast, it shows stronger binding to Alpha, Beta, Gamma, and Omicron RBDs containing N501Y. Biophysical assays, gel filtration chromatography, and cryo-EM confirm stable complex formation between elk ACE2 and Alpha RBD, but not RBD from the ancestral strain. Despite weak binding, elk ACE2 supports viral entry and replication in vitro. However, surveillance revealed limited evidence of infection in the United States, contrasting with widespread WTD transmissions. These findings demonstrate that ACE2 compatibility alone is insufficient to predict reservoir potential and provide a framework for assessing species susceptibility to emerging coronaviruses.

Source: 

Link: https://www.nature.com/articles/s41467-026-71623-5

____

#Genetic and #biological characterization of a #duck-origin clade 2.3.4.4b #H5N6 avian #influenza virus reveals partial #mammalian #adaptation

 

Highlights

• Duck-origin H5N6 virus A/Duck/Jiangsu/628/2022 shares high homology with the human strain A/Yangzhou/125/2022.

• The 628 strain shows mammalian adaptation markers: HA mutations enhance human receptors affinity and NA mutations reduce sensitivity to neuraminidase inhibitors.

• Limited airborne transmission but detectable droplet-mediated spread suggests increased mammalian transmission risk.

Abstract

Clade 2.3.4.4b H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused extensive outbreaks in poultry worldwide. H5 HPAIVs have caused sporadic but severe human infections in China, representing a persistent zoonotic threat. Here, we identified a duck-origin H5N6 HPAIV (A/Duck/Jiangsu/628/2022) through routine surveillance and assessed its biological characteristics and mammalian pathogenesis. Phylogenetic analysis revealed > 98% nucleotide identity between strain 628 and the concurrent human H5N6 strain A/Yangzhou/125/2022. Molecular characterization identified multiple mammalian adaptation markers: hemagglutinin substitutions (S137A, T160A, T192I) associated with enhanced human receptor binding; neuraminidase mutations (I117T, D198N) linked to reduced neuraminidase inhibitor susceptibility; and polymerase complex changes (PB1-D622G, PA-K142Q) conferring increased mammalian cell replication. In vitro studies demonstrated that 628 virus replicated more efficiently in mammalian than in avian cells and exhibited dual receptor-binding specificity. Mouse pathogenicity assays revealed moderate virulence with progressive lung pathology. Critically, transmission experiments confirmed both direct contact and airborne transmission capabilities of 628 in guinea pigs. These findings demonstrate that circulating H5N6 viruses have acquired partial mammalian adaptation while retaining avian fitness, significantly elevating pandemic potential. Enhanced surveillance of wild bird populations, poultry farms, and live poultry markets is urgently needed to develop effective prevention and control strategies.

Source: 

Link: https://www.sciencedirect.com/science/article/abs/pii/S037811352600146X?via%3Dihub

____

Q1020R in the #spike proteins of #MERS-CoV from Arabian #camels confers resistance against soluble #human #DPP4

 

ABSTRACT

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a pre-pandemic coronavirus that is transmitted from camels, the natural reservoir, to humans and can cause severe disease. MERS cases have been documented in Arabia but not Africa, although the virus is circulating in both Arabian and African camels. Further, evidence has been provided that viruses in African camels might have a reduced capacity to cause disease. However, the underlying determinants are incompletely understood. Here, employing pseudotyped particles as model systems for MERS-CoV entry into cells, we compared cell entry of viruses from African and Arabian camels and its inhibition. We show that viruses found in Arabian camels and recent human cases are less susceptible to inhibition by human soluble DPP4 (sDPP4) than viruses from African camels, although both enter human cells efficiently and are comparably sensitive to inhibition by interferon-induced transmembrane (IFITM) proteins and neutralizing antibodies. Furthermore, relative resistance to sDPP4 was linked to mutation Q1020R, present in the spike proteins of recent Arabian but not African viruses. Finally, indirect evidence was obtained that sDPP4 in human plasma can inhibit MERS-CoV cell entry. These results support the concept that soluble DPP4 might constitute a natural barrier against human infection that is more efficiently overcome by viruses currently circulating in Arabian camels than those in African camels.

Source: 

Link: https://journals.asm.org/doi/10.1128/jvi.00282-26

____

#Bovine #H5N1 #influenza viruses have adapted to more efficiently use #receptors abundant in #cattle

 

Abstract

Sustained mammal-to-mammal transmission of high pathogenicity H5N1avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected early in 2024. Genomic analysis revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identifies alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

Competing Interest Statement

JDB and BD are inventors on Fred Hutch licensed patents related to deep mutational scanning. JDB consults for Pfizer, GSK, Apriori Bio, and Invivyd.

Funder Information Declared

Medical Research Council, MR/Y03368X/1, MR/R010757/1, MC_UU_0034/2, MC_UU_0034/3

BBSRC, BB/Y007298/1, BB/X006123/1, BB/X006166/1, BBS/E/PI/230002A, BBS/E/PI/230002B

BBSRC, BBS/E/PI/23NB0004, BBS/E/PI/23NB0003, UKRI2253, BB/V004697/1

Defra, SE2227

Royal Society, https://ror.org/03wnrjx87, RGS\R2\242118

Houghton Trust, HT/SPRG/23/04

Flanders, G005323N, G051322N, G010326N

CEIRR, 75N93021C00045

The Rockefeller Foundation, PC-2022-POP-005

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.04.02.715584v1

____

Clade C #MERS-CoV #camel #strains vary in #protease utilization during viral entry

 

Significance

Clade A/B Middle East Respiratory Syndrome coronavirus (MERS-CoV) outbreaks have caused over 957 deaths since the first spillover in 2012; meanwhile, Clade C strains have been found in camels across Africa but have not yet been reported to cause outbreaks. Investigating why these viruses do not successfully transmit to humans will be key to understanding the pandemic potential of the African MERS-CoV camel reservoir. Our study indicates that clade C viruses exhibit less spike cleavage and that East African clade C isolates are less able to utilize the TMPRSS2 for viral entry of both human cell lines and primary nasal cells. Differences in viral entry pathways could alter cellular and organ tropism and contribute to differential pandemic potential.

Abstract

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a lethal pathogen with pandemic potential. Clade A and B MERS-CoV viruses have caused outbreaks in the Middle East since 2012 when they initially spilled over from camels to humans. Clade C viruses, however, are only found in camels across Africa and the spillover potential of these viruses seems to be lower than for clade A/B strains but remains to be fully understood. Here, we report that clade C spikes are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes and that most clade C spikes induce reduced syncytium formation. Additionally, we demonstrate that several East African clade C strains are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures. We map the molecular basis of this reduced TMPRSS2 usage to the N-terminal domain and subdomain 2 of East African clade C MERS-CoV. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may underlie the reduced replication of East African clade C strains in humans, while the reduced replication of West African strains remains to be further investigated. Altered protease usage may contribute to differential tropism of East African clade C strains and indicate geographically distinct selection pressures on spike between MERS-CoV strains circulating in camels.

Source: 

Link: https://www.pnas.org/doi/10.1073/pnas.2525313123

____

#AI - guided multi-omics #analysis identifies NPC1-modulated susceptibility to #SARS-CoV-2 #infection under #PM2.5 exposure

 

Abstract

Exposure to airborne fine particulate matter (PM2.5) has been linked to increased risk of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, yet the underlying mechanisms remain unclear. Here, by leveraging a fine-tuned foundation model of single-cell transcriptomics, we uncover shared transcriptional signatures between PM2.5 exposure and SARS-CoV-2 infection. We further validate this association using population-level epidemiological analyses and perform genome-wide association studies (GWAS) to identify genetic variants that modulate infection risk under PM2.5 exposure. In addition, we identify NPC1 as a key modulator involved in SARS-CoV-2 infection efficiency under virus-laden PM2.5 exposure through integrative functional genomic analyses and in vitro experiments. Our findings suggest that PM2.5 facilitates viral entry through an NPC1-modulated endo-lysosomal pathway, providing a mechanistic explanation for observed pollution-related susceptibility. By integrating artificial intelligence (AI)-guided transcriptomics, epidemiology, GWAS, functional genomics, and in vitro verification, our study elucidates how environmental and genetic factors jointly influence SARS-CoV-2 susceptibility. This work highlights how AI-assisted multi-omics integration systematically decodes the health impacts of environmental exposures from molecular to population levels and informs air quality policy and infectious disease preparedness.

Source: 

Link: https://www.nature.com/articles/s41467-026-71196-3

____

Host-specific functional #evolution of #seal #influenza A virus #NS1 protein following #avian-to-seal #transmission

 

ABSTRACT

Marine mammals, particularly seals, are susceptible to both avian and human influenza A viruses (IAVs), making them potential intermediates for zoonotic virus emergence. In recent decades, repeated transmissions of avian influenza viruses (AIVs) from wild aquatic birds, their natural reservoir, have caused significant mortality in seals. Defining the molecular determinants of viral adaptation in marine mammals, and their implications for replication in human cells, is therefore essential. The non-structural protein 1 (NS1) of AIV, a key antagonist of the interferon (IFN) response, plays a central role in host adaptation. Here, we analyzed NS1 proteins from seal influenza viruses (H3, H4, H5, H7, and H10 subtypes) and their closest avian relatives isolated between 1980 and 2023, and evaluated their function in seal, avian, and human cells. Phylogenetic analysis confirmed multiple bird-to-seal transmission events. Seal-derived NS1 proteins generally contained few strain-specific amino acid substitutions and showed comparable expression and IFN antagonism to their avian precursors. A notable exception was the seal H10N7 virus isolated in 2014 in Northeastern Europe, which harbored three previously uncharacterized substitutions at NS1 amino acid residues 94, 104, and 171. These amino acid substitutions markedly altered NS1 properties to enhance protein stability, suppress IFN induction, mediate host transcription shut-off, and increase polymerase activity in human cells, without affecting NS1 expression or reducing virus replication in avian cells. Overall, these results reveal how NS1 undergoes host-specific functional evolution following avian-to-seal transmission and provide mechanistic insight into the adaptation of influenza A viruses to mammalian hosts.

IMPORTANCE

Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds but occasionally infect mammals, including seals, where they can cause severe outbreaks. Seals are of particular concern because they can harbor both avian and human influenza viruses, creating opportunities for reassortment and the emergence of novel zoonotic strains. Understanding how AIVs adapt to mammalian hosts is therefore critical for anticipating and mitigating future influenza threats. Here, we investigated the role of the NS1 protein, a key viral factor that suppresses host immune responses, in seal-derived AIVs. Overall, NS1 expression and function were conserved across different subtypes and host cells. However, we identified unique amino acid substitutions in the NS1 of a seal H10N7 virus that enhanced protein stability, interferon antagonism, and viral adaptation in human cells. These findings illustrate how minor changes in NS1 protein can drive host adaptation and underscore the need for continued surveillance of AIVs in seals.

Source: 

Link: https://journals.asm.org/doi/full/10.1128/jvi.01650-25?af=R

____

Characterisation of Naturally Occurring #MERS-CoV #Spike #Mutations and Their Impact on #Fusion and Neutralisation

 

Abstract

In this study, the phenotypic consequences of naturally occurring single nucleotide polymorphisms (SNPs) in the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein were investigated. The impact of Spike mutations on the syncytia formation and neutralisation of contemporary MERS-CoV strains is not currently well understood. Mutations were identified by aligning 584 MERS-CoV Spike sequences from either human clinical isolates collected between 2012 and 2024 or from a clinical isolate that had been passaged in human cells. Fifteen SNPs of interest occurring in the N-terminal domain (NTD), receptor binding domain (RBD) and adjacent to the S1/S2 cleavage site were selected for further characterisation based on their location in the Spike protein, frequency and identification in previous studies. A contemporary clade B, lineage 5 wildtype Spike sequence, obtained from a human MERS-CoV clinical isolate, was used as the backbone in this study. The mutations of interest were introduced to the wildtype backbone to generate Spike variants. Spike variants were characterised via cell–cell fusion assays, and a lentiviral pseudotyping system was used to investigate the impact of these Spike mutations on neutralisation. The I529T, E536K and L745F mutations were shown to increase fusion and syncytia formation. The L411F, T424I, L506F, L745F and T746K mutations were found to increase resistance to neutralisation by pooled patient sera. This study has identified novel naturally occurring Spike mutations that resulted in phenotypic differences in the syncytia formation and neutralisation of contemporary MERS-CoV strains. Continued investigation of the phenotypic consequences of MERS-CoV Spike mutations is essential for assessing the risk to public health, especially given the pandemic potential of this virus.

Source: 

Link: https://www.mdpi.com/1999-4915/18/3/377

____

Identification of #thermostability-enhancing #mutations in #H9N2 avian #influenza virus hemagglutinin

 

ABSTRACT

H9N2 avian influenza viruses (AIVs) remain a significant economic burden on poultry production and a persistent zoonotic threat. Hemagglutinin (HA), a surface glycoprotein mediating viral entry and pathogenesis, critically depends on thermostability for its function. Our previous study indicated that recent H9N2 AIVs have experienced a reduction in hemagglutination activity and exhibit low HA thermostability; however, the underlying molecular determinants for this instability remain poorly defined. To address this gap, we employed an in vitro-directed evolution approach to identify HA mutations that enhance thermostability. By subjecting a diverse HA mutant library to iterative heat selection at 48°C, we isolated several HA-stabilizing mutations, including L29I, N133S, N210D, G266R, D387N, A423T, and E509G, and confirmed their effect by site-directed mutagenesis. Further characterization revealed a complex interplay between HA stability, receptor binding specificity, and acid tolerance. Our findings demonstrate that enhancing HA stability can exert pleiotropic effects on key viral properties, highlighting the importance of understanding these relationships for developing effective mitigation strategies against H9N2 AIVs.

Source: 

Link: https://journals.asm.org/doi/full/10.1128/jvi.00168-26?af=R

____

Systematic Identification of the Functional lncRNAs During #H7N9 Avian #Influenza Virus #Infection in Mice

 

Abstract

Accumulating studies have identified the pivotal role of long non-coding RNAs (lncRNAs) in participating in host–virus interactions during virus infections. However, the regulatory roles of lncRNAs in influenza A virus (IAV) infection are still not fully elucidated. In this study, using high-throughput sequencing, we comprehensively compared the expression profiles of lncRNAs and mRNAs in mouse lungs infected either with the nonpathogenic parental (SDL124) H7N9 virus or its moderately pathogenic mouse-adapted (S8) variant. A total of 7636 significantly differentially expressed (SDE) lncRNAs were obtained in the S8-infected group compared to the mock group. As for the SDL124 group, 1042 SDE lncRNAs were identified. Subsequently, the mRNAs co-expressed with SDE lncRNAs were subjected to functional annotation and pathway enrichment analysis. The results indicated that the target mRNAs regulated by the S8 virus were mainly enriched in various immunological processes and exhibited a strong correlation with inflammatory-related signaling pathways. Moreover, 12 lncRNAs and 10 mRNAs co-expressed with SDE lncRNAs were selected and successfully verified by RT-qPCR. Among these lncRNAs, NONMMUG032982.2 and NONMMUG032328.2 exhibited strong antiviral activity against IAV. Additionally, these two lncRNAs were chosen for further in-depth bioinformatics analysis, including transcription factor prediction, coding capacity assessment, genomic location, construction of secondary structure, and prediction of potential interacting proteins. Taken together, these findings provide a cluster of lncRNAs probably associated with the virulence of IAV in mice and shed light on the anti-IAV effects of two functional lncRNAs, establishing a molecular foundation for further exploring the regulatory mechanisms of lncRNAs in IAV infection.

Source: Viruses, https://www.mdpi.com/journal/viruses

Link: https://www.mdpi.com/1999-4915/18/3/353

____

Structures of #Marburgvirus #glycoprotein and its complex with NPC1 #receptor

 

Abstract

Marburgviruses (MBVs) cause severe haemorrhagic fever with higher fatality rates than Ebola virus (EBOV). Here we show that the MBV glycoprotein (GP) mediates viral entry more efficiently than EBOV GP. Using cryo-EM, we determined structures of MBV GP in three states: (1) unbound; (2) bound to its endosomal receptor NPC1; and (3) complexed with a neutralizing nanobody. The glycan cap shields the receptor-binding site from NPC1 but only partially from the nanobody, enabling limited immune evasion. After glycan cap cleavage, NPC1 binds to MBV GP in a distinct orientation compared with EBOV GP, providing an additional anchor and enhancing receptor affinity. NPC1 engagement also induces substantial conformational changes in MBV GP, probably facilitating membrane fusion. Furthermore, MBV GP is susceptible to the neutralizing nanobody, which mimics NPC1 at the receptor-binding site. Together, our findings reveal MBV GP as a highly efficient entry mediator and suggest structural mechanisms that may contribute to its enhanced entry efficiency.

Source: Nature, https://www.nature.com/

Link: https://www.nature.com/articles/s41586-026-10240-0

____

#H5N1 2.3.4.4b HA E190D and Q226H #mutations, picked up as minority #variants in a #patient, result in an inability to bind #sialic acid.

 

Abstract

A human infection with clade 2.3.4.4b H5N1 influenza A virus in Canada revealed minority variants E190D and Q226H in the hemagglutinin (HA) receptor-binding site (RBS). Because mutations at positions 190 and 226 have been associated with altered receptor specificity in other influenza subtypes, we investigated their impact on receptor binding in H5 HA. Using a recombinant protein approach and an ELISA-based glycan-binding assay, we assessed binding to representative avian- and human-type sialylated glycans. Both single mutations and their combination resulted in a complete loss of detectable binding to the tested glycans. To evaluate whether this phenotype was background-dependent, Q226H was additionally introduced into two other H5 HA proteins, each representing a distinct clade. In both cases, the mutation similarly abolished receptor binding. These findings independently validate recent glycan microarray observations and demonstrate that the patient-derived E190D and Q226H substitutions severely impair receptor-binding capacity across multiple H5 backgrounds. Single mutations at key RBS residues in H5 often disrupt receptor binding rather than confer human-type receptor specificity, confirming complex mutational pathways required for adaptation to human-type receptors.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

ICRAD, n°862605 (Flu-Switch)

NWO, OCENW.M20.106

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.03.06.710037v1

____

Mechanism of co-transcriptional cap snatching by #influenza #polymerase

 

Abstract

Influenza virus mRNAs are stable and competent for nuclear export and translation because they receive a 5′ cap(1) structure in a process called cap snatching1. During cap snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that subsequently acts as a primer for the transcription of viral genes4,5. Here we present the cryogenic electron microscopy structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, positioning the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure, trapped after cap snatching, shows that the cleaved capped RNA rearranges within FluPol, directing the capped RNA 3′ end toward the FluPol polymerase active site for viral transcription initiation. Together, our results provide the molecular mechanisms of co-transcriptional cap snatching by FluPol.

Source: 

Link: https://www.nature.com/articles/s41586-026-10189-0

____