Biology subjects

Bayoumi, M.

Publications and source records attributed to Bayoumi, M..

14 recordsLinked to original sources

bioRxiv2026

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. SignificanceInfluenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

microbiology↗

bioRxiv2026

A single PA-X mutation in bovine-origin H5N1 influenza virus reduces pathogenicity in mice

Dairy cows have emerged as a reservoir for human infection with highly pathogenic avian influenza (HPAI) H5N1. At the bovine-human interface, H5N1 strains may acquire adaptive mutations that influence their zoonotic potential. Sequence analysis identified a K142E substitution (bovine to human) in the PA and PA-X proteins, with the potential to affect both polymerase activity and host shutoff. Here, we used a loss-of-function approach to investigate how the bovine substitution (E142K) in PA/PA-X impacts viral replication, host shutoff activity, and pathogenicity in the human H5N1 background. Viral growth kinetics demonstrated that the virus containing the E142K substitution is attenuated, with reduced replication compared to wild-type (WT) virus. Consistently, PA-X-mediated host shutoff activity was reduced, resulting in increased induction of interferon (IFN) responses relative to WT. In vivo, mice infected with the E142K mutant virus survived, whereas infection with the WT virus was uniformly lethal. Despite comparable viral titers and inflammation score in mouse lungs, cytokine and chemokine profiling revealed distinct immune responses, with reduced CCL2 and increased CCL5 and IFN-{gamma} in mice infected with the E142K mutant virus compared to mice infected with the WT virus. These findings indicate that increased virulence of the human-adapted strain is driven by a PA-X mutation that modulates inflammatory responses, producing distinct immune signatures linked to host survival or viral lethality rather than changes in polymerase activity by PA. Collectively, these results highlight PA-X as a key determinant of pathogenicity of H5N1 and a potential target for the rational design of antiviral strategies. IMPORTANCEHighly pathogenic avian influenza (HPAI) H5N1 viruses have recently expanded beyond their traditional avian hosts to infect mammals, where they are acquiring mutations associated with mammalian adaptation. These changes raise the concern that influenza H5N1 viruses could evolve the capacity for sustained human infection and human-to-human transmission and pose a pandemic threat. Therefore, it is critical to identify and functionally characterize emerging mutations that influence viral pathogenicity and host interactions. Such studies will enhance our understanding of the requirements for efficient infection and disease in mammalian hosts and inform the rational design of antiviral strategies. In this study, we present data characterizing a bovine-to-human substitution (K142E) in the viral PA-X impacting viral replication, host shutoff activity, and pathogenicity. Our results demonstrate the key role of PA-X in H5N1 viral pathogenicity and the feasibility of targeting PA-X for the rational design of antivirals to control influenza infections.

microbiology↗

bioRxiv2026

Fluorescent-expressing Modified Vaccinia Ankara Encoding T7 RNA Polymerase

Efficient recovery of negative-stranded RNA viruses from plasmid cDNA requires robust bacteriophage T7 RNA polymerase expression, commonly supplied by Modified Vaccinia Ankara expressing T7 polymerase (MVA T7). Here, we describe the generation and characterization of two fluorescent MVA T7 viruses expressing either enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1). Both fluorescent-expressing MVA T7 viruses retain plaque morphology, growth kinetics, and transgene expression comparable to parental MVA T7. Importantly, both fluorescent-expressing MVA T7 viruses support rescue of replication-competent recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. Fluorescent protein expression from MVA T7 viruses facilitates stock generation and titration and allows detection of remaining helper virus during rVSV recovery and amplification. Altogehter, EGFP- and mRFP1-expressing MVA T7 viruses provide a practical tool for monitoring T7-driven rescue of recombinant RNA viruses and identifying helper virus carryover. IMPORTANCEReverse genetics systems for negative-stranded RNA viruses rely on robust bacteriophage T7 RNA polymerase expression, commonly provided by a Modified Vaccinia Ankara virus expressing T7 polymerase (MVA T7). However, generation of virus stocks, monitoring MVA T7 helper virus infection and detecting residual virus during rescue of negative-stranded RNA viral can be challenging. We generated fluorescent-expressing MVA T7 variants that enable direct visualization of helper virus infection without compromising viral growth kinetics, transgene expression, or efficient rescue of recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. MVA T7 viruses expressing fluorescent proteins simplify stock generation and titration, facilitate optimization of negative-stranded RNA rescue conditions, and allow rapid identification of helper virus carryover during generation and amplification of recombinant virus, improving the efficiency, reproducibility, and quality control in reverse genetics systems relying on T7 expression widely used across virology.

microbiology↗

bioRxiv2026

Development and characterization of mouse-adapted recombinant SARS-CoV-2 expressing reporter genes

Transgenic K18-hACE2 mice are a standard model for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), albeit with limitations. A mouse-adapted 30 (MA30) SARS-CoV-2 has been developed to allow infection of wild-type (WT) mice strains. However, SARS-CoV-2 MA30 cannot be easily tracked in vitro, ex vivo, or in vivo. To address the problem, we developed a recombinant (r)SARS-CoV-2 based on the MA30 strain expressing fluorescent (mCherry) and luciferase (nanoluciferase, Nluc) reporter genes, alone or in combination, that enable tracking of viral infection in WT C57BL/6 and BALB/c mice. Insertion of the reporter genes resulted in minor viral attenuation in vitro, with [~]0.5-1.0-log lower titers than rSARS-CoV-2 MA30 WT in A549 hACE2 cells, while maintain similar plaque morphology and replication kinetics in Vero AT cells. In vivo, reporter-expressing rSARS-CoV-2 MA30 caused transient weight loss, contrasting with lethal rSARS-CoV-2 MA30 WT infection. Bioluminescence imaging of rSARS-CoV-2 MA30 Nluc in C57BL/6 and BALB/c mice revealed peak pulmonary replication at 2 days post-infection, with resolution by day 4, and correlated with tissue viral loads. Our results demonstrate the feasibility of using rSARS-CoV-2 MA30 expressing reporter genes to track viral infection in vitro, ex vivo, and in vivo without a need for secondary approaches to monitor viral infection as are required for rSARS-CoV-2 MA30 WT. Our system is highly suitable to evaluate prophylactic vaccines and therapeutic antibodies or antiviral approaches in WT or transgenic C57BL/6 and BALB/c mice without the shortcomings of K18-hACE2 mice and with the added advantage of non-invasive monitoring of treatment efficacy. ImportanceThe K18-hACE2 transgenic mouse model limits the capability to study SARS-CoV-2. While a mouse adapted 30 (MA30) has been developed to study SARS-CoV-2 in wild-type (WT) mice, it does not allow non-invasive tracking of viral infections. Recombinant viruses expressing reporter genes enable real-time monitoring of infection dynamics, opening an avenue to study viral tropism and easily evaluate prophylactic and therapeutic approaches. They furthermore support longitudinal studies, which reduces the number of research animals required. Here, we show that a recombinant (r)SARS-CoV-2 expressing fluorescent (mCherry) and nanoluciferase (Nluc) reporter genes, alone or in combination, can be used to track viral infections in vitro, ex vivo, and in vivo without the need for secondary approaches that are required to detect SARS-CoV-2 MA30 in WT mice. These reporter-expressing rSARS-CoV-2 MA30 may accelerate vaccine development and antiviral drug discovery in WT or transgenic mice bypassing the need for hACE2 overexpression in K18-hACE2 transgenic mice.

microbiology↗

bioRxiv2025

A cocktail of SARS-CoV-2 spike stem helix domain and receptor binding domain human monoclonal antibodies prevent the emerge of viral escape mutants

Neutralizing antibodies (NAbs) targeting the spike (S) glycoprotein remain a crucial therapeutic strategy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, emerging viral variants have escaped all Food and Drug Administration (FDA)-approved NAb treatments, underscoring the urgent need for effective therapeutic alternatives. Using a nanoluciferase (Nluc)-expressing attenuated recombinant SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b ({Delta}3a7b-Nluc), we characterized resistance profiles of two broadly protective NAbs targeting the S receptor binding domain (RBD) in S1 (1301B7) and the stem helix domain (SH) in S2 (1249A8). Serial passaging of {Delta}3a7b-Nluc under selective pressure identified a 1301B7 antibody-resistant mutants (ARM-B7) harboring an RBD mutation (S371F) that conferred resistance to 1301B7 and other RBD-directed NAbs (Casirivimab, SC27 and Sotrovimab). In contrast, no ARM emerged under treatment with 1249A8, or an antibody cocktail of 1301B7 (RBD) + 1249A8 (SH). These findings demonstrate that S2 SH-targeting NAbs shows higher genetic barrier to resistance than S1 RBD-targeting NAbs, and that a NAbs cocktail therapy targeting the SARS-CoV-2 S1 RBD and S2 SH offers the most effective strategy to prevent the emergence of escape mutations. Together, our findings provide critical insights into developing next-generation resistance-evading NAb therapies against SARS-CoV-2, and potentially other coronaviruses, and demonstrate the value of using our attenuated viral platforms for the safe identification of ARM without the potential biosafety concerns of doing these experiments using wild-type (WT) forms of SARS-CoV-2. SIGNIFICANCEThe clinical efficacy of early SARS-CoV-2 NAbs has been challenged by the emergence of escape viral variants, highlighting an urgent need to anticipate resistance. Using a luminescent attenuated SARS-CoV-2 platform, we profiled resistant mutations against two broadly protective SARS-CoV-2 NAbs. Passage of {Delta}3a7b-Nluc in the presence of a NAb targeting the RBD S1 domain (1301B7) readily selected for an ARM, whereas passage in the presence of an SH S2 domain NAb (1249A8) did not. Notably, a cocktail of 1301B7 and 1249A8 created a high barrier of selecting SARS-CoV-2 ARM, preventing the emergence of resistant variants. We identified an S371F mutation in the S1 RBD of ARM-B7 that confers resistance to 1301B7 and other S1 RBD-targeting NAbs. These results highlight the importance of combination therapies targeting both variable RBD S1 and conserved SH S2 domain for the efficient treatment of SARS-CoV-2 and to prevent the emerge of NAb-induced escape mutations.

microbiology↗

bioRxiv2025

Identification and characterization of a SARS-CoV-2 Mpro G23 deletion ensitrelvir-resistant mutant

Ensitrelvir is an antiviral drug that specifically targets the main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been approved for the treatment of coronavirus disease 2019 (COVID-19) due to the conservation of its target protein which is essential in the viral lifecycle. However, SARS-CoV-2 could introduce mutations in the viral proteins to confer resistance to antivirals. Thus, screening for drug-resistant SARS-CoV-2 mutants and elucidating their resistant mechanisms are critical for guiding the selection of effective antiviral therapies. Here, we utilized a luminescent attenuated SARS-CoV-2 ({Delta}3a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a G23 deletion (G23del) in Mpro with high resistance (>1,000 fold) to ensitrelvir, but not to the other Mpro inhibitor (nirmatrelvir) or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del was confirmed by generating a recombinant luminescent attenuated SARS-CoV-2 containing G23del in the non-structural protein 5 (NSP5) gene ({Delta}3a7b-Nluc G23del). {Delta}3a7b-Nluc G23del exhibited significant resistance to ensitrelvir in both cultured cells an in K18 hACE2 transgenic mice. Binding affinity revealed that G23del mutation substantially altered Mpro binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in Mpro can confer high resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating infections with SARS-CoV-2 containing G23del with these other approved antivirals. SIGNIFICANCEThe clinical use of SARS-CoV-2 antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for FDA-approved antivirals. Our study addresses this by employing a luminescent attenuated virus platform ({Delta}3a7b-Nluc WT) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we have identified a G23 deletion (G23del) in SARS-CoV-2 Mpro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer more than 1,000-fold increased resistance to ensitrelvir, SARS-CoV-2 containing G23del remained sensitive to other Mpro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using {Delta}3a7b-Nluc to safely identify and characterize drug resistant viruses without the biosafety concern of using virulent SARS-CoV-2 and advance the design of next-generation antiviral drugs.

microbiology↗

bioRxiv2025

A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 {Delta}3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. IMPORTANCESmall-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins ({Delta}3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

microbiology↗

bioRxiv2025

Targeting ROCK Signaling Mitigates Influenza Virus-Induced Fibrogenesis in Human Airway Organoids

Influenza A virus (IAV) pandemics continue to pose serious global health threats, particularly to immunocompromised individuals, children, and the elderly. IAV infections trigger inflammation and tissue damage, promoting lung fibrosis. Unraveling these mechanisms is key to preventing and treating viral-induced pulmonary fibrosis and its lasting impact on respiratory health. Despite available antivirals and vaccines, there is a lack of FDA-approved therapeutics for severe or prolonged IAV pathogenesis. We modeled infection with a recombinant highly pathogenic human A/Texas/37/2024 H5N1 (rHPh-TX H5N1) strain using human airway organoids (HAO) to investigate viral replication, innate immune response, infection-induced fibrogenesis, and therapeutic approaches. The rHPh-TX H5N1 replicated efficiently, triggering a potent interferon (IFN) response and pro-inflammatory cytokine expression in HAO. Prolonged infection led to increased fibroblast-like cells surrounding infected areas, characterized by increased alpha-smooth muscle actin (-SMA) expression and upregulation of transforming growth factor-beta (TGF-{beta}), which caused fibroblast activation and extracellular matrix remodeling. Fibrosis-associated markers (FN, COL1A, COL3A, MMP2, MMP9) were significantly higher than in HAO infected with a pandemic recombinant A/California/04/09 H1N1 (pH1N1). Notably, Rho-associated coiled-coil-forming protein kinase (ROCK) pathway inhibition reduced fibrogenesis, with ROCK1 inhibition proving more effective than ROCK2 inhibition. These findings highlight the potential of targeting ROCK signaling to mitigate IAV-induced lung fibrosis.

microbiology↗

bioRxiv2025

A Live Attenuated Vaccine Candidate against Emerging Highly Pathogenic Cattle-Origin 2.3.4.4b H5N1 Viruses

Influenza viruses present a significant public health risk, causing substantial illness and death in humans each year. Seasonal flu vaccines must be updated regularly, and their effectiveness often decreases due to mismatches with circulating strains. Furthermore, inactivated vaccines do not provide protection against shifted influenza viruses that have the potential to cause a pandemic. The highly pathogenic avian influenza H5N1 clade 2.3.4.4b is prevalent among wild birds worldwide and is causing a multi-state outbreak affecting poultry and dairy cows in the United States (US) since March 2024. In this study, we have generated a NS1 deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and validated its safety, immunogenicity, and protection efficacy in a prime vaccination regimen against wild-type (WT) A/Texas/37/2024 H5N1. The attenuation of LPhTXdNS1 in vitro was confirmed by its reduced replication in cultured cells and inability to control IFN{beta} promoter activation. In C57BL/6J mice, LPhTXdNS1 has reduced viral replication and pathogenicity compared to WT A/Texas/37/2024 H5N1. Notably, LPhTXdNS1 vaccinated mice exhibited high immunogenicity that reach its peak at weeks 3 and 4 post-immunization, leading to robust protection against subsequent lethal challenge with WT A/Texas/37/2024 H5N1. Altogether, we demonstrate that a single dose vaccination with LPhTXdNS1 is safe and able to induce protective immune responses against H5N1. Both safety profile and protection immunity suggest that LPhTXdNS1 holds promise as a potential solution to address the urgent need for an effective vaccine in the event of a pandemic for the treatment of infected animals and humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/646033v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca0d0borg.highwire.dtl.DTLVardef@1e5232org.highwire.dtl.DTLVardef@a6694corg.highwire.dtl.DTLVardef@14b0832_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Identification of Amino Acid Residues Responsible for Differential Replication and Pathogenicity of Avian Influenza Virus H5N1 Isolated from Human and Cattle in Texas, US

Highly pathogenic avian influenza viruses (HPAIV) pose a serious public health concern. In March 2024, a first-time outbreak of HPAIV H5N1 in dairy cattle herds was reported in the United States (US). Since then, the virus has continued to spread in cattle herds and spilt over into humans. We recently showed that the first human isolate reported in the US in Texas (HPhTX) from a dairy worker in an affected cattle farm has enhanced replication kinetics and pathogenicity in mice compared to a closely related bovine isolate (HPbTX). However, the molecular determinants of differential pathogenicity have not yet been identified. Herein, we show that HPhTX has enhanced polymerase activity, compared with HPbTX, in human cells and that the polymerase basic 2 (PB2) protein is the main factor responsible for this difference. Through single and combined site-directed mutagenesis and swapping the three amino acids different between HPhTX and HPbTX, we found that PB2 mutation E627K is the major contributor to the enhanced polymerase activity of HPhTX. E362G substitution in HPhTX PB2 affected the polymerase, although to a lesser extent than E627K. Moreover, M631L mutation in HPhTX PB2 enhanced polymerase activity. Rescue of a loss-of-function recombinant HPhTX (rHPhTX) containing mutations at residues 627 and 362, alone or in combination, revealed a contribution of PB2 E362G and K627E in morbidity, mortality, and viral replication as compared to rHPhTX wild-type (WT), and significantly reduced viral pathogenicity to levels comparable to rHPbTX WT. These findings indicate that HPAIV H5N1 of cattle origin isolated from the first human case has post-transmission amino acid changes that increase viral replication in human cells and pathogenicity in mice.

microbiology↗

bioRxiv2024

Expression divergence in response to sex-biased selection

It remains debated whether greater degrees of sexual dimorphism would evolve if not for intersexual genetic constraints. Here we used experimental evolution to partially break the intersexual genetic constraint in Drosophila melanogaster to investigate the effects of a shared gene pool on the evolution of sexual dimorphism in gene expression. In six replicate populations of 1000 flies, a dominant marker (DsRed) was used to force a "Red" pool of genetically variable Chromosome 2 copies through exclusive father-to-son inheritance, while a complimentary pool of "NonRed" chromosomes was inherited primarily from mothers to daughters. After 100 generations, we demonstrated the effect of Red male-limited chromosomes in increasing male mating success. Differentially expressed genes between flies with and without Red chromosomes had on average higher intersexual genetic correlations (rMF), as expected if such correlations represent a constraint to sex-specific adaptation under normal inheritance. If conflict hinders the evolution of further dimorphism, the transcriptomes of male-selected Red chromosomes were predicted to evolve to be "masculinized" relative to female-selected NonRed chromosomes. Consistent with this, splicing patterns in Red males (but not Red females) were masculinized relative to NonRed males. Contrastingly, gene expression levels were largely feminized in Red flies of both sexes compared to NonRed. We discuss alternative forms of intralocus sexual conflict that may explain these patterns.

evolutionary biology↗

bioRxiv2024

Replication Kinetics, Pathogenicity and Virus-induced Cellular Responses of Cattle-origin Influenza A(H5N1) Isolates from Texas, United States

The host range of HPAIV H5N1 was recently expanded to include ruminants, particularly dairy cattle in the United States. Shortly after, human H5N1 infection was reported in a dairy worker in Texas following exposure to infected cattle. Herein, we rescued the cattle-origin influenza A/bovine/Texas/24-029328-02/2024(H5N1, rHPbTX) and A/Texas/37/2024(H5N1, rHPhTX) viruses, identified in dairy cattle and human, respectively, and their low pathogenic forms, rLPbTX and rLPhTX, with monobasic HA cleavage sites. Intriguingly, rHPhTX replicated more efficiently than rHPbTX in mammalian and avian cells. Still, variations in the PA and NA proteins didnt affect their antiviral susceptibility to PA and NA inhibitors. Compared to rHPbTX and rLPbTX, the rHPhTX and rLPhTX exhibited higher pathogenicity and efficient replication in infected C57BL/6J mice. The lungs of rHPhTX-infected mice produced higher inflammatory cytokines/chemokines than rHPbTX-infected mice. Our results highlight potential risk of HPAIV H5N1 virus adaptation in human and/or dairy cattle during the current multistate/multispecies outbreak.

microbiology↗

bioRxiv2019

In silico analysis of Missense Variants in Human MPL Gene Associated with Essential Thrombocythemia

IntroductionThe proto-oncogene (MPL) gen encodes the receptor for thrombopoietin (TPO-R), a member of hematopoietic receptor superfamily. Thrombopoietin (TPO), the primary cytokine regulating self-renewal of hematopoietic stem cells, thrombopoiesis and megakaryocytopoiesis. TPO binding to TPO-R induces activation of Janus Kinase 2 (JAK2). Activated JAK2 triggers the activation of downstream positive signaling pathways, leading to the survival, proliferation, and differentiation of hematopoietic cells. Mutations in MPL gene possibly will alter the normal regulatory mechanisms. Numerous MPL mutations have been observed in various hematopoietic cancers such as essential thrombocythemia and primary myelofibrosis and leukemias. In this study, we performed a comprehensive in silico analysis of the functional and structural impact of non-synonymous (nsSNP) that are deleterious to TPO-R structure and function.\n\nMethodologyThe data on human MPL gene was retrieved from dbSNP/NCBI. Nine prediction algorithms; SIFT, Polyphen, PROVEAN, SNAP2, Condel, PhD-SNP, I-Mutant, Mutpred. RaptorX and Chimera were used to analyzing the effect of nsSNPs on functions and structure of the TPO-R. STRING and KEGG database were used for TPO-R protein-protein interaction.\n\nResults and DiscussionAs per dbSNP database, the human MPL gene contained 445 missense mutations. A total 5 nsSNPs (D295G, R257C, Y252H, R537W and D128Y) were predicted to have the most damaging effects on TPO-R structure and function. STRING and KEGG revealed that MPL had strong interactions with proteins that involved in cell growth, apoptosis, signal transduction pathway, some cancers pathways such as colorectal cancer, lung cancer, pancreas cancers, and skin cancer. A literature search revealed that Y252H has contribute to the development of essential thrombocythemia.\n\nConclusionThese in silico predictions will provide useful information in selecting the target SNPs that are likely to have functional impact on the TPO-R and moreover could act as potential targets in genetic association studies. Keywords: In Silico analyses; JAK2; Missense Variants; MPL gene; Thrombopoietin (TPO); Single nucleotide polymorphism (SNP).

bioinformatics↗

bioRxiv2019

Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (A{beta}) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimers disease (AD). It is therefore critical to characterize A{beta} oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an A{beta} oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an A{beta}(1-42) tetramer. It comprises a {beta}-sheet core made of six {beta}-strands, connected by only two {beta}-turns. The two faces of the {beta}-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of A{beta}(1-42), we prepared a sample enriched in A{beta}(1-42) octamers, formed by two A{beta}(1-42) tetramers facing each other forming a {beta}-sandwich structure. Notably, samples enriched in A{beta}(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core {beta}-sheets edges of the A{beta}(1-42) tetramers and octamers.

biophysics↗