Biology subjects

Scheibner, D.

Publications and source records attributed to Scheibner, D..

6 recordsLinked to original sources

bioRxiv2025

Different genetic determinants for high virulence, transmission and replication of high pathogenicity H7N7 avian influenza virus in turkeys and chickens

High pathogenicity (HP) avian influenza viruses (AIV) generally evolve from low pathogenicity (LP) precursors after transmission from wild birds to chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), causing severe economic losses worldwide. Turkeys are more susceptible to AIV infection than chickens and are considered potential bridging hosts that facilitate the emergence of HPAIV. Beyond the polybasic cleavage site (pCS) in hemagglutinin (HA), little is known about other virulence determinants of HPAIV in these species. In 2015, HPAIV H7N7 and its LP ancestor were isolated from the same chicken farm, which differed by 16 nonsynonymous mutations across all eight gene segments, in addition to the pCS. Here we identify the genetic determinants, including the pCS, that contributed to the HPAIV H7N7 virulence, transmission, replication, and tissue distribution in chickens and turkeys. Notably, the non-structural (NS1) or matrix (M) proteins encoding segments in turkeys, or NS segment in chickens, rendered viruses as virulent and transmissible as the original HPAIV. Endotheliotropism, observed exclusively in chickens, was driven by the pCS and, to a lesser extent, the neuraminidase (NA). In vitro, the M2-V68L mutation influenced NS1 expression and virus morphology in chicken and turkey cells. Additionally, HPAIV NS1 enhanced polymerase activity and effectively suppressed interferon induction, a process further modulated by M2-V68L. These findings underscore the critical role of turkeys as a "hub" in the evolution of HPAIV from LP precursors, offering crucial insights into the genotypic and phenotypic factors that facilitate viral adaptation in different poultry species. ImportanceHigh pathogenicity avian influenza viruses (HPAIV) cause severe economic losses for the poultry industry worldwide. HPAIV generally evolve from low pathogenicity (LP) ancestors in galliform birds, with turkeys being more susceptible to severe disease and death than chickens. The mechanisms underlying HPAIV emergence in these species remain unclear. This study reveals two distinct evolutionary pathways for HPAIV. In turkeys, both the polybasic hemagglutinin cleavage site (pCS) and mutations in the NS or M segments contributed to high virulence and transmission. In chickens, only the NS segment was critical, in addition to the pCS. These segments increased virus replication in both chicken and turkey cells. However, unlike chicken cells, the M and NS segments did not play a role in blocking the innate immune response. Understanding these species-specific mechanisms highlights the role of turkeys as a bridging host and provides insights into the molecular evolution of HPAIV from LP precursors.

microbiology↗

bioRxiv2025

Synergistic interference with SARS-CoV-2 replication by Molnupiravir-derived N4 hydroxycytidine and inhibitors of CTP synthetase

N4-hydroxycytidine (NHC), the active metabolite of Molnupiravir, is incorporated into nascent RNA of SARS-CoV-2 and interferes with subsequent virus replication. We have previously described synergy between NHC and inhibitors of dehydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine synthesis. Upon DHODH inhibition, the lack of endogenous pyrimidines conceivably enhances NHC incorporation. However, the question remains whether preventing the synthesis of just one pyrimidine base, cytidine, might as well augment the antiviral efficacy of NHC. We tested this by inhibiting CTP synthetases (CTPSs), the cellular enzymes that directly catalyze the synthesis of a cytidine nucleotide. We observed that inhibitors of CTP synthetase (CTPSis), namely cyclopentenyl cytosine (CPEC) as well as STP938 and STP720, display a strong synergy with NHC for diminishing SARS-CoV-2 replication in cell culture, as shown earlier for DHODH inhibitors. NHC and CTPSis in combination prevented the cytopathic effect of SARS-CoV-2 and strongly reduced the release of viral RNA and infectious particles, as well as the synthesis of viral proteins. This combination was also active against an Omicron variant of SARS-CoV-2. Addition of cytidine, but not uridine, rescued virus growth under these conditions. Of note, treating SARS-CoV-2-infected hamsters with the CTPS1 inhibitor STP938 strongly diminished COVID pathology. We propose that CTPS inhibition has the potential to increase the efficacy of antiviral cytidine analogues and to treat coronavirus infections. HIGHLIGHTSO_LIThe efficacy of NHC against SARS-CoV-2 replication in cell culture models is intensified by several orders of magnitude through targeting cellular CTP-Synthetase. C_LIO_LIThe drug combination still displays its effect against SARS-CoV-2 replication in the presence of uridine, suggesting that serum uridine cannot counteract its efficacy. C_LIO_LICTPS inhibition diminishes COVID-19-like pathology in an established animal model. C_LI

microbiology↗

bioRxiv2024

Inhibitors of dihydroorotate dehydrogenase synergize with the broad antiviral activity of 4'-fluorouridine

RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4'-Fluorouridine (4'-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4'-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4'-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4'-FlU-resistant mutant influenza A virus was re-sensitized towards 4'-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4'-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4'-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616778v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@398504org.highwire.dtl.DTLVardef@4befaeorg.highwire.dtl.DTLVardef@1648bbcorg.highwire.dtl.DTLVardef@12169e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIStrong synergy of DHODH inhibitors with 4'-FlU C_LIO_LIActivity of the combination against previously resistant influenza virus C_LIO_LIBroadly active combination against a diverse set of RNA viruses C_LIO_LISuccessful targets include highly pathogenic Ebola and Nipah viruses C_LI

microbiology↗

bioRxiv2023

H7 influenza A viruses bind sialyl-LewisX, a potential intermediate receptor between species

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells, but not in duck cells, and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads for a longer time than chickens, although they appeared clinically healthy. To elucidate why chickens and ducks were infected by these viruses despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This revealed that mutated avian H7 HAs also bound to 2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the 2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to sialyl-LewisX enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs. (249 words) ImportanceInfluenza A viruses cause millions of deaths and illness in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and therefore the potential host species targeted. While avian and human IAVs tend to bind N-acetylneuraminic acid (a form of sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to sialyl-LewisX, another terminal epitope. These findings are important as they contribute to the understanding of the role of sialyl-LewisX in avian influenza infection. (148 words)

molecular biology↗

bioRxiv2021

Preferential selection and contribution of non-structural protein 1 (NS1) to the efficient transmission of the panzootic avian influenza H5N8 2.3.4.4 clades A and B viruses in chickens and ducks

Highly pathogenic avian influenza viruses H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade-2.3.4.4A in 2014/2015 and clade-2.3.4.4B since 2016 up to today. Both clades were highly virulent in chickens, but only clade-B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in-vitro experiments indicated that clade-2.3.4.4A and clade-2.3.4.4B viruses have a preference for NS1 of 237-aa and 217-aa, respectively over NS1 of 230-aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C-terminus of clade-B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade-A virus was observed. Remarkably, the NS1 of clade-A and clade-B, regardless of length, was efficient to block interferon induction in infected chickens and changes in the NS1 C-terminus reduced the efficiency for interferon antagonism. Together, the NS1 C-terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. ImportanceThe panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. Both clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino-acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237-aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217-aa (exclusively found in Europe) due stop-codons in the C-terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN-response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds. Subject categoryAnimal, RNA Viruses

microbiology↗

bioRxiv2020

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

microbiology↗