The panzootic spread of highly pathogenic avian influenza H5N1 sublineage 2.3.4.4b: a critical appraisal of One Health preparedness and prevention.

Changes in the epidemiology and ecology of H5N1 highly pathogenic avian influenza are devastating wild bird and poultry populations, farms and communities, and wild mammals worldwide. Having originated in farmed poultry, H5N1 viruses are now spread globally by wild birds, with transmission to many mammal and avian species, resulting in 2024 in transmission among dairy cattle with associated human cases. These ecological changes pose challenges to mitigating the impacts of H5N1 highly pathogenic avian influenza on wildlife, ecosystems, domestic animals, food security, and humans. H5N1 highly pathogenic avian influenza highlights the need for One Health approaches to pandemic prevention and preparedness, emphasising multisectoral collaborations among animal, environmental, and public health sectors. Action is needed to reduce future pandemic risks by preventing transmission of highly pathogenic avian influenza among domestic and wild animals and people, focusing on upstream drivers of outbreaks, and ensuring rapid responses and risk assessments for zoonotic outbreaks. Political commitment and sustainable funding are crucial to implementing and maintaining prevention programmes, surveillance, and outbreak responses.

Molecular plasticity of herpesvirus nuclear egress analysed in situ.

The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC-capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.

Negative-Strand RNA Virus-Vectored Vaccines.

Vectored RNA vaccines offer a variety of possibilities to engineer targeted vaccines. They are cost-effective and safe, but replication competent, activating the humoral as well as the cellular immune system.This chapter focuses on RNA vaccines derived from negative-strand RNA viruses from the order Mononegavirales with special attention to Newcastle disease virus-based vaccines and their generation. It shall provide an overview on the advantages and disadvantages of certain vector platforms as well as their scopes of application, including an additional section on experimental COVID-19 vaccines.

Immune response after oral immunization of goats and foxes with an NDV vectored rabies vaccine candidate.

Vaccination of the reservoir species is a key component in the global fight against rabies. For wildlife reservoir species and hard to reach spillover species (e. g. ruminant farm animals), oral vaccination is the only solution. In search for a novel potent and safe oral rabies vaccine, we generated a recombinant vector virus based on lentogenic Newcastle disease virus (NDV) strain Clone 30 that expresses the glycoprotein G of rabies virus (RABV) vaccine strain SAD L16 (rNDV_GRABV). Transgene expression and virus replication was verified in avian and mammalian cells. To test immunogenicity and viral shedding, in a proof-of-concept study six goats and foxes, representing herbivore and carnivore species susceptible to rabies, each received a single dose of rNDV_GRABV (108.5 TCID50/animal) by direct oral application. For comparison, three animals received the similar dose of the empty viral vector (rNDV). All animals remained clinically inconspicuous during the trial. Viral RNA could be isolated from oral and nasal swabs until four (goats) or seven days (foxes) post vaccination, while infectious NDV could not be re-isolated. After four weeks, three out of six rNDV_GRABV vaccinated foxes developed RABV binding and virus neutralizing antibodies. Five out of six rNDV_GRABV vaccinated goats displayed RABV G specific antibodies either detected by ELISA or RFFIT. Additionally, NDV and RABV specific T cell activity was demonstrated in some of the vaccinated animals by detecting antigen specific interferon γ secretion in lymphocytes isolated from pharyngeal lymph nodes. In conclusion, the NDV vectored rabies vaccine rNDV_GRABV was safe and immunogenic after a single oral application in goats and foxes, and highlight the potential of NDV as vector for oral vaccines in mammals.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Developing One Health surveillance systems.

The health of humans, domestic and wild animals, plants, and the environment are inter-dependent. Global anthropogenic change is a key driver of disease emergence and spread and leads to biodiversity loss and ecosystem function degradation, which are themselves drivers of disease emergence. Pathogen spill-over events and subsequent disease outbreaks, including pandemics, in humans, animals and plants may arise when factors driving disease emergence and spread converge. One Health is an integrated approach that aims to sustainably balance and optimize human, animal and ecosystem health. Conventional disease surveillance has been siloed by sectors, with separate systems addressing the health of humans, domestic animals, cultivated plants, wildlife and the environment. One Health surveillance should include integrated surveillance for known and unknown pathogens, but combined with this more traditional disease-based surveillance, it also must include surveillance of drivers of disease emergence to improve prevention and mitigation of spill-over events. Here, we outline such an approach, including the characteristics and components required to overcome barriers and to optimize an integrated One Health surveillance system.

Archaeome structure and function of the intestinal tract in healthy and H1N1 infected swine.

Background: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine. Aims/Methods: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics. Results and discussion: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea. Conclusions/Take home message: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.

Animals Experimentally Infected with SARS-CoV-2 Generate Functional Autoantibodies against G-Protein-Coupled Receptors.

(1) Background: SARS-CoV-2 infection has been linked to diverse clinical manifestations in humans, including cardiovascular complications. Functional autoantibodies targeting G-protein-coupled receptors have emerged as potential contributors to these effects. This study sought to investigate the production and activity of functional autoantibodies targeting G-protein-coupled receptors after SARS-CoV-2 infection of selected animal species. (2) Methods: The presence of functional autoantibodies such as 2-adrenoceptor, angiotensin II AT1 receptor, muscarinic M2 receptor, and angiotensin 1-7 MAS receptor was assessed in cattle and ferrets experimentally infected with SARS-CoV-2. Bioassays were conducted to evaluate the positive or negative chronotropic responses induced by these autoantibodies. Further experiments identified the extracellular domains to which the functional autoantibodies bind, and receptor antagonists were employed to block the induced responses. (3) Results: Only two out of six cattle that were inoculated with SARS-CoV-2 displayed viral replication and tested positive for functional autoantibodies against G-protein-coupled receptors. These functional autoantibodies specifically recognized ß2-adrenoceptor, angiotensin II AT1 receptor, muscarinic M2 receptor, and angiotensin 1-7 MAS receptor and induced distinct positive and negative chronotropic effects in the bioassay. Infected ferrets generated functional autoantibodies against ß2-adrenoceptor and muscarinic M2 receptor and presented bioactivity similar to that in cattle. (4) Conclusions: This study uncovers functional autoantibodies targeting G-protein-coupled receptors in cattle and ferrets post-SARS-CoV-2 infection, with implications for cardiovascular function.

The non-classical major histocompatibility complex II protein SLA-DM is crucial for African swine fever virus replication.

African swine fever virus (ASFV) is a lethal animal pathogen that enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, progeny virus titers and viral DNA replication. Transgene-based reconstitution of SLA-DMA/B fully restored the replication capacity demonstrating that SLA-DM, which resides in late endosomes, plays a crucial role during early steps of ASFV infection.

Mass-Spectrometric Evaluation of the African Swine Fever Virus-Induced Host Shutoff Using Dynamic Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC).

African swine fever is a viral disease of swine caused by the African swine fever virus (ASFV). Currently, ASFV is spreading over the Eurasian continent and threatening global pig husbandry. One viral strategy to undermine an efficient host cell response is to establish a global shutoff of host protein synthesis. This shutoff has been observed in ASFV-infected cultured cells using two-dimensional electrophoresis combined with metabolic radioactive labeling. However, it remained unclear if this shutoff was selective for certain host proteins. Here, we characterized ASFV-induced shutoff in porcine macrophages by measurement of relative protein synthesis rates using a mass spectrometric approach based on stable isotope labeling with amino acids in cell culture (SILAC). The impact of ASFV infection on the synthesis of >2000 individual host proteins showed a high degree of variability, ranging from complete shutoff to a strong induction of proteins that are absent from naïve cells. GO-term enrichment analysis revealed that the most effective shutoff was observed for proteins related to RNA metabolism, while typical representatives of the innate immune system were strongly induced after infection. This experimental setup is suitable to quantify a virion-induced host shutoff (vhs) after infection with different viruses.

Suid alphaherpesvirus 1 of wild boar origin as a recent source of Aujeszky's disease in carnivores in Germany.

BACKGROUND: The high susceptibility of carnivores to Suid Alphaherpesvirus 1 [SuAHV1, synonymous pseudorabies virus (PrV)], renders them inadvertent sentinels for the possible occurrence of Aujeszky's disease (AD) in domestic and wild swine populations. The aim of this study was to epidemiologically analyse the occurrence of PrV infections in domestic and wild animals in Germany during the last three decades and to genetically characterise the causative PrV isolates. METHODS: PrV in dogs was detected using standard virological techniques including conventional and real time PCR, virus isolation or by immunohistochemistry. Available PrV isolates were characterized by partial sequencing of the open gC reading frame and the genetic traits were compared with those of archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in the domestic pig population. RESULTS: During 1995 and 2022, a total of 38 cases of AD in carnivores, e.g. dogs and red foxes, were laboratory confirmed. Sequencing and subsequent phylogenetic analysis of PrV isolates established a strong connection between AD cases in carnivores and the occurrence of PrV infections in European wild boars in the end phase of and after elimination of AD from the domestic pig population. While PrV infections occur at low numbers but regularly in hunting dogs, interestingly, PrV was not observed in grey wolves in Germany. In none of 682 dead-found grey wolves and wolf-dog hybrids tested from Germany during 2006-2022 could PrV infection be detected by molecular means. CONCLUSIONS: Although PrV has been eliminated from domestic pigs, spillover infections in domestic and wild carnivores should always be expected given the endemic presence of PrV in wild pig populations. Since detection of PrV DNA and virus in carnivores is sporadic even in areas with high seroprevalence of PrV in wild pigs, it may not reflect the full diversity of PrV.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.