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.

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.

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.

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.

The Knowns and Unknowns of Herpesvirus Nuclear Egress.

Nuclear egress of herpesvirus capsids across the intact nuclear envelope is an exceptional vesicle-mediated nucleocytoplasmic translocation resulting in the delivery of herpesvirus capsids into the cytosol. Budding of the (nucleo)capsid at and scission from the inner nuclear membrane (INM) is mediated by the viral nuclear egress complex (NEC) resulting in a transiently enveloped virus particle in the perinuclear space followed by fusion of the primary envelope with the outer nuclear membrane (ONM). The dimeric NEC oligomerizes into a honeycomb-shaped coat underlining the INM to induce membrane curvature and scission. Mutational analyses complemented structural data defining functionally important regions. Questions remain, including where and when the NEC is formed and how membrane curvature is mediated, vesicle formation is regulated, and directionality is secured. The composition of the primary enveloped virion and the machinery mediating fusion of the primary envelope with the ONM is still debated. While NEC-mediated budding apparently follows a highly conserved mechanism, species and/or cell type-specific differences complicate understanding of later steps.

Zoonotic Animal Influenza Virus and Potential Mixing Vessel Hosts.

Influenza viruses belong to the family Orthomyxoviridae with a negative-sense, single-stranded segmented RNA genome. They infect a wide range of animals, including humans. From 1918 to 2009, there were four influenza pandemics, which caused millions of casualties. Frequent spillover of animal influenza viruses to humans with or without intermediate hosts poses a serious zoonotic and pandemic threat. The current SARS-CoV-2 pandemic overshadowed the high risk raised by animal influenza viruses, but highlighted the role of wildlife as a reservoir for pandemic viruses. In this review, we summarize the occurrence of animal influenza virus in humans and describe potential mixing vessel or intermediate hosts for zoonotic influenza viruses. While several animal influenza viruses possess a high zoonotic risk (e.g., avian and swine influenza viruses), others are of low to negligible zoonotic potential (e.g., equine, canine, bat and bovine influenza viruses). Transmission can occur directly from animals, particularly poultry and swine, to humans or through reassortant viruses in "mixing vessel" hosts. To date, there are less than 3000 confirmed human infections with avian-origin viruses and less than 7000 subclinical infections documented. Likewise, only a few hundreds of confirmed human cases caused by swine influenza viruses have been reported. Pigs are the historic mixing vessel host for the generation of zoonotic influenza viruses due to the expression of both avian-type and human-type receptors. Nevertheless, there are a number of hosts which carry both types of receptors and can act as a potential mixing vessel host. High vigilance is warranted to prevent the next pandemic caused by animal influenza viruses.

One Health-Key to Adequate Intervention Measures against Zoonotic Risks.

Zoonotic diseases are a heterogenous group of infections transmittable between humans and vertebrate animal species. Globally, endemic and emerging zoonoses are responsible for high social and economic costs. Due to the particular positioning of zoonoses at the human-animal-environment interface, zoonotic disease control is an integral part of One Health, which recognizes the close link between human, animal and ecosystem health. During recent years, the validity of the One Health approach has been recognized by academia and policy makers. However, gaps are still evident, particularly in the implementation of the concept as a unifying, integrated approach for different sectors and disciplines for the control of zoonoses. For example, while cooperation between human and veterinary medicine has made significant progress, networking with environmental sciences leaves room for improvement. Examination of individual intervention measures can help to gain valuable insights for future projects, and help to identify existing gaps. This is also a task for the One Health High-Level Expert Panel, which was established by WHO, OIE, FAO and UNEP to give science-based strategic advice on One Health measures. Overall, we should aim to learn from current situations, and to identify the best practice examples available, to continuously develop and improve One Health concepts for the control of zoonoses.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

CP204L Is a Multifunctional Protein of African Swine Fever Virus That Interacts with the VPS39 Subunit of the Homotypic Fusion and Vacuole Protein Sorting Complex and Promotes Lysosome Clustering.

Virus replication depends on a complex interplay between viral and host proteins. In the case of African swine fever virus (ASFV), a large DNA virus, only a few virus-host protein-protein interactions have been identified to date. In this study, we demonstrate that the ASFV protein CP204L interacts with the cellular homotypic fusion and protein sorting (HOPS) protein VPS39, blocking its association with the lysosomal HOPS complex, which modulates endolysosomal trafficking and promotes lysosome clustering. Instead, CP204L and VPS39 are targeted to virus factories and localized at the periphery of the virus DNA replication sites. Furthermore, we show that loss of VPS39 reduces the levels of virus proteins synthesized in the early phase of infection and delays ASFV replication but does not completely inhibit it. Collectively, these results identify a novel virus-host protein interaction that modulates host membrane rearrangement during infection and provide evidence that CP204L is a multifunctional protein engaged in distinct steps of the ASFV life cycle. IMPORTANCE African swine fever virus (ASFV) was first identified over a hundred years ago. Since then, much effort has been made to understand the pathogenesis of ASFV. However, the specific roles of many individual ASFV proteins during the infection remain enigmatic. This study provides evidence that CP204L, one of the most abundant ASFV proteins, modulates endosomal trafficking during virus infection. Through protein-protein interaction, CP204L prevents the recruitment of VPS39 to the endosomal and lysosomal membranes, resulting in their accumulation. Consequently, CP204L and VPS39 become sequestered in the ASFV replication and assembly site, known as the virus factory. These results uncover a novel function of viral protein CP204L and extend our understanding of complex interaction between virus and host.