The persistent threat of emerging and re-emerging viral outbreaks underscores an urgent need for broad-spectrum antivirals (BSAs). A promising strategy involves targeting host factors exploited by different viral families to develop new BSAs with a high barrier to resistance development. The present work focuses on the inhibition of the protein-protein interactions (PPIs) between viral late domains (L-domain) and the TSG101 protein from the host ESCRT pathway, which is exploited by multiple highly pathogenic viruses for their replication and budding, hence representing a crucial yet underexplored therapeutic strategy to inhibit viral replication. Starting from the previously identified hit compound UEV-10, we developed a series of simplified, more druggable quinoline analogues. These derivatives effectively inhibited the TSG101/L-domain interaction at low micromolar concentrations and were active in a series of cell-based assays, including high-content bimolecular complementation, virus-like particle release, and viral growth inhibition of several biosafety level 4 (BSL-4) pathogens (Ebola, Marburg, and Nipah viruses) and non-enveloped enteroviruses (EV-D68). Overall, our findings further support the inhibition of TSG101/L-domain PPIs as an effective and versatile strategy for the development of broad-spectrum antiviral agents with potential applicability across diverse viral families.

Airborne disinfection of high-containment facilities is mandatory for safe operations. However, sewage vent air-particularly in large-animal experimental units-can represent a critical gap and is typically not considered in the design of high-efficiency particulate air (HEPA) filter systems. Here, we present an engineering design enabling HEPA filtration of sewage vent air from primary biological containments, including large-animal facilities and necropsy suites. We additionally evaluated vaporized hydrogen peroxide (vH2O2) fumigation as an airborne disinfection approach. Disinfection efficacy was determined by quantitative carrier testing using commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus and compared with germ carriers coated with surrogate viruses, mycobacteria, and other spores. Temperature, relative humidity (RH), and vH2O2 concentration were monitored in real time to define process parameters and validate protocols targeting ≥4 log10 reduction. Inactivation efficacy differed substantially among challenge agents: CSC were least resistant to inactivation, whereas murine norovirus (MNV) on germ carriers was most challenging to inactivate. At medium-high vH2O2 concentrations (up to 520 parts per million [ppm]), increasing RH was the dominant driver of inactivation. Based on these data, we developed a biphasic disinfection cycle maintaining a saturated humidity environment with ~35% H2O2 for ≥10 h, achieving ≥4 log10 reduction of MNV infectivity and G. stearothermophilus spore viability on germ carriers. Overall, vH2O2 under high-to-saturated RH is an effective means to disinfect technical infrastructure in biological containment settings handling high-consequence pathogens.IMPORTANCEVent air from sewage systems in biological containment facilities where large animals are kept should be considered a contaminated waste stream. Therefore, effective high-efficiency particulate air (HEPA) filter systems that prevent mold or other moisture-related deterioration of the filters need to be installed. Airborne vaporized hydrogen peroxide (vH2O2) is an appropriate method to decontaminate these complex HEPA filter systems. High to saturated humidity is required for virucidal and sporicidal efficacy under medium to high concentration of vH2O2. Commercially available spore carriers are artificial in design, resulting in lower resistance to inactivation compared to custom germ carriers coated with non-enveloped viruses and thus should be avoided as read-out (validation means) by the biosafety community working with high-consequence pathogens in primary containments.

Increasing threats of viral disease underscore the urgent need for broad-spectrum antiviral drugs (BSADs). Host proteins utilized by human pathogenic viruses are key BSAD targets. The vacuolar-type H+-ATPase (V-ATPase) has been identified as a proviral factor for most pH-dependent enveloped viruses classified as pandemic threats. We report here the discovery of cladoniamide A (CA), a V-ATPase inhibitor with single-digit nanomolar antiviral activity and a high selectivity index (SI: 103-104) against human enveloped viruses [e.g., SARS-CoV-2 variants, influenza A viruses (H1N1, H5N1), respiratory syncytial virus, dengue serotypes 1-4, and Zika virus]. Transcriptome profiling, pH estimation assays, and V-ATPase bioassays indicate that CA interferes with V-ATPase-dependent acidification of the host endolysosomal network thus preventing viral entry. Using pseudoviruses derived from five pathogenic virus families, we confirmed that CA is an entry inhibitor BSAD. CryoEM revealed that CA inhibits the V-ATPase rotary motor by occupying unique binding sites in the membrane-embedded Vo motor. Importantly, intranasal CA treatment in mice infected with influenza A H1N1 significantly reduced viral load in the lung by four log orders. Together, these findings pave the way for developing next-generation BSADs targeted at unique druggable pockets that enable the reversible pharmacological modulation of the human V-ATPase.

ABSTRACT The glycoprotein (G) and fusion protein (F) of henipaviruses play crucial roles in receptor binding and entry into host cells, also enabling virus spread from cell to cell without the release of infectious particles. For Cedar virus (CedV), the proteolytic activation of the F protein precursor into F1 and F2—and thus, its biological activity—depends on clathrin-mediated endocytosis driven by classical endocytosis motifs YXXΦ and YY in the cytoplasmic tail of the F protein. Similar motifs are present in the cytoplasmic tail of CedV G protein. In this study, we investigated whether these motifs influence CedV G protein expression and transport, endocytosis from the plasma membrane, and overall the biological activity—more specifically receptor binding and mediation of fusion together with the fusion protein. Our data show that the expression of CedV G mutants is comparable to parental G in MDCK cells. Endocytosis can be detected for both parental G and its mutants. However, some G protein mutants show reduced biological activity, as indicated by a decrease in fusion when G mutants are co-expressed with CedV F protein. Interestingly, neither co-expression of CedV F and G mutants on the cell surface nor the binding of G mutants to EFNB2 receptors appears to be compromised. Consequently, the putative endocytosis motifs are not relevant for biological activity of CedV G.IMPORTANCEThe glycoprotein (G) and fusion protein (F) of henipaviruses mediate host cell entry and direct cell-to-cell virus spread. For the non-pathogenic Cedar virus (CedV), activation of F depends on clathrin-mediated endocytosis mediated by specific YXXΦ and YY motifs in its cytoplasmic tail. The presence of similar motifs in the cytoplasmic domain of CedV G suggested a potential role in G protein trafficking and function. Here, we show that mutation of these motifs does not impair CedV G surface expression, internalization, or receptor binding. However, some mutants showed reduced ability to mediate fusion with CedV F, despite unaltered surface expression and receptor binding. This suggests that these putative endocytosis motifs are not critical for the biological activity of CedV G. These insights will help dissect mechanisms of viral entry and fusion in CedV and contribute to the comparison of these processes with those of the highly pathogenic henipaviruses HeV and NiV.

Highly pathogenic Hendra and Nipah viruses encode accessory P gene products (C, V and W) that antagonize innate immunity and contribute to pathogenicity. Cedar virus (CedV), an apathogenic bat-borne henipavirus, is presumed to lack P gene mRNA editing and therefore is unable to express V and W proteins. Here, we identify CedV peptides originating from a frameshifted P gene open reading frame and demonstrate a previously unrecognized, noncanonical editing site at a homopolymeric adenine tract that introduces single-nucleotide A or G insertion. This mRNA editing produces a protein that we refer to as U protein, whose C-terminal domain shares sequence and predicted structural features with those of the henipavirus V protein. Recombinant CedV mutants defective in mRNA editing were only recoverable by trans-complementation and showed markedly reduced release of infectious virus in cell culture and attenuated replication in mice lacking type I interferon receptor. Our data revise the CedV gene expression models and reveal a noncanonical editing mechanism that supports the production of a U protein critical for efficient infectious virus release. These results expand the fundamental concepts of paramyxovirus gene expression and reveal an unexpected requirement for P-gene editing in efficient infectious-virus production, with implications for the evaluation of potentially high-consequence paramyxoviruses.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus widely distributed across Asia, Africa, the Middle East, and Southern Europe, causing severe illness and hemorrhagic fever in humans. While sheep usually show no clinical symptoms, they develop viremia and can play a key role in transmitting the virus to ticks and humans. Despite their importance in transmission, our understanding of infection dynamics in these animals remains limited. To bridge this knowledge gap, we conducted a controlled study in which six Pomeranian Coarsewool sheep were experimentally infected with CCHFV, strain Kosovo Hoti, and monitored over 21 days. All sheep developed transient fever and viremia. Necropsies at 5-7 days post infection (dpi) revealed highest viral loads in liver, spleen, testicles, and lymphoid tissues. Seroconversion occurred by 7 dpi and virus-neutralizing antibodies were detectable by day 8. These findings enhance our understanding of CCHFV infection dynamics in sheep and will aid future research, including vaccine development.

Cedar virus (CedV), closely related to the Hendra and Nipah viruses, is a novel Henipavirus that was originally isolated from flying foxes in Australia in 2012. Although its glycoprotein G exhibits relatively low sequence similarity with its counterparts of the Hendra and Nipah viruses, CedV also uses ephrin receptors, i.e., ephrins B1, B2, A2 and A5, to enters human cells. Nevertheless, the entry mechanism of CedV into bat cells remains unexplored. Considering that Rousettus aegyptiacus (Egyptian Rousette bat, ERB) is postulated to be a reservoir host for henipaviruses, we aim to reveal the receptors utilized by CedV to enable its entry into ERB cells. To this end, we cloned the class A and B ephrins of ERB and generated CHO-K1 cells stably expressing individual ephrins. We also developed a lentivirus-based pseudovirus system containing the firefly luciferase reporter. Assessment of the luciferase activity in cells expressing single ephrins demonstrated that the ERB ephrin B1 and B2 mediated CedV pseudovirus entry. Further, we generated a recombinant CedV expressing the fluorescent protein TurboFP635 (rCedV-nTurbo635). By performing high-content microscopy and flow cytometry, we unveiled that, in addition to ephrin B1 and B2, ephrin A5 was also able to mediate rCedV-nTurbo635 entry, although to a much lesser extent. In contrast to human ephrin A2, ERB ephrin A2 failed to mediate rCedV-nTurbo635 entry. Finally, we generated ERB epithelial cells with ephrin B1 and/or ephrin B2 knockdown (KD). The entry of rCedV-nTurbo635 into ERB epithelial cells was drastically impaired by ephrin B1/B2 KD, validating the importance of ephrin B1 and B2 in its entry. Altogether, we conclude that CedV primarily employs ERB ephrin B1, B2 and, possibly, A5 for its entry into ERB cells.

A lack of reagents represents a major bottleneck in pandemic preparedness and rapid vaccine development. It is therefore important to enable the design of reagents for use in the treatment and diagnosis of emerging viral diseases. Ideally, the design and identification platform is fast, can be performed by testing only a small number of candidates and enables a generally applicable strategy. In this study, we assessed the ability of recently developed computational protein design tools to establish such a workflow for validating paramyxovirus receptor-binding protein de novo binders as such reagents. The family Paramyxoviridae includes various members that cause severe disease and exhibit re-occurring zoonotic spillover events, with documented human infections over the past decades. We successfully designed, identified, and characterized mini-proteins targeting the receptor binding proteins of Nipah virus, Langya virus, and Measles virus while screening as few as 10-16 designs per target. The resulting functional binders have moderate to low nanomolar affinities and display high on-target specificity. We further showed that our most promising Nipah virus receptor-binding protein binder is able to inhibit human receptor binding in vitro and competes for an epitope that overlaps with that of the neutralizing antibody HENV-117. However, despite these promising results, this Nipah binder is only weakly neutralizing, preventing therapeutic applications. Nevertheless, we established a platform, applicable to rapidly generate diagnostically relevant proteins from only a small number of candidates, and developed novel reagents for the Paramyxoviridae family. ### Competing Interest Statement C.T.S. has received unrelated research funds from Navigo Proteins GmbH (Halle (Saale), Germany). M.B. has been employed by AI Driven Therapeutics GmbH since July 2025. All other authors declare no conflict of interest. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of any of the funding entities. Coalition for Epidemic Preparedness Innovations, https://ror.org/02j9wvt50 Medical Research Council, https://ror.org/03x94j517, MR/V031635/1, MR/S007555/1 European Molecular Biology Organization, 11899 Wellcome Trust, https://ror.org/029chgv08, 203141/Z/16/Z European Union, 101136281 German Academic Exchange Service, https://ror.org/039djdh30, 57616814

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources.

Human maximum containment facilities-also known as biosafety level 4 (BSL-4) laboratories-for zoonotic viruses such as Ebola virus or Nipah virus and veterinary maximum containment (BSL-4vet) facilities, e.g. for foot-and-mouth disease virus or peste-de-petits-ruminants virus, share many similar features but also differ in their design, standard operating procedures and operational requirements. This article summarizes the similarities and differences by addressing relevant aspects of these two types of maximum containment facilities. Construction and operation of both facilities is bound by strict regulations and regular audits by national or state authorities. The technical infrastructure is similar with respect to air handling, negative pressure differential to the outside and between rooms, as well as autoclaves and waste water handling. Both facilities require strict access control and training for entry into the area, which is more extensive on the human maximum containment side. Special personal protective equipment such as a positive pressure suits needs to be worn in the human maximum containment facility, but this is not generally necessary in veterinary facilities. Exiting the facility requires showering of personnel-a personal shower only in the veterinary containment and at least a chemical shower to decontaminate the suit in the human containment. Removal of samples from both kinds of facilities can only occur after application of strict and validated inactivation protocols. In addition, both facilities undergo room decontamination processes for maintenance or between animal studies. Overall, we would like to demonstrate that these facilities have more in common than expected at first glance and close coordination and cooperation between the individuals responsible for them is advisable.

Influenza viruses remain a major threat to both human and animal health, with seasonal outbreaks and the risk of pandemics caused by reassortant strains. Antiviral drugs are needed as a complement to vaccines, but resistance often limits their long-term efficacy. N4-hydroxycytidine (NHC), the active form of Molnupiravir, shows potent activity against influenza A viruses (IAVs) in both cell cultures and animal models, with minimal resistance observed. Building on prior work in SARS-CoV-2, we investigated whether inhibiting pyrimidine biosynthesis could enhance NHC's antiviral activity against IAVs. The combination of NHC with inhibitors of dihydroorotate dehydrogenase (DHODH) or cytidine triphosphate synthases (CTPS1/2) showed strong synergy. This was evident through reduced cytopathic effects, decreased viral RNA and protein, and a marked absence of infectious virus particles. This synergy was consistent across multiple IAV subtypes, including H1N1, H1N2, H3N2, and H5N1. This synergistic effect was reversed by exogenously supplemented pyrimidine nucleosides, confirming nucleotide depletion as a key mechanism. However, some avian IAVs were less sensitive to the treatment in mammalian cells. The PB2-K627E mutation, affecting the interaction with host factor ANP32, modulated NHC efficacy, implicating viral adaptation in drug responsiveness. In a ferret model of H5N1 infection, NHC combined with the CTPS inhibitor STP938 reduced clinical symptoms and lung pathology, with NHC mostly driving antiviral activity and STP938 contributing to disease mitigation. These findings indicate that combining NHC with pyrimidine biosynthesis inhibitors enhances antiviral efficacy against IAVs, especially in rapidly replicating viruses, and may broaden the utility of nucleoside analogues in influenza therapy.

Nairobi sheep disease virus (NSDV) is a tick-borne orthonairovirus, which is genetically related to Crimean-Congo hemorrhagic fever virus (CCHFV), and causes severe hemorrhagic gastroenteritis in infected sheep. CCHFV GP38, a cleavage product of the CCHFV glycoprotein precursor (GPC), has recently attracted attention: not only has GP38 been reported to elicit detectable anti-GP38 antibodies in CCHFV-infected patients, but anti-GP38 antibodies have also been shown to protect mice from lethal CCHFV challenge. While proteolytic cleavage of CCHFV GP38 has been described to involve the proprotein convertases furin and subtilisin/kexin-isozyme-1 (SKI-1), little is known about the processing of NSDV GPC, or the occurrence and immunogenicity of NSDV GP38 in infected sheep. Here, we provide the first evidence for the presence and immunogenicity of NSDV GP38 in infected sheep demonstrating seroconversion by the detection of anti-GP38 antibodies over the course of infection. To further characterize GPC processing in vitro, we investigated the impact of furin overexpression and the effect of a furin inhibitor on NSDV glycoprotein expression, cleavage, and viral infectivity. While virus infectivity remained unaffected, our results suggest that other proteases besides furin may play a role in the proteolytic processing of NSDV GPC at a cleavage site that remains to be explored. Taken together, our findings highlight the immunogenicity of NSDV GP38 in sheep and warrant further research into the similarities and differences in proteolytic cleavage between the glycoproteins of NSDV and other orthonairoviruses, such as CCHFV. IMPORTANCE:Nairobi sheep disease virus (NSDV) is a zoonotic orthonairovirus causing severe and often fatal hemorrhagic gastroenteritis in small ruminants. Its genetic relationship to human-pathogenic Crimean-Congo hemorrhagic fever virus (CCHFV) and striking similarities in the clinical picture between CCHFV-infected patients and NSDV-infected ruminants have led to the idea that NSDV could serve as a model organism to study CCHFV pathogenesis. However, knowledge on NSDV-host interactions has been limited. While CCHFV GP38 has recently attracted attention as vaccine candidate and possible virulence factor, the occurrence and role of putative GP38 in other orthonairoviruses has been unclear. This study provides first evidence for the presence and immunogenicity of NSDV GP38 in infected sheep. Furthermore, our data indicate that other proteases besides furin may be involved in the proteolytic cleavage of NSDV GPC. Future studies are needed to determine the proteases involved and to investigate the possible functional role of GP38 in NSDV pathogenesis.

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions.

Cedar henipavirus (CedV), which was isolated from the urine of pteropodid bats in Australia, belongs to the genus Henipavirus in the family of Paramyxoviridae. It is closely related to the Hendra virus (HeV) and Nipah virus (NiV), which have been classified at the highest biosafety level (BSL4) due to their high pathogenicity for humans. Meanwhile, CedV is apathogenic for humans and animals. As such, it is often used as a model virus for the highly pathogenic henipaviruses HeV and NiV. In this study, we challenged eight Rousettus aegyptiacus fruit bats of different age groups with CedV in order to assess their age-dependent susceptibility to a CedV infection. Upon intranasal inoculation, none of the animals developed clinical signs, and only trace amounts of viral RNA were detectable at 2 days post-inoculation in the upper respiratory tract and the kidney as well as in oral and anal swab samples. Continuous monitoring of the body temperature and locomotion activity of four animals, however, indicated minor alterations in the challenged animals, which would have remained unnoticed otherwise.

Although pigs are naturally susceptible to Reston virus and experimentally to Ebola virus (EBOV), their role in Orthoebolavirus ecology remains unknown. We tested 888 serum samples collected from pigs in Guinea during 2017–2019 (between the 2013–16 epidemic and its resurgence in 2021) by indirect ELISA against the EBOV nucleoprotein. We identified 2 hotspots of possible pig exposure by IgG titer levels: the northern coast had 48.7% of positive serum samples (37/76), and Forest Guinea, bordering Sierra Leone and Liberia, where the virus emerged and reemerged, had 50% of positive serum samples (98/196). The multitarget Luminex approach confirms ELISA results against Ebola nucleoprotein and highlights cross-reactivities to glycoprotein of EBOV, Reston virus, and Bundibugyo virus. Those results are consistent with previous observations of the circulation of Orthoebolavirus species in pig farming regions in Sierra Leone and Ghana, suggesting potential risk for Ebola virus disease in humans, especially in Forest Guinea.

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 SARSCoV-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.

Hepatitis E virus (HEV) is the main cause of acute hepatitis in humans worldwide and is responsible for a large number of outbreaks especially in Africa. Human infections are mainly caused by genotypes 1 and 2 of the genus Paslahepevirus, which are exclusively associated with humans. In contrast, viruses of genotypes 3 and 4 are zoonotic and have their main reservoir in domestic and wild pigs, from which they can be transmitted to humans primarily through the consumption of meat products. Both genotypes 3 and 4 are widespread in Europe, Asia, and North America and lead to sporadic cases of hepatitis E. However, there is little information available on the prevalence of these genotypes and possible transmission routes from animal reservoirs to humans in African countries. We therefore analysed 1086 pig sera collected in 2016/2017 in four districts in Sierra Leone for antibodies against HEV using a newly designed in-house ELISA. In addition, the samples were also analysed for HEV RNA by quantitative real-time RT-PCR. The overall seroprevalence in Sierra Leone was low with only 44 positive sera and a prevalence of 4.0%. Two serum pools were RT-PCR-positive and recovered partial sequences clustered into the genotype 3 (HEV-3) of the order Paslahepevirus, species Paslahepevirus balayani. The results are the first evidence of HEV-3 infection in pigs from Sierra Leone and demonstrate a low circulation of the virus in these animals to date. Further studies should include an examination of humans, especially those with close contact with pigs and porcine products, as well as environmental sampling to evaluate public health effects within the framework of a One Health approach.

Nipah virus (NiV) is an emerging, zoonotic paramyxovirus that is among the most pathogenic of viruses in humans. During the first reported outbreak of NiV in Malaysia and Singapore in the late 1990s, pigs served as an intermediate host, which enabled the transmission to humans. Although subsequent outbreaks in Asia only reported direct bat-to-human and human-to-human transmission, pigs are still considered a potential source for viral dissemination in the epidemiology of the disease. Thus, serological assays such as Enzyme-linked immunosorbent assay (ELISA) or virus neutralization test (VNT) represent powerful tools to characterize the serum antibody responses in NiV-infected pigs as well as to perform seroepidemiological surveillance studies on the potential circulation of NiV or NiV-related viruses among pig populations worldwide. This chapter describes both methods in detail. Furthermore, we discuss some of the major pitfalls and indicate how to avoid them.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respiratory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking in the determination of complex host response patterns in differentiated respiratory tract epithelia for the understanding of NiV replication and spread in swine. Here we characterized infection and spread of NiV in differentiated primary porcine bronchial epithelial cells (PBEC) cultivated at the air–liquid interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the apical or basal sides. Deep time course proteomics revealed pronounced upregulation of genes related to type I/II IFN, immunoproteasomal subunits, transporter associated with antigen processing (TAP)-mediated peptide transport, and major histocompatibility complex (MHC) I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II IFN host response with conversion from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.