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Currently, infections with SARS-Coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, are responsible for substantial morbidity and mortality worldwide. Older adults subjects > 60 years of age account for > 95% of the over one million fatal cases reported to date. It is unclear why in this age group SARS-CoV-2 infection causes more severe disease than in young adults. We hypothesized that differences in SARS-CoV-2 cross-reactive cellular immunity induced after infection with human coronaviruses (HCoVs), like OC43 and NL63, were at the basis of the differential mortality (and morbidity) observed after SARS-CoV-2 infection, because a small proportion of HCoV-specific T cells cross-react with SARS-CoV-2. Our data demonstrate that pre-existing T cell immunity induced by circulating human alpha- and beta-HCoVs is present in young adult individuals, but virtually absent in older adult subjects. Consequently, the frequency of cross-reactive T cells directed to the novel pandemic SARS-CoV-2 was minimal in most older adults. To the best of our knowledge, this is the first time that the presence of cross-reactive T cells to SARS-CoV-2 is compared in young and older adults. Our findings provide at least a partial explanation for the more severe clinical outcome of SARS-CoV-2 infection observed in the elderly. Moreover, this information could help to design efficacious vaccines for this age group, aiming at the induction of cell-mediated immunity.

Cryo-EM reveals the molecular basis of SARS-CoV-2 cross-neutralization by the monoclonal antibody 47D11., The emergence of SARS-CoV-2 antibody escape mutations highlights the urgent need for broadly neutralizing therapeutics. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralizing SARS-CoV-2 and SARS-CoV and protecting against the associated respiratory disease in an animal model. Here, we report cryo-EM structures of both trimeric spike ectodomains in complex with the 47D11 Fab. 47D11 binds to the closed receptor-binding domain, distal to the ACE2 binding site. The CDRL3 stabilizes the N343 glycan in an upright conformation, exposing a mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. 47D11 stabilizes a partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies targeting the exposed receptor-binding motif. Together, these results reveal a cross-protective epitope on the SARS-CoV-2 spike and provide a structural roadmap for the development of 47D11 as a prophylactic or postexposure therapy for COVID-19.

Background Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. Methods We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. Results Targeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. Conclusions Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0775-8) contains supplementary material, which is available to authorized users.

Highlights • Overexpression of porcine APN in cells does not confer susceptibility to PEDV. • Knockout APN expression in PEDV-susceptible cells has no effect on PEDV infection. • Results demonstrate that APN is not essential for PEDV cell entry., Porcine epidemic diarrhea virus (PEDV) is an emerging pathogenic coronavirus that causes a significant economic burden to the swine industry. The virus infects the intestinal epithelium and causes villous atrophy, resulting in diarrhea and dehydration. Interaction of the viral spike (S) surface glycoprotein − through its S1 subunit − with the host cell receptor is the first step in infection and the main determinant for virus tropism. As for several other alphacoronaviruses including the porcine transmissible gastroenteritis virus (TGEV) and the human coronavirus 229E (HCoV-229E), the aminopeptidase N (APN) protein was reported to be a functional receptor for PEDV. In this study we examined the role of APN as a receptor. We show that overexpression of porcine APN renders MDCK cells susceptible to TGEV, but not to PEDV. Consistently, unlike TGEV-S1, PEDV-S1 exhibited no binding to cell-surface expressed APN or to a soluble version of APN. Moreover, preincubation of these viruses with soluble APN or pretreatment of APN expressing ST cells with soluble TGEV-S1 blocked TGEV infection, but had no effect on infection by PEDV. The combined observations indicated that APN is not required for PEDV infection. To definitively prove this conclusion, we applied CRISPR/Cas9 genome engineering to knock out APN expression in PEDV-susceptible porcine (ST) and human cell lines (Huh7 and HeLa). As a consequence these cells no longer bound TGEV-S1 and HCoV-229E-S1 at their surface and were resistant to infection by the corresponding viruses. However, genetic ablation of APN expression had no effect on their infectability by PEDV, demonstrating that APN is not essential for PEDV cell entry.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

International audience; Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.

Middle East respiratory syndrome coronavirus (MERS-CoV) uses the S1(B) domain of its spike protein to bind to dipeptidyl peptidase 4 (DPP4), its functional receptor, and its S1(A) domain to bind to sialic acids. The tissue localization of DPP4 in humans, bats, camelids, pigs, and rabbits generally correlates with MERS-CoV tropism, highlighting the role of DPP4 in virus pathogenesis and transmission. However, MERS-CoV S1(A) does not indiscriminately bind to all α2,3-sialic acids, and the species-specific binding and tissue distribution of these sialic acids in different MERS-CoV-susceptible species have not been investigated. We established a novel method to detect these sialic acids on tissue sections of various organs of different susceptible species by using nanoparticles displaying multivalent MERS-CoV S1(A). We found that the nanoparticles specifically bound to the nasal epithelial cells of dromedary camels, type II pneumocytes in human lungs, and the intestinal epithelial cells of common pipistrelle bats. Desialylation by neuraminidase abolished nanoparticle binding and significantly reduced MERS-CoV infection in primary susceptible cells. In contrast, S1(A) nanoparticles did not bind to the intestinal epithelium of serotine bats and frugivorous bat species, nor did they bind to the nasal epithelium of pigs and rabbits. Both pigs and rabbits have been shown to shed less infectious virus than dromedary camels and do not transmit the virus via either contact or airborne routes. Our results depict species-specific colocalization of MERS-CoV entry and attachment receptors, which may be relevant in the transmission and pathogenesis of MERS-CoV. IMPORTANCE MERS-CoV uses the S1(B) domain of its spike protein to attach to its host receptor, dipeptidyl peptidase 4 (DPP4). The tissue localization of DPP4 has been mapped in different susceptible species. On the other hand, the S1(A) domain, the N-terminal domain of this spike protein, preferentially binds to several glycotopes of α2,3-sialic acids, the attachment factor of MERS-CoV. Here we show, using a novel method, that the S1(A) domain specifically binds to the nasal epithelium of dromedary camels, alveolar epithelium of humans, and intestinal epithelium of common pipistrelle bats. In contrast, it does not bind to the nasal epithelium of pigs or rabbits, nor does it bind to the intestinal epithelium of serotine bats and frugivorous bat species. This finding supports the importance of the S1(A) domain in MERS-CoV infection and tropism, suggests its role in transmission, and highlights its potential use as a component of novel vaccine candidates.

Positive-stranded RNA viruses extensively remodel host cell architecture to enable viral replication. Here we examined the poorly understood formation of specialized membrane compartments which are critical sites for the synthesis of the viral genome. We show that the replication compartments (RCs) of enteroviruses are created through novel membrane contact sites that recruit host lipid droplets (LDs) to the RCs. Viral proteins tether the RCs to the LDs and interact with the host lipolysis machinery to enable transfer of fatty acids from LDs, thereby providing lipids essential for RC biogenesis. Inhibiting the formation of the membrane contact sites between LDs and RCs or inhibition of the lipolysis pathway disrupts RC biogenesis and enterovirus replication. Our data illuminate mechanistic and functional aspects of organelle remodeling in viral infection and establish that pharmacological targeting of contact sites linking viral and host compartments is a potential strategy for antiviral development.

Coronaviruses (CoVs) are widespread among mammals and birds and known for their potential for cross-species transmission. In cats, infections with feline coronaviruses (FCoVs) are common. Several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. However, whether cats might become naturally infected with CoVs of other species is unknown. We analyzed coronavirus infections in cats by serological monitoring. In total 137 cat serum samples and 25 FCoV type 1 or type 2-specific antisera were screened for the presence of antibodies against the S1 receptor binding subunit of the CoV spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. Seventy-eight sera were positive for antibodies that recognized one or more coronavirus S1s whereas 1 serum exclusively reacted with human coronavirus 229E (HCoV-229E) and two sera exclusively reacted with porcine delta coronavirus (PDCoV). We observed antigenic cross-reactivity between S1s of type 1 and type 2 FCoVs, and between FCoV type 1 and porcine epidemic diarrhea virus (PEDV). Domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the CD domains of S1. The cross-reactivity of FCoV type 1 and PEDV was also observed at the level of virus neutralization. To conclude, we provide the first evidence of antigenic cross-reactivity among S1 proteins of coronaviruses, which should be considered in the development of serological diagnoses. In addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded.

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication., Author summary Enteroviruses are the most common viruses infecting humans. They cause a broad spectrum of diseases ranging from common cold to life-threatening diseases, such as poliomyelitis. To date, no effective antiviral therapy for enteroviruses has been approved yet. To ensure efficient replication, enteroviruses hijack several host factors, recruit them to the sites of virus replication, and use their physiological functions for their own purposes. Here, we characterize the complexes composed of the host protein ACBD3 and the ACBD3-binding viral proteins (called 3A) of four representative enteroviruses. Our study reveals the atomic details of these complexes and identifies the amino acid residues important for the interaction. We found out that the 3A proteins of enteroviruses bind to the same regions of ACBD3 as the 3A proteins of kobuviruses, a distinct group of viruses that also rely on ACBD3, but are oriented in the opposite directions. This observation reveals a striking case of convergent evolutionary pathways that have evolved to allow enteroviruses and kobuviruses (which are two distinct groups of the Picornaviridae family) to recruit a common host target, ACBD3, and its downstream effectors to the sites of viral replication.

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16., The adipose-specific phospholipase A2 (PLA2G16) has been identified as a pan-enterovirus host factor. Here, the authors show that the enterovirus EV-D68-947 can bypass PLA2G16 by using sulfated glycosaminoglycans as alternative glycan receptors and that the interaction induces structural changes promoting viral genome release.

[Image: see text] Multivalent carbohydrate-based ligands were synthesized and evaluated as inhibitors of the adhesion protein HA of the influenza A virus (IAV). HA relies on multivalency for strong viral adhesion. While viral adhesion inhibition by large polymeric molecules has proven viable, limited success was reached for smaller multivalent compounds. By linking of sialylated LAcNAc units to di- and trivalent scaffolds, inhibitors were obtained with an up to 428-fold enhanced inhibition in various assays.

EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry., Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.

Genetic perturbation screens using RNA interference (RNAi) have been conducted successfully to identify host factors that are essential for the life cycle of bacteria or viruses. So far, most published studies identified host factors primarily for single pathogens. Furthermore, often only a small subset of genes, e.g., genes encoding kinases, have been targeted. Identification of host factors on a pan-pathogen level, i.e., genes that are crucial for the replication of a diverse group of pathogens has received relatively little attention, despite the fact that such common host factors would be highly relevant, for instance, for devising broad-spectrum anti-pathogenic drugs. Here, we present a novel two-stage procedure for the identification of host factors involved in the replication of different viruses using a combination of random effects models and Markov random walks on a functional interaction network. We first infer candidate genes by jointly analyzing multiple perturbations screens while at the same time adjusting for high variance inherent in these screens. Subsequently the inferred estimates are spread across a network of functional interactions thereby allowing for the analysis of missing genes in the biological studies, smoothing the effect sizes of previously found host factors, and considering a priori pathway information defined over edges of the network. We applied the procedure to RNAi screening data of four different positive-sense single-stranded RNA viruses, Hepatitis C virus, Chikungunya virus, Dengue virus and Severe acute respiratory syndrome coronavirus, and detected novel host factors, including UBC, PLCG1, and DYRK1B, which are predicted to significantly impact the replication cycles of these viruses. We validated the detected host factors experimentally using pharmacological inhibition and an additional siRNA screen and found that some of the predicted host factors indeed influence the replication of these pathogens, PLoS Computational Biology, 16 (2), ISSN:1553-734X, ISSN:1553-7358

Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes., Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein., Author summary Influenza A viruses infect birds and mammals. They contain receptor-binding (HA) and receptor-destroying (NA) proteins, which are crucial determinants of host tropism and pathogenesis. It is generally accepted that the functional properties of HA and NA need to be well balanced to enable virion penetration of the receptor-rich mucus layer, binding to host cells, and release of newly assembled particles. This HA-NA-receptor balance is, however, poorly characterized resulting in part from a lack of suitable assays to measure this balance. In addition, NA is much less studied than HA. NA contains, besides its receptor-cleavage site, a 2nd receptor-binding site, which is functional in avian, but not in human viruses. We now show that this 2nd receptor-binding site prefers binding to avian-type receptors and promotes cleavage of substrates carrying this receptor. Furthermore, by using novel assays, we established an important role for this site in the HA-NA-receptor balance of virus particles as it contributes to receptor binding and cleavage by virions, the latter of which is required for virion movement and self-elution from receptors. The results may provide an explanation for the rapid loss of a functional 2nd receptor-binding site in human pandemic viruses.

The ongoing Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks pose a worldwide public health threat. Blocking MERS-CoV zoonotic transmission from dromedary camels, the animal reservoir, could potentially reduce the number of primary human cases. Here we report MERS-CoV transmission from experimentally infected llamas to naïve animals. Directly inoculated llamas shed virus for at least 6 days and could infect all in-contact naïve animals 4-5 days after exposure. With the aim to block virus transmission, we examined the efficacy of a recombinant spike S1-protein vaccine. In contrast to naïve animals, in-contact vaccinated llamas did not shed infectious virus upon exposure to directly inoculated llamas, consistent with the induction of strong virus neutralizing antibody responses. Our data provide further evidence that vaccination of the reservoir host may impede MERS-CoV zoonotic transmission to humans. info:eu-repo/semantics/publishedVersion

RNA interference (RNAi) has strong antiviral activity in a range of animal phyla, but the extent to which RNAi controls virus infection in chordates, and specifically mammals remains incompletely understood. Here we analyze the antiviral activity of RNAi against a number of positive-sense RNA viruses using Argonaute-2 deficient human cells. In line with absence of virus-derived siRNAs, Sindbis virus, yellow fever virus, and encephalomyocarditis virus replicated with similar kinetics in wildtype cells and Argonaute-2 deficient cells. Coxsackievirus B3 (CVB3) carrying mutations in the viral 3A protein, previously proposed to be a virus-encoded suppressor of RNAi in another picornavirus, human enterovirus 71, had a strong replication defect in wildtype cells. However, this defect was not rescued in Argonaute-2 deficient cells, arguing against a role of CVB3 3A as an RNAi suppressor. In agreement, neither infection with wildtype nor 3A mutant CVB3 resulted in small RNA production with the hallmarks of canonical vsiRNAs. Together, our results argue against strong antiviral activity of RNAi under these experimental conditions, but do not exclude that antiviral RNAi may be functional under other cellular, experimental, or physiological conditions in mammals.

Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in progressively infected cats. While testing/removal and vaccination led to a decreased prevalence of FeLV, recently, this decrease has reportedly stagnated in some countries. This study aimed to prospectively determine the prevalence of FeLV viraemia in cats taken to veterinary facilities in 32 European countries. FeLV viral RNA was semiquantitatively detected in saliva, using RT-qPCR as a measure of viraemia. Risk and protective factors were assessed using an online questionnaire to report geographic, demographic, husbandry, FeLV vaccination, and clinical data. The overall prevalence of FeLV viraemia in cats visiting a veterinary facility, of which 10.4% were shelter and rescue cats, was 2.3% (141/6005, 95% CI: 2.0%&ndash, 2.8%) with the highest prevalences in Portugal, Hungary, and Italy/Malta (5.7%&ndash, 8.8%). Using multivariate analysis, seven risk factors (Southern Europe, male intact, 1&ndash, 6 years of age, indoor and outdoor or outdoor-only living, living in a group of &ge, 5 cats, illness), and three protective factors (Northern Europe, Western Europe, pedigree cats) were identified. Using classification and regression tree (CART) analysis, the origin of cats in Europe, pedigree, and access to outdoors were important predictors of FeLV status. FeLV-infected sick cats shed more viral RNA than FeLV-infected healthy cats, and they suffered more frequently from anaemia, anorexia, and gingivitis/stomatitis than uninfected sick cats. Most cats had never been FeLV-vaccinated, vaccination rates were indirectly associated with the gross domestic product (GDP) per capita. In conclusion, we identified countries where FeLV was undetectable, demonstrating that the infection can be eradicated and highlighting those regions where awareness and prevention should be increased.

Discovery of novel viruses in host samples is a multidisciplinary process which relies increasingly on next-generation sequencing (NGS) followed by computational analysis. A crucial step in this analysis is to separate host sequence reads from the sequence reads of the virus to be discovered. This becomes especially difficult if no reference genome of the host is available. Furthermore, if the total number of viral reads in a sample is low, de novo assembly of a virus which is a requirement for most existing pipelines is hard to realize. We present a new modular, computational pipeline for discovery of novel viruses in host samples. While existing pipelines rely on the availability of the hosts reference genome for filtering sequence reads, our new pipeline can also cope with cases for which no reference genome is available. As a further novelty of our method a decoy module is used to assess false classification rates in the discovery process. Additionally, viruses with a low read coverage can be identified and visually reviewed. We validate our pipeline on simulated data as well as two experimental samples with known virus content. For the experimental samples, we were able to reproduce the laboratory findings. Our newly developed pipeline is applicable for virus detection in a wide range of host species. The three modules we present can either be incorporated individually in other pipelines or be used as a stand-alone pipeline. We are the first to present a decoy approach within a virus detection pipeline that can be used to assess error rates so that the quality of the final result can be judged. We provide an implementation of our modules via Github. However, the principle of the modules can easily be re-implemented by other researchers.

Icosahedral viral capsids must undergo conformational rearrangements to coordinate essential processes during the viral life cycle. Capturing such conformational flexibility has been technically challenging yet could be key for developing rational therapeutic agents to combat infections. Noroviruses are nonenveloped, icosahedral viruses of global importance to human health. They are a common cause of acute gastroenteritis, yet no vaccines or specific antiviral agents are available. Here, we use genetics and cryo-electron microscopy (cryo-EM) to study the high-resolution solution structures of murine norovirus as a model for human viruses. By comparing our 3 structures (at 2.9- to 3.1-Å resolution), we show that whilst there is little change to the shell domain of the capsid, the radiating protruding domains are flexible, adopting distinct states both independently and synchronously. In doing so, the capsids sample a range of conformational space, with implications for maintaining virion stability and infectivity., High-resolution cryo-EM reveals that the norovirus capsid continuously samples conformational space, challenging the model of conformational changes in virus structure being orchestrated along linear, irreversible pathways of assembly and disassembly, with implications for infectivity and immune evasion.

For many viruses, efficient replication is contingent on the ability to evade antiviral responses such as the ISR. Yet, for most viruses it remains poorly understood by what mechanisms the ISR is suppressed. In this thesis we set out to identify new ISR antagonists encoded by picorna- and coronaviruses, and on the elucidation of their molecular mode of action. This will increase our knowledge about evasion mechanisms employed by viruses to protect themselves against antiviral responses. From a cell biological perspective, the study of viral ISR antagonists may also teach us more about how the ISR regulates translation, one of the core processes in life. In chapter 2, we set out to investigate the function of an accessory protein (p4a) encoded by Middle-East Respiratory Syndrome coronavirus (MERS-CoV) as inhibitor of PKR-mediated ISR activation(91). We identify MERS-CoV p4a as a PKR-specific ISR antagonist that functions via dsRNA binding. Consistent with its mode of action of dsRNA sequestration, p4a also prevents the activation of another dsRNA-activated antiviral pathway, the type I IFN induction pathway. While the mode of action of p4a was described before for several viral dsRNA binding proteins, p4a is the first coronavirus protein identified with this function. In chapter 3, we assess the function of a small-molecule inhibitor of the ISR (ISRIB). We observed that ISRIB prevents ISR activation only early in infection, but failed to do so at later time points. Further investigation taught us that ISRIB prevents ISR activity only under conditions of mild stress, as defined by a limited level of eIF2 phosphorylation. High levels of p-eIF2 however may inhibit translation and enhance expression of ISR-induced proteins, even in the presence of ISRIB. Thus, ISRIB makes cells less sensitive rather than insensitive to ISR signaling. These findings are consistent with the previously reported mode of action of ISRIB, and may explain the remarkable absence of side-effects of an ISRIB treatment in vivo. In chapter 4, we investigated the function(s) of an accessory protein, AcP10, encoded by Beluga whale coronavirus. AcP10 prevents p-eIF2 from binding its target eIF2B, and thereby AcP10 ensures that eIF2B remains active even in the presence of p-eIF2. It does so via a direct interaction with eIF2B, likely at an area that overlaps the interaction site for p-eIF2, but not for native eIF2. This mode-of-action was not previously described for any viral or host cell protein, and thus AcP10 is a prototype of an entirely novel class of ISR antagonists. We classified AcP10 as a Class 4 antagonist. In chapter 5, we describe another Class 4 antagonist, the Leader protein encoded by the picornavirus Aichivirus (AiVL). AiVL, like AcP10, binds eIF2B to prevent subsequent association of p-eIF2, thereby rescuing translation in the presence of (high levels) of p-eIF2 irrespective of the eIF2 kinase involved. Yet, AiVL has no homology to AcP10. Thus, Class 4 ISR antagonists arose at least twice, in phylogenetically distinct virus families, by convergent evolution.