Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue.

Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41.IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.

The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.

Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn-to-Ala substitutions at the predicted N-glycosylation sites of the M41-RBD were evaluated along with two control Val-to-Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41-RBD construct. Six of the 10 glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented α2,3-linked sialic acid oligosaccharide ligand. LC/MSE glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall, the glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41-RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein.

Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding.

Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses.IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.

Three mutations switch H7N9 influenza to human-type receptor specificity.

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.

UNLABELLED: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. IMPORTANCE: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

Single-cell analysis of population context advances RNAi screening at multiple levels.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.

Influenza D binding properties vary amongst the two major virus clades and wildlife species.

The influenza D virus (IDV) uses a trimeric hemagglutinin-esterase fusion protein (HEF) for attachment to 9-O-acetylated sialic acid receptors on the cell surface of host species. So far research has revealed that farm animals such as cattle, domestic pigs, goats, sheep and horses contain the necessary receptors on the epithelial surface of the respiratory tract to accommodate binding of the IDV HEF protein of both worldwide clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660). More recently, seroprevalence studies have identified IDV-seropositive wildlife such as wild boar, deer, dromedaries, and small ruminants. However, no research has thus far been conducted in wildlife to reveal the distribution of acetylated sialic acid receptors that accommodate binding of IDV. Using our previously developed tissue microarray (TMA) system, we developed TMAs containing respiratory tissues of various wild and domestic species including wild boar, deer, dromedary, springbok, water buffalo, tiger, hedgehog, and Asian elephant. Protein histochemical staining of these TMAs with HEF proteins showed no receptor binding for wild Suidae, Cervidae and tiger. However, receptors were present in dromedary, springbok, water buffalo, Asian elephant, and hedgehog. In contrast to previously tested farm animals, a difference in host tropism was observed between the D/OK and D/660 clade HEF proteins in Asian elephant, and water buffalo. These results show that IDV can attach to the respiratory tract of wildlife which might facilitate transmission of IDV between wildlife and domestic animals.

The coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes.

The coronavirus nucleocapsid (N) protein is a virion structural protein. It also functions, however, in an unknown way in viral replication and localizes to the viral replication-transcription complexes (RTCs). Here we investigated, using recombinant murine coronaviruses expressing green fluorescent protein (GFP)-tagged versions of the N protein, the dynamics of its interactions with the RTCs and the domain(s) involved. Using fluorescent recovery after photobleaching, we showed that the N protein, unlike the nonstructural protein 2, is dynamically associated with the RTCs. Recruitment of the N protein to the RTCs requires the C-terminal N2b domain, which interacts with other N proteins in an RNA-independent manner.

The ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle.

The ubiquitin-proteasome system (UPS) is a key player in regulating the intracellular sorting and degradation of proteins. In this study we investigated the role of the UPS in different steps of the coronavirus (CoV) infection cycle. Inhibition of the proteasome by different chemical compounds (i.e., MG132, epoxomicin, and Velcade) appeared to not only impair entry but also RNA synthesis and subsequent protein expression of different CoVs (i.e., mouse hepatitis virus [MHV], feline infectious peritonitis virus, and severe acute respiratory syndrome CoV). MHV assembly and release were, however, not appreciably affected by these compounds. The inhibitory effect on CoV protein expression did not appear to result from a general inhibition of translation due to induction of a cellular stress response by the inhibitors. Stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) generally results in impaired initiation of protein synthesis, but the sensitivity of MHV infection to proteasome inhibitors was unchanged in cells lacking a phosphorylatable eIF2alpha. MHV infection was affected not only by inhibition of the proteasome but also by interfering with protein ubiquitination. Viral protein expression was reduced in cells expressing a temperature-sensitive ubiquitin-activating enzyme E1 at the restrictive temperature, as well as in cells in which ubiquitin was depleted by using small interfering RNAs. Under these conditions, the susceptibility of the cells to virus infection was, however, not affected, excluding an important role of ubiquitination in virus entry. Our observations reveal an important role of the UPS in multiple steps of the CoV infection cycle and identify the UPS as a potential drug target to modulate the impact of CoV infection.

Dynamics of coronavirus replication-transcription complexes.

Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus.

Coronaviruses (CoV) are enveloped positive-strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on these structures, we have performed a time-course experiment in which the mouse hepatitis virus (MHV)-induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)-electron microscopy. With our approach we were able to confirm the appearance of 6, previously reported, membranous structures during the course of a complete infection cycle. These structures include the well-characterized double-membrane vesicles (DMVs), convoluted membranes (CMs) and virions but also the more enigmatic large virion-containing vacuoles (LVCVs), tubular bodies (TBs) and cubic membrane structures (CMSs). We have characterized the LVCVs, TBs and CMSs, and found that the CoV-induced structures appear in a strict order. By combining these data with quantitative analyses on viral RNA, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of CoV infection. In particular, it provides insights in the role of each CoV-induced structure and reveals that LVCVs are ERGIC/Golgi compartments that expand to accommodate an increasing production of viral particles.

Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

Pathology and Pathogenesis of Eurasian Blackbirds (Turdus merula) Naturally Infected with Usutu Virus.

The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.

Coronavirus genetically redirected to the epidermal growth factor receptor exhibits effective antitumor activity against a malignant glioblastoma.

Coronaviruses are positive-strand RNA viruses with features attractive for oncolytic therapy. To investigate this potential, we redirected the coronavirus murine hepatitis virus (MHV), which is normally unable to infect human cells, to human tumor cells by using a soluble receptor (soR)-based expression construct fused to an epidermal growth factor (EGF) receptor targeting moiety. Addition of this adapter protein to MHV allowed infection of otherwise nonsusceptible, EGF receptor (EGFR)-expressing cell cultures. We introduced the sequence encoding the adaptor protein soR-EGF into the MHV genome to generate a self-targeted virus capable of multiround infection. The resulting recombinant MHV was viable and had indeed acquired the ability to infect all glioblastoma cell lines tested in vitro. Infection of malignant human glioblastoma U87DeltaEGFR cells gave rise to release of progeny virus and efficient cell killing in vitro. To investigate the oncolytic capacity of the virus in vivo, we used an orthotopic U87DeltaEGFR xenograft mouse model. Treatment of mice bearing a lethal intracranial U87DeltaEGFR tumor by injection with MHVsoR-EGF significantly prolonged survival compared to phosphate-buffered saline-treated (P = 0.001) and control virus-treated (P = 0.004) animals, and no recurrent tumor load was observed. However, some adverse effects were seen in normal mouse brain tissues that were likely caused by the natural murine tropism of MHV. This is the first demonstration of oncolytic activity of a coronavirus in vivo. It suggests that nonhuman coronaviruses may be attractive new therapeutic agents against human tumors.

Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation.

Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected.

Transmission Kinetics and histopathology induced by European Turkey Coronavirus during experimental infection of specific pathogen free turkeys.

Numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (PEC). In 2008 a coronavirus (Fr-TCoV 080385d) was isolated in France from turkey poults exhibiting clinical signs compatible with this syndrome. In the present study, the median infectious dose (ID50 ), transmission kinetics and pathogenicity of Fr-TCoV were investigated in 10-day-old SPF turkeys. Results revealed a titre of 104.88 ID50 /ml with 1 ID50 /ml being beyond the limit of genome detection using a well-characterized qRT-PCR for avian coronaviruses. Horizontal transmission of the virus via the airborne route was not observed however, via the oro-faecal route this proved to be extremely rapid (one infectious individual infecting another every 2.5 hr) and infectious virus was excreted for at least 6 weeks in several birds. Histological examination of different zones of the intestinal tract of the Fr-TCoV-infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of Fabricius. Viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for Fr-TCoV to replicate in antigen-presenting cells. Together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds.