63 results on '"Verheije MH"'
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2. Interference between avian corona and influenza viruses: The role of the epithelial architecture of the chicken trachea.
- Author
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Weerts EAWS, Bouwman KM, Paerels L, Gröne A, Jan Boelm G, and Verheije MH
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- Animals, Chickens, Trachea, Coinfection veterinary, Coronavirus Infections veterinary, Infectious bronchitis virus physiology, Influenza A Virus, H9N2 Subtype physiology, Poultry Diseases, Superinfection veterinary
- Abstract
Respiratory viral infections are among the major causes of disease in poultry. While viral dual infections are known to occur, viral interference in chicken airways is mechanistically hardly understood. The effects of infectious bronchitis virus (IBV) infection on tissue morphology, sialic acid (sia) expression and susceptibility of the chicken trachea for superinfection with IBV or avian influenza virus (AIV) were studied. In vivo, tracheal epithelium of chickens infected with IBV QX showed marked inflammatory cell infiltration and loss of cilia and goblet cells five days post inoculation. Plant lectin staining indicated that sialic acids redistributed from the apical membrane of the ciliated epithelium and the goblet cell cytoplasm to the basement membrane region of the epithelium. After administration of recombinant viral attachment proteins to slides of infected tissue, retained binding of AIV hemagglutinin, absence of binding of the receptor binding domain (RBD) of IBV M41 and partial reduction of IBV QX RBD were observed. Adult chicken trachea rings were used as ex vivo model to study the effects of IBV QX-induced pathological changes and receptor redistribution on secondary viral infection. AIV H9N2 infection after primary IBV infection was delayed; however, final viral loads reached similar levels as in previously uninfected trachea rings. In contrast, IBV M41 superinfection resulted in 1000-fold lower viral titers over the course of 48 h. In conclusion, epithelial changes in the chicken trachea after viral infection coincide with redistribution and likely specific downregulation of viral receptors, with the extend of subsequent viral interference dependent on viral species., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2022
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3. Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.
- Author
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Nemanichvili N, Spruit CM, Berends AJ, Gröne A, Rijks JM, Verheije MH, and de Vries RP
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- Animals, Animals, Domestic metabolism, Dogs, Ferrets metabolism, Glycolipids, Horses, Humans, Lectins, N-Acetylneuraminic Acid metabolism, Neuraminic Acids, Polysaccharides, Swine, Sialic Acids metabolism, Viruses
- Abstract
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., (© The Author(s) 2022. Published by Oxford University Press.)
- Published
- 2022
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4. Influenza D binding properties vary amongst the two major virus clades and wildlife species.
- Author
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Nemanichvili N, Berends AJ, Tomris I, Barnard KN, Parrish CR, Gröne A, Rijks JM, Verheije MH, and de Vries RP
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- Animals, Animals, Domestic virology, Animals, Wild virology, Cattle, Deer, Horses, Seroepidemiologic Studies, Sheep, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections veterinary, Orthomyxoviridae Infections virology, Receptors, Cell Surface immunology, Thogotovirus classification, Thogotovirus genetics, Thogotovirus metabolism
- Abstract
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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2022
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5. The contribution of the immune response to enhanced colibacillosis upon preceding viral respiratory infection in broiler chicken in a dual infection model.
- Author
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Weerts EAWS, Matthijs MGR, Bonhof J, van Haarlem DA, Dwars RM, Gröne A, Verheije MH, and Jansen CA
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- Air Sacs microbiology, Air Sacs pathology, Animals, Birnaviridae Infections complications, Birnaviridae Infections virology, Coinfection, Cytokines, Escherichia coli, Escherichia coli Infections immunology, Escherichia coli Infections microbiology, Female, Poultry Diseases immunology, Poultry Diseases virology, RNA, Messenger genetics, RNA, Messenger metabolism, Respiratory Tract Infections microbiology, Respiratory Tract Infections veterinary, Respiratory Tract Infections virology, Specific Pathogen-Free Organisms, Birnaviridae Infections veterinary, Chickens, Escherichia coli Infections veterinary, Infectious bursal disease virus, Poultry Diseases microbiology
- Abstract
Colibacillosis in chickens caused by avian pathogenic Escherichia coli (APEC) is known to be aggravated by preceding infections with infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). The mechanism behind these virus-induced predispositions for secondary bacterial infections is poorly understood. Here we set out to investigate the immunopathogenesis of enhanced respiratory colibacillosis after preceding infections with these three viruses. Broilers were inoculated intratracheally with APEC six days after oculonasal and intratracheal inoculation with IBV, NDV, aMPV or buffered saline. After euthanasia at 1 and 8 days post infection (dpi) with APEC, birds were macroscopically examined and tissue samples were taken from the trachea, lungs and air sacs. In none of the groups differences in body weight were observed during the course of infection. Macroscopic lesion scoring revealed most severe tissue changes after NDV-APEC and IBV-APEC infection. Histologically, persistent tracheitis was detected in all virus-APEC groups, but not after APEC-only infection. In the lungs, mostly APEC-associated transient pneumonia was observed. Severe and persistent airsacculitis was present after NDV-APEC and IBV-APEC infection. Bacterial antigen was detected by immunohistochemistry only at 1 dpi APEC, predominantly in NDV-APEC- and IBV-APEC-infected lungs. Higher numbers of CD4+ and CD8+ lymphocytes persisted over time in NDV-APEC- and IBV-APEC-infected tracheas, as did CD4+ lymphocytes in NBV-APEC- and IBV-APEC-infected air sacs. KUL01+ cells, which include monocytes and macrophages, and TCRγδ+ lymphocytes were observed mostly in lung tissue in all infected groups with transient higher numbers of KUL01+ cells over time and higher numbers of TCRγδ+ lymphocytes mainly at 8 dpi. qPCR analysis revealed mostly trends of transient higher levels of IL-6 and IFNγ mRNA in lung tissue after IBV-APEC and also NDV-APEC infection and persistent higher levels of IL-6 mRNA after aMPV-APEC infection. In spleens, transient higher levels of IL-17 mRNA and more persistent higher levels of IL-6 mRNA were observed after all co-infections. No changes in IL-10 mRNA expression were seen. These results demonstrate a major impact of dual infections with respiratory viruses and APEC, compared to a single infection with APEC, on the chicken respiratory tract and suggest that immunopathogenesis contributes to lesion persistence., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
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6. Pathology and Pathogenesis of Eurasian Blackbirds ( Turdus merula ) Naturally Infected with Usutu Virus.
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Giglia G, Agliani G, Munnink BBO, Sikkema RS, Mandara MT, Lepri E, Kik M, Ijzer J, Rijks JM, Fast C, Koopmans MPG, Verheije MH, Gröne A, Reusken CBEM, and van den Brand JMA
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- Animals, Bird Diseases pathology, Flavivirus genetics, Flavivirus isolation & purification, Flavivirus pathogenicity, Flavivirus Infections pathology, Flavivirus Infections virology, Netherlands, Phagocytes virology, Virulence, Bird Diseases virology, Flavivirus physiology, Flavivirus Infections veterinary, Passeriformes virology
- Abstract
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.
- Published
- 2021
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7. Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals.
- Author
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Nemanichvili N, Berends AJ, Wubbolts RW, Gröne A, Rijks JM, de Vries RP, and Verheije MH
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- Animals, Animals, Domestic virology, Cattle, Goats, Hemagglutinins, Viral genetics, Horses, Host Microbial Interactions, Recombinant Proteins metabolism, Sheep, Sialic Acids metabolism, Swine, Thogotovirus chemistry, Thogotovirus genetics, Viral Fusion Proteins genetics, Hemagglutinins, Viral metabolism, Respiratory System virology, Thogotovirus physiology, Tissue Array Analysis, Viral Fusion Proteins metabolism, Viral Tropism, Virus Attachment
- Abstract
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.
- Published
- 2021
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8. N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity.
- Author
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Bouwman KM, Habraeken N, Laconi A, Berends AJ, Groenewoud L, Alders M, Kemp V, and Verheije MH
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- Animals, Cell Line, Chick Embryo, Coronavirus Infections virology, Glycosylation, Infectious bronchitis virus immunology, Kidney cytology, Kidney embryology, Protein Binding, Receptors, Cell Surface metabolism, Receptors, Virus metabolism, Recombinant Proteins, Spike Glycoprotein, Coronavirus metabolism, Viral Tropism immunology, Virus Attachment, Virus Replication, Coronavirus Infections immunology, Host Specificity immunology, Infectious bronchitis virus chemistry, Protein Domains, Receptors, Virus immunology, Spike Glycoprotein, Coronavirus chemistry
- Abstract
Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.
- Published
- 2020
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9. The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.
- Author
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Figueroa T, Bessière P, Coggon A, Bouwman KM, van der Woude R, Delverdier M, Verheije MH, de Vries RP, and Volmer R
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- Animals, Animals, Wild virology, Anti-Bacterial Agents therapeutic use, Antiviral Agents, Ducks microbiology, Ducks virology, Epithelial Cells, Humans, Ileum pathology, Influenza A virus immunology, Intestines microbiology, Lung pathology, Microbiota drug effects, Poly I-C therapeutic use, Respiratory System virology, Viral Load, Influenza in Birds immunology, Influenza in Birds microbiology, Influenza in Birds therapy, Influenza in Birds virology, Microbiota physiology, Virus Replication physiology
- Abstract
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., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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10. miRNA repertoire and host immune factor regulation upon avian coronavirus infection in eggs.
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Kemp V, Laconi A, Cocciolo G, Berends AJ, Breit TM, and Verheije MH
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- Animals, Chickens, Coronavirus Infections genetics, Coronavirus Infections immunology, Coronavirus Infections virology, Cytokines genetics, Cytokines immunology, Gammacoronavirus genetics, Lung immunology, Lung pathology, MicroRNAs genetics, Ovum immunology, Poultry Diseases genetics, Poultry Diseases pathology, Poultry Diseases virology, Spleen immunology, Spleen pathology, Coronavirus Infections veterinary, Gammacoronavirus physiology, MicroRNAs immunology, Ovum virology, Poultry Diseases immunology
- Abstract
Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.
- Published
- 2020
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11. Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation.
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Laconi A, Weerts EAWS, Bloodgood JCG, Deniz Marrero JP, Berends AJ, Cocciolo G, de Wit JJ, and Verheije MH
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- Animals, Chickens, Cloaca virology, Coronavirus Infections prevention & control, Female, Kidney pathology, Kidney virology, Male, Tissue Distribution, Trachea pathology, Trachea virology, Vaccines, Attenuated administration & dosage, Vaccines, Attenuated pharmacokinetics, Viral Vaccines administration & dosage, Virus Replication, Infectious bronchitis virus immunology, Infectious bronchitis virus pathogenicity, Infectious bronchitis virus physiology, Viral Vaccines pharmacokinetics
- Abstract
Infectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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12. Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue.
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Bouwman KM, Parsons LM, Berends AJ, de Vries RP, Cipollo JF, and Verheije MH
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- Amino Acid Substitution, Animals, Chickens virology, HEK293 Cells, Humans, Kidney metabolism, Kidney pathology, Protein Domains, Respiratory Mucosa metabolism, Respiratory Mucosa pathology, Infectious bronchitis virus physiology, Kidney virology, Mutation, Missense, Respiratory Mucosa virology, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus metabolism, Viral Tropism genetics, Virus Replication genetics
- Abstract
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., (Copyright © 2020 Bouwman et al.)
- Published
- 2020
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13. N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.
- Author
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Broszeit F, Tzarum N, Zhu X, Nemanichvili N, Eggink D, Leenders T, Li Z, Liu L, Wolfert MA, Papanikolaou A, Martínez-Romero C, Gagarinov IA, Yu W, García-Sastre A, Wennekes T, Okamatsu M, Verheije MH, Wilson IA, Boons GJ, and de Vries RP
- Subjects
- Animals, Chickens, Dogs, Erythrocytes metabolism, Erythrocytes virology, Hemagglutinins chemistry, Hemagglutinins metabolism, Horses, Influenza A virus pathogenicity, Neuraminic Acids chemistry, Orthomyxoviridae Infections veterinary, Protein Binding, Host Specificity, Influenza A virus metabolism, Neuraminic Acids metabolism, Orthomyxoviridae Infections metabolism
- Abstract
A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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14. Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.
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Parsons LM, Bouwman KM, Azurmendi H, de Vries RP, Cipollo JF, and Verheije MH
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- Amino Acid Substitution, Animals, Chickens virology, Glycosylation, HEK293 Cells, Humans, Infectious bronchitis virus genetics, Infectious bronchitis virus metabolism, Mutation, Missense, Protein Structure, Secondary, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus metabolism, Infectious bronchitis virus chemistry, Molecular Docking Simulation, Spike Glycoprotein, Coronavirus chemistry
- Abstract
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/MS
E 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.- Published
- 2019
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15. Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding.
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Bouwman KM, Delpont M, Broszeit F, Berger R, Weerts EAWS, Lucas MN, Delverdier M, Belkasmi S, Papanikolaou A, Boons GJ, Guérin JL, de Vries RP, Ducatez MF, and Verheije MH
- Subjects
- Animals, Coronavirus metabolism, Coronavirus pathogenicity, Coronavirus Infections virology, Enteritis metabolism, Enteritis virology, France, Galliformes virology, Gammacoronavirus physiology, Genetic Variation, Phenotype, Polysaccharides, Receptors, Virus metabolism, Sialic Acids, Spike Glycoprotein, Coronavirus metabolism, Virus Attachment, Gammacoronavirus genetics, Gammacoronavirus metabolism, Viral Tropism genetics
- Abstract
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., (Copyright © 2019 Bouwman et al.)
- Published
- 2019
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16. Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties.
- Author
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Nemanichvili N, Tomris I, Turner HL, McBride R, Grant OC, van der Woude R, Aldosari MH, Pieters RJ, Woods RJ, Paulson JC, Boons GJ, Ward AB, Verheije MH, and de Vries RP
- Subjects
- Animals, Binding Sites physiology, Birds, Humans, Influenza in Birds virology, Influenza, Human virology, Polysaccharides metabolism, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Influenza A virus metabolism, Influenza in Birds metabolism, Influenza, Human metabolism, Protein Binding physiology, Receptors, Cell Surface metabolism
- Abstract
Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2019
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17. Transmission Kinetics and histopathology induced by European Turkey Coronavirus during experimental infection of specific pathogen free turkeys.
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Brown PA, Courtillon C, Weerts EAWS, Andraud M, Allée C, Vendembeuche A, Amelot M, Rose N, Verheije MH, and Eterradossi N
- Subjects
- Animals, Antigens, Viral analysis, Coronavirus Infections pathology, Coronavirus Infections transmission, Coronavirus Infections virology, France, Poultry Diseases pathology, Poultry Diseases virology, Specific Pathogen-Free Organisms, Basic Reproduction Number, Coronavirus Infections veterinary, Coronavirus, Turkey physiology, Poultry Diseases transmission, Turkeys
- Abstract
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 (ID
50 ), 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., (© 2018 Blackwell Verlag GmbH.)- Published
- 2019
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18. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.
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Peng W, Bouwman KM, McBride R, Grant OC, Woods RJ, Verheije MH, Paulson JC, and de Vries RP
- Subjects
- Cell Line, Epithelial Cells virology, HEK293 Cells, Hemagglutinin Glycoproteins, Influenza Virus genetics, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Humans, Influenza A Virus, H1N1 Subtype genetics, Influenza A Virus, H3N2 Subtype genetics, Influenza A Virus, H5N1 Subtype genetics, Influenza, Human virology, Mutation genetics, Polysaccharides chemistry, Protein Binding physiology, Sialic Acids chemistry, Trachea cytology, Trachea virology, Influenza A Virus, H1N1 Subtype metabolism, Influenza A Virus, H3N2 Subtype metabolism, Influenza A Virus, H5N1 Subtype metabolism, Receptors, Virus metabolism, Virus Attachment
- Abstract
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., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
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19. Ranavirus genotypes in the Netherlands and their potential association with virulence in water frogs (Pelophylax spp.).
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Saucedo B, Hughes J, Spitzen-van der Sluijs A, Kruithof N, Schills M, Rijks JM, Jacinto-Maldonado M, Suarez N, Haenen OLM, Voorbergen-Laarman M, van den Broek J, Gilbert M, Gröne A, van Beurden SJ, and Verheije MH
- Subjects
- Animals, DNA Virus Infections virology, Genotype, Netherlands, Phylogeny, Ranavirus classification, Ranavirus isolation & purification, Ranavirus pathogenicity, Virulence, DNA Virus Infections veterinary, Ranavirus genetics, Ranidae virology
- Abstract
Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011-2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.
- Published
- 2018
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20. Recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens.
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van Beurden SJ, Berends AJ, Krämer-Kühl A, Spekreijse D, Chenard G, Philipp HC, Mundt E, Rottier PJM, and Verheije MH
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- Animals, Chick Embryo, Chickens, Gene Deletion, Infectious bronchitis virus genetics, Poultry Diseases virology, RNA, Viral genetics, Vaccines, Attenuated immunology, Vaccines, Synthetic immunology, Viral Load, Viral Vaccines administration & dosage, Coronavirus Infections prevention & control, Coronavirus Infections veterinary, Infectious bronchitis virus immunology, Poultry Diseases prevention & control, Viral Vaccines immunology
- Abstract
Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, causing severe economic losses in poultry industry worldwide. Live attenuated viruses are widely used in both the broiler and layer industry because of their efficacy and ability to be mass applied. Recently, we established a novel reverse genetics system based on targeted RNA recombination to manipulate the genome of IBV strain H52. Here we explore the possibilities to attenuate IBV in a rational way in order to generate safe and effective vaccines against virulent IBV (van Beurden et al., 2017). To this end, we deleted the nonessential group-specific accessory genes 3 and/or 5 in the IBV genome by targeted RNA recombination and selected the recombinant viruses in embryonated eggs. The resulting recombinant (r) rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab could be rescued and grew to the same virus titer as recombinant and wild type IBV strain H52. Thus, genes 3ab and 5ab are not essential for replication in ovo. When administered to one-day-old chickens, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab showed reduced ciliostasis as compared to rIBV H52 and wild type H52, indicating that the accessory genes contribute to the pathogenicity of IBV. After homologous challenge with the virulent IBV strain M41, all vaccinated chickens were protected against disease based on reduced loss of ciliary movement in the trachea compared to the non-vaccinated but challenged controls. Taken together, deletion of accessory genes 3ab and/or 5ab in IBV resulted in mutant viruses with an attenuated phenotype and the ability to induce protection in chickens. Hence, targeted RNA recombination based on virulent IBV provides opportunities for the development of a next generation of rationally designed live attenuated IBV vaccines., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
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21. Chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus.
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Zhang W, Bouwman KM, van Beurden SJ, Ordonez SR, van Eijk M, Haagsman HP, Verheije MH, and Veldhuizen EJA
- Subjects
- Animals, Cell Line, Chickens, Cricetinae, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Immunity, Innate, Microscopy, Electron, Transmission, Protein Binding, Real-Time Polymerase Chain Reaction, Spike Glycoprotein, Coronavirus metabolism, Virion metabolism, Antiviral Agents metabolism, Infectious bronchitis virus immunology, Infectious bronchitis virus physiology, Mannose-Binding Lectin metabolism, Virus Attachment drug effects
- Abstract
Mannose binding lectin (MBL) is a collagenous C-type lectin, which plays an important role in innate immunity. It can bind to carbohydrates on the surface of a wide range of pathogens, including viruses. Here we studied the antiviral effect of recombinant chicken (rc)MBL against Infectious Bronchitis Virus (IBV), a highly contagious coronavirus of chicken. rcMBL inhibited in a dose-dependent manner the infection of BHK-21 cells by IBV-Beaudette, as detected by immunofluorescence staining of viral proteins and qPCR. ELISA and negative staining electron microscopy showed that rcMBL bound directly to IBV, resulting in the aggregation of viral particles. Furthermore, we demonstrated that MBL bound specifically to the spike S1 protein of IBV which mediates viral attachment. This subsequently blocked the attachment of S1 to IBV-susceptible cells in chicken tracheal tissues as shown in protein histochemistry. Taken together, rcMBL exhibits antiviral activity against IBV, based on a direct interaction with IBV virions., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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22. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors.
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de Vries RP, Tzarum N, Peng W, Thompson AJ, Ambepitiya Wickramasinghe IN, de la Pena ATT, van Breemen MJ, Bouwman KM, Zhu X, McBride R, Yu W, Sanders RW, Verheije MH, Wilson IA, and Paulson JC
- Subjects
- Animals, Chickens, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Hemagglutinins metabolism, Humans, Influenza A virus metabolism, Influenza in Birds genetics, Influenza in Birds virology, Influenza, Human genetics, Influenza, Human virology, Poultry Diseases genetics, Poultry Diseases virology, Protein Binding, Receptors, Virus genetics, Taiwan, Hemagglutinin Glycoproteins, Influenza Virus genetics, Hemagglutinins genetics, Influenza A virus genetics, Influenza in Birds metabolism, Influenza, Human metabolism, Point Mutation, Poultry Diseases metabolism, Receptors, Virus metabolism
- Abstract
In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2017
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23. A novel cetacean adenovirus in stranded harbour porpoises from the North Sea: detection and molecular characterization.
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van Beurden SJ, IJsseldijk LL, van de Bildt MWG, Begeman L, Wellehan JFX Jr, Waltzek TB, de Vrieze G, Gröne A, Kuiken T, Verheije MH, and Penzes JJ
- Subjects
- Amino Acid Sequence, Animals, Feces virology, Metagenomics, North Sea, Phylogeny, Adenoviridae genetics, Adenoviridae isolation & purification, Adenoviridae Infections veterinary, Animal Diseases virology, Phocoena virology
- Abstract
Harbour porpoises (Phocoena phocoena) are the most prevalent cetaceans in the North Sea. The fecal viral flora of 21 harbour porpoises stranded along the Dutch coastline was analyzed by a metagenomics approach. Sequences of a novel cetacean mastadenovirus, designated harbour porpoise adenovirus 1 (HpAdV-1), were detected. The sequence of a 23-kbp genomic region, spanning the conserved late region, was determined using primer walking. Phylogenetic analysis indicated that HpAdV-1 is most closely related to bottlenose dolphin adenovirus and clusters with Cetartiodactyla adenoviruses. The prevalence of HpAdV-1 was low (2.6%) based on targeted PCR-screening of the intestinal contents of 151 harbour porpoises stranded between 2010 and 2013.
- Published
- 2017
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24. Three mutations switch H7N9 influenza to human-type receptor specificity.
- Author
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de Vries RP, Peng W, Grant OC, Thompson AJ, Zhu X, Bouwman KM, de la Pena ATT, van Breemen MJ, Ambepitiya Wickramasinghe IN, de Haan CAM, Yu W, McBride R, Sanders RW, Woods RJ, Verheije MH, Wilson IA, and Paulson JC
- Subjects
- Amino Acid Sequence, Animals, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Host Specificity, Humans, Influenza A Virus, H3N2 Subtype chemistry, Influenza A Virus, H3N2 Subtype genetics, Influenza A Virus, H3N2 Subtype metabolism, Influenza A Virus, H7N9 Subtype chemistry, Influenza A Virus, H7N9 Subtype metabolism, Influenza in Birds genetics, Influenza in Birds metabolism, Influenza, Human genetics, Influenza, Human metabolism, Molecular Sequence Data, Mutation, Poultry, Poultry Diseases genetics, Poultry Diseases metabolism, Protein Binding, Receptors, Virus genetics, Receptors, Virus metabolism, Sequence Alignment, Hemagglutinin Glycoproteins, Influenza Virus genetics, Influenza A Virus, H7N9 Subtype genetics, Influenza in Birds virology, Influenza, Human virology, Poultry Diseases virology
- Abstract
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.
- Published
- 2017
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25. A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination.
- Author
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van Beurden SJ, Berends AJ, Krämer-Kühl A, Spekreijse D, Chénard G, Philipp HC, Mundt E, Rottier PJM, and Verheije MH
- Subjects
- Animals, Cell Line, Chickens, Gene Targeting methods, Mice, Infectious bronchitis virus genetics, Infectious bronchitis virus growth & development, RNA, Viral genetics, Recombination, Genetic, Reverse Genetics methods
- Abstract
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.
- Published
- 2017
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26. The 150-Loop Restricts the Host Specificity of Human H10N8 Influenza Virus.
- Author
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Tzarum N, de Vries RP, Peng W, Thompson AJ, Bouwman KM, McBride R, Yu W, Zhu X, Verheije MH, Paulson JC, and Wilson IA
- Subjects
- Animals, Binding Sites, Birds, Crystallography, X-Ray, Hemagglutinins genetics, Humans, Influenza A Virus, H10N8 Subtype chemistry, Influenza A Virus, H10N8 Subtype pathogenicity, Influenza in Birds genetics, Influenza, Human genetics, Mutant Proteins chemistry, Mutant Proteins genetics, Pandemics, Protein Conformation, Hemagglutinins chemistry, Influenza A Virus, H10N8 Subtype genetics, Influenza in Birds virology, Influenza, Human virology
- Abstract
Adaptation of influenza A viruses to new hosts are rare events but are the basis for emergence of new influenza pandemics in the human population. Thus, understanding the processes involved in such events is critical for anticipating potential pandemic threats. In 2013, the first case of human infection by an avian H10N8 virus was reported, yet the H10 hemagglutinin (HA) maintains avian receptor specificity. However, the 150-loop of H10 HA, as well as related H7 and H15 subtypes, contains a two-residue insert that can potentially block human receptor binding. Mutation of the 150-loop on the background of Q226L and G228S mutations, which arose in the receptor-binding site of human pandemic H2 and H3 viruses, resulted in acquisition of human-type receptor specificity. Crystal structures of H10 HA mutants with human and avian receptor analogs, receptor-binding studies, and tissue staining experiments illustrate the important role of the 150-loop in H10 receptor specificity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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27. Expression and characterization of recombinant chicken mannose binding lectin.
- Author
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Zhang W, van Eijk M, Guo H, van Dijk A, Bleijerveld OB, Verheije MH, Wang G, Haagsman HP, and Veldhuizen EJ
- Subjects
- Agglutination immunology, Amino Acid Sequence, Animals, Cell Line, Chickens, Cloning, Molecular, Complement Activation immunology, Hemagglutination immunology, Humans, Immunity, Innate, Mannose-Binding Lectin chemistry, Mannose-Binding Lectin isolation & purification, Mannose-Binding Lectin metabolism, Mass Spectrometry, Models, Molecular, Protein Conformation, Sequence Analysis, DNA, Gene Expression, Mannose-Binding Lectin genetics, Recombinant Proteins
- Abstract
Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species., (Copyright © 2016 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
- Published
- 2017
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28. Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity.
- Author
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Guo H, de Vries E, McBride R, Dekkers J, Peng W, Bouwman KM, Nycholat C, Verheije MH, Paulson JC, van Kuppeveld FJ, and de Haan CA
- Subjects
- Alleles, Amino Acid Substitution, Animals, Ducks, Genotype, Hemagglutinin Glycoproteins, Influenza Virus genetics, Humans, Influenza A virus isolation & purification, Influenza A virus pathogenicity, Influenza in Birds epidemiology, Influenza in Birds pathology, Influenza in Birds virology, Mutation, Phylogeny, Reassortant Viruses, Receptors, Virus chemistry, Structure-Activity Relationship, Influenza A virus classification, Influenza A virus physiology, Influenza, Human epidemiology, Influenza, Human virology, Receptors, Virus metabolism, Viral Tropism, Virus Attachment
- Abstract
Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses.
- Published
- 2017
- Full Text
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29. Identification of a novel gammaherpesvirus associated with (muco)cutaneous lesions in harbour porpoises (Phocoena phocoena).
- Author
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van Beurden SJ, IJsseldijk LL, Ordonez SR, Förster C, de Vrieze G, Gröne A, Verheije MH, and Kik M
- Subjects
- Animals, Female, Gammaherpesvirinae classification, Gammaherpesvirinae genetics, Herpesviridae Infections pathology, Herpesviridae Infections virology, Male, Molecular Sequence Data, Phylogeny, Gammaherpesvirinae isolation & purification, Herpesviridae Infections veterinary, Phocoena virology
- Abstract
Herpesviruses infect a wide range of vertebrates, including toothed whales of the order Cetacea. One of the smallest toothed whales is the harbour porpoise (Phocoena phocoena), which is widespread in the coastal waters of the northern hemisphere, including the North Sea. Here, we describe the detection and phylogenetic analysis of a novel gammaherpesvirus associated with mucocutaneous and skin lesions in stranded harbour porpoises along the Dutch coast, tentatively designated phocoenid herpesvirus 1 (PhoHV1). Phylogenetically, PhoHV1 forms a monophyletic clade with all other gammaherpesviruses described in toothed whales (Odontoceti) to date, suggesting a common evolutionary origin.
- Published
- 2015
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30. Novel Receptor Specificity of Avian Gammacoronaviruses That Cause Enteritis.
- Author
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Ambepitiya Wickramasinghe IN, de Vries RP, Weerts EA, van Beurden SJ, Peng W, McBride R, Ducatez M, Guy J, Brown P, Eterradossi N, Gröne A, Paulson JC, and Verheije MH
- Subjects
- Animals, Chickens virology, Coronavirus Infections virology, Enteritis virology, Galactans metabolism, Infectious bronchitis virus metabolism, Intestines virology, Poultry Diseases virology, Protein Binding genetics, Turkeys virology, Coronavirus Infections veterinary, Coronavirus, Turkey metabolism, Receptors, Virus metabolism, Viral Tropism genetics
- Abstract
Unlabelled: Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly-N-acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host., Importance: Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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31. Identification of anti-proliferative kinase inhibitors as potential therapeutic agents to treat canine osteosarcoma.
- Author
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Mauchle U, Selvarajah GT, Mol JA, Kirpensteijn J, and Verheije MH
- Subjects
- Animals, Cell Line, Tumor, Cell Survival, Dogs, Osteosarcoma drug therapy, Antineoplastic Agents pharmacology, Dog Diseases drug therapy, Osteosarcoma veterinary, Protein Kinase Inhibitors pharmacology
- Abstract
Osteosarcoma is the most common primary bone tumour in dogs but various forms of therapy have not significantly improved clinical outcomes. As dysregulation of kinase activity is often present in tumours, kinases represent attractive molecular targets for cancer therapy. The purpose of this study was to identify novel compounds targeting kinases with the potential to induce cell death in a panel of canine osteosarcoma cell lines. The ability of 80 well-characterized kinase inhibitor compounds to inhibit the proliferation of four canine osteosarcoma cell lines was investigated in vitro. For those compounds with activity, the mechanism of action and capability to potentiate the activity of doxorubicin was further evaluated. The screening showed 22 different kinase inhibitors that induced significant anti-proliferative effects across the four canine osteosarcoma cell lines investigated. Four of these compounds (RO 31-8220, 5-iodotubercidin, BAY 11-7082 and an erbstatin analog) showed significant cell growth inhibitory effects across all cell lines in association with variable induction of apoptosis. RO 31-8220 and 5-iodotubercidin showed the highest ability to potentiate the effects of doxorubicin on cell viability. In conclusion, the present study identified several potent kinase inhibitors targeting the PKC, CK1, PKA, ErbB2, mTOR and NF-κB pathways, which may warrant further investigations for the treatment of osteosarcoma in dogs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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32. Host tissue and glycan binding specificities of avian viral attachment proteins using novel avian tissue microarrays.
- Author
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Wickramasinghe IN, de Vries RP, Eggert AM, Wandee N, de Haan CA, Gröne A, and Verheije MH
- Subjects
- Animals, Chickens virology, Columbidae virology, Coronavirus metabolism, Disease Susceptibility, Host Specificity, Respiratory System virology, Tissue Array Analysis, Coronavirus pathogenicity, Galliformes virology, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Host-Pathogen Interactions, Spike Glycoprotein, Coronavirus metabolism, Virus Attachment
- Abstract
The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens.
- Published
- 2015
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33. ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.
- Author
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Burkard C, Verheije MH, Haagmans BL, van Kuppeveld FJ, Rottier PJ, Bosch BJ, and de Haan CA
- Subjects
- Animals, Bufanolides pharmacology, Cell Line, Chlorocebus aethiops, Coronavirus, Feline physiology, Gene Silencing, Humans, Hydrogen-Ion Concentration, Mice, Murine hepatitis virus physiology, Ouabain pharmacology, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Vero Cells, Cardiac Glycosides pharmacology, Coronaviridae Infections physiopathology, Signal Transduction physiology, Sodium-Potassium-Exchanging ATPase metabolism, Virus Attachment drug effects, Virus Internalization drug effects
- Abstract
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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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34. Protein histochemistry using coronaviral spike proteins: studying binding profiles and sialic acid requirements for attachment to tissues.
- Author
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Wickramasinghe IN and Verheije MH
- Subjects
- Animals, Chickens, Histocytochemistry, Membrane Glycoproteins chemistry, Protein Binding, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Spike Glycoprotein, Coronavirus biosynthesis, Spike Glycoprotein, Coronavirus isolation & purification, Tissue Fixation, Infectious bronchitis virus physiology, Sialic Acids chemistry, Spike Glycoprotein, Coronavirus chemistry, Virus Attachment
- Abstract
Protein histochemistry is a tissue-based technique that enables the analysis of viral attachment patterns as well as the identification of specific viral and host determinants involved in the first step in the infection of a host cell by a virus. Applying recombinantly expressed spike proteins of infectious bronchitis virus onto formalin-fixed tissues allows us to profile the binding characteristics of these viral attachment proteins to tissues of various avian species. In particular, sialic acid-mediated tissue binding of spike proteins can be analyzed by pretreating tissues with various neuraminidases or by blocking the binding of the viral proteins with specific lectins. Our assay is particularly convenient to elucidate critical virus-host interactions for viruses for which infection models are limited.
- Published
- 2015
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35. Complete genome sequence of a common midwife toad virus-like ranavirus associated with mass mortalities in wild amphibians in the Netherlands.
- Author
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van Beurden SJ, Hughes J, Saucedo B, Rijks J, Kik M, Haenen OL, Engelsma MY, Gröne A, Verheije MH, and Wilkie G
- Abstract
A ranavirus associated with mass mortalities in wild water frogs (Pelophylax spp.) and other amphibians in the Netherlands since 2010 was isolated, and its complete genome sequence was determined. The virus has a genome of 107,772 bp and shows 96.5% sequence identity with the common midwife toad virus from Spain., (Copyright © 2014 van Beurden et al.)
- Published
- 2014
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36. The avian coronavirus spike protein.
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Wickramasinghe IN, van Beurden SJ, Weerts EA, and Verheije MH
- Subjects
- Animals, Chickens, Infectious bronchitis virus physiology, Spike Glycoprotein, Coronavirus metabolism, Viral Tropism, Virus Attachment, Virus Internalization
- Abstract
Avian coronaviruses of the genus Gammacoronavirus are represented by infectious bronchitis virus (IBV), the coronavirus of chicken. IBV causes a highly contagious disease affecting the respiratory tract and, depending on the strain, other tissues including the reproductive and urogenital tract. The control of IBV in the field is hampered by the many different strains circulating worldwide and the limited protection across strains due to serotype diversity. This diversity is believed to be due to the amino acid variation in the S1 domain of the major viral attachment protein spike. In the last years, much effort has been undertaken to address the role of the avian coronavirus spike protein in the various steps of the virus' live cycle. Various models have successfully been developed to elucidate the contribution of the spike in binding of the virus to cells, entry of cell culture cells and organ explants, and the in vivo tropism and pathogenesis. This review will give an overview of the literature on avian coronavirus spike proteins with particular focus on our recent studies on binding of recombinant soluble spike protein to chicken tissues. With this, we aim to summarize the current understanding on the avian coronavirus spike's contribution to host and tissue predilections, pathogenesis, as well as its role in therapeutic and protective interventions., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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37. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
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Burkard C, Verheije MH, Wicht O, van Kasteren SI, van Kuppeveld FJ, Haagmans BL, Pelkmans L, Rottier PJ, Bosch BJ, and de Haan CA
- Subjects
- Animals, Cats, Chlorocebus aethiops, Dogs, Endosomes virology, HeLa Cells, Humans, Lysosomes virology, Madin Darby Canine Kidney Cells, Membrane Fusion, Mice, Murine hepatitis virus genetics, Spike Glycoprotein, Coronavirus genetics, Vero Cells, Endosomes metabolism, Lysosomes metabolism, Murine hepatitis virus metabolism, Proteolysis, Spike Glycoprotein, Coronavirus metabolism, Virus Internalization
- Abstract
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.
- Published
- 2014
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38. Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus.
- Author
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Promkuntod N, van Eijndhoven RE, de Vrieze G, Gröne A, and Verheije MH
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Chickens, Coronavirus Infections metabolism, Coronavirus Infections virology, Infectious bronchitis virus chemistry, Infectious bronchitis virus genetics, Molecular Sequence Data, Poultry Diseases virology, Protein Binding, Spike Glycoprotein, Coronavirus genetics, Coronavirus Infections veterinary, Infectious bronchitis virus metabolism, Poultry Diseases metabolism, Receptors, Virus metabolism, Sialic Acids metabolism, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus metabolism
- Abstract
The infection of the avian coronavirus infectious bronchitis virus (IBV) is initiated by the binding of the spike glycoprotein S to sialic acids on the chicken host cell. In this study we identified the receptor-binding domain (RBD) of the spike of the prototype IBV strain M41. By analyzing the ability of recombinantly expressed chimeric and truncated spike proteins to bind to chicken tissues, we demonstrate that the N-terminal 253 amino acids of the spike are both required and sufficient for binding to chicken respiratory tract in an α-2,3-sialic acid-dependent manner. Critical amino acids for attachment of M41 spike are present within the N-terminal residues 19-69, which overlap with a hypervariable region in the S1 gene. Our results may help to understand the differences between IBV S1 genotypes and the ultimate pathogenesis of IBV in chickens., (© 2013 Elsevier Inc. All rights reserved.)
- Published
- 2014
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39. Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus.
- Author
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Promkuntod N, Wickramasinghe IN, de Vrieze G, Gröne A, and Verheije MH
- Subjects
- Animals, Cell Line, Chick Embryo, Coronavirus Infections metabolism, Humans, Infectious bronchitis virus chemistry, Infectious bronchitis virus genetics, Protein Structure, Tertiary, Receptors, Virus metabolism, Spike Glycoprotein, Coronavirus genetics, Coronavirus Infections virology, Host Specificity, Infectious bronchitis virus physiology, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus metabolism, Virus Attachment
- Abstract
The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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40. Adaptation of novel H7N9 influenza A virus to human receptors.
- Author
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Dortmans JC, Dekkers J, Wickramasinghe IN, Verheije MH, Rottier PJ, van Kuppeveld FJ, de Vries E, and de Haan CA
- Subjects
- Epithelial Cells metabolism, Fetuins chemistry, HEK293 Cells, Hemagglutinins genetics, Humans, Lung metabolism, Lung pathology, Lung virology, Mutation, Neuraminidase genetics, Polysaccharides metabolism, Protein Binding, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Trachea metabolism, Trachea pathology, Trachea virology, Fetuins metabolism, Hemagglutinins metabolism, Influenza A Virus, H7N9 Subtype metabolism, Neuraminidase metabolism
- Abstract
The emergence of the novel H7N9 influenza A virus (IAV) has caused global concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of this virus using a recombinant protein approach in combination with fetuin-binding, glycan array and human tissue-binding assays demonstrates increased binding of H7 to both α2-6 and α2-8 sialosides as well as reduced binding to α2-3-linked SIAs compared to a closely related avian H7N9 virus from 2008. These differences could be attributed to substitutions Q226L and G186V. Analysis of the enzymatic activity of the neuraminidase N9 protein indicated a reduced sialidase activity, consistent with the reduced binding of H7 to α2-3 sialosides. However, the novel H7N9 virus still preferred binding to α2-3- over α2-6-linked SIAs and was not able to efficiently bind to epithelial cells of human trachea in contrast to seasonal IAV, consistent with its limited human-to-human transmission.
- Published
- 2013
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41. Expression of epidermal growth factor receptor in canine osteosarcoma: association with clinicopathological parameters and prognosis.
- Author
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Selvarajah GT, Verheije MH, Kik M, Slob A, Rottier PJ, Mol JA, and Kirpensteijn J
- Subjects
- Animals, Blotting, Western veterinary, Cell Line, Tumor, Dogs, Female, Imidazoles, Immunohistochemistry veterinary, Male, Osteosarcoma metabolism, Prognosis, RNA, Messenger metabolism, Real-Time Polymerase Chain Reaction veterinary, Tissue Array Analysis veterinary, Bone Neoplasms metabolism, Dog Diseases metabolism, ErbB Receptors metabolism, Gene Expression Regulation, Neoplastic, Osteosarcoma veterinary
- Abstract
Expression of epidermal growth factor receptor (EGFR) is associated with aggressive growth and metastasis of a range of tumours, including osteosarcomas (OS), although some studies have reported no relevance to clinicopathological events or prognosis. The present study evaluated EGFR mRNA and protein expression in a panel of OS cell lines, normal bones, frozen primary OS and tissue microarrays. EGFR expression was significantly elevated in primary OS compared to normal bones and in metastases of OS to the lungs in comparison with extrapulmonary sites. However, there were no clinical or pathological associations with mRNA expression levels in frozen tumours. Tissue microarray analysis demonstrated that a subset of canine OS with high EGFR expression was associated with significantly shorter survival times and disease-free intervals. Cytoplasmic expression of EGFR was present in 75% of metastases and was similar to expression in primary tumours. EGFR expression alone is not a reliable predictor of outcome and other markers are necessary for further prognostic stratification of dogs with OS. However, these findings suggest that a subset of dogs may benefit from anti-EGFR adjuvant therapies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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42. Single-cell analysis of population context advances RNAi screening at multiple levels.
- Author
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Snijder B, Sacher R, Rämö P, Liberali P, Mench K, Wolfrum N, Burleigh L, Scott CC, Verheije MH, Mercer J, Moese S, Heger T, Theusner K, Jurgeit A, Lamparter D, Balistreri G, Schelhaas M, De Haan CA, Marjomäki V, Hyypiä T, Rottier PJ, Sodeik B, Marsh M, Gruenberg J, Amara A, Greber U, Helenius A, and Pelkmans L
- Subjects
- Bayes Theorem, Cellular Microenvironment, Computer Simulation, Genomics methods, HeLa Cells, Humans, Image Processing, Computer-Assisted methods, Models, Biological, RNA, Small Interfering, RNA, Viral isolation & purification, Reproducibility of Results, Systems Biology methods, Viral Proteins genetics, Viral Proteins isolation & purification, Virus Diseases metabolism, Viruses isolation & purification, Viruses pathogenicity, RNA Interference, Single-Cell Analysis methods, Virus Diseases genetics
- Abstract
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.
- Published
- 2012
- Full Text
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43. Retargeting of viruses to generate oncolytic agents.
- Author
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Verheije MH and Rottier PJ
- Abstract
Oncolytic virus therapy is based on the ability of viruses to effectively infect and kill tumor cells without destroying the normal tissues. While some viruses seem to have a natural preference for tumor cells, most viruses require the modification of their tropism to specifically enter and replicate in such cells. This review aims to describe the transductional targeting strategies currently employed to specifically redirect viruses towards surface receptors on tumor cells. Three major strategies can be distinguished; they involve (i) the incorporation of new targeting specificity into a viral surface protein, (ii) the incorporation of a scaffold into a viral surface protein to allow the attachment of targeting moieties, and (iii) the use of bispecific adapters to mediate targeting of a virus to a specified moiety on a tumor cell. Of each strategy key features, advantages and limitations are discussed and examples are given. Because of their potential to cause sustained, multiround infection-a desirable characteristic for eradicating tumors-particular attention is given to viruses engineered to become self-targeted by the genomic expression of a bispecific adapter protein.
- Published
- 2012
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44. Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity.
- Author
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Wickramasinghe IN, de Vries RP, Gröne A, de Haan CA, and Verheije MH
- Subjects
- Animals, Chickens, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Influenza A Virus, H5N1 Subtype pathogenicity, Protein Binding, Respiratory Mucosa virology, Spike Glycoprotein, Coronavirus, Coronavirus pathogenicity, Host-Pathogen Interactions, Membrane Glycoproteins metabolism, Receptors, Virus metabolism, Viral Envelope Proteins metabolism, Viral Tropism
- Abstract
The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.
- Published
- 2011
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45. The coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes.
- Author
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Verheije MH, Hagemeijer MC, Ulasli M, Reggiori F, Rottier PJ, Masters PS, and de Haan CA
- Subjects
- Animals, Coronavirus Nucleocapsid Proteins, Mice, Protein Binding, Protein Structure, Tertiary, Protein Transport, Nucleocapsid Proteins metabolism, Transcription, Genetic, Virus Replication
- Abstract
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.
- Published
- 2010
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46. The ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle.
- Author
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Raaben M, Posthuma CC, Verheije MH, te Lintelo EG, Kikkert M, Drijfhout JW, Snijder EJ, Rottier PJ, and de Haan CA
- Subjects
- Animals, Boronic Acids pharmacology, Bortezomib, Cats, Cell Line, Chlorocebus aethiops, Leupeptins pharmacology, Mice, Oligopeptides pharmacology, Protease Inhibitors pharmacology, Proteasome Inhibitors, Pyrazines pharmacology, Virus Internalization, Virus Release, Virus Replication, Coronavirus Infections virology, Coronavirus, Feline pathogenicity, Murine hepatitis virus pathogenicity, Proteasome Endopeptidase Complex metabolism, Severe acute respiratory syndrome-related coronavirus pathogenicity, Ubiquitin metabolism
- Abstract
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.
- Published
- 2010
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47. Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication.
- Author
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Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, and Molinari M
- Subjects
- Animals, Cells, Cultured, Cytoplasmic Vesicles chemistry, Membrane Proteins metabolism, Microscopy, Confocal, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Cytoplasmic Vesicles virology, Endoplasmic Reticulum virology, Microtubule-Associated Proteins analysis, Murine hepatitis virus physiology, Ubiquitins analysis, Virus Replication
- Abstract
Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question. We show that MHV co-opts the host cell machinery for COPII-independent vesicular ER export of a short-living regulator of ER-associated degradation (ERAD), EDEM1, to derive cellular membranes for replication. MHV infection causes accumulation of EDEM1 and OS-9, another short-living ER chaperone, in the DMVs. DMVs are coated with the nonlipidated LC3/Atg8 autophagy marker. Downregulation of LC3, but not inactivation of host cell autophagy, protects cells from CoV infection. Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes an autophagy-independent role for nonlipidated LC3-I., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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48. Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus.
- Author
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Ulasli M, Verheije MH, de Haan CA, and Reggiori F
- Subjects
- Animals, Cell Line, Humans, Microscopy, Immunoelectron, Models, Biological, Murine hepatitis virus ultrastructure, Time Factors, Cell Membrane metabolism, Cell Membrane ultrastructure, Murine hepatitis virus physiology, Virus Assembly, Virus Release
- Abstract
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.
- Published
- 2010
- Full Text
- View/download PDF
49. Dynamics of coronavirus replication-transcription complexes.
- Author
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Hagemeijer MC, Verheije MH, Ulasli M, Shaltiël IA, de Vries LA, Reggiori F, Rottier PJ, and de Haan CA
- Subjects
- Animals, Base Sequence, Cats, Cell Line, Coronavirus Infections metabolism, Coronavirus Infections virology, Cytoplasmic Vesicles metabolism, Cytoplasmic Vesicles ultrastructure, Cytoplasmic Vesicles virology, DNA Primers genetics, DNA, Viral genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, HeLa Cells, Host-Pathogen Interactions, Humans, Macromolecular Substances, Mice, Microscopy, Electron, Transmission, Murine hepatitis virus pathogenicity, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transcription, Genetic, Viral Nonstructural Proteins chemistry, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins physiology, Virus Replication genetics, Virus Replication physiology, Murine hepatitis virus genetics, Murine hepatitis virus physiology
- Abstract
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.
- Published
- 2010
- Full Text
- View/download PDF
50. Coronavirus genetically redirected to the epidermal growth factor receptor exhibits effective antitumor activity against a malignant glioblastoma.
- Author
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Verheije MH, Lamfers ML, Würdinger T, Grinwis GC, Gerritsen WR, van Beusechem VW, and Rottier PJ
- Subjects
- Animals, Brain pathology, Cell Line, Tumor, ErbB Receptors metabolism, Female, Genetic Vectors genetics, Genetic Vectors physiology, Glioblastoma genetics, Glioblastoma pathology, Humans, Mice, Mice, Nude, Murine hepatitis virus physiology, Oncolytic Viruses physiology, ErbB Receptors genetics, Glioblastoma therapy, Murine hepatitis virus genetics, Oncolytic Virotherapy adverse effects, Oncolytic Viruses genetics
- Abstract
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.
- Published
- 2009
- Full Text
- View/download PDF
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