488 research outputs found
Tissue interactions of avian viral attachment proteins
No full textViruses can infect a wide range of hosts; varying from bacteria and plants to animals and humans. While many viral infections may pass unnoticed, some are of major importance due to their implications on health and welfare of plants, animals and/or humans. In particular, viruses that can infect avian hosts have been studied intensively due the occurrence of the pandemics of highly pathogenic influenza A virus infection or “bird flu’’. Viral infections in domesticated birds can result in huge economic losses in the poultry industry. In wild birds, infections may be asymptomatic, but these viruses may remain in these birds and thus they may act as reservoirs. Particularly wild birds may be ideal reservoirs because of their ability fly over long distance and hence they can effectively spread viruses along the migration routes. Coronaviruses in birds are considered economically important pathogens worldwide. They are classified in the genus Gammacoronaviridae, family Coronaviridae. Infectious bronchitis virus (IBV) is the prototype of this group and can cause severe respiratory disease in chickens. In addition, coronaviruses have been reported to induce respiratory disease in other avian species, including pheasants, pigeons and peafowls. Interestingly, apart from infecting the respiratory system IBV is capable of replicating in other organ systems within a specific host, indicating that IBV has an extended virus tropism. On the other hand, some other gammacoronaviruses, for an example turkey, guineafowl and quail coronaviruses have been observed to possess a more restricted tropism by limiting the infection to the gastrointestinal system. Viral and host factors that enable the attachment of a virus to the target host cell are crucial in defining viral tropism. This thesis was aimed to identify these factors for avian coronaviruses in view of understanding the mechanism behind viral tropism. To this end several novel methods were established to study the first step in the avian coronavirus infection cycle. While Chapter 1 gives a introduction to the aims of this thesis chapter 2 provides an overview on the function of the gammacoronavirus spike proteins and its role in defining the tropism and susceptibility. In addition it summarizes the reported data on tissue interactions of the coronaviral spike. Chapter 3 describes the use of recombinant spike proteins as a tool to understand the tropism of gammacoronaviruses using infectious bronchitis virus, the prototype of the group, as an example. The novel avian tissues microarray and its benefits in view of defining organ and susceptibility to a particular coronavirus are demonstrated in Chapter 4. Chapter 5 reveals a novel receptor for enterotropic gammacoronaviruses and receptor specificity towards defining their restricted enterotropism. While differences in the tissue interactions of mutated influenza virus attachment proteins, HAs, have been shown in Chapter 6 the detailed protocol of evaluating tissue interactions with protein histochemistry is described in Chapter 7. Overall we provide evidence for viral and host determinants that mediate avian coronavirus tropism. In the final discussion (Chapter 8) remaining gaps in defining the tropism and pathogenicity of avian coronaviruses will be discussed
Onderzoeksresultaten gestrande witsnuitdolfijn te Wijk aan Zee: Pathologie, dieet en plastic
No full textOp 7 december 2017 werd in de ochtend een gestrande dode witsnuitdolfijn (Lagenorhynchus albirostris) te Wijk aan Zee ontdekt. De Minister van het Ministerie van Landbouw, Natuur en Voedselkwaliteit (LNV) is verantwoordelijk voor de invulling van internationale verplichtingen en afspraken omtrent de biodiversiteit en de bescherming van in het wild levende bedreigde diersoorten. Vanuit die verantwoordelijkheid is er door het Ministerie van LNV een onderzoeksopdracht uitbesteed naar de strandingsoorzaak en herkomst van deze dolfijn. Strandingsonderzoek vindt in Nederland sinds 2008 plaats bij het Departement Pathobiologie van de Faculteit Diergeneeskunde van de Universiteit Utrecht met als hoofddoel het vaststellen van doodsoorzaken en hierbinnen het onderscheidt tussen natuurlijke en antropogene oorzaken. Daarnaast worden weefsels verzameld waarmee aanvullende onderzoeken kunnen worden uitgevoerd. Hieronder valt onder andere onderzoek naar dieet en plastics. Deze onderzoeken worden respectievelijk uitgevoerd door Wageningen Marine Research (WMR) en Bureau Waardenburg (BuWa). De gestrande witsnuitdolfijn was een volwassen mannelijk dier van 10 jaar oud. De witsnuitdolfijn was waarschijnlijk levend gestrand te Wijk aan Zee. Het dier was in een zeer goede voedingstoestand ten tijden van stranding. De laatste maaltijd van deze dolfijn bestond voornamelijk uit twee prooisoorten: wijting en zandspiering. Hoewel de tweede wellicht secundaire prooi was, vormde de wijting prooimassa duidelijk de belangrijkste prooisoort voor deze witsnuitdolfijn. Het dieet had een gereconstrueerde massa van ruim 19 kg vis. Dit was 6,84% van het gewicht van de dolfijn zelf, terwijl een dolfijn van deze omvang gemiddeld per dag ongeveer de helft van deze hoeveelheid moet eten. Er werd geen plastic in de maag en darm aangetroffen. Op basis van het postmortaal onderzoek kon worden vastgesteld dat de deze witsnuitdolfijn hoogstwaarschijnlijk acuut is overleden door een bacteriemie en sepsis (bloedvergiftiging) door bacteriële infectie met Clostridium sordelli. Dit resulteerde in hersen schade, shock, en uiteindelijk de dood
Postmortaal onderzoek van bruinvissen (Phocoena phocoena) uit Nederlandse wateren, 2017 : biologische gegevens, gezondheidsstatus en doodsoorzaken
No full textThis annual report present the results of post-mortem examinations of porpoises (Phocoena phocoena) in 2017. One of the main objectives of the research is to quantify human-induced causes of death. In 2017, 55 dead harbour porpoises were examined: 25 males and 30 females, including 22 adults, 26 juveniles and 7 neonates. Most of the harbour porpoises examined died as a result of infectious diseases (36%), bycatch (20%) or grey seal attacks (18%). The proportion of animals dying of infectious diseases was higher in 2017 than in previous years
Stranding van een gewone spitssnuitdolfijn (Mesoplodon bidens) in de Oosterschelde: Resultaten van het strandingsonderzoek, met speciale aandacht voor onderzoek naar gehoorschade
No full textOp 31 augustus 2017 strandde er in de Oosterschelde een jonge, levende gewone spitssnuitdolfijn (Mesoplodon bidens) welke later ter plaatsen overleed. Dit dier werd verzameld voor onderzoek naar de doodsoorzaak en in het bijzonder de aanwezigheid van gehoorschade als oorzaak van de stranding, omdat spitssnuitdolfijn strandingen in Nederland zeldzaam zijn en er weinig bekend is over deze walvisachtigen. Er werden geen indicaties gevonden dat dit dier een infectieuze ziekte of gehoorschade had. Het dier had recentelijk niet gegeten en was ondervoed. Op de flank werden parallelle krassen gevonden, waarbij de afstand tussen de krassen overeenkwamen met de afstand tussen de tanden van tuimelaar dolfijnen. Over waarom de spitssnuitdolfijn de Noordzee is ingezwommen kan alleen worden gespeculeerd. Een scenario kan zijn dat de nog jonge en onervaren spitssnuitdolfijn de zuidelijke Noordzee is ingevlucht of in gedwaald als gevolg van de interactie met één of meerdere tuimelaars, waar het vervolgens door voedselgebrek en verzwakking strandde en overleed. Strandingen van spitssnuitdolfijnen op de Nederlandse kust zijn nog altijd uniek met twaalf gerapporteerde strandingen op www.walvisstrandingen.nl. Wel lijkt er een plotselinge toename, met vijf strandingen van deze soort in alleen al de afgelopen twee jaar (2016 en 2017). Strandingsonderzoek van deze en andere walvisachtigen zullen noodzakelijk zijn om eventuele trends in aantallen en oorzaken te achterhalen. Sterfte door akoestische vervuiling van het marine ecosysteem kan alleen worden uitgevoerd wanneer snel en adequaat wordt gehandeld tijdens strandingen. Onderzoek naar natuurlijke en antropogene doodsoorzaken, inclusief akoestisch trauma, kan zonder projectfinanciering bij toekomstige strandingen niet worden gegarandeerd
The ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle
No full textThe ubiquitin-proteasome system (UPS) is a key player in regulating the intracellular sorting and degradation of proteins. In this study we investigated the role of the UPS in different steps of the coronavirus (CoV) infection cycle. Inhibition of the proteasome by different chemical compounds (i.e., MG132, epoxomicin, and Velcade) appeared to not only impair entry but also RNA synthesis and subsequent protein expression of different CoVs (i.e., mouse hepatitis virus [MHV], feline infectious peritonitis virus, and severe acute respiratory syndrome CoV). MHV assembly and release were, however, not appreciably affected by these compounds. The inhibitory effect on CoV protein expression did not appear to result from a general inhibition of translation due to induction of a cellular stress response by the inhibitors. Stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) generally results in impaired initiation of protein synthesis, but the sensitivity of MHV infection to proteasome inhibitors was unchanged in cells lacking a phosphorylatable eIF2alpha. MHV infection was affected not only by inhibition of the proteasome but also by interfering with protein ubiquitination. Viral protein expression was reduced in cells expressing a temperature-sensitive ubiquitin-activating enzyme E1 at the restrictive temperature, as well as in cells in which ubiquitin was depleted by using small interfering RNAs. Under these conditions, the susceptibility of the cells to virus infection was, however, not affected, excluding an important role of ubiquitination in virus entry. Our observations reveal an important role of the UPS in multiple steps of the CoV infection cycle and identify the UPS as a potential drug target to modulate the impact of CoV infection
Dynamics of coronavirus replication-transcription complexes
No full textCoronaviruses 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
Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation.
No full textContains fulltext :
70648.pdf (Publisher’s version ) (Open Access)Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected
Advanced multicentric lymphoma in a Belgian Draft Horse mare
No full textA 15-year old draft horse mare was presented to the University Clinic for evaluation of lethargy, anorexia, ptyalism, weight loss and ventral oedema. Clinical examination and rectal palpation revealed generalised lymphadenopathy and numerous firm subcutaneous and abdominal masses of various sizes. Transcutaneous ultrasonography revealed significant bilateral pleural fluid accumulation and a single hypoechoic structure in the abdomen lateral to the liver. Blood analysis showed several abnormalities including a marked leucocytosis with an increased number of segmented neutrophils, atypical lymphocytes and monocytosis, suggestive of leukaemic lymphoma. A significant hyperproteinaemia with a hypoalbuminemia and a monoclonal gammopathy was identified. At necropsy myriad masses presented through the whole body. Histology confirmed the suspicion of lymphoma, which was classified as a T-cell rich B-cell multicentric lymphoma. This article describes the clinical and pathologic findings of this case of leukaemic lymphoma
Infectious Bronchitis Virus QX Field Progenitor Loses Nephropathogenicity After Attenuation into a Live Viral Vaccine
No full textIntroduction: Infectious bronchitis (IB) is a gammacoronavirus-induced respiratory disease in chickens. IB virus strain QX is additionally known for its detrimental nephropathogenicity. In this study we compared the phenotypic differences between an IB QX vaccine virus and its field progenitor with focus on viral replication and dissemination through the host. Materials and Methods: Specific pathogen free chickens were inoc-ulated intratracheally with either 10 3 EID 50 vaccine strain Nobilis IB Primo QX (MSD Animal Health) or field strain IBV-D388 and killed at 1 day intervals over an 8-day period. Histopathology was studied over time in the trachea, kidney, cloaca and gastrointestinal tract. Immunohistochemistry was used to demonstrate viral protein expression in these tissues. qPCR was performed on tracheal and cloacal swabs and kidney to reveal viral RNA loads. Results: IB Primo QX and IBV-D388 induced comparable tracheal le-sions, characterized by epithelial cell death and desquamation and heter-ophilic and lymphohistiocytic infiltration. Changes were earlier and more severe with IBV-D388, while tracheal viral RNA loads were comparable over time for both viruses. In contrast, degenerative and inflammatory le-sions and viral protein and RNA were only found after IBV-D388 infection in the kidneys. This renal viral presence was preceded by viral RNA detection in the cloaca and viral protein expression in the gastrointestinal tract, albeit without associated lesions in these tissues. Discussion: Dissemination to the kidneys by IBV-D388 might result from cloacal ascending infection. In contrast to its field progenitor, IB Primo QX was unable to cause renal infection, which likely considerably contributes to attenuation of its phenotype
Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus
No full textThe 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
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