Passive inhalation of dry powder influenza vaccine formulations completely protects chickens against H5N1 lethal viral challenge
Tomar, Jasmine ; Biel, Carin ; Haan, Cornelis A.M. de; Rottier, Peter J.M. ; Petrovsky, Nikolai ; Frijlink, Henderik W. ; Huckriede, Anke ; Hinrichs, Wouter L.J. ; Peeters, Ben - \ 2018
European Journal of Pharmaceutics and Biopharmaceutics 133 (2018). - ISSN 0939-6411 - p. 85 - 95.
Adjuvants - Challenge - Influenza - Inhalation - Passive - Powders - Protection - Pulmonary
Bird to human transmission of high pathogenicity avian influenza virus (HPAIV) poses a significant risk of triggering a flu pandemic in the human population. Therefore, vaccination of susceptible poultry during an HPAIV outbreak might be the best remedy to prevent such transmissions. To this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. Our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. The aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. Two adjuvants, namely, Bacterium-like Particles (BLP) and Advax, were spray freeze dried with influenza vaccine into dry powder formulations. Delivery of dry formulations directly at the syrinx revealed that BLP and Advax had the potential to boost either systemic or mucosal immune responses or both. Upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, BLP and Advax/BLP adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. Remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the Advax group. Overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings.
Genetic versus antigenic differences among highly pathogenic H5N1 avian influenza A viruses : Consequences for vaccine strain selection
Peeters, Ben ; Reemers, Sylvia ; Dortmans, Jos ; Vries, Erik de; Jong, Mart de; Zande, Saskia van de; Rottier, Peter J.M. ; Haan, Cornelis A.M. de - \ 2017
Virology 503 (2017). - ISSN 0042-6822 - p. 83 - 93.
Antigenic distance - Genetic variation - H5N1 - Hemagglutinin - Influenza A virus - Vaccine performance
Highly pathogenic H5N1 avian influenza A viruses display a remarkable genetic and antigenic diversity. We examined to what extent genetic distances between several H5N1 viruses from different clades correlate with antigenic differences and vaccine performance. H5-specific antisera were generated, and cross-reactivity and antigenic distances between 12 different viruses were determined. In general, antigenic distances increased proportional to genetic distances although notable exceptions were observed. Antigenic distances correlated better with genetic variation in 27 selected, antigenically-relevant H5 residues, than in the complete HA1 domain. Variation in these selected residues could accurately predict the antigenic distances for a novel H5N8 virus. Protection provided by vaccines against heterologous H5N1 challenge viruses indicated that cross-protection also correlates better with genetic variation in the selected antigenically-relevant residues than in complete HA1. When time is limited, variation at these selected residues may be used to accurately predict antigenic distance and vaccine performance.
The postfusion 3D-structure of the Spodoptera exigua multiple nucleopolyhedrovirus envelope fusion protein F
Wang, Q. ; Vasiliauskaite, I. ; Bosch, B.J. ; Krey, T. ; Rottier, P.J. ; Vlak, J.M. ; Rey, F. - \ 2016
- p. 91 - 91.
Budded baculovirus particle structure revisited
Wang, Qiushi ; Bosch, Berend Jan ; Vlak, J.M. ; Oers, M.M. van; Rottier, P.J. ; Lent, J.W.M. van - \ 2016
Journal of Invertebrate Pathology 134 (2016). - ISSN 0022-2011 - p. 15 - 22.
Baculovirus - Budded virus - Cryo-EM - Spike structure - Ultrastructure
Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈6-7 nm, which is significantly thicker than a usual biological membrane (
Structural and functional analysis of the baculovirus envelope fusion protein F
Wang, Q. - \ 2015
Wageningen University. Promotor(en): Just Vlak; J.P.M. Rottier; Monique van Oers, co-promotor(en): B.J. Bosch. - Wageningen : Wageningen University - ISBN 9789462575639 - 242
baculovirus - envelopeiwitten - fusie-eiwitten - virusmorfologie - virale structurele eiwitten - baculovirus - envelope proteins - fusion proteins - viral morphology - viral structural proteins
Baculoviruses are enveloped, double-stranded DNA viruses that are pathogenic predominantly to insects. Insects, such as caterpillars, become infected by oral feeding on proteinaceous occlusion bodies (OBs) containing the occlusion-derived virus (ODV) form of the virus. The ODV infects epithelial cells in the midgut of the host insect. In infected cells, a budded virus (BV) form of the virus is first produced, which is then responsible for the systemic infection of the host. The BV entry into target insect cells is mediated by envelope fusion proteins that are present on the surface of BVs upon low pH activation. In this thesis the structure and activation mechanism of the baculovirus envelope fusion protein F were studied. The low-pH triggered, postfusion structure of soluble baculovirus F protein revealed remarkable resemblance with mammalian RNA virus respiratory syncytial virus (RSV). Furthermore, an F homolog of mosquito Anopheles darlingi (Ad-F) was shown to share structural features and amino acid homology with the baculovirus F protein and appears to have fusion function. It was further found that histidines may involved in low pH sensing to trigger conformational changes of baculovirus F protein, yet the highly conserved histidine residues are not pH sensors but some of them may participate in fusion pore expansion. Moreover, the baculovirus BV morphology was reanalyzed using cryo-electron microscopy to main the native virus structure. The study emphasized on several new features of BV structure including ovoid-shaped virion with polar distributed envelope (fusion) proteins and the ‘empty’ lateral space between the nucleocapsid and envelope of the BVs. This thesis provides novel structural details of the low-pH triggered baculovirus F protein in relation to its fusion mechanism. The gained structural knowledge sheds new light on the evolutionary relationship of the baculovirus F proteins with homologs found in mammalian viruses and insects.
Pulmonary immunization of chickens using non-adjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic H5N1 avian influenza virus.
Peeters, B.P.H. ; Tonnis, W.F. ; Murugappan, S. ; Rottier, P. ; Koch, G. ; Frijlink, H.W. ; Huckriede, A. ; Hinrichs, W.L.J. - \ 2014
Vaccine 32 (2014)48. - ISSN 0264-410X - p. 6445 - 6450.
a h5n1 - aerosol vaccination - immune-responses - mass vaccination - powder - transmission - delivery - poultry - inulin - birds
Highly pathogenic avian influenza (HPAI) H5N1 virus is a major threat to public health as well as to the global poultry industry. Most fatal human infections are caused by contact with infected poultry. Therefore, preventing the virus from entering the poultry population is a priority. This is, however, problematic in emergency situations, e.g. during outbreaks in poultry, as there are currently no mass application methods to effectively vaccinate large numbers of birds within a short period of time. To evaluate the suitability of needle-free pulmonary immunization for mass vaccination of poultry against HPAI H5N1, we performed a proof-of-concept study in which we investigated whether non-adjuvanted spray-freeze-dried (SFD) whole inactivated virus (WIV) can be used as a dry powder aerosol vaccine to immunize chickens. Our results show that chickens that received SFD-WIV vaccine as aerosolized powder directly at the syrinx (the site of the tracheal bifurcation), mounted a protective antibody response after two vaccinations and survived a lethal challenge with HPAI H5N1. Furthermore, both the number of animals that shed challenge virus, as well as the level of virus shedding, were significantly reduced. Based on antibody levels and reduction of virus shedding, pulmonary vaccination with non-adjuvanted vaccine was at least as efficient as intratracheal vaccination using live virus. Animals that received aerosolized SFD-WIV vaccine by temporary passive inhalation showed partial protection (22% survival) and a delay in time-to-death, thereby demonstrating the feasibility of the method, but indicating that the efficiency of vaccination by passive inhalation needs further improvement. Altogether our results provide a proof-of-concept that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens from lethal HPAI challenge. If the efficacy of pulmonary vaccination by passive inhalation can be improved, this method might be suitable for mass application.
|Acid activated fusion mechanism of baculovirus mediated by F protein
Wang, Q. ; Rottier, P.J.M. ; Bosch, B.J. ; Oers, M.M. van; Vlak, J.M. ; Vasliauskaite, I. ; Rey, F. - \ 2013
In: Abstract book of the 32nd Annual Meeting of the American Society for Virology, Pennsylvania, USA, July 20-24, 2013. - - p. 164 - 164.
Genome-wide gene expression analysis of anguillid herpesvirus 1
Beurden, S.J. van; Peeters, B.P.H. ; Rottier, P.J.M. ; Davison, A.A. ; Engelsma, M.Y. - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164 - 11 p.
channel catfish virus - time rt-pcr - dna microarray - european eel - murine gammaherpesvirus-68 - transcription - persistence - proteins - carp
Background Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR. Results Four immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins. Conclusions Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.
Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin
Vries, R.P. de; Smit, C.H. ; Bruin, E. de; Rigter, A. ; Vries, E. de; Cornelissen, A.H.M. ; Eggink, D. ; Chung, N.P.Y. ; Moore, J.P. ; Sanders, R.W. ; Hokke, C.H. ; Koopmans, M.P.G. ; Rottier, P.J.M. ; Haan, C.A.M. de - \ 2012
Journal of Virology 86 (2012)21. - ISSN 0022-538X - p. 11735 - 11744.
influenza-virus hemagglutinin - receptor-binding - carbohydrate moiety - dc-sign - glycosylation - glycoprotein - proteins - antibody - recognition - specificity
Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH53) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2 moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.
Acid-activated structural reorganization of the Rift Valley fever virus Gc fusion protein
Boer, S.M. de; Kortekaas, J.A. ; Spel, L. ; Rottier, P.J.M. ; Moormann, R.J.M. ; Bosch, B.J. - \ 2012
Journal of Virology 86 (2012)24. - ISSN 0022-538X - p. 13642 - 13652.
semliki-forest-virus - conserved histidine-residues - triggering membrane-fusion - cell-fusion - uukuniemi-virus - mediated endocytosis - hemorrhagic-fever - enveloped viruses - mammalian-cells - influenza-virus
Entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and its pH-dependent activation mechanism in particular using our recently developed nonspreading RVFV particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (
Heparan sulfate facilitates Rift Valley fever virus entry into the cell
Boer, S.M. de; Kortekaas, J.A. ; Haan, C.A. de; Rottier, P.J.M. ; Moormann, R.J.M. ; Bosch, B.J. - \ 2012
Journal of Virology 86 (2012)24. - ISSN 0022-538X - p. 13767 - 13771.
hamster ovary cells - protein - receptor - binding - infection - biosynthesis - glycoprotein - replication - phlebovirus - expression
Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly-sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.
Anguillid herpesvirus 1 transcriptome
Beurden, S.J. van; Gatherer, D. ; Kerr, K. ; Galbraith, J. ; Herzyk, P. ; Peeters, B.P.H. ; Rottier, P.J.M. ; Engelsma, M.Y. ; Davidson, A.J. - \ 2012
Journal of Virology 86 (2012)18. - ISSN 0022-538X - p. 10150 - 10161.
channel catfish virus - human cytomegalovirus transcriptome - european eel - structural proteins - genome sequences - gene-expression - koi herpesvirus - common carp - identification - persistence
We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5' untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5' and 3' ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5' untranslated exons or, because of alternative splicing, 5' untranslated and 5' translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome.
Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice
Cornelissen, A.H.M. ; Leeuw, O.S. de; Tacken, M.G.J. ; Klos, H.C. ; Vries, R.P. de; Boer-Luijtze, E.A. de; Zoelen-Bos, D.J. van; Rigter, A. ; Rottier, P.J.M. ; Moormann, R.J.M. ; Haan, C.A.M. de - \ 2012
PLoS ONE 7 (2012)8. - ISSN 1932-6203
avian influenza - fusion protein - neutralizing antibodies - respiratory-tract - lethal challenge - vaccine vectors - fowlpox virus - recombinant - immunization - virulence
Background: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. Methodology/Principal Findings: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5 3). A single intramuscular immunization with NDV-sH5 3 or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5 3 was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5 3 was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. Conclusions/Significance: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.
|Acid activation of the budded virus fusion protein F of Spodoptera exiqua multicapsid nucleopolyhedrovirus
Wang, Q. ; Weijer, M. van de; Hoeven, T. van den; Oers, M.M. van; Vlak, J.M. ; Rottier, P. ; Bosch, B.J. - \ 2012
In: Abstract Book of the 45th Annual meeting of the Society for Invertebrate Pathology, Buenos Aires, Argentina, August 5-9, 2012. - Buenos Aires, Argentina : - p. 94 - 94.
Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus to high titres
Loeffen, W.L.A. ; Vries, R.P. de; Stockhofe, N. ; Zoelen-Bos, D.J. van; Maas, H.A. ; Koch, G. ; Moormann, R.J.M. ; Rottier, P.J.M. ; Haan, C.A.M. de - \ 2011
Vaccine 29 (2011)8. - ISSN 0264-410X - p. 1545 - 1550.
genetic reassortment - a viruses - infection - transmission - pathogenesis - antibodies - adjuvant - ferrets
In 2009 a new influenza A/H1N1 virus strain (“pandemic (H1N1) 2009”, H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 106 TCID50 per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 105 TCID50 per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.
Virulence of Newcastle disease virus: what is known so far?
Dortmans, J.C.F.M. ; Koch, G. ; Rottier, P.J.M. ; Peeters, B.P.H. - \ 2011
Veterinary Research 42 (2011)1. - ISSN 0928-4249 - 11 p.
hemagglutinin-neuraminidase protein - vesicular stomatitis-virus - strand rna viruses - paramyxovirus-v-proteins - pigeon pmv-1 viruses - influenza-a virus - fusion protein - cleavage-site - matrix protein - nucleotide-sequence
In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors
Identification and localization of the structural proteins of anguillid herpesvirus 1
Beurden, S.J. van; Leroy, B. ; Wattiez, R. ; Haenen, O.L.M. ; Boeren, S. ; Vervoort, J.J.M. ; Peeters, B.P.H. ; Rottier, P.J.M. ; Engelsma, M.Y. ; Vanderplasschen, A.F. - \ 2011
Veterinary Research 42 (2011). - ISSN 0928-4249 - 15 p.
herpes-simplex-virus - channel catfish virus - koi herpesvirus - proteomic analysis - genome sequences - european eel - virions - peptides
Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.
A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: Evaluation of clinical signs, virus shedding and seroconversion
Dortmans, J.C.F.M. ; Koch, G. ; Rottier, P.J.M. ; Peeters, B.P.H. - \ 2011
Avian Pathology 40 (2011)2. - ISSN 0307-9457 - p. 125 - 130.
newcastle-disease-virus - inactivated aqueous-suspension - oil-emulsion vaccines - fusion protein gene - racing pigeons - monoclonal-antibodies - great-britain - biological characterization - phylogenetic analysis - nucleotide-sequence
The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons
Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence
Dortmans, J.C.F.M. ; Rottier, P.J.M. ; Koch, G. ; Peeters, B.P.H. - \ 2011
Journal of General Virology 92 (2011)2. - ISSN 0022-1317 - p. 336 - 345.
avian paramyxovirus type-1 - fusion protein - great-britain - cleavage site - influenza-virus - molecular-basis - rna-polymerase - pmv-1 viruses - pathogenicity - host
Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.
Complete genome sequence and taxonomic position of anguillid herpesvirus 1
Beurden, S.J. van; Bossers, A. ; Voorbergen-Laarman, H.A. ; Haenen, O.L.M. ; Peters, S.A. ; Abma-Henkens, M.H.C. ; Peeters, B.P.H. ; Rottier, P.J.M. ; Engelsma, M.Y. - \ 2010
Journal of General Virology 91 (2010)4. - ISSN 0022-1317 - p. 880 - 887.
dna-polymerase gene - european eel - japanese eels - hva - evolution - japonica - regions - taiwan - virus - koi
Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.