Ben Peeters - Academia.edu (original) (raw)

Papers by Ben Peeters

Journal of General Virology, 1997

Envelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not ... more Envelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not required for cell-to-cell spread. When animals are inoculated with a phenotypically complemented PRV gD mu- tant, the virus is able to spread locally by means of direct cell-to-cell transmission, but progeny virions released by infected cells are non-infectious be- cause they lack gD. Therefore, the

Journal of virology, 1996

Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is... more Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is not required for the subsequent steps in virus replication. Phenotypically complemented gD mutants can infect cells and can spread, both in vitro and in mice, by direct cell-to-cell transmission. Progeny virions released by infected cells are noninfectious because they lack gD. The aim of this study was to determine the role of gD in the neuropathogenicity of PRV in its natural host, the pig. We investigated whether gD-negative PRV can spread transneuronally via synaptically linked neurons of the olfactory and trigeminal routes. High doses of a phenotypically complemented gD mutant and gD mutants that are unable to express either gI or gI plus gE were inoculated intranasally in 3- to 5-week-old pigs. Compared with the wild-type virus, the virulence of the gD mutant was reduced. However, pigs inoculated with the gD mutant still developed fever and respiratory signs. Additional inactivati...

Journal of Virology, 2002

is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains a... more is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N-or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).

Journal of Virology, 2000

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due... more Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A-or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system.

Journal of General Virology, 1995

ABSTRACT We examined the influence of inactivation of various genes located in the unique short (... more ABSTRACT We examined the influence of inactivation of various genes located in the unique short (U(S)) region of pseudorabies virus on virus replication and assembly in porcine nasal mucosa explant cultures. The following strains were used: the virulent wild-type strain NIA-3, and strains derived from NIA-3 containing a mutation inactivating the genes encoding either the US3-encoded protein kinase (PK), gG, gD, gI, gE, the 28 kDa ('28K') protein (single mutant), or the 28K and 11 kDa ('11K') proteins (double mutant). In addition a wild-type rescuant was used, which was generated by marker rescue from a PK- mutant. All virus strains infected nasal epithelium and had invaded the stroma after approximately 24 h. The morphogenesis in nasal epithelium cells of two PK- mutants showed the most striking differences compared to the parent NIA-3 strain and the other mutant strains. The changes could be ascribed to the US3-encoded PK because the rescue mutant showed a similar morphogenesis to wild-type NIA-3. The transmembrane transport of the PK- mutants was impaired at the outer nuclear membrane which resulted in an accumulation of virions in the perinuclear space. These results suggest that proteins, phosphorylated by the US3-encoded PK, are involved in debudding of virus particles at the outer nuclear membrane. This defect in the transport of the US3 mutant probably explains their reduced replication in vitro. The gG-, gD-, gI-, gE-, 28K- and 11K- mutant strains showed minor or no changes in viral assembly. Thus the reported decreased virulence of the gD-, gI- and gE- mutants was, in contrast to that of the PK- mutants, not associated with clear alterations in morphogenesis.

Avian Diseases, 2006

The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino ac... more The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino acid sequence at the fusion cleavage activation site. To extend the understanding of the role of the fusion cleavage activation site in NDV virulence, the pathogenesis in chickens of a lentogenic LaSota isolate and two infectious clones, NDFL and NDFLtag, were compared. NDFL is an infectious clone of a lentogenic NDV strain (LaSota E13-1), and NDFLtag is the infectious clone with the fusion cleavage site sequence mutated to the virulent motif. NDFL and NDFLtag were described by Peeters et al. The viruses were inoculated intraconjunctivally into groups of 4-wk-old white leghorn chickens and compared in a pathogenesis study for determination of disease causation (clinical signs of disease, gross lesions, histology, virus isolation, and serology) and viral distribution (presence of viral nucleoprotein and mRNA was detected by immunohistochemistry and in situ hybridization, respectively). The modification of the fusion cleavage activation site to the virulent motif in the infectious clone only slightly increased disease severity and viral distribution in the pathogenesis assessment, even though dramatically increased pathogenicity of NDFLtag was confirmed by standard pathogenicity index tests. The result, that the mutated fusion cleavage site of NDV-NDFLtag had only a small influence on pathogenesis in chickens compared to either E13-1 or NDFL, suggests that the pathogenic effects of NDV are not dependent on the fusion cleavage site alone.

Journal of the Royal Society, Interface / the Royal Society, 2016

Highly pathogenic avian influenza (HPAI) H5N1 epidemics in poultry cause huge economic losses as ... more Highly pathogenic avian influenza (HPAI) H5N1 epidemics in poultry cause huge economic losses as well as sporadic human morbidity and mortality. Vaccination in poultry has often been reported as being ineffective in preventing transmission and as a potential driving force in the selection of immune escape mutants. We conducted transmission experiments to evaluate the transmission dynamics of HPAI H5N1 strains in chickens vaccinated with high and low doses of immune escape mutants we have previously selected, and analysed the data using mathematical models. Remarkably, we demonstrate that the effect of antigenic distances between the vaccine and challenge strains used in this study is too small to influence the transmission dynamics of the strains used. This is because the effect of a sufficient vaccine dose on antibody levels against the challenge viruses is large enough to compensate for any decrease in antibody titres due to antigenic differences between vaccine and challenge stra...

International journal of oncology, 2008

A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2... more A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was produced in high amounts by tumor cells infected with rec(IL-2). Tumor vaccine cells infected by rec(IL-2) stimulated human T cells to exert anti-tumor activity in vitro in a tumor neutralization assay. These effects were significantly increased when compared to vaccine infected by rec(-) virus without IL-2 gene. After incubation with rec(IL-2) infected tumor cells, T cells showed increased expression of the activation marker CD69 and produced increased amounts of IFNgamma when compared to T cells co-incubated with rec(-) infected tumor cells. CD8 T cells incubated with rec(IL-2) infected tumor cells showed upregulation of perforin, cell surface exposure of the degranulation marker CD107a and...

International journal of oncology, 2006

The aim of the study was: i) to specifically target tumor tissue by Newcastle disease virus (NDV)... more The aim of the study was: i) to specifically target tumor tissue by Newcastle disease virus (NDV) with oncolytic properties, ii) to improve the delivery system for systemic application of NDV via a bispecific adapter protein and iii) to investigate anti-tumor activity and side-effects. We selected two oncolytic virus strains, one native and the other recombinant, which showed multicyclic replication patterns in tumor cells. In order to reduce normal cell binding, they were modified by preincubation with a recombinant bispecific protein which blocks the viral native cell binding site and introduces a new binding site for a tumor-associated target (in this study, the interleukin-2-receptor, IL-2R). After intravenous transfer to mice, uptake of modified NDV in liver, spleen, kidney and lung was greatly reduced in comparison to unmodified NDV as determined by RRT-PCR of viral M gene copies. In IL-2R+ tumor bearing mice, the same assay revealed a high replication efficiency of the modifi...

International journal of oncology, 2005

Previously we have demonstrated that a recombinant Newcastle disease virus (NDV) carrying the tra... more Previously we have demonstrated that a recombinant Newcastle disease virus (NDV) carrying the transgene EGFP can be retargeted to IL-2 receptor positive tumor cells by a bispecific fusion protein alphaHN-IL-2 in vitro. The purpose of the present study was to investigate the specificity and efficiency of gene delivery to tumor cells in vivo via this modified RNA virus. Prior ex vivo infection of murine lymphoma cells by the modified virus resulted in selective EGFP expression in IL-2R+ target tumor cells in vivo. Direct fluorescence microscopy and immunohistology showed viral replication in target positive tumor tissue resulting in much more EGFP expression than in target negative tumor tissue, 24 h after intratumoral injection of the alphaHN-IL-2 modified NDV. A quantitative real-time RT-PCR for EGFP mRNA. confirmed the selective gene expression in IL-2R+ tumor cells. Biodistribution studies showed that EGFP transgene delivery was reduced by 35-100% in liver, spleen, kidney, lung an...

The Journal of general virology, 2000

Little is known about the intermolecular interactions between the viral proteins of infectious bu... more Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 ...

The Journal of general virology, 1999

We have completely sequenced the genome of Newcastle disease virus (NDV) vaccine strain LaSota. T... more We have completely sequenced the genome of Newcastle disease virus (NDV) vaccine strain LaSota. The sequences of the 3'- and 5'-terminal ends of the RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends. The entire genomic sequence, which was established by sequencing cDNA fragments generated by high-fidelity RT-PCR, consists of 15186 nt. Comparison of the 5'-terminal sequence of NDV LaSota with the 5'-terminal sequences of ten members of the Paramyxovirinae showed that NDV LaSota has an unusually long 5' untranscribed region. Comparison of the entire genomic sequences showed that NDV is only distantly related to the other members of the genus Rubulavirus, to which NDV has been assigned. In this paper we present data which suggest that NDV should not be classified in the genus Rubulavirus, but instead should be considered as a member of a new genus within the subfamily Paramyxovirinae.

Vaccine, 2010

In the past decade, the use of Newcastle disease virus (NDV) as a vaccine vector for the preventi... more In the past decade, the use of Newcastle disease virus (NDV) as a vaccine vector for the prevention of economically important livestock diseases as well as for human diseases has been extensively explored. In this study, we have constructed a recombinant NDV vaccine virus, named NDFL-Gn, that produces the Rift Valley fever virus (RVFV) Gn glycoprotein. Calves were immunized via either the intranasal route or the intramuscular route. Delivery via the intranasal route elicited no detectable antibody responses, whereas delivery via the intramuscular route elicited antibodies against both NDV and the Gn protein.

Virology, 2003

Infectious bursal disease virus (IBDV), a non-enveloped double-stranded RNA virus of chicken, enc... more Infectious bursal disease virus (IBDV), a non-enveloped double-stranded RNA virus of chicken, encodes five proteins. Of these, the RNA-dependent RNA polymerase (VP1) is specified by the smaller genome segment, while the large segment directs synthesis of a non-structural protein (VP5) and a structural protein precursor from which the capsid proteins pVP2 and VP3 as well as the viral protease VP4 are derived. Using the recently redefined processing sites of the precursor we have reevaluated the homotypic interactions of the viral proteins using the yeast two-hybrid system. Except for VP1, which interacted weakly, all proteins appeared to selfassociate strongly. Using a deletion mutagenesis approach we subsequently mapped the interacting domains in these polypeptides, where possible confirming the observations made in the two-hybrid system by performing coimmunoprecipitation analyses of tagged protein constructs co-expressed in avian culture cells. The results revealed that pVP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein.

Phenotypically complemented pseudorabies virus gpSO nullmutants are abletoproduce plaques on nonc... more Phenotypically complemented pseudorabies virus gpSO nullmutants are abletoproduce plaques on noncomplementing cell lines despite thefact that progenyvirions arenoninfectious. Todetermine whether gp5O null mutantsandgpSO+gp63 null mutants arealso abletoreplicate andspread inanimals, micewere infected subcutaneously orintraperitoneally. Surprisingly, bothgp5Omutants andgpSO+gp63 double mutants proved tobelethal formice. Incomparison withthewild-type virus, gpSOmutants were still highly virulent, whereas thevirulence ofgp5O+gp63 mutants was significantly reduced. Severe signs ofneurological disorders, notably pruritus, were apparent inanimals infected withthewild-type virus or a gpSOmutantbutwere muchless pronounced inanimals infected witha gp5O+gp63 or gp63mutant.Immunohistochemical examination of infected animals showedthatallviruses wereable toreach, andreplicate in,thebrain. Examination ofvisceral organsofintraperitoneally infected animals showed that viral antigen was predominantl...

Virus Research, 1994

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal ... more We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase ('IX) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TIC gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, re~mbination stilt occurred. No re~mbination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isoIated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that implementation may ~ntribute to pathogenici~ of PRV. * Corresponding author. Fax. (+ 31) 3200-42804. Old-1702/94/$07.~ 0 1994 Elsevier Science B.V. All rights reserved SSDI 0168-17a2t94)00055-H 116 KL. Glazenburg et al. /I&us Research 34 (1994) 115-126

Virology Journal, 2012

Background: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) an... more Background: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens. Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses. Methods: To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains.

Veterinary Research, 2011

Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HP... more Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8α+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found.

Veterinary Microbiology, 2010