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T G Kimman

Publications and source records attributed to T G Kimman.

At least 55 records · Page 3Linked to original sources
PubMed1996

An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.

Animals↗
PubMed1996

Transmission of two pseudorabies virus strains that differ in virulence and virus excretion in groups of vaccinated pigs.

OBJECTIVE: To determine whether 2 pseudorabies virus (PRV) strains that differ in virulence differ in transmission among vaccine strain 783-inoculated pigs. DESIGN: Three experiments were conducted, each with 2 groups of 10 pigs inoculated with the glycoprotein E-negative vaccine 783. In the first 2 experiments, half of one group was inoculated with the mildly virulent strain Sterksel, and half of the other group was inoculated with the virulent strain NIA-3. The remaining pigs were contact-exposed to the inoculated pigs. In the third experiment, 2 pigs in each group were inoculated with strain NIA-3, and the other 8 pigs in each group were contact-exposed to these 2 pigs. ANIMALS: Specific-pathogen-free pigs that were sero-negative for antibodies to PRV. PROCEDURE: Estimation of the transmission of virus in each group was based on a stochastic model, in which the observable variable was the number of contact infections. The sole parameter of the model was the reproduction ratio theta. A glycoprotein E ELISA was used to determine whether infection occurred. Virus excretion in the oropharyngeal fluid was determined by plaque assay to measure infectivity. RESULTS: Vaccinated pigs had a significant difference in virus excretion between the mildly virulent and virulent strains. Sterksel strain-inoculated pigs shed significantly more virus than did NIA-3 strain-inoculated pigs. Despite this difference in virus excretion, the 2 strains did not differ in transmission. CONCLUSIONS: Both PRV strains would be eliminated from a population of vaccine strain 783-inoculated pigs.

Analysis of Variance↗
PubMed1996

Lack of serum antibodies against glycoprotein E in pseudorabies virus-immune pigs infected with wild-type virus.

OBJECTIVE: To examine whether pigs with solid immunity against pseudorabies virus (PRV) could harbor latent infection with wild-type PRV without developing antibodies against glycoprotein E (gE), which is used as a marker protein to differentiate pigs that have been vaccinated from pigs infected with wild-type PRV. ANIMALS: Specific-pathogen-free pigs that were seronegative for antibodies to PRV. PROCEDURE: Oropharyngeal swab specimens were collected, and virus content was measured, Serum samples were obtained 1 week before and 2 and 4 weeks after challenge exposure. Four weeks after challenge exposure, pigs were slaughtered; various tissues were collected for virus isolation, and DNA was analyzed by polymerase chain reaction. RESULTS: Of the intranasally inoculated pigs, only 1 pig shed virus (for 1 day) but none developed antibodies directed against gE. Of the inoculated IM pigs, 3 pigs shed virus (for several days) and 5 developed antibodies directed against gE. One pig did not shed virus and did not develop detectable gE antibodies. However, variable amounts of wild-type virus DNA were detected in various tissues. CONCLUSIONS: Immunized pigs can be infected with wild-type PRV without being detected by the gE-specific ELISA, which is routinely used to discriminate between infected and vaccinated pigs. The implication for eradication programs is that these pigs might be a source of new outbreaks.

Animals↗
PubMed1995

Genetic recombination of pseudorabies virus: evidence that homologous recombination between insert sequences is less frequent than between autologous sequences.

We studied in vivo recombination between a thymidine kinase (TK) negative, glycoprotein E (gE) negative, attenuated strain and a virulent strain of pseudorabies virus (PRV) in pigs. To simplify the detection of recombination we inserted different but overlapping (375 bp) parts of the E1 gene of classical swine fever virus into the gG locus of both virus strains. Recombination between the E1 sequences of these viruses results in reconstitution of the complete E1 coding sequence and expression of the E1 protein. Since E1 is highly immunogenic, we expected to detect in vivo recombination in co-inoculated pigs by the presence of serum antibodies against E1. However, after co-inoculation of pigs with high doses of both virus strains, we were unable to detect antibodies against E1, suggesting that in vivo recombination did not occur or remained below the detection limit. Analysis of individual progeny viruses showed that 13 out of 995 (1.3%) possessed a recombinant TK-negative gE-positive phenotype. In contrast, no E1-positive viruses were detected among 5000 analyzed. This result showed that in vivo recombination between the two virus strains did occur, but was much more frequent between the TK and gE loci than between the E1 sequences. Similar results were obtained in in vitro recombination experiments in which possible growth differences between the various virus strains were excluded. The different recombination frequencies could not be attributed to the difference in distance of the genetic loci since recombination between mutations at a distance of 266 bp in the TK gene occurred as frequent as recombination between the TK and gE genes which are separated by approximately 60 kilobasepairs. These results indicate that some property of the E1 sequence and/or the location of the E1 sequence within the PRV genome affects the frequency of recombination.

Animals↗
PubMed1995

Interaction of pseudorabies virus with immortalized porcine B cells: influence on surface class I and II major histocompatibility complex and immunoglobulin M expression.

We examined whether the L14 cell line, an immortalized B cell line originating from inbred miniature pigs of the MHC haplotype d/d, could be useful to study T cell responses of pigs to pseudorabies virus (PRV). Compared with porcine kidney cells, the replication of PRV in L14 cells was slower and yielded lower quantities of infectious virus, which agrees with the reported poor replication of PRV in peripheral blood lymphocytes of swine. The virus yield and the number of L14 cells expressing the viral glycoprotein gE were both maximal at 48 h postinfection, when approximately 90% of all viable L14 cells expressed gE. Morphologically detectable effects of PRV replication in L14 cells were not obvious, but the number of viable cells at 72 h postinfection was lower in infected cultures than in uninfected cultures. Major histocompatibility complex (MHC) class I and II antigen expression was significantly higher at different time points postinfection on infected than on uninfected L14 cells. In contrast, expression of IgM appeared very slightly reduced on infected L14 cells, indicating a selective influence of PRV on cellular protein expression. PRV-infected L14 cells were lysed by lymphocytes from PRV-immune minipigs of MHC haplotype d/d, indicating their usefulness in in vitro cytolytic assays.

Animals↗
PubMed1995

Experimental quantification of transmission of genetically engineered pseudorabies virus.

There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent different PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PRV mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PRV vector vaccine (gE-, TK-, gG-) that expresses the glycoprotein E1 (E1) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when R1, the infection can spread; when R < 1, the infection will disappear. We found that R for a gE-negative strain was 10.1, and R for a TK-negative strain was 5. Furthermore, the R for the vector vaccine (gE-, TK-, gG-) expressing E1 was 0.18, and did not differ significantly from the R for the control strain without E1. The R of gE-negative strain was significantly 1 (P = 0.0005). Co-inoculation with a gE-positive field strain did not prevent the transmission of a gE-negative strain. This study shows that a small-scale experiment can be used to estimate the transmission of genetically engineered organisms in their host species. The results of this study indicate that the deletion of gE alone or TK alone is not enough to prevent spread of PRV among susceptible pigs, and that transmission of gE-negative PRV is not firmly limited by co-presence of a gE-positive strain.

Administration, Intranasal↗
PubMed1995

Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells.

We examined the capability of pseudorabies virus (PRV) to replicate in vitro in porcine peripheral blood mononuclear cells (PBMC) and characterized the phenotype of infected cells. In addition, we investigated whether inactivation of various PRV proteins or the expression of a foreign gene affected this replication. Finally, we studied the replication of PRV strains in concanavalin A (Con A)-stimulated lymphocytes. The replication of PRV mutants with inactivated glycoproteins gE or gG, thymidine kinase (TK), ribonucleotide reductase (RR) or US3-encoded protein kinase (PK), and the replication of PRV vector strains expressing the envelope glycoprotein E1 of hog cholera virus (HCV) were studied. By adherence of PBMC to plastic, monocytes and lymphocytes were largely separated. Infected monocytes were analysed with an immunostaining monolayer assay and infected lymphocytes were analysed with immunofluorescence staining and flow cytometry. We found that the wild-type NIA-3 virus replicated in both lymphocyte and monocyte cultures. NIA-3 infected relatively more monocytes (> 90%) than non-adherent B cells (46-65%) and T cells (17-28%); approximately equal numbers of CD4+ and CD8+ T cells were infected. Although E1 is probably involved in adsorption of HCV to host cells, the expression of E1 by PRV vector strains did not change the level of replication. Inactivation of TK and RR, but not inactivation of gE, gG or PK, severely affected the replication in both monocytes and lymphocytes. Con A stimulation of lymphocytes restored the reduced replication of the TK mutant, but not of the RR mutant. Moreover, Con A stimulation of lymphocytes reduced the replication of the wild-type NIA-3 virus. We concluded that both viral TK and RR activity are important for efficient replication of PRV in resting lymphocytes. Furthermore, Con A-stimulated lymphocytes can restore the viral TK defect and PRV replication can also be influenced by cellular metabolism.

Animals↗
PubMed1995

The US3-encoded protein kinase from pseudorabies virus affects egress of virions from the nucleus.

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.

Animals↗
PubMed1995

Development and antigen specificity of the lymphoproliferation responses of pigs to pseudorabies virus: dichotomy between secondary B- and T-cell responses.

To better understand the contribution of T cells to the immunity of pigs to pseudorabies virus (PRV), we examined the lymphoproliferation response to this virus. Depletion studies demonstrated that both CD2+CD8+ and CD2+CD4+ cells contributed to lymphoproliferation, but to varying degrees upon stimulation with live and ultraviolet (UV) light-inactivated PRV. Flow cytometric analysis revealed the emergence of both CD2+CD8+ and CD2+CD4+ lymphoblastoid cells. To examine the contribution of specific viral proteins, we prepared immortalized porcine B cells of haplotype d/d that stably expressed a single PRV protein, and used these cells for in vitro stimulation of lymphocytes from PRV-immune miniature pigs of the same haplotype. Cells expressing PRV gB or gC induced proliferation. An immunization/challenge experiment showed that the lymphoproliferation response was stronger upon immunization with the virulent NIA-3 strain than with the attenuated Bartha strain. Upon challenge inoculation, the NIA-3-immunized pigs were almost completely immune, in contrast to the Bartha-immunized pigs. Such poorly protected pigs showed secondary B- and T-cell immune responses upon challenge. In contrast, the better protected NIA-3-immunized pigs did not show a secondary B-cell response. However, they developed a secondary lymphoproliferation response, which was quicker and stronger than in the Bartha-immunized pigs. This dichotomy between secondary B- and T-cell responses indicates that an effective T-cell memory response is able to quickly eliminate challenge virus in immune pigs, so preventing a secondary B-cell response.

Animals↗
PubMed1994

Mutagenesis and characterization of a 41-kilobase-pair region of the pseudorabies virus genome: transcription map, search for virulence genes, and comparison with homologs of herpes simplex virus type 1.

We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragments. More than half of these transfections yielded viable virus mutants. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated from cells infected with wild-type PRV. In this way we were able to construct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes simplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants were assayed for virulence in mice. Mutagenesis of the identified homologs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DNA polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsid-associated protein), and UL12 (alkaline nuclease) completely abrogated or strongly reduced virulence.

Animals↗
PubMed1994

Spread of Aujeszky's disease virus within pig herds in an intensively vaccinated region.

An intensive vaccination programme with the glycoprotein I (gI) and thymidine kinase-deleted vaccine strain 783 was applied on all the pig farms in a region with a high pig density. To monitor the spread of Aujeszky's disease virus within breeding herds in that region, all the breeding stock in nine herds were examined for antibodies to gI six times at intervals of four months. The prevalence of gI-seropositive sows decreased greatly in all nine herds. The mean percentage of gI-seropositive sows decreased from 56.4 per cent (range 80.0 to 13.6 per cent) at the start, to 20.3 per cent (range 29.1 to 1.6 per cent) after two years. Nevertheless, seroconversions to gI were detected in all the herds, and in six out of the nine breeding herds even during the second year of the study. The intensive regional vaccination apparently did not completely prevent Aujeszky's disease virus infections within these herds. The source of the virus responsible for these infections was not identified. However, because in most herds only a few sows seroconverted, the virus either circulated at a low level within the herds, or its introduction or reactivation did not lead to an extensive spread of the virus.

Animals↗
PubMed1994

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity.

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.

Animals↗
PubMed1994

In vivo recombination of pseudorabies virus strains in mice.

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 (TK) 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 TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination 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 isolated 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 complementation may contribute to pathogenicity of PRV.

Animals↗
PubMed1994

Vaccine properties of pseudorabies virus strain 783 are not affected by a deletion of 71 base pairs in the promoter/enhancer region of the viral immediate early gene.

Strain 783 of pseudorabies virus (PRV) is a genetically engineered vaccine which contains three deletions. The purpose of this study was to examine the effect of one of the deletions, which until now has not been characterized. The deletion occurs within the inverted repeats. Seventy-one base pairs (bp) were deleted, including one of the repeat sequence elements related to the TAATGARATTC boxes detected within the promoter and enhancer region of the immediate early (IE) genes of herpes simplex virus. The deletion affected neither the transcription of the IE gene nor viral growth in vitro. In our animal experiments, one group of pigs was inoculated with the original strain 783 and another with strain 783 which had had the repeat sequences restored. These two groups were then compared to determine the protective efficacy of the two vaccine strains against PRV infection. The deletion in the inverted repeats does not affect the vaccine properties of PRV strain 783: strain 783, with and without the 71 bp deletion in the repeats, protected pigs equally well.

Animals↗
PubMed1994

Experimental quantification of vaccine-induced reduction in virus transmission.

Although reduction in transmission of an agent in the host population is an important goal of many vaccinations, suitable experimental methods to measure transmission have been lacking. Therefore, we designed and tested an animal experiment to quantify transmission among vaccinated and unvaccinated animals. We used Aujeszky's disease virus (ADV) in pigs, because a serological test was available to detect infection in vaccinated pigs and because vaccination against ADV will be used in an attempt to eliminate ADV from the Netherlands. Our experiments showed that vaccinating twice with vaccine 783 significantly reduces ADV transmission. In unvaccinated groups, the estimated maximum number of secondary cases per infectious individual, i.e. the basic reproduction ratio R0, was 10.0. In contrast, the reproduction ratio for the vaccinated groups R, i.e. the average number of secondary cases per infectious individual in a totally vaccinated population, was 0.5. These results show that it is possible to measure transmission experimentally. Therefore, such measurements should be obtained for all vaccines that are intended to eliminate agents causing animal diseases, either on a single farm or in a whole country.

Animals↗
PubMed1994

Intensive regional vaccination with a gI-deleted vaccine markedly reduces pseudorabies virus infections.

The development of marker vaccines against pseudorabies virus (PRV) and companion diagnostic tests have enabled us to perform a unique field trial. In this study, the effect of intensive regional vaccination on pig-finishing herd immunity was directly measured by comparing the seroprevalence of antibodies to glycoprotein I in trial and control groups. The seroprevalence of infected finishing herds in the trial region decreased from 81% at the start of the trial to 19% after 2 years (p < 0.001). The mean seroprevalence of infected pigs in these herds diminished from 49 to 5% (p < 0.001). In the control group, representing routine PRV control, no significant change in seroprevalences was noticed.

Animals↗