Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G.

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection.

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.

An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.

Use of PCR and immunofluorescence to detect bovine herpesvirus 1 recombinants.

Homologous recombination occurs frequently between strains of the same alphaherpesvirus species. Studies of this phenomenon require techniques that can differentiate parental strains from putative recombinant progeny viruses. Usually, progeny viruses generated by co-infection of two distinguishable parental strains are first cloned by selection of a single plaque and then characterised by PCR. An assay designed to investigate recombination between two bovine herpesvirus 1 (BHV-1) strains lacking either the glycoprotein gC or gE ORF is described. A PCR assay was developed in which a single step co-amplifies both BHV-1 glycoprotein-encoding sequences. Because the usual procedure for virus isolation, viral plaque picking, can lead to polyclonal virus preparations, a PCR protocol alone does not differentiate between samples containing recombinant viruses (gC+/gE+) and those containing a mixture of both single deleted parental strains (gC-/gE+ and gC+/gE-), and false positives resulting from recombination could occur. To reduce this possibility, double-label immunofluorescence staining of isolated plaques was developed, which coupled with PCR, allows straightforward discrimination between parental strains and progeny recombinant viruses. This assay will be useful for further studies of recombination, especially those evaluating the potential emergence of recombinants between BHV-1 marker vaccine and wildtype strains.

Development of an ELISA for detection of antibodies to glycoprotein I of Aujeszky's disease virus: a method for the serological differentiation between infected and vaccinated pigs.

A blocking ELISA was developed to distinguish between Aujeszky's disease virus (ADV)-infected and vaccinated pigs, on the basis of presence or absence of serum antibodies to glycoprotein I (gI) of ADV. The gI-ELISA detects antibodies that block the reaction of monoclonal antibodies to one or two epitopes on gI of ADV. The ADV-gI antibody response appeared between one and two weeks post-infection and persisted at a high level for at least seven months. Five of the nine ADV-vaccine strains examined were found to be "gI-negative". Pigs vaccinated with a gI-negative vaccine did not develop an ADV-gI antibody response until they were challenge-exposed to a virulent strain of ADV. The gI-ELISA is highly specific, sensitive and suitable for large-scale sero-epidemiological studies to identify infected pigs in populations vaccinated with gI-negative vaccines. The gI-ELISA provides, therefore, a basis for ADV-eradication programmes, which introduces a novel concept in the control of animal virus diseases.

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen.

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk.

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.

Diva vaccines that reduce virus transmission.

This brief review deals with the effect of diva (Differentiating Infected from VAccinated individuals) vaccines (also termed marker vaccines) on transmission of herpesviruses and pestiviruses in swine and cattle. Pseudorabies and bovine herpesvirus 1 diva vaccines have been demonstrated to reduce transmission of wild-type virus in populations of pigs and cattle in the laboratory as well as in the field. A subunit diva vaccine based on the immunodominant E2 protein of classical swine fever virus that is expressed in the baculovirus system may reduce transmission of wild-type virus among pigs and also transmission from mother to foetuses. A similar diva vaccine against bovine virus diarrhoea infections protected sheep against transplacental transmission of antigenically homologous wild-type virus. Diva vaccines along with their companion diagnostic tests can play a role in control of infections, ultimately leading to eradication of viruses.

The use of marker vaccines in eradication of herpesviruses.

Marker vaccines are vaccines that allow serological differentiation between infected and vaccinated individuals. This differentiation is based on the absence of one or more microbial proteins in the vaccine that are present in the wild-type micro-organism. Consequently, after infection, but not after vaccination, an antibody response against that specific protein(s) can be detected. With a protein-specific antibody test infected individuals can thus be distinguished from vaccinated individuals. Marker vaccines against pseudorabies virus (PRV) and against bovine herpesvirus 1 (BHV1) infections have been developed, along conventional routes and by recombinant DNA technology. These vaccines have been shown to be efficacious in reducing (a) clinical signs after infection, (b) wild-type virus replication after infection, and (c) transmission of wild-type virus in the laboratory and in the field. At present, PRV vaccines that lack the gene for the glycoprotein gE are used worldwide in novel eradication programmes. The first phase of such a programme consists of systematic vaccination of pigs on a farm, in a region or an entire country. Experiences in the Netherlands show that it is feasible to eradicate PRV by the intensive use of marker vaccines. Whether, this also holds true for BHV1 is now under investigation.

Respiratory syncytial virus infections in human beings and in cattle.

Respiratory syncytial virus (RSV) causes yearly outbreaks of respiratory disease in human beings and cattle all over the world. Most severe human respiratory syncytial virus (HRSV)-associated disease is observed in children less than 1 year of age while most severe bovine respiratory syncytial virus (BRSV)-associated disease is observed in calves less than 6 months of age. Two subgroups of HRSV have been identified. The existence of two subgroups of BRSV has been repeatedly suggested but is not yet well established. BRSV and HRSV are closely related antigenically but antigenic differences have been observed. Seasonal periodicity of RSV infection is usual with highest incidences in autumn and winter. Stress such as caused by movement, crowding and temperature changes are considered to play a role in bovine outbreaks. Human beings and cattle are the natural hosts of HRSV and BRSV, respectively. Primarily infected individuals are the most important source of RSV during outbreaks. The role of other species in the spread of HRSV and BRSV is unknown. Protective efficacy of maternally derived antibodies is considered to be incomplete. Such antibodies do not reduce shedding of virus after HRSV and BRSV infection. RSV is often transmitted by contact with nasal secretions but may also be transmitted by aerosols. Seroprevalence of HRSV and BRSV among adult human beings and cattle is over 70% and is always higher than it is among younger individuals. Both human beings and cattle of all ages may be reinfected with RSV. During BRSV reinfections, signs of respiratory tract disease and shedding of virus are seldom observed whereas these are common during HRSV reinfections. Persistent HRSV and BRSV infections in human beings and cattle have been suggested but have not so far been reported.

Effect of infections with swine fever virus on immune functions. III. Antibody response to lipopolysaccharide and sheep red blood cells.

In order to determine the effect of infections with low-virulent swine fever virus (SFV) on antibody responses, pigs were administered lipopolysaccharide (LPS) or sheep red blood cells (SRBC), 2 days after infection. Infected pigs showed an enhanced primary response to LPS late during infection. The secondary response to LPS seemed to be unaffected. Both the primary and secondary antibody response to SRBC appeared to be enhanced rather than depressed in infected pigs. These in vivo findings suggest that pigs infected with low-virulent SFV do not develop a depression of B lymphocyte function.

Congenital infections with nonarbo togaviruses.

The present review deals with the similarities and differences of selected aspects of prenatal pestivirus infections of domestic animals and congenital rubella of man. Hog cholera virus, bovine virus diarrhoea virus and border disease virus are antigenically closely related, but unrelated to rubella virus. The nonarbo togaviruses are capable of producing congenital infections resulting in a wide spectrum of abnormalities. The infected foetus can die in utero, in the neonatal period, or it may be born with teratogenic defects. In addition, apparently healthy progeny can be delivered that develop a late onset disease, months, or years after birth, or remain clinically normal for life. The ultimate outcome of a congenital infection is mainly determined by the stage of foetal development, at which infection occurs. Foetuses exposed to rubella virus raise an antibody response to the virus, whereas domestic animals frequently fail to respond immunologically to a congenital pestivirus infection. In congenital rubella the virus usually disappears from the host's body 1-2 years after birth. However, congenital pestivirus infections may be characterized by a lifelong and widespread persistence of virus in clinically healthy animals. Such animals are of significance in the epizootiology of bovine virus diarrhoea, border disease or hog cholera.

Intranasal vaccination of pigs against Aujeszky's disease: protective immunity at 2 weeks, 2 months and 4 months after vaccination in pigs with maternal antibodies.

The purpose of the study was to evaluate the short- and long-term immunity after intranasal vaccination in pigs with maternally derived antibodies (MDA). In two experiments, 10-week-old pigs with moderate MDA titres against Aujeszky's disease virus (ADV) were vaccinated intranasally with the Bartha strain of ADV to evaluate the protective immunity conferred at 2 weeks, 2 months and 4 months after vaccination. Protection was evaluated on the basis of severity of clinical signs, periods of fever and growth arrest, and duration and amount of virus excreted after challenge with a virulent ADV. During the first 2-3 weeks after vaccination, antibodies to ADV continued to decline as in unvaccinated control pigs. After that, antibody titres stabilized or gradually increased. At 2 weeks, 2 months and 4 months after vaccination, vaccinated pigs were significantly better protected than unvaccinated controls. The vaccinated pigs challenged 2 weeks after vaccination hardly developed any sign of disease. Mild signs of Aujeszky's disease and a growth arrest period of 5 days were observed in vaccinated pigs challenged 2 months after vaccination, whereas vaccinated pigs challenged 4 months after vaccination developed severe signs of disease and a growth arrest period of 13 days. Vaccinated pigs challenged 2 weeks after vaccination did not excrete challenge virus, and pigs challenged 2 or 4 months after vaccination excreted far less virus than unvaccinated controls. The results demonstrate that intranasal ADV vaccination of pigs with moderate MDA titres protected them from 2 weeks to at least 4 months after vaccination. Immunity steadily declined, however, after vaccination.

Vaccinology of classical swine fever: from lab to field.

There are two types of classical swine fever vaccines available: the classical live and the recently developed E2 subunit vaccines. The live Chinese strain vaccine is the most widely used. After a single vaccination, it confers solid immunity within a few days that appears to persist lifelong. The E2 subunit vaccine induces immunity from approximately 10-14 days after a single vaccination. The immunity may persist for more than a year, but is then not complete. The Chinese strain vaccine may establish a strong herd immunity 1-2 weeks earlier than the E2 vaccine. The ability of the Chinese vaccine strain to prevent congenital infection has not been reported, but the E2 subunit vaccine does not induce complete protection against congenital infection. Immunological mechanisms that underlie the protective immunity are still to be elucidated. Both types of vaccine are considered to be safe. A great advantage of the E2 subunit vaccine is that it allows differentiation of infected pigs from vaccinated pigs and is referred to as a DIVA vaccine. However, the companion diagnostic E(rns) ELISA to actually make that differentiation should be improved. Many approaches to develop novel vaccines have been described, but none of these is likely to result in a new DIVA vaccine reaching the market in the next 5-10 years. Countries where classical swine fever is endemic can best control the infection by systematic vaccination campaigns, accompanied by the normal diagnostic procedures and control measures. Oral vaccination of wild boar may contribute to lowering the incidence of classical swine fever, and consequently diminishing the threat of virus introduction into domestic pigs. Free countries should not vaccinate and should be highly alert to rapidly diagnose any new outbreak. Once a new introduction of classical swine fever virus in dense pig areas has been confirmed, an emergency vaccination programme should be immediately instituted, for maximum benefit. The question is whether the time is ripe to seriously consider global eradication of classical swine fever virus.

Intranasal vaccination of pigs against Aujeszky's disease. 4. Comparison with one or two doses of an inactivated vaccine in pigs with moderate maternal antibody titres.

Intranasal (IN) vaccination of pigs with low levels of maternally-derived antibody (MDA) has previously been shown to confer good protection against challenge with virulent Aujeszky's disease virus (ADV). The objective of the present study was to determine the efficacy of IN vaccination with an attenuated ADV, in comparison with that of an inactivated vaccine given parenterally, in pigs with higher MDA titres at the time of vaccination. In one experiment, vaccinations were done at 6 weeks of age, and in another experiment pigs were vaccinated at 4 and/or 9 weeks of age. Two months after (the last) vaccination pigs were challenged intranasally with a virulent ADV. Protection was evaluated on the basis of mortality, periods of growth arrest, fever and virus shedding after challenge. The presence of MDA markedly depressed the serum-neutralizing antibody response after vaccination. Sensitisation occurred after parenteral vaccination with an inactivated vaccine despite high MDA levels. Although the intranasally-vaccinated pigs had lower levels of neutralizing antibody at the time of challenge, they were significantly better protected than pigs given 1 or 2 doses of the inactivated vaccine. Comparing the present results with those of a previous study, it appears that the efficacy of parenteral as well as intranasal ADV vaccination decreases with increasing levels of MDA at the time of vaccination.

Early immunity induced by a live gE-negative bovine herpesvirus 1 marker vaccine.

We studied the early immunity induced by a live glycoprotein E (gE) negative bovine herpesvirus 1 (BHV1) marker vaccine. Three groups of specific-pathogen-free calves were either not vaccinated, or vaccinated two days or two hours before the introduction of a calf that was intranasally infected with wild-type BHV1 the day before. We quantified the shedding of gE-negative vaccine virus and of wild-type virus, using a double-staining immunoassay. In calves vaccinated two hours before the introduction of the infected calf, the shedding of wild-type virus was reduced, compared with that of the unvaccinated control calves. The shedding of wild-type virus was most significantly reduced in the calves that were vaccinated two days before: only very small amounts of wild-type virus were isolated. Wild-type virus was not detected at all in the samples from one of the five calves of that group. Furthermore, this calf was the only one in which we did not detect antibodies against gE. Hence, intranasal vaccination with a live gE-negative vaccine induced early immunity against a BHV1 contact infection. This suggests that this vaccine can be used efficaciously in the early stages of a BHV1 outbreak.

Effect of infections with swine fever virus on immune functions. II. Lymphocyte response to mitogens and enumeration of lymphocyte subpopulations.

Peripheral blood and spleen lymphocytes from pigs infected with a low-virulent strain of swine fever virus (SFV) were transiently hyporesponsive to the mitogenic action of PHA, PWM and Con A. The mitogenic reactivity of lymphocytes from lymph nodes from such pigs appeared to be enhanced rather than depressed at that time. In addition, hyper-responsiveness of peripheral blood lymphocytes (PBL) to these mitogens occurred in some pigs. PBL from pigs lethally infected with virulent SFV showed a persistent depression of the response to these mitogens, whereas lymphocytes from lymph nodes had a high responding capacity. A lymphocyte response to SFV antigens could not be demonstrated in infected pigs. These SFV infections did not markedly affect the percentage of lymphocytes in the blood and most lymphoid organs rosetting with sheep red blood cells. On the other hand, surface immunoglobulin-bearing lymphocytes were markedly increased in lymph nodes from pigs exposed to virulent SFV. The sum of both lymphocyte subpopulations in the lymph nodes from these pigs often considerably exceeded the 100% value, which strongly suggests the presence of cells bearing both surface immunoglobulin and receptors for dextran-treated sheep red blood cells. Possible correlations between these findings are discussed. The results suggest that infections with SFV induce systemic alterations in the process of lymphocyte recirculation in the pig.

Advances in the development and evaluation of bovine herpesvirus 1 vaccines.

This review deals with conventional and modern bovine herpesvirus 1 (BHV1) vaccines. Conventional vaccines are widely used to prevent clinical signs of infectious bovine rhinotracheitis. The use of conventional vaccines, however, does not appear to have resulted in reduction of the prevalence of infection. Novel BHV1 marker vaccines comprise either mutants with a deletion in one of the non-essential genes, or subunit vaccines that contain one or more glycoproteins. These marker vaccines can be used in conjunction with companion diagnostic tests to differentiate between infected and vaccinated cattle. Their efficacy has been evaluated in vaccination-challenge experiments, transmission experiments and in field trials. The results demonstrate that the marker vaccines can contribute to the eventual eradication of BHV1. However, there remains room for improvement of BHV1 marker vaccines.

A European inter-laboratory trial to evaluate the reliability of serological diagnosis of bovine herpesvirus 1 infections.

The detection of cattle latently infected with bovine herpesvirus 1 (BHV1) is of importance in control programs and in international trade activities. Therefore, tests to detect specific antibodies in serum must be highly sensitive. To evaluate the reliability of serological diagnosis of BHV1 infections in Europe, seventeen laboratories in 15 European countries were asked to determine BHV1-specific antibodies in a panel of bovine serum samples using the serological tests available in their laboratory. Laboratory tests included virus neutralisation tests (1, 2 and 24 h), indirect immunofluorescence tests and ELISAs. The serum panel consisted of 12 duplicate lyophilised samples which were randomly coded from 1 to 24 and included negative, weak- and strong-positive samples as well as international reference sera. Virus neutralisation tests and ELISAs showed a high specificity. All participants using neutralisation tests (n = 13) scored the negative samples correctly. Twenty three of 25 ELISAs showed 100% specificity. A serum sample obtained at day 7 after infection was scored as negative by all tests except one home-made blocking ELISA. Samples obtained at day 9 and at day 11 after infection were scored as positive by approximately half and by all tests, respectively. To score the weak-positive European reference standard (EU2) correctly, 24 h neutralisation tests (positive by 8 of 9 laboratories) and home-made blocking ELISAs (positive by 5 of 6 laboratories) were the most reliable. The results indicate that standardisation is urgently needed to ensure that BHV1-infected animals with low antibody titres are recognised.

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA.

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.