Implementation of two-step vaccination in the control of bovine viral diarrhoea (BVD).

Bovine viral diarrhoea (BVD) control/eradication programmes based on the test and removal of persistently infected cattle without use of vaccination were first introduced by the Scandinavian countries in the early 1990s. Within the last 10 years the programmes have proven to be very successful and have served as a blueprint for several other European regions. However, in areas with high cattle densities, intense animal trade and high BVD prevalence this control approach is risky, because there is a high probability that herds, which have been cleared of persistently infected (PI) animals and have become partly or fully susceptible to reintroduction of the virus, will come in contact with a BVD virus (BVDV) infected animal. A combination of the test and removal strategy with subsequent systematic vaccination of cattle could overcome this problem. The goals of vaccination in such a programme is protection against reintroduction of BVDV into herds free from PI cattle and foetal protection of pregnant animals accidentally exposed to the virus. Two-step vaccination is based on the use of inactivated BVDV-1 vaccine for priming followed by a live attenuated vaccine booster 4 weeks later. The immune response elicited by such a vaccination scheme has proven to be long lasting and foetal infection after challenge with BVDV-1 and BVDV-2 was prevented in pregnant animals 5 months after vaccination. These findings suggest that the implementation of a two-step vaccination in the initial phase of control programmes in addition to test and removal of PI animals in areas with high cattle densities and endemic BVD is practical and efficacious.

[Kinetics and persistence of neutralizing antibodies against bovine viral diarrhea virus-1 and -2 and border disease virus after two step vaccination of cattle]. / Kinetik und Persistenz neutralisierender Antikörper gegen das Bovine Virusdiarrhoe Virus-1 und 2 und das Border Disease Virus nach zweistufiger Impfung von Rindern.

The aim of the study was the assessment of rise and persistence of neutralizing antibodies (nAb) to bovine viral diarrhea virus (BVDV) and border disease virus (BDV) after a two step vaccination using an inactivated BVDV/BDV (Mucobovin) and a modified live BVDV vaccine (Vacoviron). In a first experiment eight heifers were kept in isolation and were serologically surveyed regularly over a three year period after vaccination. The same experiment was done with 80 vaccinated cattle kept under field conditions. Neutralizing antibody titres were monitored using homologous as well as heterologous BVDV and one BDV strain, respectively. Maximum titres were obtained two to three months after vaccination. During the three years of monitoring the antibody titres decreased but never fell below the detection limit. This slow antibody regression demonstrates that a single two step vaccination elicited high nAb titres which persist over at least three years. These results might serve as a decision tool when considering the necessity and time of revaccination of cattle, which have been vaccinated using the two step method.

Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation.

A virological survey was carried out to establish the distribution of classical swine fever (CSF) virus among wild boar in the Federal State of Brandenburg, Germany. Organ materials and blood samples were collected from 11,670 wild boar shot or found dead during the period March 1995 to December 1997. In total 211 (1.8%) wild boar were positive for CSF virus or antigen. The incidence of CSF-positive animals decreased continuously from 4.6% at the beginning of the epidemic in 1995 to 0.7% in 1997. The highest incidence of positive animals (22%) was found in wild-boar piglets younger than 3 months of age in 1995. The findings were indicative for the decisive role which young wild boar play in the epidemiology of CSF. Following intrauterine transfer some of the wild-boar piglets were probably persistently infected with CSF virus as experienced experimentally. Such piglets can be held responsible for CSF virus perpetuation within the wild-boar population. No CSF virus was isolated from adult wild boar weighing more than 75 kg. During 3 years of monitoring a sufficient number of susceptible wild boar, in particular young animals, was available to maintain the infection chain in that area. It was concluded that persistently infected piglets and the high population density of wild boar in the Brandenburg region offered optimal conditions for the establishment of an CSF epidemic.

Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

[Immunohistochemical studies on organ tropism of different biotypes of BVD virus in experimentally infected sheep fetuses]. / Immunhistochemische Untersuchungen zum Organtropismus unterschiedlicher Biotypen des BVD-Virus bei experimentell infizierten Schaffeten.

Paraffin sections from various organs of sheep fetuses following transplacental infection with non-cytopathogenic (ncp) bovine viral diarrhoea virus (BVDV) or cytopathogenic (cp) BVDV were stained immunohistochemically with BVDV-specific monoclonal antibodies. Comparison of the distribution of viral antigen in sections from fetuses of experiment A revealed that in organs such as parotid, thyroid, thymus, lung, spleen, kidney, liver and skin from 20 days post inoculation (p.i.) onwards numerous antigen-containing cells were present. In organs of fetuses infected with cp BVDV, however, antigen-positive cells were only detectable until days 10 and 14 p.i. These findings suggest that the ncp BVDV used in experiment A replicated considerably faster and more efficient than the cp BVDV used in experiment B and that the two virus biotypes differ considerably concerning their tropism for fetal ovine organs.

Evaluation of a gp 55 (E2) recombinant-based ELISA for the detection of antibodies induced by classical swine fever virus.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against classical swine fever virus (CSFV) based on the competition of the serum antibodies with a CSFV-specific monoclonal antibody directed against the viral glycoprotein gp 55 (E2), has been evaluated. A total of 553 sera obtained from pigs experimentally infected in groups with different pestiviruses were included in this study. The ELISA was applied to a group of sera collected from pigs prior to pestivirus inoculation and therefore expected to have no detectable CSFV neutralizing antibodies. The specificity of the ELISA was calculated to range between 93% and 98%. The sensitivity of the ELISA was determined by testing predominantly sera of pigs exposed to CSFV and exhibiting CSFV neutralizing antibodies. When the cut-off level was reduced by 10%, the average sensitivity increased from 60% to 70% for the groups of sera tested. The ELISA was able to discriminate between CSFV and bovine viral diarrhoea virus (BVDV) induced antibodies.

Canine distemper virus infection in Serengeti spotted hyenas.

Clinical signs suggestive of canine distemper virus (CDV) infection were observed among a group of spotted hyenas (Crocuta crocuta) in the Serengeti, Tanzania. Virus antigen was detected immunohistologically in a brain sample from a diseased cub. The presence of virus RNA could be demonstrated in this brain as well as in intestine and lymph node of the animal by RT-PCR. Sequence comparison of brain-derived amplicons showed that the virus was related to recent CDV field isolates. The closest homology (>99 percent) was to a recently described CDV which caused high mortality in sympatric lions.

Characterization of phocid herpesvirus-1 and -2 as putative alpha- and gammaherpesviruses of North American and European pinnipeds.

To study the relationships between herpesvirus recently isolated from different pinniped species, antigenic and genetic analyses were performed. First, herpesviruses isolated from North American harbour seals (Phoca vitulina), a Californian sea lion (Zalophus californianus) and a European grey seal (Halichoerus grypus) were examined in an enzyme immunoassay (EIA) with a panel of monoclonal antibodies which had previously been shown to allow typing of herpesviruses from European harbour seals into two distinct virus types: phocid herpesvirus type-1 and type-2 (PhHV-1 and PhHV-2). The EIA data showed that all but one of the isolates from seals ranging in United States coastal waters were PhHV-2-like while the European grey seal herpesvirus was PhHV-1-like. Genetic characterization was facilitated by PCR analysis using primers based on conserved regions of the glycoprotein B and D (gB and gD) genes of the antigenically closely related canid (CHV) and felid (FHV) herpesvirus. Specific amplified products were obtained with five isolates antigenically characterized as PhHV-1-like but not with five PhHV-2-like isolates. Sequence analysis of the PCR products confirmed greatest similarity to members of the genus Varicellovirus of the Alphaherpesvirinae and in particular to CHV. Sequence analysis of two EcoRI fragments of the PhHV-2 genome (European isolate 7848) revealed greatest similarity to gammaherpesviruses and in particular equine herpesvirus-2. Although an unambiguous subgrouping was not feasible, this is the first evidence that PhHV-2 may be a putative gammaherpesvirus of pinnipeds.

Variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of bovine-virus diarrhoea virus.

Pregnant Merino ewes were inoculated intravenously between days 63 and 65 of gestation with a non-cytopathogenic (ncp) bovine-virus diarrhoea-virus (BVDV) isolate (experiment A). The histomorphological findings and the distribution of viral antigen, as revealed by immunohistochemistry in brains of fetuses from experiment A, were compared with those seen in fetal brains from a previous study (experiment B), in which pregnant ewes had been intravenously infected between days 65 and 68 of gestation with the cytopathogenic (cp) BVDV strain Indiana. The two viruses showed remarkable variations concerning their pathogenicity for the developing fetal brain. The cp BVDV had a much higher neuropathogenic potential than the ncp BVDV and induced severe intracranial malformations in most fetuses. In experiment A, exclusively relatively mild leucoencephalomalacic lesions occurred. Between fetuses of the two experiments, significant differences concerning the distribution of viral antigen and the inflammatory response were found. In the majority of fetal brains from experiment B examined at days 10, 14 and 21 post inoculation (p.i.), antigen-containing differentiated brain cells (neurons, astrocytes, oligodendrocytes) and undifferentiated cells in the periventricular germinal zones were seen throughout the different zones of the developing telencephalon and cerebellum. At 21 days p.i., a marked inflammatory response consisting of brain macrophages and other mononuclear cells occurred in the meninges and in the brain parenchyma of fetuses from experiment B. In brain sections of fetuses infected with ncp BVDV, in contrast to fetuses infected with cp BVDV, viral antigen was not detectable during the early stages (days 10 and 20) p.i., and histopathological lesions were not seen at this stage. At days 41 and 47 p.i., antigen-positive astrocytes and oligodendrocytes were found in the developing white matter of the telencephalon and cerebellum. Furthermore, antigen-containing neurons were seen in the developing cerebral cortex. Cellular infiltrations in fetal brains from experiment A were limited to the leucoencephalomalacic areas in the developing cerebral and cerebellar white matter and consisted exclusively of brain macrophages. Immunohistochemical staining in brain sections of fetuses from both experiments revealed that numerous perivascular cells contained viral antigen, whilst positive endothelial cells were exclusively found in fetuses from experiment A. From the findings of this study it was concluded that the cp BVDV stain used in experiment B has a marked tropism for the fetal brain and both its already differentiated and undifferentiated cell populations, and that the resulting brain lesions primarily are the consequence of a direct cytolysis of these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Pathogenesis of intrauterine infections with bovine viral diarrhea virus.

BVDV shares with other Pestiviruses the ability to cross the placenta of pregnant host animals. The effects of fetal infections are complex and depend on a number of factors, e.g., age of the zygote/embryo stage, no infection seems to occur. During the last one third of gestation the infection is terminated by the ontogeny of the fetal immune system. This leaves a window of susceptibility during early stages of fetal development allowing establishment of viral persistence and/or the development of a number of fetopathologic effects. Additionally, fertility problems and abortions are observed. Calves that are born immunotolerant to BVDV and persistently viremic display a wide variety of abnormalities. However, there is an unknown proportion of calves born without any clinical signs indicative of persistent infection. The time of fetal infection during the first stages of pregnancy seems to play a crucial role with respect to the lesions induced. Generally, early infections seem to induce less damage compared with late infections, suggesting an indirect, possibly immune-mediated pathogenesis. Additionally, direct virus-cell interactions may play a role. Few data exist about the influence of differences in viral virulence on fetal pathology. Likewise the role of the viral target cell range is not clear.

Classical swine fever in wild boar (Sus scrofa)--experimental infections and viral persistence.

A pregnant wild boar and two wild boar weaners were inoculated intranasally with a field isolate of classical swine fever virus (CSFV) recently derived from a diseased domestic pig. The clinical, pathological and haematological findings noted in the young wild boars were comparable to those in domestic weaner pigs inoculated with the same virus isolate. Both wild boars showed the acute haemorrhagic form of CSF, one animal died 18 days post inoculation (p. i.) and the second one had to be euthanized when moribund two days later. The wild boar sow did not show any signs of illness p. i. but seroconversion was noticed. Twenty-eight days p. i. birth was given to six clinically healthy offsprings. One of the newborn proved to be viraemic until death when 39 days of age. Except for poor growth no other symptoms were noticed in this piglet. The non-viraemic litter mates remained healthy, although they had close contact to the persistently infected piglet. High titres of neutralizing antibodies against CSFV were measured in the serum samples of these offsprings. All findings were more or less in accordance with observations previously made in domestic pigs when infected with CSFV around 85 to 90 days of gestation. The wild boar was calculated to have been inoculated at about 87 to 92 days of gestation.

Experimentally induced "late-onset" mucosal disease--characterization of the cytopathogenic viruses isolated.

Antigenic and genetic analyses were performed in order to establish relationships between the noncytopathogenic (ncp) and the cytopathogenic (cp) bovine viral diarrhoea viruses (BVDV) involved in the induction of a case of experimentally induced "late-onset" mucosal disease (MD) symptoms. The persistent ncpBVDV, the cpBVDV used for superinfection (strain TGAC) and the virus isolates from faeces (cpX) were examined using an immunoplaque test (IPT) to distinguish between cp and ncp virus populations. The cp populations were cloned by plaque purification and found to be free of ncpBVDV when using the IPT. The cpBVDV clones and the persistent ncpBVDV were analysed in an enzyme immunoassay on heat-fixed infected cells (IM-EIA) and in a neutralization test using a panel of 27 monoclonal antibodies against the E0 (gp48) and E2 (gp53) viral glycoproteins. It was found that strain TGAC contained two antigenically distinct subpopulations of cpBVDV (TGAC-B1 and TGAC-B2). The endogenous ncpBVDV and the cpX clones had the same reactivity pattern in both tests. In addition, p80 gene duplications in the genomes of the cpBVDV clones were analysed using the polymerase chain reaction and subsequent restriction enzyme analysis of the amplicons. The clones analysed from TGAC-B1 and those from cpX had gene duplications of identical sizes showing the same restriction enzyme patterns. Our results suggest that the cpBVDV which finally lead to "late-onset" MD arose by recombination and/or by mutations of the cpBVDV used for superinfection.

Evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs.

A workshop was convened, at which seven enzyme-linked immunosorbent assays (ELISAs) were compared with virus isolation for the detection of viraemia in serial blood samples collected from six pigs at up to fourteen days after inoculation with classical swine fever virus. All ELISAs were of the double antibody sandwich type, using monoclonal and/or polyclonal antibodies to detect a variety of viral proteins in leukocytes, or in anti-coagulated blood or serum. Compared to virus isolation, specificity of the ELISA was good: only one sample found negative by virus isolation yielded a positive result in a single ELISA. Some false-negative results occurred with samples collected at up to eight days after inoculation, but all tests found samples collected between nine and fourteen days post-inoculation to be positive. The ELISAs require less-specialised facilities and can be performed much more rapidly than virus isolation. They are therefore extremely promising tools for screening large numbers of live pigs.

Brain malformations in ovine fetuses associated with the cytopathogenic biotype of bovine viral-diarrhoea virus.

A total of six ewes were intravenously inoculated at between 65 and 68 days of gestation with the Indiana strain of bovine viral diarrhoea virus (BVDV), containing both non-cytopathogenic (ncp) and cytopathogenic (cp) biotypes. Eight transplacentally infected fetuses were sequentially removed from the infected ewes and were found to have inflammatory lesions and malformations of the brain. BVDV RNA was isolated from formalin-fixed, paraffin-embedded brain tissue sections and detected by nested polymerase chain reaction after reverse transcription. The two biotypes of BVDV were distinguished by the fact that a sequence insertion in the RNA of the cp biotype of the inoculum results in larger amplicons. Only RNA from cp BVDV was detected in three of the brains removed up to 14 days post-inoculation (p.i.), and no BVDV RNA was detected after more than 14 days p.i. These findings suggest that, in critical phases of development, cp BVDV may transplacentally infect the ovine fetal brain and cause malformations.

Pathomorphological and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity.

Experimental infection of nine cattle with seven rinderpest virus strains of different pathogenicity resulted in significant variations of clinical signs, morphological lesions and distribution of viral antigen in tissues. The severity of clinical disease was correlated with the extent of tissue alterations and the amount of immunohistologically detectable viral antigen. Both mild and virulent strains of rinderpest share essentially the same tissue tropisms in vivo, i.e. epithelio- and lympho-tropism. However, rinderpest virus isolates of higher pathogenicity showed a more rapid and wider distribution with more extensive lesions than milder strains, which probably accounts for the higher mortality.

Characterisation of morbilliviruses isolated from Lake Baikal seals (Phoca sibirica).

Sequence analysis of the haemagglutinin protein (H) gene of the morbillivirus (PDV-2) isolated from a Siberian seal (Phoca sibirica) during the 1987/1988 epizootic in Lake Baikal revealed that it was most closely related to two recent isolates of canine distemper virus (CDV) from Germany and different from CDV vaccines currently in use in that region. The virus continued to circulate in seals in Lake Baikal after the 1987/1988 epizootic since sera collected from culled seals in the spring of 1992 were positive in morbillivirus ELISA tests, reacting most strongly with the CDV antigen.

Spot synthesis of overlapping peptides on paper membrane supports enables the identification of linear monoclonal antibody binding determinants on morbillivirus phosphoproteins.

In order to map antigenic domains on the P-protein of morbillivirus, a series of overlapping peptides, representing the P-protein sequences of phocid distemper virus strain 2558/Han88 and canine distemper virus strain Onderstepoort, were synthesized on a paper support by the spot-technique. The reactivity of six monoclonal antibodies with the peptides was tested in an enzyme immunoassay and compared to their reactivity in Western blots and in an ELISA using detergent extracts from virus-infected cells. Three linear determinants could be localized on the P-protein. Two antibody-binding sites were delineated within the C-terminal (between amino acids 307-322 and 382-400, respectively), and a third one was located on the N-terminal part (amino acids 13-31) of the protein. Fine mapping of this binding site revealed that this was a part of an antigenic domain. In Western blots, the monoclonal antibodies reacting with this domain also reacted with a second protein which was possibly the V-protein.

Editing of morbillivirus P gene transcripts in infected animals.

RNA editing in the Morbillivirus genus in vivo was investigated by applying a polymerase chain reaction-based primer extension technique to measure the edited and non-edited mRNA transcripts. In this genus of the Paramyxoviridae the P gene transcript is altered by the co-transcriptional addition of one extra G residue to produce the mRNA for the V non-structural protein. Using tissues of phocine distemper virus (PDV) infected seals, canine distemper virus (CDV) infected dogs and rinderpest virus (RPV) infected cattle, it was demonstrated that editing occurs in vivo. The P:V mRNA ratios were generally similar to those found in tissue culture infections with the same virus and a minor fraction of transcripts had 2-4 extra G residues. In one seal brain infected with PDV the ratio of P:V mRNA was reversed but no differences were found in the levels of mRNA editing in different tissues from the same animal infected with CDV or RPV. However, variation was seen between animals infected with different isolates of RPV and even between animals infected with the same isolate of RPV.

[Epidemiological characterization of a pestivirus isolate from a viremic pig out of a mixed pig-cattle herd]. / Epidemiologische Charakterisierung eines Pestivirus-Isolates von einem virämischen Schwein in einem gemischten Schweine-Rinderbestand.

A pestivirus isolated from a healthy pig out of a mixed pig-cattle holding was identified by use of monoclonal antibodies as a porcine virus related or identical to bovine viral diarrhoea (BVD) virus. About 7% of the pigs of this herd showed neutralizing antibodies (nab) against BVD and Border Disease (BD) virus and against the homologous porcine nonclassical swine fever (CSF) pestivirus isolate 10421/Han94. The nab titres against this virus were clearly higher than against CSF virus strain Alfort/187. Amongst the cattle kept in the farm no BVD viraemic animal was detected. About 13% of them showed nab to BVD virus with higher titres against the BVD virus strain NADL than against the porcine pestivirus virus isolate 10421/Han94. There was no of a BVD virus transmission from the cattle to the pigs within this herd. The problem of misinterpretation of non-CSF virus isolation from pigs is discussed. The necessity of identifying pestiviruses of porcine origin by use of species-specific monoclonal antibodies is stressed in order to prevent erroneous declarations of CSF outbreaks.