Multiple neural cell types are infected in vitro by border disease virus.

Border disease (BD) of sheep results from a congenitally acquired nonarbotogavirus infection which causes a highly selective central nervous system (CNS) pathological lesion consisting of diffuse decreased myelination without inflammation or neuronal destruction. Thus, a selective disruption of oligodendroglial function appears to occur. In order to investigate the in vitro cell tropism of BD virus, primary cultures derived from fetal and adult ovine CNS and peripheral nervous system were inoculated with BD virus. Infected cell types were determined by dual immunofluorescent labeling for viral and cell type specific antigens. Infection of all the major cell types represented in these cultures, including oligodendrocytes, astrocytes, fibroblasts, dorsal root ganglion neurons and Schwann cells was found. Oligodendrocytes were only infected earlier and appeared to remain infected longer than astrocytes and fibroblasts. Infectious virus was produced by all cultures and continued to be produced even after the disappearance of nearly all immunocytochemically detectable viral antigen within cells. These studies suggest that the selective dysfunction of the oligodendrocyte in BD is not based on a selective viral tropism.

Intracellular virus-induced polypeptides of pestivirus border disease virus.

Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness.

An ELISA for detecting pestivirus antigen in the blood of sheep persistently infected with border disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (ELISA) has been developed to detect a pestivirus-specific antigen in leucocytes of sheep persistently infected with border disease virus. A blind trial was conducted to compare the specificity of the ELISA with conventional tissue culture virus isolation on blood samples from 58 sheep, aged 3 to 48 months. There was total agreement between the two tests; 27 sheep were shown to be BDV-infected. The ELISA OD values of the positive samples ranged from 0.12 to 0.86 and were not related to age, strain of virus with which they were infected or presence of serum neutralising antibody. Negative samples had OD values between 0 and 0.02.

Characterization of porcine and some ruminant pestiviruses by cross-neutralization.

Serologic relationships between 11 pestivirus strains that originated from pigs and five that originated from cattle or sheep were studied by cross-neutralization. Experiments were performed with pig and sheep sera raised against the strains. The results were analysed by a computerized taxonomic procedure. The 16 viruses were classified into four distinct serologic groups. All hog cholera virus (HCV) strains were classified in one group; the other three groups consisted of strains that can infect pigs, but that are identified as bovine viral diarrhoea virus (BVDV) or border disease virus (BDV), or showed a closer relationship to BVDV and BDV than to HCV.

The production and survival of lambs persistently infected with border disease virus.

From 1985 to 1989 lambs persistently infected with border disease virus (BDV) were produced for comparative immunological studies by infecting 57 susceptible pregnant ewes between 50 and 60 days' gestation with Moredun or Oban strains of BDV. Ewes were infected either by injection with virus grown in cell culture or by housing with lambs excreting BDV. There was no significant difference in the outcomes of these different methods of infection. There was a significant difference in the number of viable lambs born to ewes receiving the two viruses. Of 41 ewes infected with Moredun virus 21 produced 32 live lambs of which 17 were reared to 1 month old (53% viability). Of 16 ewes receiving Oban virus 10 gave birth to 17 live lambs of which 15 were reared to 1 month old (88% viability). All the lambs born to ewes infected with Moredun BDV had varying signs of tremor and increased hairiness ("hairy-shakers") while those born to ewes infected with the Oban virus had no obvious clinical signs. Survival of the lambs was poor. Up until February 1991, 14 Moredun and 10 Oban sheep between the ages of 4 months and 5.5 yr had died from a variety of causes. The two commonest causes were a chronic wasting syndrome and a mucosal disease-like syndrome which was associated with the recovery of cytopathic BDV. Mating of unrelated persistently infected sheep was largely unproductive although 2 lambs were reared.

Variable nature of border disease on a single farm: clinical and pathological description of affected sheep.

The clinical, pathological and virological findings in cases of Border disease occurring on a farm were studied. Fourteen abnormal lambs approximately three months old were obtained and were necropsied within 18 months at death or slaughter. Another lamb born during the study to a persistently infected ewe was also examined. The findings of these 15 animals are presented in three groupings. The animals of group A were persistently viraemic and hypomyelinogenesis was present in two lambs at one and 14 weeks old. Systemic, chronic, multifocal inflammation including nephritis, myocarditis and pneumonitis was found in older sheep. Group B lambs were non-viraemic and the predominant necropsy finding was cystic cerebral cavitation of variable severity. Histological examination showed cerebellar dysplasia and in two animals the retinas also showed multifocal photoreceptor cell atrophy. Lambs in group C were also non-viraemic and although these failed to thrive, no consistent pathological changes were found. It is concluded that the dams of animals in group A were infected in early pregnancy while those of animals in groups B and C were infected at mid-gestation or later.

Variable nature of border disease on a single farm: the infection status of affected sheep.

Study of the virological and serological status of abnormal sheep derived from a single outbreak of Border disease indicated a relationship between infection status and diverse pathological lesions. Persistent infection was associated with typical Border disease whereas an active, even exaggerated, serological response correlated with severe intracranial malformations which probably resulted from fetal infections occurring during the early stages of acquisition of immune responsiveness.

Effects of experimental infection with border disease virus on lymphocyte subpopulations in the peripheral blood of lambs.

Experimental infection with Border disease virus was characterised by significant changes in the total numbers of leucocytes and neutrophils, and in the proportions and numbers of the different lymphocyte subpopulations. Three days after experimental infection there was significant leucopenia due to lymphocytopenia and neutropenia (P < 0.001). The lymphocytopenia and neutropenia lasted for up to seven days after inoculation. The lymphocytopenia was due to a reduction in the number of both T cells and B cells. During the early period of infection, the reduction in T cells was mainly due to a reduction in the number of OvCD4+ and T-19+ lymphocytes as the number of circulating OvCD8+ cells was not significantly affected. The cells expressing the OvCD4 and OvWC1 epitopes returned to pre-inoculation values 10 and 14 days after inoculation, respectively. In contrast, during the same period, the number of T cells expressing the OvCD8 molecule became significantly higher than the corresponding pre-inoculation values. There were no significant changes in all the T cell subsets in the control lambs.

[Asymptomatic carriage of Pestivirus in ruminants]. / Le portage asymptomatique des Pestivirus chez les ruminants.

Pestiviruses are enveloped single-chain ribonucleic acid viruses with a positive polarity. Pestiviruses include the viruses of classical swine fever (hog cholera), Border disease of sheep, mucosal disease of cattle, and isolates obtained from wild animals, such as red deer (Cervus elaphus). Among ruminants, pestiviruses have developed a remarkable strategy for assuring their persistence. Through epigenetic transmission, they lead to the birth of asymptomatic carrier animals harbouring non-cytopathic variants, which become immunotolerant to the strain of virus present. The presence of a small number of asymptomatic carriers enables the virus to circulate within a herd by horizontal transmission, leading to the birth of a new generation of asymptomatic carriers.

Genetic comparison of ovine and bovine pestiviruses.

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.

Some antigenic and cultural properties of border disease and bovine viral diarrhoea viruses.

In vitro comparison of the replication cycles of three strains of border disease virus and one strain of bovine viral diarrhoea virus showed similar growth curve patterns and a tendency of cell-free virus to exceed cell-associated virus. Each virus was antigenically distinguishable from the others in cross-neutralisation tests.

Cytopathic and non-cytopathic biotypes of border disease virus induce polypeptides of different molecular weight with common antigenic determinants.

Ten monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non-cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent Mr 80,000 and 130,000 from cells infected with cytopathic virus and a single polypeptide of apparent Mr 120,000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.

Immunocytochemical localization of border disease virus in the spinal cord of fetal and newborn lambs.

The peroxidase-antiperoxidase technique was used to determine the cellular localization of Border Disease (BD) virus in cryostat sections of fetal and newborn lamb spinal cord following experimental infection by maternal inoculation in early gestation. Viraemic fetuses and lambs with hypomyelinogenesis showed BD viral antigen in neurons, glia, ependymal cells, vascular endothelial cells and fibrocytes within the dura mater. Double immunolabelling demonstrated co-expression of BD viral antigen and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) in both fetal and newborn lamb glia. In fetal lambs there was a pia-associated population of glia in which viral antigen was also co-expressed with GFAP or MBP. The results suggest that BD virus infects myelinating oligodendroglia, astroglia and probably also transitional cells and pluripotential glioblasts. The relationship between infection of specific cell types and hypomyelinogenesis was not resolved but infection of transitional cells and oligodendroglia may affect oligodendroglial function and permit morphologically inapparent perturbations leading to hypomyelinogenesis. A single nonviraemic lamb with a precolostral antibody titre to BD virus and cystic cerebral cavities but no hypomyelinogenesis showed BD viral antigen confined to glia of the spinal cord white matter. This suggests that oligodendroglia may require to be infected before a critical period in their development or factors additional to oligodendroglia infection are necessary for hypomyelinogenesis.

The ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses.

The morphology of border disease virus and bovine virus diarrhoea virus in infected bovine embryonic testis cells was examined by electron microscopy. Particles which appeared to be mature virions of both viruses were similar, being roughly circular and approximately 46 nm in diameter with a 20 to 25 nm core. Virus replication took place totally within the cytoplasm in association with structures formed from modified endoplasmic reticulum.

Protection of piglets born from ruminant pestivirus experimentally infected sows, and their contacts, to the challenge with hog cholera virus.

Two groups, A and B, of two specific pathogen-free pregnant sows were experimentally infected between the 25th and 29th days post-breeding with two strains of ruminant pestivirus: NADL cytopathic bovine viral diarrhoea virus for group A and Aveyron non-cytopathic border disease virus French strain for group B. Two other pregnant sows (group C) were kept uninoculated as control. When 7 weeks old, 8 piglets of group C were put in contact with 4 piglets of group A (group D), and 8 other piglets of group C with 4 piglets of group B (group E) in two separate pens with the purpose of testing the horizontal transmission of the viruses. All animals were kept under observation and serologically controlled at weekly intervals; two pigs of each group were finally submitted to a challenge with hog cholera virus. The two pigs of group E which were put in contact with the offspring of the border disease virus infected sow were protected; all other animals developed typical hog cholera symptoms and died. The relation between neutralizing titres of the sera to ruminant pestiviruses and protection to the challenge with hog cholera virus is discussed. The two protected pigs had high neutralizing antibody titres to border disease virus but no antibody to hog cholera virus at the time of the challenge. Though the two viruses look serologically distant, we surprisingly observed that infection with border disease virus protects against a superinfection with hog cholera virus.

Border disease. Virus-induced decrease in thyroid hormone levels with associated hypomyelination.

Border disease (BD) was induced in lambs by inoculation of their dams at 50 days gestation with Border disease virus (BDV) isolate #31. At birth, the clinically affected lambs had diffuse spinal cord hypomyelination, confirmed by immunocytochemical staining for myelin-associated glycoprotein and myelin basic protein. In the BD lambs, large numbers of thyroid follicular epithelial cells and scattered pituitary cells contained BDV antigen by immunofluorescence staining. Double labeling techniques demonstrated the BDV-infected pituitary cells to contain growth hormone, adrenocorticotrophic hormone, prolactin, or luteinizing hormone. Cells containing thyroid stimulating hormone were rare and were not positive for BDV antigen. Infection of the pituitaries and thyroid glands caused no detectable morphologic changes as compared to controls. The BD lambs had statistically significantly (p less than 0.05) lower mean serum concentrations of thyroxine and L-3,3',5-triiodothyronine as compared to age-matched uninfected controls. Similar significant differences in the mean plasma levels of growth hormone and thyroid stimulating hormone were not found. In addition, the BD lambs had a statistically significant (p less than 0.05) lower mean activity of the myelin-associated, thyroid hormone dependent enzyme, 2',3'-cyclic nucleotide-3'-phosphodiesterase in spinal cord tissue. Although not conclusive, these results indicate that the hypomyelination in BD may be due to depressed levels of circulating thyroid gland hormones secondary to a noninflammatory and noncytolytic infection of the thyroid gland by BDV. This is one of the first reports indicating that a virus-induced hormonal alteration may cause a congenital lesion in animals.

Viral persistence and abnormalities of the central nervous system after congenital infection of sheep with border disease virus.

Lambs congenitally infected with border disease (BD) virus and sheep exposed to BD virus as adults were studied for one year to determine the pathogenesis of congenital exposure compared with adult exposure to the virus. Persistent BD virus was isolated in tissue culture and detected by immunofluorescence of the peripheral white blood cells, urine, and cerebrospinal fluid of lambs with congenital BD up to one year of age. These animals had no detectable serum neutralizing antibody response to the virus for the same interval. BD virus antigen was also detected by immunofluorescence in many central nervous system tissues of these lambs with congenital BD. Dysmyelination and glial proliferation in the central nervous system and microencephaly were noted in the lambs with congenital BD, and these lesions appeared to remain the same over a 12-month period.