Pestivirus Infection Research Articles

Immunohistochemical diagnosis of pestivirus infection associated with bovine and ovine abortion and perinatal death

SUMMARY Objective To establish a reliable, rapid, economical method for detection of pestivirus infection in bovine and ovine fetuses and to examine participation of these viruses in abortions and neonatal mortality. Animals 213 bovine and 31 ovine fetuses, as well as 36 newborn calves and 25 lambs, which had died within 3 days after birth, were tested for bovine viral diarrhea virus (BVDV) and border disease virus by use of different methods. Procedure Detection of BVDV in fetuses was performed by immunohistochemical methods, using a panel of monoclonal antibodies against pestivirus antigens on cryostat and paraffin sections and by virus isolation in cell culture; in some instances, an antigencapture ELISA was performed. Results of the various methods were compared. Results Sensitivity of BVDV detection by immunohistochemical methods and virus isolation in cell culture was equal; however, it decreased in association with autolysis. In autolytic fetuses, use of formalin-fixed, paraffin-embedded brain sections was the most favorable method. Antigen detection by ELISA was less sensitive. Conclusions Immunohistochemical analysis of cryostat sections of brain, skin, thyroid gland, abomasum, and placenta is a rapid, sensitive method for detecting pestiviruses in fetuses. In the presence of advanced autolysis, this method used on formalin-fixed, paraffin-embedded brain sections is recommended over the other described methods. (Am J Vet Res 1997;58:1371–1375)

Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors.

Pure preparations of envelope glycoproteins E(rns) and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV). Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 microg/ml E(rns). After removal of the virus no E(rns) was needed in the overlay medium (growth medium) to maintain this level of inhibition. In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 microg/ml E2 was only achieved when E2 was added to the overlay medium. When E2 was omitted, a maximum of 50% inhibition was achieved. This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface. Treatment with 100 microg/ml E(rns) released these particles from the cell surface. Furthermore, E(rns) bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by E(rns). These results demonstrated that E(rns) and E2 interacted with different cell surface receptors. Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor. BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV E(rns), whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of E(rns) plays a role in host tropism.

The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log 10 TCID 50/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 ±2.31, of which 4.00 ± 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 ±0.34 ova/embryos, of which 0.23 ± 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.

Immunology of Bovine Pestivirus Infection

The bovine pestiviruses (here referred to as bovine viral diarrhea viruses-BVDV) cause three types of disease: bovine viral diarrhea (BVD), mucosal disease (MD) or fetal disease (FD). In each case, the immune system of the host responds in a characteristic fashion (Nettleton and Entrican, 1995).

Swine and ruminant pestiviruses require the same cellular factor to enter bovine cells.

Pestiviruses initiate infection of susceptible cells by receptor-mediated endocytosis. Cellular plasma membrane or endosomal molecules involved in translocation of these viruses into the cytosol have not been unequivocally identified. We reported previously that a mutant cell line derived from Madin-Darby bovine kidney (MDBK) cells, termed CRIB-1, was resistant to infection with bovine viral diarrhoea virus. CRIB-1 cells were also resistant to infection with classical swine fever virus and border disease virus of sheep, suggesting that entry of these three different pestiviruses into bovine cells requires a common cell membrane function. The resistance is pestivirus-specific: CRIB-1 cells were as susceptible as the parental MDBK cells to 14 other viruses of cattle and swine belonging to unrelated families. The resistance of CRIB-1 cells to pestivirus infection involves a block in virus entry since transfection of virus RNA or virus inoculation in the presence of PEG resulted in productive infection. Furthermore, quantitative analyses of the outcome of PEG-mediated infection of CRIB-1 cells indicated that the intracellular milieu was fully permissive for pestivirus replication. Binding studies revealed that virus attachment to CRIB-1 cells was not completely abrogated. These results indicate that entry of pestiviruses into MDBK cells depends on a common plasma membrane or endosomal function, which is lacking in CRIB-1 cells.

The outcome of widespread use of semen from a bull persistently infected with pestivirus

During the certification of the bulls at an artificial breeding centre for freedom from pestivirus infection, a single viraemic bull was identified, and further testing confirmed that it was persistently infected. The two-year-old bull was healthy and of similar bodyweight to its peers. Its semen was of normal quality on the basis of density, motility and morphological criteria. Approximately 600 doses of semen had been distributed for sire evaluation purposes to 97 dairy farms. An examination of the breeding records indicated a first service conception rate of 38 per cent. All but one of the 162 cows inseminated with the bull's semen were seropositive compared with 95 of 143 cows (66.4 per cent) inseminated with semen from other bulls. Virological studies of the 61 calves sired by the persistently infected bull revealed that two were persistently infected, but that the others were healthy and uninfected. It was concluded that the semen from this bull was a potential source of pestivirus infection for 'clean' herds.

Arachidonic acid immunoregulation in lambs persistently infected with border disease virus

To evaluate arachidonic acid-related immunoregulatory mechanisms during long-term persistent pestivirus infection, we measured plasma contents of leukotriene C4 (LTC4), prostaglandin D2 (PGD2) and their plasma fatty acid (FA) precursor, arachidonic acid (AA), in six lambs with congenital border disease (BD). Significantly elevated average plasma LTC4 during the first half year of life was associated with increased PDG2 when compared to uninfected control lambs. Significantly elevated total plasma esterified AA stores suggest an effective BDV-mediated prostenoid immunostimulation. However, at 1 yr old, prostenoid secretion had fallen to normal (LTC4) or below normal (PGD2) levels. In contrast, there remained significantly elevated plasma esterified AA, present as available substrate for formation of these anti-viral immunoregulatory agents. These results suggested that preventing mobilization of AA from lipid stores for effective immune responses may be a viral strategy of BD virus that is associated with long term border disease effects.

Pestivirus infection in identical twins discordant for schizophrenia

Pestivirus infection in identical twins discordant for schizophrenia

Pestivirus infection of ruminants in Australia.

Pestivirus infections are commonly diagnosed in cattle but are relatively uncommon in other ruminant species in Australia. Virus isolation is a very reliable technique for detecting pestivirus in specimens, especially when group reactive monoclonal antibodies are used with immunoperoxidase staining to detect non-cytopathogenic virus. Care must be taken to prevent adventitious pestivirus contamination of serum or cells used for cell culture. A recently developed antigen capture enzyme-linked immunosorbent assay has been extensively evaluated and found to be extremely accurate. This test is also much quicker and less expensive than virus isolation. Procedures are outlined to reliably certify animals to be free of pestivirus infection for export or as donors of semen or embryos.

Pestivirus infection in ruminants in Norway

Serological surveys in Norway have demonstrated neutralising antibodies against bovine virus diarrhoea (BVD) virus in cattle, sheep and goats. The prevalences were 18.5%, 4.5% and 3.6%, respectively. Occurrence of pestivirus-induced disease in Norway is described. Outbreaks of reproductive failure and mucosal disease have been reported, and the number of persistently-infected animals detected has increased considerably in recent years. Acute BVD occurs rarely. Border disease (BD) in sheep, first diagnosed in 1981, has subsequently been demonstrated sporadically. In goats, typical BD was diagnosed in 1982, with three later occurrences of reproductive failure. Experimental infections in pregnant goats induced a high rate of severe foetopathogenic effect. Signs and lesions in offspring were comparable to ovine BD. Similar findings were demonstrated in goats given a pestivirus-contaminated vaccine. In newborn kids, experimental infection had an adverse influence on growth and health. Persistent infection in goats is probably rare.

Induced sero‐conversion in heifers with a field strain of bovine pestivirus–a comparison of methods and doses

Losses from pestivirus infection in a closed herd of cattle occurred over several years. In order to prevent further losses, controlled exposure of non-pregnant heifers to pestivirus from viraemic carrier animals was undertaken. Two initial experiments were conducted using either intra-nasal EDTA blood or field contact. Subsequently, other yearling heifers were inoculated with various dilutions of serum using subcutaneous, conjunctival and intra-nasal routes. Effective doses were determined. Neither inoculation nor contact infection produced any clinical illness. The highest dilutions of serum at which sero-conversion occurred were conjunctival, undiluted; intranasal, 10(-1) and subcutaneous 10(-5). With the subcutaneous route all heifers sero-converted at 10(-3). The results for the subcutaneous inoculations showed that the 50% infectious dose for cattle was not distinguishable from that determined in cell culture. Inoculation with a field strain of pestivirus in freeze-thawed serum has effectively and safely induced sero-conversion in heifers. Inoculation of all cattle at risk is considered necessary because no secondary transmission from inoculated heifers was observed.

Pestivirus infections in Norway. Epidemiological studies in goats

During one breeding season, 2335 female goats in 39 herds in different parts of Norway were examined for pestivirus infection and for reproductive performance. Before breeding, all animals were examined for neutralizing antibodies against the NADL strain of pestivirus, 83 (3.6 per cent) positive animals in 12 herds being demonstrated. The herd prevalences ranged from 1 to 63 per cent. Antibody titres varied from 1 in 4 to 1 in 2048. Of 1816 females in 30 herds for which post-breeding information was available, a total of 178 (9.8 per cent) animals in 25 of the herds demonstrated gestation failure. Three of these goats began to produce antibodies against the NADL strain during gestation. Sera from the 83 animals with NADL antibodies were titrated for neutralizing antibodies against three additional strains of pestivirus, the highest geometrical mean titre being found for antibodies against a Norwegian bovine strain.

The impact of pestivirus on an artificial breeding program for cattle

After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial insemination program, substantial post-weaning calf losses occurred. The predominant clinical feature was severe respiratory disease, in contrast to the commonly recognised mucosal disease. Thirty-one of 76 calves were affected, with a case fatality rate of 58%. All calves which were persistently infected with pestivirus died during the study period. There was a significant association in the surviving calves between the occurrence of recent pestivirus infection and respiratory disease. The losses on this property clearly indicate the need to routinely screen animals in an artificial breeding program for freedom from pestivirus infection.

Incidence, epidemiology and control of bovine pestivirus infections and disease in Australia and New Zealand

Pestivirus infection of cattle is widespread and common in both Australia and New Zealand. The majority of adult animals, of the order of 60%, carry antibody. Associated disease is almost entirely that resulting from infection in utero. This includes death of the conceptus, at any stage from conception through pregnancy, or, in those which are born as persistently infected carriers, mucosal disease, most commonly in a chronic form. Little or no disease is recognised as a result of the post-natal infection of non-pregnant animals and these appear to be of little consequence as spreaders of infection. Transmission and enzootic maintenance depend primarily on the persistently infected carriers that are immunotolerant after early in utero infection and range clinically from normal, or nearly normal, to overtly mucosal diseased. The expulsion of an infected conceptus, and associated discharges, also provides an effective source of infection. There is generally little active control attempted. Vaccines are not available in Australia and are not widely used in New Zealand. However, interest in control is growing in those areas of the industry, especially in breeding by artificial insemination and embryo transfer, where it is perceived that the pathogenic impact of the virus may be amplified.

Ruminant pestivirus infection in pigs

Ruminant pestivirus infections of pigs have a worldwide distribution. The prevalence is varied and depends mainly on (i) contact with cattle, (ii) age of pigs and (iii) degree of homology of virus strains used for serology, with field strains of bovine virus diarrhoea virus (BVDV) infecting pigs. Emphasis should be laid on sources of BVDV other than cattle, e.g. contaminated vaccines and fetal calf serum. The need for differentiation of pestiviruses (hog cholera, bovine virus diarrhoea and Border disease viruses) is highlighted by the fact that clinical disease syndromes, e.g. growth retardation and wasting, are reminiscent of hog cholera. Monoclonal antibodies are available which differentiate between hog cholera virus (HCV) and ruminant pestiviruses, presumably BVDV. An up-to-date account of the antigenic relationship between pestiviruses is included in the review. Analysis of the in vitro host range of these viruses is considered to be important and may explain infections of pigs with pestiviruses other than HCV. Recent results have shown the existence of "specialists" amongst BVDV strains for bovine cells, and a few isolates also performed well in cultures of the PK15 cell line. In contrast, multipotent BVDV strains presumably have additional attachment sites for ovine and porcine cells. Identification of receptors on ovine and porcine cells could contribute to a clear distinction between BVDV and HCV infections of pigs. Immediate control measures for BVDV infections of pigs are not required. However, such infections may interfere with serologic surveys and surveillance on a herd basis and, therefore, impair eradication programmes and efforts to maintain the status in countries declared free of hog cholera.

INFANTILE GASTROENTERITIS ASSOCIATED WITH EXCRETION OF PESTIVIRUS ANTIGENS

Faeces from children under 2 years old who had gastroenteritis that could not be attributed to recognised enteric pathogens were examined with a monoclonal-antibody-based immunoassay for Pestivirus antigens. Such antigens were detected in 30 of 128 episodes of gastroenteritis. Children without diarrhoeal disease and children infected with rotaviruses had little evidence of Pestivirus infection (faeces positive in 1 of 28 and 1 of 31, respectively). The diarrhoeal disease in children excreting Pestivirus antigens resembled that in other children except that it was more commonly associated with signs and symptoms of respiratory inflammation.

Bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine

On eight farms a congenital pestivirus infection in piglets was detected which could be traced to vaccination of the dams against swine fever (SF). Viruses isolated from the piglets were not recognised by monoclonal antibodies (MCAs) against swine fever virus (SFV) and were shown to be bovine viral diarrhoea virus (BVDV) or Border disease virus. The expected 'Chinese' strain of SFV could not be demonstrated in the batch of vaccine that had been used on these farms. Instead, a contaminating pestivirus was recovered which was not recognised by the MCAs against SFV. The contaminant had an unexpectedly high virulence for lambs and induced antibodies against BVDV in lambs and pigs. It could, therefore, be characterised as BVDV or Border disease virus.

BVD virus infection: prospects for control.

Losses resulting from pestivirus infections in cattle are of considerable importance to the livestock industry yet, until relatively recently, they have been poorly understood. The escalation of research effort in this field has started to clarify the pathogenesis and epidemiology of the associated diseases but the evolution of understanding is far from complete. Sufficient information exists to indicate the means by which pestivirus infection is introduced to and maintained in populations of cattle. A brief review of current knowledge is given placing particular emphasis on the role played by persistent infections which have resulted from intrauterine infection. The crux of the problem of control is seen to be the avoidance of fetal infection in early gestation. Ways of achieving this which include the immunisation of female cattle with live or inactivated virus vaccines are discussed and areas requiring further work are indicated.

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.