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Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide., Competing Interests: The authors declare no conflict of interest.

Bovine viral diarrhoea virus (BVDV) causes persistent infections by infecting the fetus of susceptible animals during gestation. These persistently infected (PI) animals are important sources of infection. On the contrary, transiently infected (TI) animals are believed to be less important, but transient infections with a severe BVDV-2 strain can spread explosively. To assess the importance of TI cattle in the epidemiology of BVDV, two experimental infections were performed to determine basic reproduction ratios (R0). In each experiment three calves were infected via intranasal inoculation and housed together with seven susceptible animals. Two strains isolated in Belgium were used, a virulent BVDV-1b and a virulent BVDV-2a field isolate, resulting in an R0 of 0.25 (95% CI 0.01; 1.95) and 0.24 (95% CI 0.01; 2.11), respectively. A PI animal was then introduced to the remaining uninfected animals and produced an R of +∞ (95% CI 1.88; +∞). These results support the suggestion that TI animals, compared to PI animals, contribute only a limited amount to BVDV spread. Additionally, the severe clinical symptoms observed in the field with these isolates could not be reproduced during these experiments, suggesting that other factors besides strain virulence influence the clinical manifestations evoked by BVDV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

The seroprevalence of bovine viral diarrhea virus (BVDV) infection in yaks was investigated in Qinghai and Tibet of China during the year 2011. A total of 549 (Tibet 287, Qinghai 262) serum samples was collected from Tibet and Qinghai and were examined for BVDV p80 antibody by ELISA. The results of the experiment showed that 145 (53.65 %) of Tibetan samples and 189 (72.14 %) of Qinghai's samples were positive for BVDV. The observations of the present study suggest that bovine viral diarrhea is common in yaks in Tibet and Qinghai, China.

Enterocytozoon bieneusi known as a causative agent of opportunistic infections instigating diarrhoea in AIDS patients was identified also in a number of immunocompetent patients and in a wide range of animals, including cattle. In the present study we tested if the Bovine Viral Diarrhea Virus (BVDV), the most common pathogen underlying immunosuppressive Bovine Viral Diarrhoea (BVD), can enhance the occurrence of opportunistic infections with E. bieneusi in cattle. Six dairy farms were investigated using ELISA to detect antibodies against or antigens arising from BVDV in collected sera. A total of 240 individual faecal samples from four age groups were examined for the presence of E. bieneusi by nested PCR. Sequence analysis of six E. bieneusi positive samples revealed the presence of the genotype I of E. bieneusi, previously described in cattle. The hypothesis expecting higher prevalence of E. bieneusi in BVDV positive cattle herds was not confirmed in this study; however this is the first description about E. bieneusi in cattle in the Czech Republic., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: The goal of this study was to determine whether calves persistently infected with bovine viral diarrhea virus (BVDV) and inoculated with Escherichia coli O157 will shed the organism at a higher concentration and for a longer duration than calves not infected with BVDV., Materials: Nine calves, 6-8 weeks old, persistently infected with noncytopathic BVDV and eight calves not infected with BVDV obtained from separate cow-calf operations were used in this study. BVDV status, positive or negative, of all calves was confirmed by repeated testing throughout the study. Both groups were orally inoculated with 10(9) colony-forming units (CFU) of five nalidixic acid-resistant strains of E. coli O157. All calves were examined daily, and fecal samples were collected three times a week for 55 days for detection and enumeration of the nalidixic acid-resistant E. coli O157. Calves were then necropsied, and samples from the gastrointestinal tract were taken for the detection of the nalidixic acid-resistant E. coli O157., Results: Data analysis indicated no statistical difference in the concentration of E. coli O157 shed or the duration of shedding between the persistently infected BVDV calves and the control calves throughout the length of the study., Conclusions: Our data indicate that persistent infection with noncytopathic BVDV does not play a role in the level or duration of shedding of E. coli O157 in cattle.

The primary purpose of this research was to determine associations among seropositivity for bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV), Mycobacterium avium ssp. paratuberculosis (MAP), and Neospora caninum (NC) and each of 3 outcome variables (305-d milk, fat, and protein production) in Canadian dairy cattle. Serum samples from up to 30 randomly selected cows from 342 herds on monthly milk testing were tested for antibodies against BLV (IDEXX ELISA; IDEXX Corporation, Westbrook, ME), MAP (IDEXX or Biocor ELISA; Biocor Animal Health, Inc., Omaha, NE), and NC (IDEXX or Biovet ELISA; Biovet Inc., St. Hyacinthe, Quebec, Canada). Up to 5 unvaccinated cattle over 6 mo of age were tested for virus-neutralizing antibodies to the Singer strain of type 1 BVDV. Dairy Herd Improvement records were obtained electronically for all sampled cows. Linear mixed models with herd and cow as random variables were fit, with significant restricted maximum likelihood estimates of outcome effects being obtained, while controlling for potential confounding variables. Bovine leukemia virus seropositivity was not associated with 305-d milk, 305-d fat, or 305-d protein production. Cows in BVDV-seropositive herds (at least one unvaccinated animal with a titer > or =1:64) had reductions in 305-d milk, fat, and protein of 368, 10.2, and 9.5 kg, respectively, compared with cows in BVDV-seronegative herds. Mycobacterium avium ssp. paratuberculosis seropositivity was associated with lower 305-d milk of 212 kg in 4+-lactation cows compared with MAP-seronegative 4+-lactation cows. Neospora caninum seropositivity in primiparous cows was associated with lower 305-d milk, fat, and protein of 158, 5.5, and 3.3 kg, respectively, compared with NC-seronegative primiparous cows. There were no interactions among seropositivity for any of the pathogens and their effects on any of the outcomes examined, although the low MAP seroprevalence limited this analysis. Results from this research will contribute to understanding the economic impacts of these pathogens and justify their control.

Thirty-three pestiviruses isolated from bovines on different farms in Chile were characterized at the molecular level. The 5'-untranslated region (5'UTR) of the isolates was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently digested with restriction enzymes (RE) Bgl I, Xho I and Pst I. Furthermore, the isolates were amplified by differential RT-PCR, which selectively amplified bovine viral diarrhea virus type 2 (BVDV-2) but not bovine viral diarrhea virus type 1 (BVDV-1). Of the 33 isolates, 16 were classified as BVDV-1 and 17 as BVDV-2. Phylogenetic analysis of the PCR-amplified fragments from 14 isolates showed the presence of viruses belonging to the BVDV-1a, BVDV-1b, BVDV-1c, and BVDV-2 types. Antigenicity of some viruses belonging to both genotypes was studied by cross-seroneutralization, revealing great antigenic differences among them. It is concluded that BVDV viruses circulating in Chile are genetically and antigenically heterogeneous, comprising isolates of genogroups 1 and 2.

Our objective in this cross-sectional study was to determine the seroprevalence in 1996 of commercial dairy herds in central Chile positive to bovine viral diarrhea (BVD) virus, by the detection of antibodies in bulk-tank milk samples. Samples were obtained from 501 dairy farms ( approximately 50,000 cows) from the Metropolitan, VI, VII and VIII regions of Chile. An indirect ELISA was used to detect BVD antibodies. An absorbance (A450) > or = 0.55 was considered positive. The apparent proportion of positive herds ranged from 71.2 to 83% by region and did not differ by region. We concluded that exposure to BVD was widely distributed in the central area of Chile in 1996.

To characterise the bovine virus diarrhoea virus (BVDV) isolates circulating in the southeastern region of Austria, namely in the province of Styria, 71 blood samples collected between 1998 and 2000 from persistently infected cattle in 62 herds were subjected to genetic typing. For this, 288bp fragments from the 5' untranslated region (5'-UTR) were amplified by polymerase chain reaction after reverse transcription (RT-PCR). The products were sequenced and used for phylogenetic analysis. Seventy virus isolates were typed as BVDV species 1 (BVDV-1). Only one isolate was typed as BVDV species 2 (BVDV-2), representing the first isolate of this pestivirus genotype found in Austria. In addition, phylogenetic analysis revealed that viruses belonging to five genetic groups within BVDV-1 are circulating in Styria. Most viruses (53) were found in group BVDV-1f, nine viruses in BVDV-1h, four viruses in BVDV-1b, three viruses in BVDV-1d and one virus in BVDV-1g. No virus was found in genetic group BVDV-1a, which is dominant in the UK and widely distributed in USA. Likewise, the BVDV isolates predominating in a neighbouring country, namely Germany, belonged to different genogroups than those circulating in Styria. We conclude that in a particular region and environment certain BVDV-1 genetic groups predominate. New groups, including BVDV-2, can be introduced, e.g. by trade of animals. The low incidence of BVDV-2 in Styria is in concert with the sporadic occurrence of these viruses in other regions of Europe.

Cytopathogenic and non-cytopathogenic bovine viral diarrhea viruses (BVDVs) were isolated from cattle with mucosal disease or persistent infection in Japan. These isolates were compared for antigenic properties by cross-neutralization tests with Japanese reference strains of BVDV belonging to classical type 1. Significantly low cross-reactivity to reference strains was noted, indicating the viruses to possibly represent a new serotype in Japan. Thus, to determine the genotype of the isolates, nucleotide sequences of the 5' untranslated region were determined and compared with those of previously reported BVDV 1 and 2. The isolates were clearly shown to belong to BVDV 2, not to BVDV 1.

Bovine viral diarrhea viruses (BVDV) are segregated into two genotypes, BVDV 1 and BVDV 2. Viruses within both genotypes may exist as one of two biotypes, cytopathic or noncytopathic. A highly fatal form of BVDV termed mucosal disease (MD) occurs when an animal persistently infected with noncytopathic BVDV becomes superinfected with cytopathic BVDV. In this study, we characterized a noncytopathic (BVDV2-125nc)/cytopathic (BVDV2-125c) viral pair isolated from an animal that died of MD 3 months after vaccination with modified-live BVDV1-NADL. In comparison to BVDV2-125nc, BVDV2-125c contained a 366-nucleotide insertion. The insertion was in the correct reading frame for the large open reading frame of the BVDV genome and occurred in the portion of the genome that codes for the p125 viral polypeptide. There was a 99% identity between the inserted sequences found in BVDV2-125c and sequences from the vaccine virus BVDV1-NADL. These data suggest that MD was induced after a recombination between noncytopathic BVD2-125nc and cytopathic vaccine virus BVDV1-NADL created the cytopathic virus BVDV2-125c.

Three monoclonal antibodies directed against cell surface molecules of bovine cells inhibited subsequent infections with bovine viral diarrhoea virus (BVDV). They specifically blocked the infectivity of three non-cytopathogenic and three cytopathogenic BVDV strains. These results showed that an important mechanism for virus uptake was inhibited. The ligand of the monoclonal antibody BVD/CA 17, which blocked infectivity most efficiently, was found on leukocytes from a wide range of domestic and wild even-toed ungulates using flow cytometric analysis. In contrast, the monoclonal antibodies BVD/CA 26 and BVD/CA 27 appeared to be specific for bovine cells. Immunoprecipitation of labelled bovine cell surface proteins showed that the three monoclonal antibodies bound to proteins with identical relative molecular masses (M(r)). Proteins of an apparent M(r) of 93 K and 60 K were precipitated from lysates of fetal bovine kidney cells irrespectively of the MAbs used.

New BVDV strains associated with very high mortalities, which killed about 25% of the veal calves in Quebec in 1993, have been isolated. In this study, characterization of the last two-thirds of the 5' untranslated region (5'UTR) of their genome and virus neutralization experiments with polyvalent antisera raised in different animals both demonstrated that these strains formed a distinct group. Despite a difference of about 25% in the 5'UTR sequence with that of the classical strains, these 5'UTRs maintained the same secondary structure albeit with a higher stability. Serological crossreactivity between the classical and new BVDV strains was relatively low and suggest that new strains should also be included to obtain efficient BVDV vaccines. Based upon the distinct characteristics of these new BVDV strains, we propose to divide BVDV into two groups. Group I comprises the classical BVDV isolates including commonly used laboratory and vaccine strains, and group II comprises the newly described BVDV strains and those associated with thrombocytopenia and hemorrhaging.

Cytopathogenic bovine viral diarrhea virus (BVDV) arises by RNA recombination in animals persistently infected with noncytopathogenic BVDV. Such animals develop fatal mucosal disease. In this report, the genome of a cytopathogenic BVDV isolate, termed CP9, is characterized. CP9-infected cells contained not only viral genomic RNA of 12.3 kb but also a BVDV-specific RNA of 8 kb. cDNA cloning and sequencing revealed that the 8-kb RNA is a BVDV genome with an internal deletion of 4.3 kb. The 8-kb RNA represents the genome of a typical defective interfering particle (DI), since its replication was strictly dependent on the presence of a helper virus and strongly interfered with the replication of the helper. Cell culture experiments demonstrated that the CP9 virus stock contains two viruses, namely, a helper virus and DI9. While the helper virus alone was noncytopathogenic, the presence of the DI conferred cytopathogenicity. Expression experiments demonstrated that p80, the marker protein of cytopathogenic BVDV, is translated from the defective genome. The occurrence of this cytopathogenic DI is linked to a fatal disease in cattle.

In order to verify the role of immunosuppression in the pathogenesis of Bovine Viral Diarrhea Virus (BVDV) infection, two experiments have been carried out. In one experiment, calves previously infected with cytopathogenic (CP) or non-cytopathogenic (NCP) BVDV were treated with dexamethasone (DMS) 30 days later. In the other experiment, calves were simultaneously exposed to BVDV infection and to DMS treatment. In both experiments the DMS treated calves developed a more serious disease which in one calf was fatal. It was speculated that immunosuppression represents one of the most significant "key factors" in the occurrence of the bovine viral diarrhea/mucosal disease in cattle.

During 1993 outbreaks of diarrhoea in adult dairy cows in three geographically unrelated herds were found to be caused by bovine viral diarrhoea virus (BVDV). The affected animals showed signs of acute watery diarrhoea, agalactia and pyrexia (39.4 to 42 degrees C). Ulceration of the buccal mucosa, a mucoid nasal discharge and stiffness were inconsistent signs. The disease spread rapidly in each case. The diagnosis was confirmed by the isolation of non-cytopathic BVDV from blood and tissues and by the demonstration of significantly rising titres to BVDV by an ELISA. The highest morbidity recorded was 40 per cent with one herd experiencing a 10 per cent mortality. There was no increased incidence of abortion in any of the herds, either at the time of or subsequent to the outbreaks of diarrhoea. In one herd the purchase of a persistently viraemic heifer 14 days before the outbreak was thought to be the source of infection, but in the other two herds the source was not established.

Infections with the Bovine Viral Diarrhea/Mucosal Disease Virus (BVDV) are widespread and cause a variety of diseases including reproductive disorders, abortion and malformation, pneumoenteritis, thrombocytopenia and mucosal disease. Together with the closely related border disease virus of sheep (BDV) and European Swine fever virus (CSFV), also referred to as Hog Cholera virus, BVDV is now classified in the genus pestivirus of the Flaviviridae family. The BVDV exists in two biotypes, noncytopathic and cytopathic, the latter differing in structural proteins from the noncytopathic biotypes. In virus-free animals infection is transient and mostly subclinical or mild but may also lead to an array of diverse symptoms such as pneumoenteritis (often in combination with other microorganisms). Infection of the developing fetus early in gestation with a noncytopathic biotype of BVDV may result in persistent infection and birth of apparently healthy calves. Such calves may later in their lives develop Mucosal Disease, a lethal course of infection associated with a mutation to the cytopathic biotype or superinfection with a cytopathic BVDV antigenically similar to the non-cytopathic virus already present in these animals. Diagnosis of infections with BVDV is based on the clinical symptoms and demonstration of virus. Paired serum samples allow the detection of seroconversion in acute infections while persistently infected animals are immunotolerant and generally lack antiviral antibody. Although generally found in their respective host species, pestiviruses of cattle, sheep and pigs are capable of crossing the species barrier into the other species. The existence of pestiviruses in wild ruminants and boars may complicate control strategies that are aimed at removing virus carriers and the control of animal movements.

Monoclonal antibodies to bovine viral diarrhea virus (BVDV) were examined for binding with a large number of North American BVDV isolates and eight strains of the serologically related pestivirus, hog cholera virus (HCV). No single BVDV monoclonal antibody reacted with all BVDV isolates. The most cross-reactive monoclonal antibody was an anti-p80/p125 antibody which showed a positive reaction with 173 of 180 (96%) North American isolates. From a fewer number of isolates tested, one anti-gp53 monoclonal antibody also showed a high cross-reactivity (94%). All BVDV isolates showed a positive reaction with at least one of the seven monoclonal antibodies in the panel. Thus, the results indicated that a pool of these monoclonal antibodies may be used in place of polyclonal antisera for the detection of BVDV contamination of cell lines or for virus isolation. For HCV, all three anti-p80/p125 monoclonal antibodies reacted positively with all eight virus strains. In contrast, none of the anti-gp53 monoclonal antibodies were reactive to HCV strains. Thus, the anti-gp53 monoclonal antibodies may be useful for distinguishing between usually innocuous BVDV infections and the highly significant HCV infections in swine for foreign animal disease surveillance.

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle that induces economically important diseases affecting multiple organ systems. In the United States, over 150 biological products are licensed for control of BVDV. These products contain live or killed BVDV, and many products contain other viruses or bacteria. Potency tests for these vaccines are based on animal inoculation and serology. For live virus vaccines, titration of viral infectivity in cell culture is an accepted alternative to animal inoculation. The immunogens in a killed virus vaccine may be measured by enzyme linked immunoabsorbent assay. Immunogens of BVDV that stimulate a protective immune response have not been conclusively identified. Epitopes on a putative viral envelope glycoprotein, gp53, are involved in viral neutralization. Other viral glycoproteins, gp48 and gp25, are immunogenic but epitopes on these proteins do not stimulate production of antibodies that efficiently neutralize virus. Progress in developing meaningful in vitro assays for quantitation of BVDV immunogens awaits identification of viral proteins that stimulate a protective immunity.

Bovine viral diarrhoea (BVD) virus is a cosmopolitan pestivirus of animals which is associated with diarrhoea, immunosuppression and synergy with other pathogens. This study was conducted to establish the prevalence of anti-BVD virus antibodies in healthy Zambian adults and those with asymptomatic and symptomatic HIV disease. Sera from 1159 adults were tested for anti-BVD virus antibodies using the indirect immunofluorescence test and the confirmatory Western blot. Of the 1159 sera examined, 180 (15.5%) showed significantly elevated titres of anti-BVD antibodies. These included 70 out of 477 (14.7%) HIV-negative healthy adults; 73 out of 480 (15.2%) of HIV-positive asymptomatic individuals; 23 out of 129 (17.8%) HIV-seropositive patients with associated illnesses excluding diarrhoea; and 14 out of 73 (19.2%) of HIV-seropositive patients with chronic diarrhoea. HIV-seropositive patients with chronic diarrhoea or associated illnesses appear to have significantly increased seroprevalence of anti-BVD virus antibodies (P = > 0.01). The mechanism of interaction between BVD virus and HIV infections and the synergistic effects with other opportunistic pathogens in humans requires definition.

Cultural isolation, identification and biotype characterization of BVD virus is based on monolayer tissue culture techniques using mainly organs of, e.g., bovine fetuses. These might have experienced a transplacental transmission and are therefore of no use for tissue cultures produced, e.g., from kidneys of such fetuses. In order to rule out BVD virus infections, fetuses must be tested for BVD virus, viral antigen or specific nucleotide sequences. BVD virus and other ruminant pestiviruses have been detected in Turkey by cultural isolation. In bovine fetuses BVD virus has not been demonstrated so far. Cultural isolation was attempted in the present investigation in 50 randomly selected pregnant cows which were brought to an abattoir. Of these cows serum samples were assayed for neutralizing antibodies to BVD virus and 40 (80%) found positive with titres > or = 1:40. In contrast fetuses 120 days of ontogenic age and therefore supposedly immunocompetent were derived from seropositive dams (n = 16) but did not show any neutralizing antibodies to BVD virus. Leucocyte fractions of peripheral blood samples of the 50 cows as well as 353 fetal blood and organ samples were inoculated and submitted to three passages in fetal calf kidney cell cultures before testing for BVD viral antigen by immunofluorescence technique. BVD virus was thus detected in the fetus of one seropositive cow and also in the leucocyte fraction of one seronegative dam including organs of her fetus. The number of fetuses found positive for BVD virus amongst fetuses collected in abattoirs corresponds well to figures gained by other investigators outside Turkey.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine viral diarrhoea (BVD)/mucosal disease (MD) is an economically important infectious disease of cattle, and persistently infected animals are of central epidemiological significance. The identification of these animals is a prerequisite for further sanitary measures in affected herds. In order to process large numbers of samples an antigen capture enzyme immunoassay (AC-ELISA) for the detection of bovine viral diarrhoea viral antigen in peripheral blood leukocytes of cattle of all ages has been developed. In this communication an improved method for the preparation of samples is described. A total of 563 blood samples was tested using the AC-ELISA and a routine virus isolation procedure on susceptible bovine cells. When compared to virus isolation in cell culture, the sensitivity of the AC-ELISA was 97%. From the samples that were virus negative in cell culture, 99.1% were also negative in the AC-ELISA. There is some evidence that the AC-ELISA discriminated between transiently and persistently infected animals.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

A bull persistently viremic with noncytopathogenic (ncp) BVDV was inoculated with the cytopathic (cp) BVDV strain TGAC, which had been found to be antigenically different from the endogenous ncpBVDV (ncpW8). Neutralizing antibodies against strains NADL and TGAC were detectable 12 days and four weeks post infection, respectively. The animal developed fever and diarrhea 15 weeks post infection. On days 3 and 8 after onset of diarrhea a cpBVDV (cpX) was isolated from feces. Antigenic analysis using monoclonal antibodies (MoAbs) showed that cpX and the endogenous ncpBVDV (ncpW8) had identical reactivity patterns except for one epitope that was neither expressed on TGAC nor on ncpW8. Using polymerase chain reaction analysis it was shown that both TGAC and cpX contained a p8 phi gene duplication combined with genomic insertions of identical size. Restriction enzyme analysis of the TGAC and cpX amplicons using four enzymes showed an identical cleavage pattern, except for HaeIII digestion where an additional fragment was observed with cpX. These results suggest that cpBVDV strain TGAC persisted in the viremic animal and apparently caused disease after 15 weeks.

The role played by contaminated hypodermic needles and nose tongs in the spread of bovine viral diarrhoea virus was investigated by using a persistently viraemic animal as a virus donor. Susceptible cattle were infected by using contaminated needles and tongs and in some instances they in turn infected animals kept with them. Non-biting flies which fed on the ocular secretions of a persistently viraemic animal were also shown to be contaminated with the virus.

Cattle infected in utero with bovine viral diarrhoea virus (BVDV) often develop a lifelong persistent infection (PI). During this PI, BVDV infects many cell types including peripheral blood mononuclear cells (PBMNC). To define the lymphoid cell populations in which BVDV persists PBMNC subpopulations were separated using monoclonal antibodies to cell surface markers. Separated cells were analysed by a sensitive PCR assay for BVDV, in conjunction with flow cytometry to identify antigen-containing cells and with viral infectivity assays. The results indicate that BVDV establishes a productive PI in monocytes and T cells bearing the marker BoCD4, BoCD8 or gamma-delta T cell receptor. BVDV was not detected in B cells as a productive nor a latent infection.

Cytopathogenic (cp) and noncytopathogenic (ncp) biotypes of bovine virus diarrhoea virus (BVDV) could be clearly distinguished by hollow plaques with peripheral immunostaining in infected fetal calf kidney (FCK) monolayers cultures or the appearance of homogenously stained immunoplaques (IPs), respectively. Of 70 BVDV isolates formerly classified as cytopathogenic according to microscopic observations in FCK cell cultures, 46 produced IPs of both types while the remaining 24 isolates induced only the IP type characteristic of the ncp BVDV biotype. The same was true for 24 additional isolates classified by light microscopy as ncp after ten blind passages in FCK cell cultures. Thus it appeared that in 1/3 of the cases changes in FCK cell cultures had been formerly misinterpreted as BVDV induced cytopathic effects (CPE). Of the 46 isolates which produced both ncp and cp immunoplaques 5 BVDV isolates were selected and successfully subjected to cloning. Clones of the cp biotype were developed by picking native plaques from unfixed FCK cell monolayers. In case of ncp IP, cloning was attempted by picking randomly in the spaces between visible native plaques. Pairs of clones from each of the field isolates proved to consist of pure plaque populations of cp BVDV and ncp BVDV biotypes, respectively, at the dilution of viral suspension which allowed the classification of a single IP according to morphology. Analysis of the clones using 15 monoclonal antibodies against the major viral glycoprotein revealed far-reaching antigenic homogeneity for each pair with only few but distinct exceptions.

In 5 herds in which bovine virus diarrhoea virus (BVDV) had been isolated, all animals were bled for virological and serological examination. After the herd blood test, follow up blood tests were made on calves born up to 6 months later in 1 herd, 9 months later in 1 herd and up to 12 months later in 3 herds. Persistently infected animals (PI animals) were removed and after a time period a small herd sample of 10 animals that were born after removal of the PI animals were examined for BVDV antibodies. At the herd blood test a total of 21 PI animals were detected. During the follow up period another 25 PI animals were born. Among animals in the small herd samples collected after removal of the PI animals, antibody positive animals were found in the 2 herds with the shortest follow up period. In the 3 herds with a 1 year follow up period there were no antibody carriers in the herd sample. It seems possible to prevent further spread of infection with BVDV if all animals in the herds as well as animals born during the following year are examined and PI animals removed.

A comparative analysis of the distribution of cytopathogenic (cp) and noncytopathogenic (ncp) bovine virus diarrhea disease (BVD) virus in tissues from a calf with experimentally induced mucosal disease was performed using immunohistology and polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. For immunohistology, an antigenic marker on the superinfecting cp BVD virus defined by a monoclonal antibody (mab) was used, and overall presence of antigen was assessed with a pestivirus specific mab. The primers selected for RT-PCR detected the genomic insertion in the p125 region of the superinfecting cp BVD virus. Both methods gave consistent results.

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.

Two cytopathogenic isolates of bovine viral diarrhea virus (cpBVDV) have been analyzed. For both viruses two regions of their genomic RNAs were found to be duplicated and rearranged. The viral genomes contain a small duplicated element (SD) derived from the genomic 5' end far downstream of its original context. This sequence is followed by a larger duplication which encompasses the region coding for the protein p80(LD), a molecular marker for cpBVDV. The SD element codes for the viral protease p20. In the case of the viruses analyzed here the aminoterminus of p80 is generated by autoproteolytic removal of the preceding SD-encoded protease. For one of the cpBVDV isolates a specific fusion protein (p28) could be identified which is composed of p20 and part of p10, another viral protein. Molecular characterization of the respective noncytopathogenic counterpart revealed that duplication and rearrangement of sequences as well as the expression of p28 and p80 are specific for the cytopathogenic virus.

A noncytopathic bovine viral diarrhea virus (BVDV), BVDV-890, isolated from a yearling heifer that died with extensive internal hemorrhages, was compared for virulence in calves with noncytopathic BVDV-TGAN, isolated from an apparently healthy persistently infected calf. After challenge exposure with BVDV-890, nonimmune calves (n = 7) developed fever > 40 C, diarrhea, leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Most calves (n = 6) died or were euthanatized by 19 days after challenge exposure. Challenge exposure with BVDV-890 did not induce disease in 2 calves that had congenital persistent infection with BVDV or in 3 calves that had neutralizing antibody titer > 4 against BVDV-890. After challenge exposure with BVDV-TGAN, nonimmune calves (n = 7) developed fever > 40 C and, rarely, diarrhea or lymphopenia. All of those calves survived challenge exposure. The average maximal titer of BVDV-890 isolated from serum was 1,000 times that of BVDV-TGAN. In calves infected with BVDV-890, the average maximal percentages of lymphocytes and platelets associated with virus were greater than those found in calves infected with BVDV-TGAN. Additional findings of epidemiologic significance were prolonged shedding of virus and delayed production of viral-neutralizing antibody in 1 calf challenge-exposed with BVDV-890. Also, after production of neutralizing antibody, mutant virus that was refractory to neutralization was isolated from calves challenge-exposed with BVDV-TGAN.

Distribution of bovine viral diarrhoea virus (BVDV) antigens in the central nervous system (CNS) of 26 cattle persistently BVDV infected, 11 cattle with mucosal disease (MD), and 32 calves with congenital brain malformations was studied using monoclonal antibodies against BVDV epitopes. In persistently infected cattle and in cattle with MD, a widespread infection of neurons was present. Predilection sites for BVDV antigens were the cerebral cortex and the hippocampus. In calves with congenital encephalopathies, viral antigen-containing neurons could only be detected in the CNS of four animals. From the topographical distribution of BVDV antigens in these four postnatal cases with end-stage lesions, no conclusions could be drawn concerning the pathogenesis of BVDV-induced encephalopathies.

Four Japanese black cattle and four Holstein-Friesian cows were diagnosed with diabetes mellitus. Based on intravenous glucose tolerance tests, all these animals were believed to be insulin-dependent diabetics. Moreover, bovine viral diarrhea-mucosal disease (BVD-MD) virus was isolated and mucosal lesions and diarrhea were recognized in all cases. There was no genetic relation among the affected cattle. The breeding places of these cattle were different to each other. Therefore, it is suggested that BVD-MD virus induced the diabetes mellitus.

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced. Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus. From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.

Deaths within a litter of sucking pigs led to a suspicion of hog cholera, but pestiviruses isolated from both dead and live piglets appeared to be bovine viral diarrhoea virus. Persistent viraemia with bovine viral diarrhoea virus was demonstrated in living littermates and also in a bought-in calf, housed in a nearby pen on the same farm. Only two of the littermates survived, both of which had been virus negative and seropositive from the outset of testing. Porcine and bovine virus isolates grew well in calf testicular cells and were neutralised equally by sera collected at the farm from cattle and pigs. However, a comparison by means of their reactivity to monoclonal antibodies showed that they were similar but not identical, and only the porcine isolates grew well in a porcine kidney cell line.

This presentation summarizes the results of a study on the pathogenesis of bovine viral diarrhea (BVDV) infection. The cytopathic (CP) strain TVM-2 of BVDV induced in calves an overt clinical disease which is usually recorded as the acute primary BVDV infection observed under natural conditions. In contrast the non-cytopathic (NCP) strain New York-1 of BVDV did not cause any significant signs of disease. However, when the calves were immunosuppressed by treatment with dexamethasone (DMS) the biotype of BVDV involved did not seem to be as important as it appeared to be in an immunologically normal animal. This was shown in this study by the NCP BVDV which caused a fatal disease in calves treated with DMS. A mixed infection given to calves by injecting them with both CP and NCP BVDV, did not result in any particularly serious disease. So, the potential immunosuppressive activity of BVDV itself for the host has not been proven under the experimental procedures used in this experiment. Finally, a modified-live CP BVDV vaccine was unable to cause clinical disease when injected into calves that had been infected previously with strain New York-1 of BVDV.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.

We used the polymerase chain reaction (PCR) technique to detect bovine viral diarrhea virus (BVDV) infections in cattle. Of 120 cattle screened in this study, 29 were scored positive for BVDV with both PCR and conventional virus isolation. Ninety cattle were negative in both assays. One cow was scored positive for BVDV with the PCR but was negative with virus isolation. In dilution experiments PCR analysis was at least 10 times more sensitive than BVDV isolation.

The clinical course, post mortem lesions as well as virological and serological results after simultaneous intranasal inoculation of pigs with bovine viral diarrhoea virus (BVDV) and hog cholera virus (HCV) are described. Five groups of four weaners received constant doses of BVDV strain OSLOSS/2482 and tenfold decreasing doses of HCV strain ALFORT/187. Doses of 1,000 and 100 TCID50 of HCV in groups A and B of pigs led to fever and severe clinical signs in all animals of two groups, whereas at higher dilution of inoculum two, three or four animals survived without any clinical signs in the respective groups (C-E). Leucocyte samples taken from febrile animals and from normal pigs on five consecutive days were inoculated into both fetal calf kidney (FCK) and PK (15) cell cultures. Virus isolates were differentiated with BVDV and HCV specific monoclonal antibodies. HCV viraemia was detected in febrile animals exclusively, and BVDV viraemia occurred in not affected animals on days 3 to 7 post inoculation. Neutralizing antibodies (nab) against BVDV appeared before HCV nab in surviving animals of groups C and D after receiving low doses of HCV (10 or 1 TCID50). No BVDV nab were detected in group E that had received such a high dilution of HCV in addition to BVDV that theoretically no HCV was applied.

Since 1976 nineteen naturally occurring and eight experimentally induced incidents of congenital bovine viral diarrhoea virus (BVDV) infections were detected in routine laboratory diagnosis of swine fever. In nearly all naturally occurring cases, the disease was limited to one litter only. Clinical signs of disease and gross pathological lesions resembled those of chronic swine fever. Nearly all piglets born infected with the disease died within four months of birth. One pig remained persistently infected and immunotolerant until slaughtered at the age of 24 months; one of its litter mates seroconverted when six months old. In contrast, postnatal infections of pigs with BVDV strains isolated from swine ran a nearly or completely subclinical course. A serological survey of slaughter sows and boars showed that 20 per cent of the animals had antibodies against BVDV. The percentage of seropositive pigs was significantly higher on farms where cattle were present, and the percentage increased with age regardless of whether cattle were present. Sheep, pigs and humans can also transmit BVDV infections to pigs.

A technique for detection of bovine viral diarrhea virus (BVDV) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of BVDV and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral RNA in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of BVDV by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.

Samples of sera were obtained from 5,725 cows in a semiclosed herd. In each of the preceding 7 years, the herd was vaccinated against bovine viral diarrhea (BVD) with killed virus. Neutralizing antibody tests were done on all samples of sera, using cytopathic virus, BVD-TGAC virus, that was antigenically distinct from the vaccine virus. Most samples of sera had high titers of neutralizing antibodies against BVD-TGAC virus. In 48 samples of sera, neutralizing antibodies were not detected against BVD-TGAC virus, but were detected against the vaccine virus. Neutralizing antibodies against selected noncytopathic BVD viruses were not detected in several samples of serum that had neutralizing antibodies against the vaccine virus and BVD-TGAC virus. Noncytopathic BVD virus was isolated from sera obtained from 3 cows less than 4 years old. Two cows were available for further testing, and persistent infection with BVD virus was confirmed in both cows. The BVD viruses isolated from those cows were not neutralized by several samples of sera. Immunoprecipitation of polypeptides induced by the vaccine virus was done with selected samples of serum. Two patterns of immuno-precipitated viral-induced polypeptides were identified. One pattern was consistent with exposure of cows with live virus. The other pattern was consistent with exposure of cows with only the killed virus vaccine.

Five mature bulls were studied during an acute transient infection with bovine viral diarrhoea virus (BVDV). The bulls had been infected experimentally by the intranasal instillation of blood and serum from a cow which was a persistent carrier of the virus. Infection was confirmed by the demonstration of a low titred viraemia in four of the five animals and by the seroconversion of all five. Semen samples were collected from each bull on four occasions between seven and 14 days after infection. The virus was isolated from the semen of three of the five bulls and from nine of 12 batches of semen from them. In contrast to other studies of the infection of semen, BVDV was isolated with similar efficiency from raw, unprocessed semen and from diluted, extended semen. The titres of virus in the semen ranged from 5 to 75 TCID50/ml. The infection did not appear to affect the quality of the semen. Shedding of virus continued after the end of the period of viraemia and appeared to be a consequence of the replication of the virus in the reproductive tract and its subsequent excretion in the seminal fluid. Virological studies of the reproductive tracts of these bulls suggested that the most productive sites of virus replication were the seminal vesicles and the prostate gland. Concurrent studies in a persistently infected bull supported these findings.

Antigenic and genomic relationships between five pairs of non-cytopathic and cytopathic bovine viral diarrhoea (BVD) viruses isolated from cattle with mucosal disease were examined. Antigenic similarity was evaluated by studying the binding characteristics of 10 monoclonal antibodies (MAbs) directed against the gp53 BVD virus glycoprotein. The MAb binding match between members of the same virus pair ranged from 10/10 to 7/10. The genomic relationships was evaluated by studying the hybridization characteristics of two cDNA probes and six complementary 20 base oligomer probes with virus DNA, selected on the basis of conservation between published BVD virus sequences; cDNA probes were hybridized at two stringencies (60 degrees C and 45 degrees C). The match of hybridization results between members of the same virus pair ranged from 4/4 to 1/4 with the cDNA probes and from 6/6 to 3/6 with the oligomer probes. The results indicate that members of the same virus pair can differ at both the antigenic and genomic levels.