Cytopathic bovine viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction

Bovine viral diarrhea virus (BVDV), a Flaviviridae pestivirus, is arguably one of the most widespread cattle pathogens worldwide. Each of its two genotypes has two biotypes, non-cytopathic (ncp) and cytopathic (cp). Only the ncp biotype of BVDV may establish persistent infection in the fetus when infecting a dam early in gestation, a time point which predates maturity of the adaptive immune system. Such fetuses may develop and be born healthy but remain infected for life. Due to this early initiation of fetal infection and to the expression of interferon antagonistic proteins, persistently infected (PI) animals remain immunotolerant to the infecting viral strain. Although only accounting for some 1% of all animals in regions where BVDV is endemic, PI animals ensure the viral persistence in the host population. These animals may, however, develop the fatal mucosal disease, which is characterized by widespread lesions in the gastrointestinal tract. Cp BVD virus, in addition to the persisting ncp biotype, can be isolated from such animals. The cp viruses are characterized by unrestrained genome replication, and their emergence from the persisting ncp ones is due to mutations that are unique in each virus analyzed. They include recombinations with host cell mRNA, gene translocations and duplications, and point mutations. Cytopathic BVD viruses fail to establish chains of infection and are unable to cause persistent infection. Hence, these viruses illustrate a case of “viral emergence to extinction” – irrelevant for BVDV evolution, but fatal for the PI host.     

Cytopathic and non-cytopathic bovine viral diarrhoea virus biotypes affect fluid phase uptake and mannose receptor-mediated endocytosis in bovine monocytes

We have used non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea viruses (BVDV) to determine how the two biotypes affect mannose receptor (MR)-mediated endocytosis and fluid phase uptake in bovine monocytes. We have demonstrated that endocytosis in uninfected monocytes after 1 h of culture was mediated by the MR and fluid phase uptake, and after 24 h of culture it was mediated via fluid phase uptake only. Both cp and ncp BVDV affected the mechanisms of antigen uptake in monocytes. Endocytosis in BVDV infected monocytes, unlike in uninfected cells, was MR-independent and mediated by fluid phase uptake after 1 h of infection. The 24-h-BVDV infection changed the antigen uptake mechanisms to become MR- and fluid phase uptake-dependent. We conclude that antigen uptake, an important antigen presenting cell (APC) function, is affected in the early stage of BVDV infection during the first 24 h, with both BVDV biotypes, cp and ncp, having similar effects on monocyte antigen uptake in cattle. By influencing the early antigen uptake function of APC, BVDV might disrupt the function of monocytes as professional APC and contribute to the specific immunotolerance to BVDV.     

Bovine viral diarrhea viruses modulate toll-like receptors, cytokines and co-stimulatory molecules genes expression in bovine peripheral blood monocytes

We have used noncytopathic (ncp) and cytopathic (cp) Bovine Viral Diarrhea Viruses (BVDV) to determine the expression levels of TLR genes, type I IFN, pro-inflammatory and Th1/Th2 cytokine gene expression in bovine monocytes. In general, both BVDV strains had similar effects. However, we found some significant differences that could be due to biological differences between cp and ncp BVDV strains. TLR3 was significantly up-regulated in 1h ncp, but not in cp BVDV- infected monocytes, whereas TLR7 expression dominated in 24h infection with both BVDV strains. Type I IFN and IL-12 gene expression was also significantly up-regulated in 1h ncp, but not cp BVDV infection that correlated with the enhanced TLR3 gene expression. Both BVDV biotypes suppressed pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, co-stimulatory molecules CD80 and CD86, but did not change Th1 type cytokine IL-12 and INF-gamma, gene expression after 24h infection. We hypothesize that BVDV may escape immune responses by altering the expression of TLR 3 and 7 and their signaling pathways.     

Apoptosis inhibitors delay the cytopathic effect of bovine viral diarrhoea virus (BVDV)

Based on their action in cell culture, two biotypes of bovine viral diarrhoea virus (BVDV) can be distinguished. The noncytopathic (ncp) BVDV isolated from persistently infected animals cause no visible damage to cultured bovine cells. In contrast, cytopathic (cp) BVDV induces severe damage and apoptosis in cell cultures. Cp BVDV can be isolated from cattle suffering from mucosal disease (MD) and is associated with the severe lesions that primarily affect the gastrointestinal tract. To get an insight into the molecular events during BVDV induced cytopathic effect (CPE), the effect of three chemical reagents (3-aminobenzamide, ascorbic acid and N-acetyl-leucyl-leucyl-methional) with completely different mode of actions in infected cells was analysed. All three substances were able to delay the cytopathic effect induced in permissive bovine cells.     

Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates

When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was first described for strain NADL. To find out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV field isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600-850bp in size, were amplified from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were confirmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90-140 amino acids. The 5' and 3' ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 5'-end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 3'-end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No significant matches for these stretches were found in the EMBL and Swissprot databases.     

Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana.

Bovine viral diarrhea (BVD) is one of the economically important diseases of cattle. For many years, different types of vaccines have been commercially available, yet this disease is hard to control in high-density population areas. Detection and isolation of bovine viral diarrhea virus (BVDV) from any potential reservoir is vital, especially when considering virus eradication from a herd or locale. One potential source is wild ruminants. Ear notches and lymph nodes were collected from the wild population of white-tailed deer (Odocoileus virginianus) during deer hunting season in Indiana and tested for BVDV with a commercial BVD antigen capture enzyme-linked immunosorbent assay. Two samples out of 745 collected samples were positive, and subsequently cp and ncp BVDV was isolated from 1 ear notch and 1 lymph node. These isolates were genotyped as type 1a and 1b based on sequence analysis of the 5' untranslated region (UTR). The results of the present study indicate that the prevalence of BVDV in the white-tailed deer population of Indiana is about 0.3%. Wild ruminants infected with BVDV should be taken into consideration during an eradication program of BVDV from the livestock population.     

Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus.

The in vitro cell tropism of non-cytopathogenic (ncp) and cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) was studied in primary dissociated brain cell cultures derived from ovine fetuses of different gestational ages. The cell types infected were identified by double immunofluorescence using antibodies against BVDV and cell type-specific markers. In cultures infected with ncp BVDV viral antigen was present in neurofilament (NF 200 kDa)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes and fibronectin-expressing cells. Estimation of the percentages of individual cell types infected with ncp BVDV indicated a tropism for NF 200-positive neurons. In cultures infected with cp BVD virus cytopathic changes were observed beginning at 40 hours post infection. Viral antigen was present in vacuolated NF 200-, GFAP- and fibronectin-positive cells. In comparison with non-infected control cultures a considerable reduction of the number of the different cell types was seen.     

No caspase activation but overexpression of Bcl-2 in bovine cells infected with noncytopathic bovine virus diarrhoea virus

Cytopathic bovine viral diarrhoea viruses (cp BVDV) induce apoptosis in permissible cell cultures via the intrinsic pathway, which involves the mitochondria as key organelles. An important event is the irreversible opening of the permeability transition pore (PTP) and the breakdown of the transmembrane potential DeltaPsi(m). The resulting release of cytochrome C from the mitochondria serves as a trigger to form the apoptosome which then leads to caspase activation and cell death. In contrast, noncytopathic (ncp) BVDV do not seem to affect cells in vivo or in vitro, suggesting that they inhibit apoptosis. Interestingly, inhibition of caspases in cells infected with cp BVDV delayed the apoptotic cascade but did not prevent the cytopathic effect (CPE). This suggests that the induction of apoptosis and the processes finally leading to the CPE may proceed separately, implying that the inhibition of apoptosis by ncp BVDV has to start earlier in the cascade. In this study we show that in fact apoptosis inhibition in cells infected with ncp BVDV must occur at the mitochondrial level, before the activation of the caspase cascade occurs. To elucidate the role of mitochondria after infection of cells with ncp BVDV, expression of Bcl-2 and Bax were analysed. It was shown that while Bax expression was not affected, the anti-apoptotic Bcl-2 protein was upregulated, presumably suppressing initiation of cell death and enabling persistent infection in vitro.     

Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus

A panel of 40 monoclonal antibodies (MAb) specific for bovine viral diarrhea virus (BVDV) was produced, and each MAb was characterized and grouped according to its viral protein specificity, immunoglobulin subclass, virus-neutralizing activity, and immunoreactivity with a large collection of BVDV isolates. The MAb were found to be specific for 1 of 3 sets of related viral-induced proteins found in cells infected with the Singer strain of BVDV. Group-1 MAb were specific for the 80- and 118-kilodalton (kD) proteins of BVDV. Group-2 MAb recognized 3 proteins with molecular sizes of 54, 56, and 58 kD. Group-3 MAb recognized a 43- and a 65-kD protein. The MAb belonged to either the IgG1, IgG2a, IgG2b, IgG3 subclasses or the IgE class of mouse immunoglobulin. All MAb in group 2 were able to neutralize BVDV and had neutralization titers that ranged from 24 to 1,600,000. The reactivity of the MAb with numerous field isolates of BVDV was highly variable. Both cytopathic and noncytopathic biotypes of BVDV were examined and had the same degree of antigenic variation. The greatest degree of variation was detected with group-2 MAb. The data demonstrate that BVDV isolates have a high degree of antigenic variation that is largely confined to the envelope glycoproteins associated with virus neutralization. The results also suggest that antigenic variability of this virus is important in the development and severity of the disease it causes.     

Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine viral diarrhea virus biotypes

The specificity of the humoral IgG response of cattle naturally or experimentally infected with bovine viral diarrhea virus (BVDV) was studied by radioimmunoprecipitation. Serum samples were tested against radiolabeled lysates of cells infected with cytopathic and noncytopathic biotypes of BVDV. A biotype-specific serologic marker was not detected. The specificity of the IgG induced in cows naturally or experimentally infected with either BVDV biotype was essentially the same. A strong IgG response to the 2 glycoproteins (56 to 58 kilodaltons, [kD] and 48 kD) of both biotypes and to the major polypeptides was induced in infected cells: 118 kD and 80 kD by cytopathic BVDV and only 118 kD by noncytopathic BVDV. The most consistent difference among cattle was the presence of IgG specific for the 37-kD polypeptide. Sequential serum sample collection after spontaneous and induced infections with either BVDV biotype did not indicate specific IgG markers for determining infection history. Sera from cattle with a confirmed diagnosis of mucosal disease and lacking neutralizing antibodies to BVDV usually lacked (greater than 80%) nonneutralizing BVDV-specific IgG. One animal had substantial amounts of IgG to the 80-kD polypeptide. Other cattle had less readily detectable amounts of IgG specific for 80-kD or 37-kD viral polypeptides.     

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3- and NS3Delta50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3Delta50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3Delta50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3Delta50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.     

Virus neutralizing antibodies against a panel of 18 BVDV isolates in calves vaccinated with Rispoval RS-BVD

Seven of nine colostrum-deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval RS-BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non-cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine-induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1:log2), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum-deprived BVDV seronegative calves, Rispoval RS-BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

The genetic basis for cytopathogenicity of pestiviruses

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by their effects on tissue culture cells. Identification of cp bovine viral diarrhea virus (BVDV) has been frequently reported since antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system, only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed a second way for the development of a cp pestivirus which is based on the introduction of a set of point mutations within the NS2 gene.     

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.     

Antigenic variability in bovine viral diarrhea virus (BVDV) isolates from alpaca (Vicugna pacos), llama (Lama glama) and bovines in Chile

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities where they have been introduced worldwide. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus and mainly to bovine viral diarrhea virus (BVDV). Seventeen Chilean BVDV isolates were analyzed by serum cross neutralization with samples obtained from five llama, six alpacas, three bovines, plus three reference strains belonging to different subgroups and genotypes. The objective was to describe antigenic differences and similarities among them. Antigenic comparison showed significant differences between different subgroups. Consequently, antigenic similarities were observed among isolates belonging to the same subgroup and also between isolates from different animal species belonging the same subgroup. Among the analyzed samples, one pair of 1b subgroup isolates showed significant antigenic differences. On the other hand, one pair of isolates from different subgroups (1b and 1j) shared antigenic similarities indicating antigenic relatedness. This study shows for the first time the presence of antigenic differences within BVDV 1b subgroup and antigenic similarities within 1j subgroup isolates, demonstrating that genetic differences within BVDV subgroups do not necessary corresponds to differences on antigenicity.     

Virus Neutralizing Antibodies Against a Panel of 18 BVDV Isolates in Calves Vaccinated with Rispoval™ RS‐BVD

Seven of nine colostrum‐deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval^? RS‐BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non‐cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine‐induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1 : log₂), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum‐deprived BVDV seronegative calves, Rispoval^? RS‐BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Cytopathogenicity markers in the genome of Hungarian cytopathic isolates of bovine viral diarrhoea virus

Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.     

Attempts to characterize bovine viral diarrhea virus isolated from cattle after immunization with a contaminated vaccine

Bovine viral diarrhea virus (BVDV) was isolated from 28 animals with a history of immunization against respiratory disease with a vaccine contaminated with BVDV. The vaccine-derived parental virus strain and the 28 isolates were analyzed using 10 monoclonal antibodies (MAbs) directed against different epitopes and antigenic domains on the major envelope glycoprotein of BVDV. None of the isolates displayed a reaction pattern identical with the parental virus. Instead, seven different reaction patterns (#A-G) emerged. Circumstantial evidence indicated that six of these were vaccine related whereas in one case (pattern #F) the origin of the isolate was unclear. The results indicated that BVDV rapidly changed during animal passages and that the tracing of the vaccine contaminant using Mabs was impossible.