Cytopathogenic Bovine Viral Diarrhea Virus Research Articles

A Recent Non cytopathogenic bovine viral diarrhea virus detected by immunofluorescence and reverse transcriptase polymerase chain reaction

A Recent Non cytopathogenic bovine viral diarrhea virus detected by immunofluorescence and reverse transcriptase polymerase chain reaction

Expression of the NS3 protease of cytopathogenic bovine viral diarrhea virus results in the induction of apoptosis but does not block activation of the beta interferon promoter

Bovine viral diarrhea virus (BVDV; genus Pestivirus) can exist as two biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP). The CP form differs from NCP by the continual expression of free non-structural protein 3 (NS3). CP BVDV infection of cultured cells induces apoptosis, whereas NCP BVDV infection has been reported to block the induction of beta interferon (IFN-beta). To investigate the viral mechanisms underlying these effects, NS3 or NS2-3 proteins of NCP and CP BVDV biotypes, together with the cognate NS3 co-factor NS4A, were expressed in cells, and their effect on apoptosis and induction of IFN-beta was investigated. Expression of NS3/4A resulted in increased activity of caspase-9 and caspase-3, indicating induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease-inactive NS3/4A was unable to induce apoptosis, suggesting that NS3 protease activity is required for initiation of apoptosis during CP BVDV infection. The ability of NS2-3 to modulate activation of the IFN-beta promoter was also investigated. These studies confirmed that, unlike the related hepatitis C virus and GB virus-B, BVDV proteases are unable to inhibit TLR3- and RIG-I-dependent activation of the IFN-beta promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with CP biotype pathogenesis.

Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is Crucial for Efficient Viral Replication and Cytopathogenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.

Experimental Superinfection of a Lesser Malayan Mousedeer (Tragulus javanicus) Persistently Infected with Bovine Viral Diarrhea Virus

A Lesser Malayan mousedeer (Tragulus javanicus), persistently infected with noncytopathogenic bovine viral diarrhea virus (BVDV) type 1f, was experimentally superinfected with a cytopathogenic isolate of BVDV type 1c, which antigenically partially matched the endogenous strain. Within the observational period of 125 days after superinfection, the animal did not demonstrate any clinical signs of the disease and/or significant changes in blood values. Neutralizing antibodies were detected at 35 and 42 days postinfection. The isolate causing the superinfection was found in feces, nasal swabs, and saliva starting from day 29 and at various times postchallenge. Macroscopic or histologic examination did not reveal mucosal disease-like lesions, despite the detection of the cytopathogenic isolate in the salivary gland, rumen, abomasum, kidney, and superficial prescapular lymph node. Results indicate that the cytopathogenic BVDV strain, which was used in the superinfection, persisted in the viremic animal without causing disease within the observation period.

Bovine Viral Diarrhea Virus: Prevention of Persistent Fetal Infection by a Combination of Two Mutations Affecting ErnsRNase and NproProtease

Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N(pro) and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N(pro) and E(rns) RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

Genetic and Pathobiological Characterization of Bovine Viral Diarrhea Viruses Recently Isolated from Cattle in Japan

The 475 strains of bovine viral diarrhea virus (BVDV) isolated from cattle in 12 prefectures of Japan in the last 7 years were phylogenetically classified as BVDV-1 or BVDV-2 on the basis of the nucleotide sequence of the 5'-untranslated region. BVDV-1 strains were further subtyped as 1a (101 strains), 1b (163), 1c (128), 1j (3), and So CP/75-like (1), and all of the 79 BVDV-2 strains belonged to subtype 2a. These 2a BVDVs contain two isolates that had high nucleotide identities with those of highly pathogenic BVDV-2 strains reported in North America (Pellerin et al., 1994). However, acute infection with severe mortality like North American outbreak was not observed and most of the present BVDV-2 strains were isolated from persistently infected (PI) cattle showing mild or no clinical sign. Moreover, it was revealed that 61.5% of the 39 PI cattle with cytopathogenic BVDVs did not show typical mucosal disease and 54.6% of the 405 PI animals only with non-cytopathogenic BVDVs were apparently healthy. The present results indicate that the prevention of the infection with an appropriate vaccine and active surveillance covering healthy cattle are required for the control of BVD.

The double-stranded RNA-induced apoptosis pathway is involved in the cytopathogenicity of cytopathogenic Bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV), which is classified in the genus Pestivirus, family Flaviviridae, can be divided into two biotypes according to its ability to induce a cytopathic effect in tissue culture cells. The mechanisms through which cytopathogenic (cp) BVDV induces cell death and non-cytopathogenic (ncp) BVDV causes persistent infection without producing cell death remain unclear. Here, it was found that the overexpression of four apoptosis-related cellular mRNAs in cells infected with cpBVDV could also be caused by synthetic dsRNA. In fact, it was found that the amount of dsRNA produced by cpBVDV considerably exceeded the amount yielded by ncpBVDV. To evaluate the possible involvement of dsRNA in the induction of apoptosis, this study examined whether RNAi-mediated depletion of two dsRNA-reactive cellular factors, dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase 1, resulted in the prevention of cpBVDV-induced apoptosis. Although the induction of apoptosis was reduced after the suppression of either factor alone, the simultaneous silencing of both factors resulted in an almost complete inhibition of apoptosis without affecting viral titre. These results showed that dsRNA is the main trigger of apoptosis in cpBVDV-infected cells and that the cytopathogenicity of BVDV depends on the yield potential of dsRNA. In contrast, ncpBVDV yielded minimal levels of dsRNA, thereby establishing a persistent infection without inducing apoptosis. This report supports the significance of viral dsRNA as a trigger of innate immune responses.

Identification of a Cell Population That Produces Alpha/Beta Interferon In Vitro and In Vivo in Response to Noncytopathic Bovine Viral Diarrhea Virus

In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN-alpha/beta), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN-alpha/beta in vitro. Similarly, cpBVDV induces IFN-alpha/beta in the early bovine fetus, but ncpBVDV does not. However, acute infection of naive cattle with ncpBVDV results in IFN-alpha/beta production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN-alpha in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN-alpha in the absence of detectable productive infection.

Differential activation of interferon regulatory factors-3 and -7 by non-cytopathogenic and cytopathogenic bovine viral diarrhoea virus.

Non-cytopathogenic bovine viral diarrhoea virus (ncpBVDV) has previously been shown to inhibit the function of interferon regulatory factor-3 in cultured cells [J. Virol. 76 (2002) 8979]. In this study, we show that, like ncpBVDV, when cells were previously exposed to cytopathogenic BVDV (cpBVDV) the appearance of an IRF-3-DNA complex from nuclear extracts that can be induced by heterologous virus infection was not observed. Infection of cells with ncpBVDV or cpBVDV resulted in neither the translocation of IRF-7 from the cytoplasm to the nucleus of infected cells, nor an inhibition of its nuclear translocation in cells super-infected by Semliki Forest Virus. We conclude that cpBVDV and ncpBVDV both share the ability to inhibit the full function of IRF-3 but neither stimulate or block the nuclear uptake of IRF-7.

Isolation and confirmation of bovine viral diarrhoea virus in Serbia and comparative typing with recent Slovenian isolates

The results of an investigation on bovine viral diarrhoea virus (BVDV) in fetal calf serum (FCS), whole blood and pathological material obtained from sick or dead cattle in Serbia are presented. Whole blood and FCS from sick animals were screened for BVDV antigen (Erns) by ELISA. ELISA positive samples and pathological material from dead animals were inoculated into primary cell cultures of fetal calf testis (FTTe). After threefold passage in FTTe cells, BVDV was detected by direct immunofluorescence and indirect immunoperoxidase tests and by reverse transcriptase-polymerase chain reaction (RT-PCR). Among 64 individual samples of FCS, two were positive for noncytopathogenic BVDV. One cytopathogenic BVDV was isolated from a whole blood sample from a heifer with clinical signs of mucosal disease. The 5'-untranslated region (5'-UTR) of three Serbian BVDV isolates was amplified by RT-PCR, sequenced and, together with 15 recent Slovenian BVDV isolates characterized by phylogenetic analysis. All isolates were classified as BVDV genotype 1 viruses. The majority of the BVDV isolates were of the 1f (Serbia - 2 isolates, Slovenia - 7 isolates) and 1d subtypes (Slovenia - 7 isolates) whilst one Serbian and one Slovenian isolate were genotyped as BVDV 1b.

Insertion of cellular sequence and RNA recombination in the structural protein coding region of cytopathogenic bovine viral diarrhoea virus.

The cytopathogenic bovine viral diarrhoea virus (cp BVDV) strain KS86-1cp was isolated from a calf persistently infected with the noncytopathogenic (ncp) strain KS86-1ncp after it was exposed to cp BVDV strain Nose and developed mucosal disease (MD). Molecular analysis revealed that an insertion of a cellular gene and a duplication of the viral RNA encoding the nucleocapsid protein C and part of N(pro) had occurred in the C coding region of the Nose and KS86-1cp genomes. The inserted cellular gene was closely related to the cINS sequence. Remarkably, the 5' upstream region from the insertion of KS86-1cp had high sequence identity to that of Nose, but differed from that of KS86-1ncp. In contrast, the region downstream from the insertion of KS86-1cp showed high identity to KS86-1ncp, but not to Nose. These data reveal that KS86-1cp is a chimeric virus generated by homologous RNA recombination in a calf with MD.

牛腎由来株化細胞を用いた牛ウイルス性下痢ウイルスのウイルス分離法および抗体検査法

牛腎由来株化細胞を用いた牛ウイルス性下痢ウイルスのウイルス分離法および抗体検査法

Experimental induction of mucosal disease: consequences of superinfection of persistently infected cattle with different strains of cytopathogenic bovine viral diarrhea virus.

Mucosal disease (MD) can be induced in cattle persistently infected with noncytopathogenic bovine viral diarrhea virus (ncp BVD virus) by superinfecting them with antigenically related cytopathogenic (cp) BVD virus strains. While some of these animals succumb to early onset MD after 2 to 3 weeks post infectionem (p.i.), others only react by producing neutralizing antibodies against the cp BVD virus strain and may develop late onset MD after longer incubation periods. The aim of this study was to determine if an increasing degree of antigenic homology between the ncp and the superinfecting cp BVD virus strains as determined by their comparative reactivity with E2 glycoprotein specific monoclonal antibodies (mabs) increases the probability of inducing early or late onset MD, respectively. For this, each two of eight clinically healthy animals from the same herd and persistently infected with the same ncp BVD viruses were superinfected with four different cp BVD virus strains. As only two of these animals developed late onset MD, one animal from a different herd that developed early onset MD was included in the study. Besides clinical observation and testing for antibody production, virus isolation and characterization of the cp BVD virus isolates were performed. The results indicate that antigenic similarity as determined by comparative mab analysis alone is not sufficient to allow prediction of the outcome of the disease.

Serologic evidence of bovine viral diarrhea virus in free-ranging rabbits from Germany.

From 1993 to 1995, 100 blood samples and 97 spleen samples of free-ranging rabbits (Oryctolagus cuniculus) from northern Germany were tested for prevalence of different bovine viral diarrhea virus (BVDV) antibodies and BVDV antigen, respectively. Forty sera (40%) were positive for antibodies to one or more of three cytopathogenic BVDV strains (NADL, Grub 313/83, and roe deer isolate SH9/11). Microneutralization test (NT) titers ranged from 5 to 13. Moreover, 13 of 35 NT positive sera also were positive by enzyme-linked immunosorbent assay for detection of anti-BVDV antibodies. Therefore, we assume that the rabbits either had passively acquired antibodies or had experienced natural infection with BVDV. Furthermore, 97 spleen samples were used for attempts to isolate BVDV and tested by reverse transcription polymerase chain reaction for viral nucleic acid using various protocols. Bovine viral diarrhea virus was not identified in these samples.

Variation from cytopathogenic biotype to non-cytopathogenic biotype is correlated with the deletion of cellular sequence from bovine viral diarrhea viruses.

Non-cytopathogenic (NCP) viruses of bovine viral diarrhea (BVD) virus were detected at a low ratio by the reverse plaque formation method from virus samples after several plaque clonings of cytopathogenic (CP) BVD viruses; NADL and Osloss strains. This phenomenon suggests that the NCP BVD viruses are produced at a low ratio during the propagation of CP BVD viruses in vitro. To investigate the differences between the parent CP BVD virus and the NCP BVD virus as a real progeny, the regions flanking the insertion of cellular mRNA in the p125 domain of NADL and Osloss strains were amplified by RT-PCR and cloned into pGEM 3Z plasmid vector, and then sequenced. Consequently, it was confirmed that sequences of cellular mRNA insertion of CP BVD viruses, NADL and Osloss strains, were completely and exactly deleted from the NCP BVD viruses which were real progeny of CP BVD viruses, NADL and Osloss strains. These results suggest that NCP BVD viruses may revert from CP biotype to NCP biotype by the deletion of cellular mRNA insertion in the viral genome of CP BVD viruses (NADL and Osloss strains).

Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.

Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhoea virus infection.

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.

Experimental mucosal disease in cattle: changes of lymphocyte subpopulations in Peyer's patches and in lymphoid nodules of large intestine

Changes in the number and distribution of lymphocyte subtypes were investigated in Peyer's patches in the jejunum and ileum, and mucosa-associated lymphoid nodules in the proximal colon and rectum of cattle with end-stage mucosal disease. Mucosal disease had been induced experimentally in seven of 13 animals by inoculation with cytopathogenic bovine viral diarrhea virus (cp BVD-virus). For comparison, six clinically healthy, persistently viremic cattle were used. IgM +, IgA +, BoCD4 +, BoCD8 + and γδTCR + lymphocytes, and the cp BVD-viral antigen were visualized in tissue sections by immunohistochemistry. In cattle with mucosal disease, the size of lymphoid follicles was significantly decreased in all localizations resulting in decreased numbers of B-lymphocytes per average follicular area. In most animals domes were missing and epithelium was invaginated into the lymphoid follicles. Numbers of BoCD4 + and BoCD8 + T-lymphocytes were increased per mm 2 of lymphoid follicle. Conversion of these counts into number of cells per average follicular area revealed, however, that the absolute number of BoCD4 + T-lymphocytes had decreased within lymphoid follicles and there was no distinct change of BoCD8 + T-lymphocytes in comparison to the controls. Interfollicular areas were less densely populated due to reduced numbers of BoCD4 + and BoCD8 + T-lymphocytes. cp BVD-viral antigen was detected predominantly in epithelial cells and in cells with dendritic morphology within lymphoid follicles. This may indicate that the severe depletion of B-lymphocytes in the lymphoid follicles is due to alterations of the microenvironment. The decrease of BoCD4 + and BoCD8 + T-lymphocytes does not support the hypothesis of T-cell-mediated tissue damage. Destruction of mucosa-associated lymphoid nodules does not only lead to local disruption of the gastrointestinal barrier, but will reduce the seeding of effector cells to the mucosa and therefore impair the defense mechanisms of the gastrointestinal barrier.

Isolation of different non-cytopathogenic bovine viral diarrhoea (BVD) viruses from cytopathogenic BVD virus stocks using reverse plaque formation method

Non-cytopathogenic (NCP) bovine viral diarrhoea (BVD) viruses were isolated from three cytopathogenic (CP) BVD virus stocks using the reverse plaque formation method, which was based on intrinsic interference. By means of an exaltation of Newcastle disease virus (END) test, these NCP BVD viruses were divided into two groups; END phenomenon positive (END +) and END phenomenon negative (END −) viruses. Additionally, the END + NCP BVD viruses interfered only with CP BVD virus whereas the END − NCP BVD viruses interfered with vesicular stomatitis virus as well as CP BVD virus. Differences in antigenicity existed among the three CP strains, however, each group of parent CP BVD virus and derivative NCP BVD virus was antigenically indistinguishable.

Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences

The RNA genomes of cytopathogenic bovine viral diarrhea virus (BVDV) isolates contain insertions highly homologous to cellular sequences. For two of them the insert was identified as ubiquitin coding sequence. The genome of BVDV Osloss contains exactly one ubiquitin gene monomer. In the case of BVDV CP1 the cellular insertion comprises one complete ubiquitin gene and part of a second monomer. The host cell-derived element in the CP1 genome is embedded in a large duplication of about 2.4 kb of viral sequences. Cellular insertion and duplication were not found in the genome of NCP1, the noncytopathogenic counterpart of CP1. These results strongly suggest that recombination between viral and cellular RNA is responsible for development of the cytopathogenic viruses, which is linked to pathogenesis of a lethal disease in cattle.