CD46 is a cellular receptor for bovine viral diarrhea virus

Various monoclonal antibodies (MAbs) that recognize cell surface proteins on bovine cells were previously shown to efficiently block infection with bovine viral diarrhea virus (BVDV) (C. Schelp, I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess, Arch. Virol. 140:1997-2009, 1995). With one of these MAbs, a 50- to 58-kDa protein was purified from calf thymus by immunoaffinity chromatography. Microchemical analysis of two internal peptides revealed significant sequence homology to porcine and human CD46. The cDNA of bovine CD46 (CD46(bov)) was cloned and further characterized. Heterologously expressed CD46(bov) was detected by the MAb used for purification. A putative function of CD46(bov) as a BVDV receptor was studied with respect to virus binding and susceptibility of nonpermissive cells. While the expression of CD46(bov) correlated well with the binding of [(3)H]uridine-labeled BVDV, the susceptibility of cells nonpermissive for BVDV was not observed. However, the expression of CD46(bov) resulted in a significant increase in the susceptibility of porcine cells to BVDV. These results provide strong evidence that CD46(bov) serves as a cellular receptor for BVDV.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Cell-protective monoclonal antibodies to bovine enterovirus-3 and partial or no activity against other serotypes.

Preparation of monoclonal antibodies to bovine virus diarrhea virus (BVDV) yielded some hybridoma cells that secreted monoclonal antibodies against the Madin-Darby bovine kidney cells. The anti-cellular monoclonal antibodies reacted with other bovine cells (bovine turbinate and testicle) but not with cell lines derived from other animal species. Subclones derived from one hybridoma partially blocked the infectivity of BVDV, possibly through the binding of the monoclonal antibodies with an epitope close to the receptor site of BVDV and not by way of steric hindrance. Unexpectedly, these same subclones completely blocked the infectivity of bovine enterovirus-3 (BEV-3) strain 240A and partially blocked the infectivity of BEV-2 and BEV-3 (ATCC strain) but not that of other serotypes. Other subclones derived from two other hybridomas, although cell membrane specific, did not have a protective activity against BEV or BVDV.     

Effect of bovine viral diarrhea virus infection on fertility of dairy heifers

A prospective field study in heifers from birth to first breeding was undertaken on two commercial dairies to assess the effect of bovine viral diarrhea virus (BVDV) congenital and post-natal infection (PNI) on fertility. A high BVDV Type 2 antibody titer (1:4096) at 10 months of age was associated with 32 more days to conceive, compared with a low titer (1:128). Conversely, infection with BVDV by 5-6 months of age and high BVDV Type 2 titers 1 month before conception or breeding was associated with improved fertility. Heifers with evidence of congenital BVDV infection had lower fertility than non-infected heifers (15-42 days longer time-to-first AI), which depended on BVDV Type 2 titers at 10 months of age. Neospora caninum infection was associated with additional services per conception (SPC) and Leptospira interrogans infection was associated with a delay in the time-to-first breeding. It appears that under field conditions, the effect of subclinical BVDV infection on subsequent heifer fertility may be due to a complex of interrelationships among multiple BVDV infections that depend on the type and timing of infection relative to reproductive development and events.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

BVD Virus Antigens in Tissues of Persistently Viraemic,Clinically Normal Cattle: Implications for the Pathogenesis of Clinically Fatal Disease

The cellular events involved in precipitation of the clinically fatal outcome of an infection with bovine viral diarrhoea virus (BVDV) remain unresolved, though it is now known that this course of the infection, Mucosal Disease (MD), only occurs in calves persistently infected with non-cytopathic BVDV. In studies aimed at elucidating the pathogenesis of MD, the distribution of BVDV antigens and infectious virus in tissues of persistently infected, clinically normal calves was investigated. Virus antigen was detected in most tissues, in epithelial and immune cells. No signs of an inflammatory response were detected and cytopathological changes were subtle or absent. The infection may nevertheless create a cell-environment which will enhance replication of cytopathic virus. Variations in the clinical, pathomorphologies and virological appearance of MD-cases may depend on both the host-reactions, including virus-induced immunopathology, and the virus-strain combinations in a putative mixed infection.     

Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer.

Neutralizing monoclonal antibodies directed against hog cholera virus (HCV) precipitated two HCV-encoded glycoproteins, HCV gp55 and HCV gp33. Immunoassay with bacterial fusion proteins and Western immunoblotting with extracts from infected cells revealed that the antibodies recognized only HCV gp55. Coprecipitation of HCV gp33 was shown to be due to intermolecular disulfide bridges. One of the antibodies also reacted with the major glycoprotein of another pestivirus, bovine viral diarrhea virus (BVDV). The analogous BVDV glycoproteins exhibited a distribution of cysteine residues which was almost identical to that of HCV gp55 and gp33. The two BVDV glycoproteins were also linked by disulfide bridges.     

Patterns of cellular gene expression in cells infected with cytopathic or non-cytopathic bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection in cattle is responsible for mucosal disease; an invariably fatal syndrome characterized by the recovery of two BVDV strains: cytopathic (cp) or noncytopathic (ncp). To understand the cellular responses to cp BVDV infection, we carried out differential display-polymerase chain reaction (DD-PCR) analysis of gene expression in infected cells. Altered expression of 14 genes involved in several functions was observed in cells infected with cp BVDV: (1) immune regulation, such as CD46, FKBP-12, and osteopontin (OPN); (2) apoptosis-related cysteine proteases like calpain; (3) signaling plasma membrane proteins such as integrin beta1, and prion protein; and (4) unknown function genes. Northern blot analysis of the expression of these genes in ncp BVDV infected cells revealed that while the expression of some genes was affected as in cp BVDV infected cells, others show a clearly contrary change. We postulate that a cause-effect relationship may exist between the differential gene expression alterations that characterize cp and ncp BVDV infections and the unique diseases associated with each BVDV biotype.     

Transdominant inhibition of bovine viral diarrhea virus entry

Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100- to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor.     

Bovine viral diarrhea virus entry is dependent on clathrin-mediated endocytosis

Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.     

Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV)

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro.The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos.These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

Processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.     

Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.     

BVDV and innate immunity.

Infection with bovine viral diarrhea virus (BVDV) is prevalent in the cattle population worldwide. The virus exists in two biotypes, cytopathic and non-cytopathic, depending on the effect of the viruses on cultured cells. BVDV may cause transient and persistent infections which differ fundamentally in the host's antiviral immune response. Transient infection may be due to both cytopathic and non-cytopathic biotypes of BVDV and leads to a specific immune response. In contrast, only non-cytopathic BVD viruses can establish persistent infection as a result of infection of the embryo early in its development. Persistent infection is characterized by immunotolerance specific for the infecting viral strain. In this paper we discuss the role of innate immune responses in the two types of infection. In general, both transient and persistent infections are associated with an increased frequency of secondary infections. Associated with the increased risk of such infections are, among others, impaired bacteria killing and decreased chemotaxis. Interestingly, non-cytopathic BVDV fails to induce interferon type I in cultured bovine macrophages whereas cytopathic biotypes readily trigger this response. Cells infected with non-cytopathic BVDV are also resistant to induction of interferon by double stranded RNA, a potent interferon inducer signalling the presence of viral replication in the cell. Thus, non-cytopathic BVDV may dispose of a mechanism suppressing a key element of the antiviral defence of the innate immune system. Since interferon is also important in the activation of the adaptive immune response, suppression of this signal may be essential for the establishment of persistent infection and immunotolerance.     

Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus

Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.     

A sensitive and efficient detection method for bovine viral diarrhea virus (BVDV) in single preimplantation bovine embryos

The objective was to develop a method to accurately and efficiently detect minute amounts of bovine viral diarrhea virus (BVDV) associated with a single embryo. There are two major challenges for BVDV detection in a single embryo: the test sensitivity and the efficiency of viral molecule recovery. These become even more critical when attempts are made to detect BVDV infections that occurred naturally, not through artificial exposure of the embryos to high affinity BVDV strains. We have developed a one-step sample preparation method that has increased the viral molecule recovery rate compared to the standard RNA isolation procedure by 7-100-fold. Instead of using the traditional virus exposure approach, we generated BVDV positive embryos via somatic cell nuclear transfer (SCNT) technology using BVDV positive donor cells. By combining the highly efficient sample preparation procedure with a sensitive one-step, real-time PCR system, we have developed a sensitive test that allows detection of as low as two copies of BVDV in a single embryo. This method will allow systematic risk assessment for BVDV transmission during in vitro embryo production via IVF or SCNT procedures.     

RNA Recombination between Persisting Pestivirus and a Vaccine Strain: Generation of Cytopathogenic Virus and Induction of Lethal Disease

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.