DDIT3 Targets Innate Immunity via the DDIT3-OTUD1-MAVS Pathway To Promote Bovine Viral Diarrhea Virus Replication.

DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.

A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line-A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV).

Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.

Evolutionary selection of pestivirus variants with altered or no microRNA dependency.

Host microRNA (miRNA) dependency is a hallmark of the human pathogen hepatitis C virus (HCV) and was also described for the related pestiviruses, which are important livestock pathogens. The liver-specific miR-122 binds within the HCV 5' untranslated region (UTR), whereas the broadly expressed let-7 and miR-17 families bind two sites (S1 and S2, respectively) in the pestiviral 3' UTR. Here, we dissected the mechanism of miRNA dependency of the pestivirus bovine viral diarrhea virus (BVDV). Argonaute 2 (AGO2) and miR-17 binding were essential for viral replication, whereas let-7 binding was mainly required for full translational efficiency. Furthermore, using seed site randomized genomes and evolutionary selection experiments, we found that tropism could be redirected to different miRNAs. AGO cross-linking and immunoprecipitation (CLIP) experiments and miRNA antagonism demonstrated that these alternative variants bound and depended on the corresponding miRNAs. Interestingly, we also identified miRNA-independent variants that were obtained through acquisition of compensatory mutations near the genomic 3' terminus. Rescue experiments demonstrated that miRNA binding and 3' mutagenesis contribute to replication through mutually exclusive mechanisms. Altogether, our findings suggest that pestiviruses, although capable of miRNA-independent replication, took advantage of miRNAs as essential host factors, suggesting a favorable path during evolutionary adaptation.

Effects of oocytes exposure to bovine diarrhea viruses BVDV-1, BVDV-2 and Hobi-like virus on in vitro-produced bovine embryo development and viral infection.

As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 × 102 - 2.50 × 103 TCID50/100 µL), BVDV-2 (6.25 × 101 - 5.20 × 102 TCID50/100 µL) or Hobi-like virus (1.90 × 102 - 1.58 × 103 TCID50/100 µL) for 22-24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50-100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2-64.1% vs 49.8%; P = 0.003-0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20-50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3-38.0% vs 49.8% for negative controls; P ≤ 0.001-0.014). Blastocyst rates were only reduced in the 7.9 × 102 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P = 0.009), when calculated by oocyte number. 50-80% of degenerated embryos tested positive for Hobi-like virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and -2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Genetic characterization of bovine viral diarrhea virus strains in Beijing, China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle.

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-ß, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-ß mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

Bovine viral diarrhea virus infection induces autophagy in MDBK cells.

Bovine viral diarrhea virus (BVDV) is an enveloped, positive-sense, single-stranded RNA virus that belongs to the genus Pestivirus (Flaviviridae). The signaling pathways and levels of signaling molecules are altered in Madin-Darby Bovine Kidney (MDBK) cells infected with BVDV. Autophagy is a conservative biological degradation pathway that mainly eliminates and degrades damaged or superfluous organelles and macromolecular complexes for intracellular recycling in eukaryotic cells. Autophagy can also be induced as an effective response to maintain cellular homeostasis in response to different stresses, such as nutrient or growth factor deprivation, hypoxia, reactive oxygen species exposure and pathogen infection. However, the effects of BVDV infection on autophagy in MDBK cells remain unclear. Therefore, we performed an analysis of autophagic activity after BVDV NADL infection using real-time PCR, electron microscopy, laser confocal microscopy, and Western blotting analysis. The results demonstrated that BVDV NADL infection increased autophagic activity and significantly elevated the expression levels of the autophagy-related genes Beclin1 and ATG14 in MDBK cells. However, the knockdown of Beclin1 and ATG14 by RNA interference (RNAi) did not affect BVDV NADL infection-related autophagic activity. These findings provided a novel perspective to elaborate the effects of viral infection on the host cells.

Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

Experimental infection of pregnant goats with bovine viral diarrhea virus (BVDV) 1 or 2.

Infections with bovine viral diarrhea virus (BVDV) of the genus pestivirus, family Flaviviridae, are not limited to cattle but occur in various artiodactyls. Persistently infected (PI) cattle are the main source of BVDV. Persistent infections also occur in heterologous hosts such as sheep and deer. BVDV infections of goats commonly result in reproductive disease, but viable PI goats are rare. Using 2 BVDV isolates, previously demonstrated to cause PI cattle and white-tailed deer, this study evaluated the outcome of experimental infection of pregnant goats. Pregnant goats (5 goats/group) were intranasally inoculated with BVDV 1b AU526 (group 1) or BVDV 2 PA131 (group 2) at approximately 25-35 days of gestation. The outcome of infection varied considerably between groups. In group 1, only 3 does became viremic, and 1 doe gave birth to a stillborn fetus and a viable PI kid, which appeared healthy and shed BVDV continuously. In group 2, all does became viremic, 4/5 does aborted, and 1 doe gave birth to a non-viable PI kid. Immunohistochemistry demonstrated BVDV antigen in tissues of evaluated fetuses, with similar distribution but reduced intensity as compared to cattle. The genetic sequence of inoculated viruses was compared to those from PI kids and their dam. Most nucleotide changes in group 1 were present during the dam's acute infection. In group 2, a similar number of mutations resulted from fetal infection as from maternal acute infection. Results demonstrated that BVDV may cause reproductive disease but may also be maintained in goats.

The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV.

BACKGROUND: Dendritic cells (DC) are important antigen presentation cells that monitor, process, and present antigen to T cells. Viruses that infect DC can have a devastating impact on the immune system. In this study, the ability of bovine viral diarrhea virus (BVDV) to replicate and produce infectious virus in monocyte-derived dendritic cells (Mo-DC) and monocytes was studied. The study also examined the effect of BVDV infection on Mo-DC expression of cell surface markers, including MHCI, MHCII, and CD86, which are critical for DC function in immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from bovine blood through gradient centrifugation. The adherent monocytes were isolated from PBMCs and differentiated into Mo-DC using bovine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GMCSF). To determine the effect of BVDV on Mo-DC, four strains of BVDV were used including the severe acute non-cytopathic (ncp) BVDV2a-1373; moderate acute ncp BVDV2a 28508-5; and a homologous virus pair [i.e., cytopathic (cp) BVDV1b TGAC and ncp BVDV1b TGAN]. The Cooper strain of bovine herpesvirus 1 (BHV1) was used as the control virus. Mo-DC were infected with one of the BVDV strains or BHV-1 and were subsequently examined for virus replication, virus production, and the effect on MHCI, MHCII, and CD86 expression. RESULTS: The ability of monocytes to produce infectious virus reduced as monocytes differentiated to Mo-DC, and was completely lost at 120 hours of maturation. Interestingly, viral RNA increased throughout the course of infection in Mo-DC, and the viral non-structural (NS5A) and envelope (E2) proteins were expressed. The ncp strains of BVDV down-regulated while cp strain up-regulated the expression of the MHCI, MHCII, and CD86 on Mo-DC. CONCLUSIONS: The study revealed that the ability of Mo-DC to produce infectious virus was reduced with its differentiation from monocytes to Mo-DC. The inability to produce infectious virus may be due to a hindrance of virus packaging or release mechanisms. Additionally, the study demonstrated that ncp BVDV down-regulated and cp BVDV up-regulated the expression of Mo-DC cell surface markers MHCI, MHCII, and CD86, which are important in the mounting of immune responses.

Laboratory diagnosis and transmissibility of bovine viral diarrhea virus from a bull with a persistent testicular infection.

Recently, in the United States, a dairy bull was diagnosed as the second confirmed case of persistent testicular infection (PTI) with bovine viral diarrhea virus (BVDV). The first objective of this study was to evaluate the testing methodologies currently used by the artificial insemination industry in order to improve the detection of bulls with PTI. This study evaluated the impact of multiple factors ([1] sample tested, [2] sample handling, [3] assay used, and [4] assay methodology) on the sensitivity of detection of BVDV. The second objective of this study was to evaluate the transmissibility of BVDV from the bull through casual or sexual contact. Results from this study indicate that straws of semen should be transported to the diagnostic laboratory in liquid nitrogen dry shippers. PCR proved to be a more sensitive assay than virus isolation; however, certain PCR protocols exhibited greater diagnostic sensitivity than others. Insemination with cryopreserved semen from this infected bull caused viral transmission to a seronegative heifer resulting in viremia and seroconversion. After 42 months of age, the bull appeared to clear the infection. In conclusion, this bull validates that natural exposure to a 1a strain of BVDV can result in a unique PTI causing contamination of semen with detectable infectious virus. Appropriate handling and testing of samples is necessary in order to detect bulls exhibiting PTI. Additionally, PTI with BVDV may potentially be cleared after an extended duration.

Antiviral activity of Bacillus sp. isolated from the marine sponge Petromica citrina against bovine viral diarrhea virus, a surrogate model of the hepatitis C virus.

The Hepatitis C virus causes chronic infections in humans, which can develop to liver cirrhosis and hepatocellular carcinoma. The Bovine viral diarrhea virus is used as a surrogate model for antiviral assays for the HCV. From marine invertebrates and microorganisms isolated from them, extracts were prepared for assessment of their possible antiviral activity. Of the 128 tested, 2 were considered active and 1 was considered promising. The best result was obtained from the extracts produced from the Bacillus sp. isolated from the sponge Petromica citrina. The extracts 555 (500 µg/mL, SI>18) and 584 (150 µg/mL, SI 27) showed a percentage of protection of 98% against BVDV, and the extract 616, 90% of protection. All of them showed activity during the viral adsorption. Thus, various substances are active on these studied organisms and may lead to the development of drugs which ensure an alternative therapy for the treatment of hepatitis C.

Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry.

Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

Distribution of bovine viral diarrhoea virus antigen in persistently infected white-tailed deer (Odocoileus virginianus).

Infection with bovine viral diarrhoea virus (BVDV), analogous to that occurring in cattle, is reported rarely in white-tailed deer (Odocoileus virginianus). This study evaluated the distribution of BVDV antigen in persistently infected (PI) white-tailed deer and compared the findings with those from PI cattle. Six PI fawns (four live-born and two stillborn) from does exposed experimentally to either BVDV-1 or BVDV-2 were evaluated. Distribution and intensity of antigen expression in tissues was evaluated by immunohistochemistry. Data were analyzed in binary fashion with a proportional odds model. Viral antigen was distributed widely and was present in all 11 organ systems. Hepatobiliary, integumentary and reproductive systems were respectively 11.8, 15.4 and 21.6 times more likely to have higher antigen scores than the musculoskeletal system. Pronounced labelling occurred in epithelial tissues, which were 1.9-3.0 times likelier than other tissues to contain BVDV antigen. Antigen was present in >90% of samples of liver and skin, suggesting that skin biopsy samples are appropriate for BVDV diagnosis. Moderate to severe lymphoid depletion was detected and may hamper reliable detection of BVDV in lymphoid organs. Muscle tissue contained little antigen, except for in the cardiovascular system. Antigen was present infrequently in connective tissues. In nervous tissues, antigen expression frequency was 0.3-0.67. In the central nervous system (CNS), antigen was present in neurons and non-neuronal cells, including microglia, emphasizing that the CNS is a primary target for fetal BVDV infection. BVDV antigen distribution in PI white-tailed deer is similar to that in PI cattle.

Suppression of bovine viral diarrhea virus replication by single and dual short hairpin RNA-mediated RNA interference.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens to the cattle industry, causing a significant economic loss throughout the world. Despite the wide use of various control measures for BVDV, the disease remains prevalent. In this study, we achieved an efficient inhibition of NADL strain replication by plasmid-mediated shRNA targeting conserved regions of the viral genome. To further enhance the inhibiting efficiency, a dual shRNA expression plasmid, which could simultaneously express two different shRNA, was established and showed stronger inhibitory effects on virus replication. Moreover, the antiviral activity induced by the dual shRNA expression system was also evident on other BVDV-1 subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). Therefore, the dual shRNA system provides a more powerful strategy for inhibiting BVDV replication in a cross-resistance manner.

An alternative -1/+2 open reading frame exists within viral N(pro)(1-19) region of bovine viral diarrhea virus SD-1.

We previously reported the engineering of an N(pro)-disrupted bovine viral diarrhea virus (BVDV), BSD1-N(pro)/eGFP2A (Fan and Bird, 2008a). Here, we report that BSD1-N(pro)/eGFP2A survives a single nucleotide missing in its C-terminal eGFP region. By using our established reverse genetics system for BVDV, we confirm that the viral mutant is rescued through a -1/+2 ORF initiated in the N(pro)(1-19)/eGFP region of the mutant viral genome. We furthermore uncover that this event occurs in the N(pro)(1-19) region of BVDV strain SD-1. The rescued viral mutant showed dramatic reductions in levels of both viral RNA and viral protein in host cells. Although the mutant is similar to the native strain in viral kinetics, the peak yield of the mutant is decreased dramatically. These findings reveal the existence of an alternative -1/+2 ORF in the N(pro)(1-19) region during the replication of BVDV and open a new avenue to understand the life cycle and pathogenesis of pestiviruses.

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.

Genetic and splice variations of Bos taurus CD46 shift cell permissivity to BVDV, the bovine pestivirus.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal the existence of ten distinct allelic versions of the CCP1 module, varying significantly in frequency among taurine and indicine races. A complex mRNA splicing pattern was also evidenced for bovine CD46, generating three different serine-threonine-proline segments and five different cytoplasmic domains. The four most frequent allelic variants and the six splice variants were then expressed in BVDV-nonpermissive porcine cells and the quantity of progeny virions generated by each cell preparation was measured 48 h post-infection. As expected, ectopic expression of the 10 bovine CD46 isoforms rendered the PK15 cells permissive to BVDV, as attested by the 100,000-fold greater recovery of virions from these cells than from non-transfected cells. This permissivity increase was significantly lower (-33%, P<0.001) when the canonical CCP1 was replaced with the variant most frequent in zebus, suggesting positive or negative selection of this allele in the latter and in the former, respectively. The predicted secondary structure of this variant suggests that the measured loss of function is due to the disappearance of one of the two beta sheets constituting the BVDV attachment platform. On the other hand we showed that for a given CCP1, the titer recovered at 48 hpi also depended on the nature of the CD46 cytoplasmic domain (P<0.001). This result implies that virus binding generates a cytoplasmic-tail-dependent outside-in signal that determines permissivity to BVDV.

Investigation of a dual fetal infection model with bovine viral diarrhoea viruses (BVDV)-1 and BVDV-2.

Two studies were performed in pregnant heifers to determine whether inoculation with two bovine viral diarrhoea viruses (BVDV), one BVDV-1 and one BVDV-2, inoculated separately into either nostril, results in fetal infection with both viruses. Dual transplacental infection of the fetus with BVDV-1 and BVDV-2 was observed in one case, but not consistently.