Carbon monoxide and biliverdin suppress bovine viral diarrhoea virus replication.

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

Cytopathic BVDV-1 strain induces immune marker production in bovine cells through the NF-κB signaling pathway.

The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1ß, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates.

Molecular biology of bovine viral diarrhea virus.

Bovine viral diarrhea viruses (BVDV) are arguably the most important viral pathogen of ruminants worldwide and can cause severe economic loss. Clinical symptoms of the disease caused by BVDV range from subclinical to severe acute hemorrhagic syndrome, with the severity of disease being strain dependent. These viruses are classified as members of the Pestivirus genus of the Flaviviridae. BVDV are considered primarily a pathogen of cattle but can infect most ruminant species. The virus particle consists of a lipid bilayer membrane surrounding the encapsidated genomic RNA. Inserted in the outer membrane are two virus-encoded glycoproteins that contain the major antigenic determinants of the virus as well as receptor binding and cell fusion functions. A third glycoprotein is weakly associated with the virion, but also possesses unique features that play important roles in suppression of innate immunity. The viral proteins are encoded in a single, large open reading frame. The viral proteins are proteolytically cleaved from the polyprotein by different proteases. The structural proteins are processed by cellular signal peptidases while the processing of the nonstructural proteins is by the viral serine protease. The virus is assembled and matures in the endoplasmic reticulum and golgi bodies of the cell. The virus is released via exocytosis, where viral proteins are not exposed on the surface of the cell.

Contamination of cell cultures with bovine viral diarrhea virus (BVDV).

The incidence of contamination of cell strains used in biological and virological studies and of fetal calf sera (FCS) manufactured by Russian and foreign companies used for cell culturing with noncytocidal bovine viral diarrhea virus (BVDV; Pestivirus, Flaviviridae) was analyzed. The virus was detected by reverse transcription PCR and indirect immunofluorescence with monoclonal antibodies to BVDV virion envelope glycoprotein in 25% of 117 cell strains and 45% of 35 tested FCS lots. The virus multiplied and persisted in a wide spectrum of human cell strains and in monkey, swine, sheep, rabbit, dog, cat, and other animal cells. The levels of BVDV genome RNA in contaminated cell cultures reached 10(2)-10(3) g-eq/cell and in serum samples 10(3)-10(7) g-eq/ml. These facts necessitate testing of cells and FCS for BVDV reproduced in cells without signs of infection detectable by light microscopy. The molecular mechanisms of long-term virus persistence in cells without manifestation of cell destruction are unknown.

Amplification of bovine viral diarrhoea virus introduced into an in vitro embryo production system via oocytes from persistently infected cattle.

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus.Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2)oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus.Virus was not isolated from any samples in treatment group 1.As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions / Amplificación in vitro de cepas de campo de virus de la diarrea viral bovina (VDVB) aisladas en Argentina: efecto de la línea celular y las condicioes de cultivo

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD- 420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.

Se estudió la interacción de aislamientos de campo de Argentina del VDVB (Pestivirus, Flaviridae) en las líneas celulares continuas MDBK, BoTur y BHK-21. Se utilizaron los virus de campo genotipo 1b, 99/134, 00/693 (casos compatibles con enfermedad de las mucosas) y 04P7016 (cuadro respiratorio) y las cepas de referencia genotipo 1a Singer y NADL. Además se evaluó la interacción de VDVB-NADL con las líneas celulares experimentales de bovino RD-420 y NCL-1 y de riñón porcino (PKZ). Se usaron 2 protocolos de infección. Los títulos virales observados dependieron del virus y del tiempo de infección y no así del modo de infección. Mientras que MDBK y BoTur resultaron susceptibles a la infección, BHK-21 y PKZ no lo fueron. Los virus NADL, 00/693 y 04/89 incrementaron su título entre las 24 y las 48 h p.i. en BoTur para mantenerlo posteriormente; los virus 99/134 y 04P7016 no presentaron variaciones y la cepa Singer presentó título máximo a las 24 h p.i para luego descender. La cinética del virus NADL en las células MDBK, RD-420 y NCL-1 tuvo un incremento de título para MDBK y NCL-1 entre las 24 y 48 h p.i que descendió a las 72 h p.i. La interacción virus-línea celular no estaría relacionada con el sub-genotipo del virus (1a o 1b), ni con el cuadro clínico; las células MDBK y NCL-1 serían más susceptibles a la replicación del VDVB.

Bovine viral diarrhea virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes.

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS(2)) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

Bovine caruncular epithelial cell line (BCEC-1) isolated from the placenta forms a functional epithelial barrier in a polarised cell culture model.

In the bovine synepitheliochorial placenta key sites of fetal-maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither alpha-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (+/-1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal-maternal communication, transport and infection.

Amino acid derivatives. Part I. Synthesis, antiviral and antitumor evaluation of new alpha-amino acid esters bearing coumarin side chain.

A series of amino acid esters bearing coumarin (3-15) were synthesized and evaluated, in vitro, against HIV-1, and bovine viral diarrhea virus (BVDV). The in vitro cytotoxicity of 3-10 and 12 was assayed against a panel of tumor cell lines consisting of CD4 human T-cells. Compound 14 showed inhibition of HIV-1 with EC50 > 1.6 microg mL(-1), meanwhile compound 9 exhibited activity against leukaemia (MT4) with CC50 = 24 micromol L(-1).

Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication.

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.

[Antiviral activity of different drugs in vitro against viruses of bovine infectious rhinotracheitis and bovine diarrhea]. / Protivovirusnaia aktivnost' razlichnykh preparatov in vitro v otnoshenii virusov infektsionnogo rinotrakheita i virusnoi diarei krupnogo rogatogo skota.

In vitro experiments studied the antiviral activity of 11 different drugs against viruses of bovine infective rhinotracheitis (BIRT) and bovine viral diarrhea (BVD). The 50% inhibiting concentrations of the test agents were determined in the monolayers of MDBK and KCT cell cultures. Only did phosprenyl show a virucidal activity against BIRT virus. All the tested drugs significantly inhibited the reproduction of BIRT virus in the sensitive MDBK cell cultures. Thus, bromuridin, acyclovir, ribavirin and methisazonum inhibited the virus by > or = 100,000 times; liposomal ribavirin, gossypolum, anandinum, polyprenolum, phosprenyl, by 1000-10,000 times; eracond and argovit, by 100 times. In experiments on BVD virus, the cultured KCT cells displayed the antiviral activity of bromuridin, phosprenil, polyprenolum, methisazonum, acyclovir, gossypolum, argovit, and ribavirin (in two variants), which caused a statistically significant (100-10,000-fold) decrease in the productive activity of this virus. Eracond and anandid proved to be ineffective.

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.

Korean medicinal plant extracts exhibit antiviral potency against viral hepatitis.

OBJECTIVES: Investigation of natural ethnopharmacologic extracts exhibiting antiviral potential may lead to the discovery of new therapeutics for the treatment of chronic viral hepatitis infections. Traditional Korean medicinal herbs have been identified that exhibit potency against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Research on the antiviral potential of naturally derived extracts is facilitated through the use of appropriate animal and liver cell culture models for these hepatotrophic pathogens. Objectives of this study were to demonstrate antiviral activity of an aqueous extract of herbal formulation KYH-1 in surrogate in vitro assays for HBV and HCV and identify mechanisms of action. METHODS: Antiviral potency of KYH-1 was measured in tissue culture systems that support replication of the woodchuck hepatitis virus (WHV), and the bovine viral diarrhea virus (BVDV). These assays serve as surrogate models for HBV and HCV, respectively. A recombinant HBV polymerase gene expression assay was used to define a molecular target. RESULTS: KYH-1 exhibited potent antiviral activity against WHV and to a lesser extent against BVDV. KYH-1 and its constituent components inhibited HBV polymerase priming in vitro. Additionally, KYH-1 suppressed HBV replication in a human hepatoblastoma cell line. CONCLUSION: Evaluation of naturally derived products for antiviral activity against HBV and HCV in standardized surrogate assays provides a scientific basis for potential use as complementary or alternative medicines. This study provides significant results justifying preclinical evaluation of KYH-1 as an antiviral therapy for HBV infections.

Virus-inhibiting surgical glove to reduce the risk of infection by enveloped viruses.

Needle puncture and other accidents that occur during surgery and other procedures may lead to viral infections of medical personnel, notably by hepatitis C (HCV) and human immunodeficiency virus (HIV), now that hepatitis B can be prevented by vaccination. A new surgical glove called G-VIR, which contains a disinfecting agent for enveloped viruses, has been developed. Herpes simplex type 1 (HSV) was used as a standard enveloped virus in both in vitro and in vivo tests of the virucidal capacity of the glove. Bovine viral diarrhea virus (BVDV) and feline immunodeficiency virus (FIV) were used as models for HCV and HIV, respectively. For in vitro study, a contaminated needle was passed through a glove and residual virus was titrated; for in vivo studies, animals were stuck with a contaminated needle through a glove. Despite variation in virus enumeration inherent in the puncture technique, statistical evaluation showed that infection was reproducibly and substantially reduced by passage through the virucidal layer. For BVDV, the amount of virus passing through the virucidal glove was reduced in 82% of pairwise comparisons with control gloves that lacked the virucidal agent; when plaque counts were adjusted to a common dilution, the median count for the virucidal glove was on the average reduced >10-fold. In experiments in which the proportion of wells infected with FIV was measured, the ratio of TCID(50) values (control glove to G-VIR) was >15, and probably much higher. For HSV, the amount of virus passing through the virucidal glove was reduced in 81% of comparisons with control gloves; the median of adjusted plaque counts was reduced on the average approximately eightfold or ninefold. In vivo tests with FIV and HSV in cats and mice, respectively, found smaller percentage reductions in infection than the in vitro tests but confirmed the virucidal effect of the gloves.

Distribution of viral antigen and development of lesions after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves.

OBJECTIVE: To correlate tissue distribution with development of lesions after experimental infection with a virulent strain of noncytopathic bovine viral diarrhea virus (BVDV) type 2 in calves. ANIMALS: Ten 14-day-old and two 2-month-old colostrum-deprived calves. PROCEDURE: Calves were intranasally inoculated with BVDV type-2 strain 1373 from an outbreak of clinically severe bovine viral diarrhea (BVD). Two 14-day-old calves served as noninfected controls. Two calves each were euthanatized on postinoculation days 3, 6, and 12, and 1 each on days 8, 9, 13, and 14. Tissues were collected for immunohistologic and histologic examination. RESULTS: Inoculated calves developed nonspecific clinical signs characterized by high fever and decreased numbers of leukocytes and thrombocytes. Viral antigen was detected focally in lymphoid tissues on day 3. On days 6, 8, 9, 12, and 14, viral antigen became increasingly widespread throughout organs and tissues. Viral antigen in lymphoid tissues was associated with severe depletion of all compartments. Lesions in other tissues were not well correlated with distribution of viral antigen. Depletion of lymphoid tissues was observed in a calf on day 13, but viral antigen had been cleared from most tissues and was detected in vascular walls only. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with a virulent BVDV strain resulted in wide dissemination of viral antigen in host tissues. Severe lymphoid depletion developed in lymphoid tissues, whereas viral antigen was generally not associated with lesions in other tissues. Findings suggest that development of lesions in acute BVD is not solely a function of viral replication and is also attributable to host reaction to infection.

Pathogenesis of transplacental virus infection: pestivirus replication in the placenta and fetus following respiratory infection.

Although transplacental virus infections account for considerable morbidity and mortality in both animals and humans, very little is so far known about the pathways whereby virus reaches the conceptus, the subsequent virus-host interactions in the early phases of the infections, and the establishment of persistent non-lethal infection. Using a natural animal model we recently demonstrated that bovine pestivirus can spread from the site of infection to the ovine fetus within 72 h, despite the expression of interferon in the reproductive tract [1]. In the present study we demonstrate that pestivirus first establishes infection and spread within the allantoic and amniotic membranes and then the fetus, followed several days later by infection of the uterine glands. However, virus replication and spread within the fetus is, at least in part, controlled by fetal developmental factors. In fetuses less than 25 days of gestational age, the virus remains restricted to the bulbis cordis, the first brachial pouch and occasionally the aorta. Over the next few days the virus spreads to multiple tissues, in addition to becoming more widespread and pronounced within the initially infected tissues. A potential role for the binucleated cells of the allantochorion in the spread of the virus from the fetal to the maternal tissues was also found. These cells expressed high levels of viral antigen just prior to and during the time period in which virus antigen became detectable in the epithelial cells of the uterine glands, in endothelial cells of uterine vessels and in scattered macrophage-like cells in the uterine stroma. Most likely this relatively late virus transfer is inconsequential for the mother, since it occurs at a time when a maternal virus-specific antibody response is becoming measurable. This is in contrast to the fetus, where the infection will have established itself widely prior to the development of lymphoid tissues and a functional immune response, thus setting the scenario for development of specific tolerance to the persisting virus.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy.

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 x 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. At the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics.

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.

An experimental multivalent bovine virus diarrhea virus E2 subunit vaccine and two experimental conventionally inactivated vaccines induce partial fetal protection in sheep.

The primary aim of a bovine virus diarrhea virus (BVDV) vaccine is to prevent transplacental transmission of virus. We studied the efficacy of two experimental conventionally inactivated vaccines, based on BVDV strain Singer and containing a different antigen amount, against three antigenically different BVDV strains in a vaccination-challenge experiment in sheep. We also studied the efficacy of an experimental multivalent E2 subunit vaccine against four antigenically different BVDV strains. The vaccine contained the glycoproteins E2 of BVDV strains that belong to antigenic groups IA, IB and II. All three vaccines induced neutralizing antibodies against all challenge strains. Only the conventional vaccine that contained the highest antigen amount induced complete protection against homologous challenge. Neither of the conventional vaccines provided complete protection against heterologous challenge. The multivalent subunit vaccine induced partial protection against the homologous challenge strains. However, the immune response did inhibit virus replication in ewes, as shown by the results of the virus titrations.