Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

Gypenoside Inhibits Bovine Viral Diarrhea Virus Replication by Interfering with Viral Attachment and Internalization and Activating Apoptosis of Infected Cells.

Bovine viral diarrhea virus (BVDV) causes a severe threat to the cattle industry due to ineffective control measures. Gypenoside is the primary component of Gynostemma pentaphyllum, which has potential medicinal value and has been widely applied as a food additive and herbal supplement. However, little is known about the antiviral effects of gypenoside. The present study aimed to explore the antiviral activities of gypenoside against BVDV infection. The inhibitory activity of gypenoside against BVDV was assessed by using virus titration and performing Western blotting, quantitative reverse transcription PCR (RT-qPCR), and immunofluorescence assays in MDBK cells. We found that gypenoside exhibited high anti-BVDV activity by interfering with the viral attachment to and internalization in cells. The study showed that BVDV infection inhibits apoptosis of infected cells from escaping the innate defense of host cells. Our data further demonstrated that gypenoside inhibited BVDV infection by electively activating the apoptosis of BVDV-infected cells for execution, as evidenced by the regulation of the expression of the apoptosis-related protein, promotion of caspase-3 activation, and display of positive TUNEL staining; no toxicity was observed in non-infected cells. Collectively, the data identified that gypenoside exerts an anti-BVDV-infection role by inhibiting viral attachment and internalization and selectively purging virally infected cells. Therefore, our study will contribute to the development of a novel prophylactic and therapeutic strategy against BVDV infection.

The evaluation of an immunoperoxidase assay applicable in antiviral drug screening.

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.

Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach.

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its genome replication and constitutes an attractive target for the identification of antivirals. With the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-house databases were evaluated and several structurally different compounds were tested in vitro for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and displayed EC50 values in the sub and low-micromolar range. Time of drug addition experiment and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis. Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the most active compounds with RdRp, which will allow future ligand optimization. These studies highlight the use of in silico screening to identify a new class of BVDV inhibitors.

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.

ANTI-VIRAL EFFECT OF HERBAL MEDICINE KOREAN TRADITIONAL CYNANCHUM PANICULATUM (BGE.) KITAG EXTRACTS.

BACKGROUND: Pestiviruses in general, and Bovine Viral Diarrhea (BVD) in particular, present several potential targets for directed antiviral therapy. MATERIAL AND METHODS: The antiviral effect of Cynanchum paniculatum (Bge.) Kitag (Dog strangling vine: DS) extract on the bovine viral diarrhea (BVD) virus was tested. First, a cytotoxicity test in MDBK (Madin-Darby bovine kidney) cells was done with all organic extract concentrations. RESULTS: The cytotoxic concentration CC50 for the ethyl acetate (EA) extracts was 18.2 ug/ml. In the tissue culture, infectious dose (TCID50) assay, the BVD virus decreased when treated with 18.2 ug/ml of the ethyl acetate extracts. CONCLUSION: Ethyl acetate extracts and fractions of the DS extract could be used as a potential antiviral for BVD.

N-Alkyldeoxynojirimycin derivatives with novel terminal tertiary amide substitution for treatment of bovine viral diarrhea virus (BVDV), Dengue, and Tacaribe virus infections.

Novel N-alkyldeoxynojirimycins (NADNJs) with two hydrophobic groups attached to a nitrogen linker on the alkyl chain were designed. A novel NADNJ containing a terminal tertiary carboxamide moiety was discovered that was a potent inhibitor against BVDV. Further optimization resulted in a structurally more stable lead compound 24 with a submicromolar EC50 against BVDV, Dengue, and Tacaribe; and low cytotoxicity.

Design, synthesis, and biological evaluation of N-alkylated deoxynojirimycin (DNJ) derivatives for the treatment of dengue virus infection.

We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10-18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC(50) against DENV in BHK cells of 6.5 µM, which limited its use in in vivo. Compound 7 presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure-activity relationship studies led to analogues 2h, 2l, 3j, 3l, 3v, and 4b-4c with nanomolar antiviral activity (EC(50) = 0.3-0.5 µM) against DENV infection, while maintaining low cytotoxicity (CC(50) > 500 µM, SI > 1000). In male Sprague-Dawley rats, compound 3l was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 ± 4%).

Antiviral treatment of calves persistently infected with bovine viral diarrhoea virus.

BACKGROUND: Animals persistently infected (PI) with bovine viral diarrhoea virus (BVDV) are a key source of viral propagation within and among herds. Currently, no specific therapy exists to treat PI animals. The purpose of this research was to initiate evaluation of the pharmacokinetic and safety data of a novel antiviral agent in BVDV-free calves and to assess the antiviral efficacy of the same agent in PI calves. METHODS: One BVDV-free calf was treated with 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) once at a dose of 1.6 mg/kg intravenously and one BVDV-free calf was treated three times a day for 6 days at 9.5 mg/kg intravenously. Subsequently, four PI calves were treated intravenously with 12 mg/kg DB772 three times a day for 6 days and two PI control calves were treated with an equivalent volume of diluent only. RESULTS: Prior to antiviral treatment, the virus isolated from each calf was susceptible to DB772 in vitro. The antiviral treatment effectively inhibited virus for 14 days in one calf and at least 3 days in three calves. Subsequent virus isolated from the three calves was resistant to DB772 in vitro. No adverse effects of DB772 administration were detected. CONCLUSIONS: Results demonstrate that DB772 administration is safe and exhibits antiviral properties in PI calves while facilitating the rapid development of viral resistance to this novel therapeutic agent.

Xanthohumol enhances antiviral effect of interferon alpha-2b against bovine viral diarrhea virus, a surrogate of hepatitis C virus.

Xanthohumol (XN) is a natural compound with multifunctional potentials, including antiviral activity. In this study, the antiviral activity of addition of XN to interferon (IFN)-alpha was examined and compared with each compound alone using bovine viral diarrhea virus (BVDV), a surrogate model of hepatitis C virus (HCV). BVDV E2 protein and the viral RNA level were determined by immunofluorescence and quantitative real-time RT-PCR, respectively. The addition of XN to IFN-alpha significantly improved CPEs induced by the virus and inhibited BVDV E2 protein and viral RNA levels. The interaction between XN and IFN-alpha was significant (P<0.001). XN at 3.13 microg/ml in combination with IFN-alpha at 50 IU/ml showed greater inhibitory effect on the viral RNA level than each compound used alone at 6.25 microg/ml and 100 IU/ml, respectively, indicating synergistic effect on BVDV replication in this combination. The inhibitory activity in all the tested combinations of XN and IFN-alpha was stronger than that of each compound used alone at the corresponding concentration. These results suggest that XN in combination with IFN-alpha exhibited a greater in vitro antiviral effect compared with each compound used alone. Further studies are deserved to investigate the anti-HCV activity of XN and the potential of XN in formulating novel anti-HCV regimen.

Discovery of diphenylmethane analogs as anti-bovine diarrhea viral agents.

Based on antiviral screening of our diphenylmethane derivatives prepared as steroid substitutes, we identified a 1,1-diphenylcyclobutane analog (9) and two diethyldiphenylsilane analogs (12 and 13) as superior lead compounds with potent anti-bovine viral diarrhea virus (BVDV) activity, having 50% effective concentration (EC(50): based on reduction of BVDV replication-induced cell destruction) and 50% cytotoxic concentration (CC(50): based on reduction of viable cell number) values of 6.2-8.4 microM and >100 microM, respectively, in Madin-Darby bovine kidney (MDBK) cells infected with BVDV.

Design, synthesis and anti flaviviridae activity of N(6)-, 5',3'-O- and 5',2'-O-substituted adenine nucleoside analogs.

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)>100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.

Effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) replication.

The effect as antiviral agents versus viral hepatitis B and C of three compounds purified from natural products commonly used as remedies in traditional Chinese medicine, cantharidin, cephalotaxine and homoharingtonine, was investigated. To assess the activity of these compounds against flavivirus, we used bovine viral diarrhoea virus (BVDV) as a surrogate for hepatitis C virus (HCV). Anti-BVDV activity was determined by reduction in BVDV-RNA production and protection of infected embryonic bovine trachea (EBTr) cells against the cytopathic effect of BVDV. The effect versus hepatitis B virus (HBV) was investigated by measuring HBsAg and HBV-DNA release from hepatoblastoma HepG2 2.2.15 cells infected with HBV. As positive control we used the standard anti-HBV and anti-HCV drugs, lamivudine and ribavirin, respectively. Up to 100 microM lamivudine and ribavirin did not induce cell toxicity, whereas they induced dose-dependent anti-HBV and anti-BVDV effects, respectively. In the same range, cantharidin, cephalotaxine and homoharringtonine induced toxicity in EBTr cells and had no protective effect against BVDV. In contrast, they were able to inhibit HBV production at concentrations 10- to 100-fold lower than those inducing cell toxicity, which suggests that they are useless for the treatment of infection by flaviviruses, but potentially useful in combined therapy against hepatitis B.

Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: from anti-pestivirus to anti-hepatitis C virus activity.

A novel class of inhibitors of the hepatitis C virus [substituted 2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridines] is described. Introduction of a fluorine in position 2 of the 2-phenyl substituent of the lead anti-pestivirus compound 1 (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) resulted in an analogue with selective activity against HCV in the subgenomic replicon system.

Antiviral effect of artemisinin from Artemisia annua against a model member of the Flaviviridae family, the bovine viral diarrhoea virus (BVDV).

The antiviral activity versus flaviviruses of artemisinin, a safe drug obtained from Artemisia annua and commonly used to treat malaria, has been investigated using as an IN VITRO model bovine epithelial cells from embryonic trachea (EBTr) infected with the cytopathic strain Oregon C24V, of bovine viral diarrhoea virus (BVDV), which is a member of the Flaviviridae family. Antiviral activity was estimated by the degree of protection against the cytopathic effect of BVDV on host cells and by the reduction in BVDV-RNA release to the culture medium. To induce an intermediate cytopathic effect in non-treated cells, EBTr cells were first exposed to BVDV for 48 h and then incubated with virus-free medium for 72 h. Ribavirin and artemisinin (up to 100 microM) induced no toxicity in host cells, whereas a slight degree of toxicity was observed for IFN-alpha at concentrations above 10 U/mL up to 100 U/mL. Treatment of infected cells with IFN-alpha, ribavirin and artemisinin markedly reduced BVDV-induced cell death. A combination of these drugs resulted in an additive protective effect. These drugs induced a significant reduction in the production/release of BVDV virions by infected EBTr cells; there was also an additive effect when combinations of them were assayed. These results suggest a potential usefulness of artemisinin in combination with current pharmacological therapy for the treatment of human and veterinary infections by flaviviruses.

Inhibitory effect of medicinal herbs against RNA and DNA viruses.

Fifteen Argentine medicinal plants were tested for their antiviral activity in vitro against herpes simplex viruses types 1 and 2 (HSV-1 and 2), bovine viral diarrhoea virus type 1 (BVDV-1), influenza virus type A (Inf A) and human immunodeficiency virus type 1 (HIV-1). Antiviral activity was evaluated by a reduction in cytopathic effect, plaque-forming units and p24 HIV-1 antigen. The Selective Index of the active extract (SI(extract) = CC50(extract)/EC50(extract)) of Coronopus didymus (SI(extract) = 110.7), Juglans australis (SI(extract) = 8.1) and Lippia alba (SI(extract) = 19.2) against BVDV-1, HSV-1 and influenza A virus, respectively, justify a further analysis. None of the seven plants assayed against HIV-1 displayed any antiviral activity. The results of this study justify the continuing isolation and characterization of the antiviral components present.

The use of phenothiazine dyes to inactivate bovine viral diarrhea virus in goat colostrum.

The objectives of this study were to determine the optimal concentration of phenothiazine dye required to inactivate bovine viral diarrhea virus (BVDV) in goat colostrum following 60 min of illumination and determine if immunoglobulin concentration is affected by this technique. In addition, the potential of continuous agitation of colostrum during illumination to affect viral kill was investigated. This experiment was designed to more closely approximate on-farm use than a previous pilot study performed by the same investigators. Bovine viral diarrhea virus was used as a model for caprine arthritis-encephalitis virus. Goat colostrum containing BVDV was illuminated for 60 min following the addition of either methylene blue (MB) or methylene violet (MV). Four different concentrations of each dye were evaluated. Illumination was performed in a small, portable chest-type freezer equipped on the inside with white fluorescent lights. Some samples were continuously rocked during illumination, while others remained stationary. Virus levels were determined before and after illumination. Immunoglobulin concentrations were determined for time 0 and 60 min. One microM MB reduced virus to undetectable levels following 60 min of illumination. A concentration of 20 microM MV was required to reduce virus levels to zero. Agitation of colostrum samples had no effect with either MB or MV on whether virus levels were reduced. High concentrations of MB and MV had no important effect on immunoglobulin concentrations.

Antiviral activity of hop constituents against a series of DNA and RNA viruses.

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.

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.

Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents.

The identification and development of new antiviral agents that can be used to combat hepatitis C virus (HCV) infection has been complicated by both technical and logistic issues. There are few, if any, robust methods by which HCV virions can be grown in vitro. The development of HCV RNA replicons has been a great breakthrough that has allowed for the undertaking of significant screening efforts to identify inhibitors of HCV intracellular replication. However, since replicons do not undergo a complete replication cycle, drug screening programs and mechanism of action studies based solely on these assays will not identify compounds targeting either early (virion attachment, entry, uncoating) or late (virion assembly, egress) stages of the viral replication cycle. Drugs that negatively affect the infectivity of new virions will also not be identified using HCV RNA replicons. Bovine viral diarrhea virus (BVDV) shares a similar structural organization with HCV, and both viruses generally cause chronic long-term infections in their respective hosts. The BVDV surrogate model is attractive, since it is a virus-based system. It is easy to culture the virus in vitro, molecular clones are available for genetic studies, and the virus undergoes a complete replication cycle. Like HCV, BVDV utilizes the LDL receptor to enter cells, uses a functionally similar internal ribosome entry site (IRES) for translation, uses an NS4A cofactor with its homologous NS3 protease, has a similar NS3 helicase/NTPase, a mechanistically similar NS5B RNA-dependent RNA polymerase, and a seemingly equivalent mechanism of virion maturation, assembly and egress. While the concordance between drugs active in either BVDV or HCV is largely unknown at this time, BVDV remains a popular model system with which drugs can be evaluated for potential antiviral activity against HCV and in studies of drug mechanism of action.