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.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

An in vitro assessment of antiviral activity for ethanol extract of Desmodium canadense against bovine herpesvirus type 1.

Herpesviruses (HV) are pathogens causing infections in humans and animals worldwide. Since it shares many common features with other HV, bovine HV type 1 (BoHV-1) was selected as a model to test the anti-herpesviral activity of medicinal plants.Fifteen plants were chosen in this study for their medical, antibacterial and antiviral proper-ties. The aim was to investigate ethanolic extracts from the selected medicinal plants for anti-BoHV-1 activity. The virucidal activities were evaluated by comparing the effect of noncy-totoxic concentrations of extracts on BoHV-1 strain 1640 replication in Madin-Darby bovine kidney (MDBK) cells. Virucidal activity was determined by means of virus titration after expo-sure to the extracts. The extract of Desmodium canadense was found to be the most effective virucide - the 50% tissue culture infective dose (TCID50) after exposure was 3.75 log10 and the virus reduction factor was ≥5.0±0.25 log10. The extract of D. canadense was therefore chosen for further studies. Virus yield reduction assays showed that D. canadense extract had time-depen-dent and dose-dependent effects. It effectively reduced virus titre from 8.33 log10 to 4.67 log10(p⟨0.01). The virucidal activity was also confirmed by real-time polymerase chain reaction (real-time PCR), where the number of threshold cycles (Ct) was inversely proportional to the virus titre in TCID50 The virucidal activity was also confirmed by real-time polymerase chain reaction (real-time PCR). This method showed that the number of threshold cycles (Ct) was inversely proportional to the virus titre (direct correlation with exposure time R=0.9321). The extract of D. canadense showed a high virus reduction capacity. In future, such active substances should be identified for the development of effective antivirals.

Immune memory induced by intranasal vaccination with a modified-live viral vaccine delivered to colostrum fed neonatal calves.

Bovine respiratory disease (BRD) remains a major health problem despite extensive use of vaccines during the post-weaning period. Apparent vaccine failure is attributed, in part, to primary vaccination during the period of greatest risk for BRD, providing inadequate time for onset of protective immunity. The current study investigated whether intranasal (IN) vaccination of 3-6 week old calves with a modified-live viral (MLV) vaccine induced sufficient immune memory to prevent respiratory disease and accelerate onset of protective immunity 5 months later. Vaccine groups included naïve controls, a single IN vaccination at 3-6 weeks of age, primary IN vaccination at 6 months, and either an IN or subcutaneous (SC) booster vaccination at 6 months (n = 10/group). All calves were challenged with BHV-1 four days after vaccination at 6 months of age. Primary IN vaccination at 6 months did not significantly reduce clinical disease but significantly (P < 0.01) reduced virus shedding. A single IN vaccination at 3-6 weeks of age significantly (P < 0.05) reduced weight loss but did not reduce fever or virus shedding. Both IN and SC booster vaccinations, significantly (P < 0.01) reduced clinical disease but virus shedding was significantly (P < 0.001) reduced only by IN booster vaccination. Reduction in virus shedding was significantly (P < 0.01) greater following booster versus primary IN vaccination at 6 months. All vaccination regimes significantly (P < 0.01) reduced secondary bacterial pneumonia and altered interferon responses relative to naïve controls. Only IN booster vaccination significantly (P < 0.05) increased BHV-1 specific IgA in nasal secretions. These results confirm primary MLV IN vaccination at 3 to 6 weeks of age, when virus neutralizing maternal antibody was present, induced immune memory with a 5 month duration. This immune memory supported rapid onset of protective immunity four days after an IN booster vaccination.

Anti-Inflammatory and antiviral effects of water-soluble crude extract from Phragmites australis in vitro.

Phragmitesaustralis (P. australis), a worldwide distributed wetland grass, is traditionally used as food-making helper and spice in China. The pharmacological effect of this plant is poorly understood. Here, we demonstrated that lipopolysaccharide (LPS)-induced production of inflammatory mediators nitric oxide (NO) and reactive oxygen species (ROS), and the pro-inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1ß (IL-1ß) in RAW264.7 macrophage were significantly inhibited by the crude extract. The inflammation pertinent signaling extra cellular signal-regulated kinase 1/2 (Erk1/2), P38MAPK, C-Jun and NF-kappaB (NF-κB) activated by LPS could be dramatically inhibited by this extract. It also remarkably inhibited bovine herpes virus type 1 (BoHV-1) replication in MDBK cells. Taken together, here, for the first time we provided P. australisa a novel natural herb as a potential candidate for the generation of antiviral and anti-inflammatory agent.

Non-cytotoxic Thymus capitata extracts prevent Bovine herpesvirus-1 infection in cell cultures.

BACKGROUND: Bovine herpes virus type 1 (BHV-1) still causes great economic loss to the livestock industry and trade because there aren't any available drugs that proved to be fully effective against it. In this study, the cytotoxicity and the antiviral activities of the Thymus capitata extracts were evaluated for the development of new, non toxic and specific anti-herpesvirus drug. Aqueous extracts (AE), ethanolic extracts (EE) and essential oil (EO) of the aerial parts of Thymus capitata were analyzed to determine their chemical compositions by gas chromatography, and high performance liquid chromatography combined with mass spectrometry. Their cytotoxicity and antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by quantifying the reduction of the viral cytopathic effect using Madin-Darby Bovine Kidney cell line with colorimetric assay. T. capitata extracts were added at different stages of the viral infection to investigate and better quantify their potential inhibitory effects. RESULTS: Polyphenols and flavonoids were the major compounds found in T. capitata EO, EE and AE. The cytotoxic concentrations at 50% were 48.70, 189 and 289 µg ml(-1) for EO, EE and AE, respectively. The inhibitor concentrations at 50% for the EO, EE and AE, were 3.36, 47.80 and 164 µg ml(-1), respectively. The selectivity index anti-BHV-1 values were 14.49, 3.95 and 1.81 for EO, EE and AE, respectively. Thus, the EO extracts were the most efficient antiviral compounds. T. capitata extracts affect mainly the adsorption of BHV-1 virus to host cells. CONCLUSION: T. capitata extracts inhibit the viral replication by interfering with the early stages of viral adsorption and replication. Thus, T. capitata is a potential candidate for anti-herpesvirus treatment.

In vitro antiviral activity from Acanthospermum australe on herpesvirus and poliovirus.

CONTEXT: The Asteraceae family has been of interest to researchers due to the presence of polyphenolic compounds, mainly flavonoids, which demonstrated antiviral activity. OBJECTIVE: The hydroethanol extract of the aerial parts of Acanthospermum australe (Loefl.) Kuntze (Asteraceae) and its fractions, were evaluated in vitro for their potential cytotoxic and antiviral activity against bovine herpesvirus and human poliovirus. MATERIALS AND METHODS: The sulforhodamine B colorimetric assay were used to evaluate the capacity of the hydroethanol extract and fractions to inhibit the lytic activity of herpes and poliovirus in infected cell cultures and their influence on the viability of uninfected cell cultures. RESULTS AND DISCUSSION: A progressive increase in the antiviral effect against herpesvirus was observed in the course of the purification process of the extract. The hydroethanol extract had a 50% antiviral effective concentration (EC(50)) at 70 µg/mL and 36 µg/mL for herpes and poliovirus, respectively, and it exhibited no cytotoxicity. The fractions F3 (dichloromethane) and F4 (dichloromethane: ethyl acetate (1:1 v/v)) both showed EC(50) at 6.25 µg/mL against herpesvirus, and these fractions showed cytotoxic concentrations (CC(50)) at 12.7 and 11.7 µg/mL, respectively. These fractions had no effect against poliovirus in the concentrations tested. From the bioactive F3, a diterpene lactone (acanthoaustralide-1-O-acetate) was isolated at a concentration of 0.5% and from F4 two flavonoids (quercetin and chrysosplenol D) were isolated at concentrations of 0.14 and 0.24%, respectively. CONCLUSION: The present study reports for the first time the antiviral activity of extracts and fractions from A. australe aerial parts.

Lactoferrin from bovine milk inhibits bovine herpesvirus 1 in cell culture but suppresses development of in vitro-produced bovine embryos.

Bovine herpesvirus 1 (BoHV-1) is widely distributed among cattle populations and has been associated with cells, fluids, and tissues collected from donor animals for use in reproductive technologies. The purpose of this study was to determine if lactoferrin would inhibit BoHV-1 in cell culture and to evaluate if embryos could develop normally when cultured in vitro with lactoferrin. In Experiment 1, lactoferrin (10 mg/mL) inhibited up to 25,000 plaque forming units (PFU)/mL of BoHV-1 in Madin Darby bovine kidney (MDBK) cell culture. In Experiment 2, lactoferrin (10 mg/mL) combined with cidofovir (62.5 microg/mL) inhibited up to 100,200 PFU/mL of virus in cell culture. In Experiment 3, following fertilization, presumptive zygotes were cultured in media containing lactoferrin (10, 5, and 2.5 mg/mL). Embryonic development and quality were assessed, and embryonic viability was determined by counting the nucleated cells of developed blastocysts. While lactoferrin did not affect the nucleated cell count of the treated embryos, it did significantly decrease blastocyst development. In conclusion, lactoferrin from bovine milk can inhibit BoHV-1 in cell culture. However, supplementation of in vitro culture medium with lactoferrin inhibits blastocyst development of in vitro-produced embryos.

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000Da. The peptide designated 2kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40-90% of the control level, after incubation with 10-50 microM of the peptide, with EC(50) and EC(90) values of 6.25 and 15.25 microM, respectively. The IC(50) value of the 2kD peptide against Vero cells was 250 microM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 microM. The presence of the 2kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC(50) values of 12.5, 6.25, and 6.25 microM, respectively. Similar results were observed when the 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca.

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal plant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 cells, and the 50% inhibitory concentration (IC50) were 22.01 microg/ml (selectivity index (SI)=2.83) and 21.10 microg/ml (SI=2.95), respectively. At the highest concentration of the drug (40 microg/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains.

Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus.

Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia Lam., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (Mart.) Coville, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp-2 cultured cells. The antiviral activity was monitored by plaque assay and immunofluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p<0.05, Student's t-test). For S. adstringens, AqF was the most efficient, inhibiting BHV-1 and P-1 by 97% and 93%, respectively (p<0.05). In the virucidal protocol, G. ulmifolia CE inhibited the replication of BHV-1 and P-1 by 60% and 26%, respectively (p<0.05), while, for S. adstringens, inhibition of 62% (p<0.05) was demonstrated only with EtOAcF for P-1. IFA demonstrated that the greatest reduction in fluorescent cell number occurred with G. ulmifolia, under the therapeutic protocol for both virus strains. However, AqF and EtOAcF of S. adstringens were most efficient with the virucidal protocol for P-1. In conclusion, we demonstrated that G. ulmifolia and S. adstringens inhibited BHV-1 and P-1 replication, as well as, blocked the synthesis of viral antigens in infected cell cultures.

Evaluation of the antiviral activity of an aqueous extract from Phyllanthus orbicularis.

The antiviral and cytotoxic activity of Phyllanthus orbicularis, a member of the Euphorbiaceae, was evaluated in tissue culture. An aqueous extract made from leaves and stems of this plant exhibited selective antiviral indexes of 12.3 and 26 against bovine herpesvirus type 1 and herpes simplex virus type 2, respectively, showing no selective antiviral activity against adenovirus type 5 and mengovirus. Incubation with this plant extract during cell culture infection, impaired the productive replication of both herpes viruses in an extract concentration-dependent manner, also depending on the multiplicity of infection (MOI) used. The results obtained suggested that the P. orbicularis antiviral activity might be partially due to a direct interaction with virus particles or their entry into the cell, instead of interfering with intracellular virus-specific macromolecular synthesis.

Antiviral activity of an extract from leaves of the tropical plant Acanthospermum hispidum.

Incubation of the alphaherpesviruses pseudorabiesvirus (PRV) and bovine herpesvirus 1 during infection of cell cultures with an extract prepared from the leaves of Acanthospermum hispidum impaired productive replication of these viruses in a concentration-dependent manner whereas propagation of classical swine fever virus, foot-and-mouth disease virus and vaccinia virus was not affected. The 50% inhibitory concentration for cell growth (IC50) was 107 +/- 5 microliters/ml, and the concentration reducing PRV yield by 1 log10 (90% effective concentration, EC90) was 8 +/- 3 microliters/ml. The selectivity index calculated as the IC50/EC90 ration was 13 +/- 4. Delineation of the mechanism of the antiviral activity demonstrated inhibition of alphaherpesvirus attachment to and, to a lesser extent, penetration into the cells. In contrast, viral gene expression was not inhibited by the extract when added after entry of virions into the target cells. Reduced antiviral activity of A.h. against PRV deletion mutants lacking glycoprotein C (gC) or glycoproteins gC, gE, gG and gI altogether indicated that gC alone and/or viral attachment complexes of which gC is a component constitute the target structures for A. hispidum.

Antiviral activity of crude extracts of Guarea guidona.

Crude extracts of leaves and fruits of Guarea guidona were tested for antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 micrograms/ml and 500 micrograms/ml. respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 micrograms/ml. Virucidal activity was not observed at 62.5 micrograms/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 micrograms/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.

Antiviral activity of crude extracts of Guarea guidona

Crude extracts of leaves and fruits of Guarea guidona were tested antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine Cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 mug/ml and 500 mug/ml, respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 mug/ml. Virucidal activity was not observed at 62.5 mug/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 mug/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.

In vivo and in vitro effect of acyclovir on pseudorabies virus, infectious bovine rhinotracheitis virus and pigeon herpesvirus.

The effect of various concentrations of acyclovir on the mean plaque size of pseudorabies virus (SHV), infectious bovine rhinotracheitis virus (IBR virus) and pigeon herpesvirus (PHV) has been studied. Acyclovir significantly reduced the mean plaque size of SHV and PHV, whereas IBR virus was less affected and did only show a reduction of the mean plaque size at the highest concentration of acyclovir used (1000 microM). In vivo effect of acyclovir was tested using pigeons and budgerigars experimentally infected with PHV and rabbits experimentally infected with a very low dose of SHV. Intramuscular injections of acyclovir (100 mg/kg/day; three injections/day) did not prevent the appearance of clinical disease in infected pigeons nor did reduce the level of viral excretion. The same treatment applied, as for the pigeons, before infection protected most of the budgerigars as long as they were treated, but most of them died soon after the end of the treatment. Only one rabbit was protected by the treatment. SHV was recovered in the lung of only one of the treated animals, whereas it was isolated in the lungs of each control animal.

Effects of crude extracts of various plants on infectious bovine rhinotracheitis virus-plaque production.

Extracts of 28 plants were tested without demonstable antiviral activity in an agar-overlay plaque-reduction antiviral assay system, using infectious bovine rhinotracheitis virus and bovine endocardial cell cultures. Ethanolic extract of Narcissus tazetta L bulb elicited antiviral activity by inhibition of viral plaque formation. Antiviral activity was demonstrated against infectious bovine rhinotracheitis and equine rhinopneumonitis viruses. Narcissus tazetta L bulb did not directly inactivate the virus extracellularly. The extract exhibited only limited toxicity to rapidly multiplying bovine endocardial cells at plaque-inhibitory levels and was not cytoxic to preformed confluent cell monolayers. Narcissus extract did not induce the formation of drug-resistant viral strains.