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.

Photodynamic inactivation of Bovine herpesvirus type 1 (BoHV-1) by porphyrins.

In this work, the photodynamic efficiency of anionic meso-tetrakis sulfonophenyl (TPPS4), cationic meso-tetrakis methylpyridiniumyl (TMPyP) and their zinc complexes (ZnTPPS4 and ZnTMPyP) in the inactivation of Bovine herpesvirus type 1 (BoHV-1) was evaluated. At a non-cytotoxic concentration, all porphyrins showed significant antiviral activity after irradiation using a halogen lamp. The efficiency of the cationic porphyrins was higher than that of the anionic ones. Porphyrin complexation with zinc increases its lipophilicity and the number of absorbed photons, dramatically reducing the time for complete virus inactivation. The high superposition of the compound optical absorption and light source emission spectra played a key role in the virus inactivation efficiency. The results demonstrated the high effectivity of the photodynamic inactivation of BoHV-1. This method can be used as an auxiliary in the treatment of disorders attributed to BoHV-1 infection, and the porphyrins are promising photosensitizers for this application.

Kaempferol inhibited bovine herpesvirus 1 replication and LPS-induced inflammatory response.

Plant-derived flavonoids contain large amount of compounds with pharmacological effects. In this study, we showed the compound Kaempferol to have robust antiviral activity against bovine herpesvirus 1 (BoHV-1) replication in vitro. Kaempferol at a concentration of 100 µmol/l completely inhibited viral replication in MDBK cells. It mainly affects the viral replication at the post-entry stages. The inhibition of Akt signaling is a potential mechanism underlying the antiviral effect of Kaempferol. In addition, at a concentration of 25 and 50 µmol/l Kaempferol could significantly reduce the expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and macrophage inflammatory protein 1 alpha (MIP-1α) in human promonocytic U937 cells-derived macrophages (dU937) in response to lipopolysaccharides (LPS) stimulation. Overall, our results indicated that Kaempferol provides a potent protection against BoHV-1 infection and LPS-induced inflammatory response.

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.

Does porphyrin suppres the apoptotic and necrotic effects of bovine herpes virus type-1(BoHV-1) and herpes simplex virus type-1(HSV-1)?

In this study, antiviral effect of porphyrin was investigated. Cooper strain of Bovine Herpes Virus type 1(BoHV-1) and Kos strain of Herpes Simplex Virus type-1 (HSV-1) were used to determine the potential of porphyrins to inhibit infection in vitro (with morphological and cytopathological criteria). Apoptotic and necrotic changes were determined by using DAPI and propidium staining. The non-cytotoxic dose of porphyrin (NCD-p) was initially calculated as 312.50µg/mL on MDBK and Vero cells. The apoptotic cell (APC) count was found 10% with BoHV-1 while it was 5.3% with BoHV-1 treated with porphyrin on MDBK cells between 6th to 24th hours post infection (hpi). Necrotic cell (NEC) count was 51% with BoHV-1 and 37.8% BoHV-1 treated with porphyrin on MDBK cells at 24th hpi. On the other hand, the APC count was found 23% with HSV-1, while 22% with the HSV-1 treated with porphyrin on Vero cells between 6th to 24th hpi. NEC count was 49% with HSV-1 and 34% HSV-1 treated with porphyrin on MDBK cells at 24th hpi. The results show that BoHV-1 was inhibited by porphyrin resulting in decreased apoptotic and necrotic changes in MDBK cells. On the contrary, porphyrine was not effective in the inhibition of HSV-1 in terms of apoptosis but it caused necrotic changes in Vero cells.

Curcumin inhibits bovine herpesvirus type 1 entry into MDBK cells.

The generation of antiviral drugs from herbs and other natural resources with traditionally long-confirmed effects is an efficient approach. So far, no herb or components from herbs that could inhibit bovine herpesvirus type 1 (BoHV-1) replication have been described. In this study, the antiviral effect of curcumin, a natural phenolic constituent of the spice turmeric, on BoHV-1 replication was evaluated in cell culture. We demonstrated that curcumin impairs BoHV-1 viral particles and affects the virus post-binding entry process. Furthermore, curcumin upregulated the proportion of the plasma membrane adopting a lipid raft conformation in MDBK cells, which supported the previous reports that curcumin can modulate the lipid bilayer. Though the antiviral mechanism of curcumin on BoHV-1 needs further study, we identified for the first time a component from herb that could inhibit BoHV-1 replication, in vitro.

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.

Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters.

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay-Bovine Gene Chip-was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons.

Polysaccharide and extracts from Lentinula edodes: structural features and antiviral activity.

BACKGROUND: Lentinula edodes, known as shiitake, has been utilized as food, as well as, in popular medicine, moreover, compounds isolated from its mycelium and fruiting body have shown several therapeutic properties. The aim of this study was to determine the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts and polysaccharide (LeP) from Lentinula edodes in the replication of poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BoHV-1). METHODS: The time-of-addition assay was performed at the times -2, -1, 0, 1 and 2 h of the infection. The virucidal activity and the inhibition of viral adsorption were also evaluated. Plaque assay was used to monitor antiviral activity throughout. RESULTS: The AqE and LeP were more effective when added at 0 h of infection, however, EtOHE was more effective at the times 1 h and 2 h of the infection. AqE, EtOHE and LeP showed low virucidal activity, and the inhibition of viral adsorption was not significant. CONCLUSIONS: The results allowed us to conclude that AqE, EtOHE and LeP act on the initial processes of the replication of both strains of virus.

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.

Antiviral properties of polysaccharides from Agaricus brasiliensis in the replication of bovine herpesvirus 1.

Natural products are an inexhaustible source of compounds with promising pharmacological activities, including antiviral action. In the present study, the antiviral potential of polysaccharide-peptide (PLS) and an extracted ß-glucan from Agaricus brasiliensis were investigated in the replication of bovine herpesvirus 1 (BoHV-1) in HEp-2 cell cultures. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the plaque reduction assay. To study the possible mode of action of PLS and ß-glucan, the following protocols were performed: the virucidal assay, adsorption assay and the time-of-addition assay. The PLS presented a selectivity index (SI) higher than 12.50 and ß-glucan 9.19. The antiviral inhibition (67.9%) in cells treated with PLS during virus infection was higher than that in cells treated prior to or post infection. The ß-glucan presented high inhibition of virus replication by plaque assay (83.2%) and by immunofluorescence assay (63.8%). Although the mechanism has yet to be defined, we suggest that PLS and ß-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. Additional studies are required for a better understanding of the mechanism of action of PLS and ß-glucan.

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.

Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model.

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 103 PFU/mL) versus untreated (8.25 ± 0.25 × 105 PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.

MG-132 interferes with iron cellular homeostasis and alters virulence of bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) requires an iron-replete cell host to replicate efficiently. BoHV-1 infection provokes an increase in ferritin levels and a decrease of transferrin receptor 1 (TfR-1) expression, ultimately lowering iron pool extent. Thus, cells try to limit iron availability for virus spread. It has been demonstrated that MG-132, a proteasome inhibitor, reduces BoHV-1 release. Since ferritin, the major iron storage protein in mammalian cells, undergoes proteasome-mediated degradation, herein, the influence of MG-132 on iron metabolism during BoHV-1 infection was examined. Following infection in bovine cells (MDBK), MG-132 reduced cell death and viral yield. Western blot analysis showed a significant ferritin accumulation, likely due to the inhibition of its proteasome-mediated degradation pathway. In addition, the concomitant down-regulation of TfR-1 expression, observed during infection, was counteracted by proteasome inhibitor. This trend may be explained by enhanced acidic vesicular organelles, detected by acridine orange staining, determining a reduction of intracellular pH, that promotes new synthesis of TfR-1 degraded in a recycling pathway. In addition, MG-132 influences cellular iron distribution during BoHV-1 infection, as revealed by Perls' Prussian blue staining. However, cellular iron content, evaluated by Atomic Absorption Spectrophotometry, resulted essentially unaltered. These findings reveal that MG-132 may contribute to limit cellular iron availability for virus replication thereby enhancing cell survival.

Dewormer drug fenbendazole has antiviral effects on BoHV-1 productive infection in cell cultures.

BACKGROUND: Fenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects. OBJECTIVES: This study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures. METHODS: The effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods. RESULTS: Fenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 (bICP0), bCP4, and bICP22, as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection. CONCLUSIONS: Overall, fenbendazole has antiviral effects on BoHV-1 replication.

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.

Bovine herpesvirus type 1 (BoHV-1) anterograde neuronal transport from trigeminal ganglia to nose and eye requires glycoprotein E.

The requirement of bovine herpesvirus type 1 (BoHV-1) envelope protein gE (Us8 homolog) for establishment of latency and reactivation in trigeminal ganglia (TG) was examined. Although BHV-1 gE-rescued and gE-deleted viruses were isolated from nasal or ocular swabs during primary infection, only the gE-rescued virus was isolated following dexamethasone-induced reactivation. Furthermore, gC protein expression, which requires viral DNA replication for its expression, was detected in TG of calves infected with either virus following reactivation. These studies suggest that gE is required for anterograde transport of BoHV-1 from neuronal cell bodies in the TG to their nerve processes.

Evaluation of trypsin treatment on the inactivation of bovine herpesvirus type 1 on in vitro produced pre-implantation embryos.

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 microl of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Co-encapsulation of acyclovir and curcumin into microparticles improves the physicochemical characteristics and potentiates in vitro antiviral action: Influence of the polymeric composition.

The present study developed and characterized microparticles formulations containing acyclovir and curcumin co-encapsulated in order to overcome the biopharmaceutical limitations and increase the antiviral effect of both drugs. The microparticles were prepared by a spray drying methodology following the ratio 1:3 (drug:polymer), which were made by hydroxypropylmethylcellulose (HPMC) and/or Eudragit® RS100 (EUD). The MP-1 formulation was composed of HPMC and EUD (1:1), MP-2 formulation was composed only of HPMC and MP-3 formulation was composed only of EUD. All formulations showed yielding around 50% and acceptable powder flowability. Drug content determination around 82.1-96.8% and 81.8-87% for acyclovir and curcumin, respectively. The microparticles had spherical shape, size within 11.5-15.3 µm, unimodal distribution and no chemical interactions among the components of the formulations. Of particular importance, the polymeric composition considerably influenced on the release profile of the drugs. The in vitro release experiment demonstrated that the microencapsulation provided a sustained release of acyclovir as well as increased the solubility of curcumin. Besides, mathematical modeling indicated that the experimental fit biexponential equation. Importantly, drugs microencapsulation promoted superior antiviral effect against BoVH-1 virus in comparison to their free form, which could be attributed to the improvement in the aforementioned physicochemical parameters. Therefore, these formulations could be promising technological drug carriers for acyclovir and curcumin, which highlight the great offering a potential alternative treatment for viral herpes.

Bovine Herpesvirus 1 Productive Infection Led to Inactivation of Nrf2 Signaling through Diverse Approaches.

Bovine herpesvirus type 1 (BoHV-1) is a significant cofactor for bovine respiratory disease complex (BRDC), the most important inflammatory disease in cattle. BoHV-1 infection in cell cultures induces overproduction of pathogenic reactive oxygen species (ROS) and the depletion of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a master transcriptional factor regulating a panel of antioxidant and cellular defense genes in response to oxidative stress. In this study, we reported that the virus productive infection in MDBK cells at the later stage significantly decreased the expression levels of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) proteins, the canonical downstream targets regulated by Nrf2, inhibited Nrf2 acetylation, reduced the accumulation of Nrf2 proteins in the nucleus, and relocalized nuclear Nrf2 proteins to form dot-like staining patterns in confocal microscope assay. The differential expression of Kelch-like ECH associated protein 1 (KEAP1) and DJ-1 proteins as well as the decreased association between KEAP1 and DJ-1 promoted Nrf2 degradation through the ubiquitin proteasome pathway. These data indicated that the BoHV-1 infection may significantly suppress the Nrf2 signaling pathway. Moreover, we found that there was an association between Nrf2 and LaminA/C, H3K9ac, and H3K18ac, and the binding ratios were altered following the virus infection. Taken together, for the first time, we provided evidence showing that BoHV-1 infection inhibited the Nrf2 signaling pathway by complicated mechanisms including promoting Nrf2 degradation, relocalization of nuclear Nrf2, and inhibition of Nrf2 acetylation.