Cellular, Molecular, Pharmacological, and Nano-Formulation Aspects of Thymoquinone-A Potent Natural Antiviral Agent.

The goal of an antiviral agent research is to find an antiviral drug that reduces viral growth without harming healthy cells. Transformations of the virus, new viral strain developments, the resistance of viral pathogens, and side effects are the current challenges in terms of discovering antiviral drugs. The time has come and it is now essential to discover a natural antiviral agent that has the potential to destroy viruses without causing resistance or other unintended side effects. The pharmacological potency of thymoquinone (TQ) against different communicable and non-communicable diseases has been proven by various studies, and TQ is considered to be a safe antiviral substitute. Adjunctive immunomodulatory effects in addition to the antiviral potency of TQ makes it a major compound against viral infection through modulating the production of nitric oxide and reactive oxygen species, decreasing the cytokine storm, and inhibiting endothelial dysfunction. Nevertheless, TQ's low oral bioavailability, short half-life, poor water solubility, and conventional formulation are barriers to achieving its optimal pharmacologic benefits. Nano-formulation proposes numerous ways to overcome these obstacles through a small particle size, a big surface area, and a variety of surface modifications. Nano-based pharmaceutical innovations to combat viral infections using TQ are a promising approach to treating surmounting viral infections.

Broccoli: A Multi-Faceted Vegetable for Health: An In-Depth Review of Its Nutritional Attributes, Antimicrobial Abilities, and Anti-inflammatory Properties.

Broccoli, Brassica oleracea var. italica, has recently gained considerable attention due to its remarkable nutritional composition and numerous health benefits. In this review, the nutritional aspects of broccoli are examined, highlighting its rich nutrient content and essential bioactive compounds. The cruciferous vegetable broccoli is a rich source of several important nutrients, including fiber, vitamins (A, C, and K), minerals (calcium, potassium, and iron), and antioxidants. It has also been shown to contain bioactive compounds such as glucosinolates, sulforaphane, and indole-3-carbinol, all of which have been shown to have significant health-promoting effects. These chemicals are known to have potent antioxidant, anti-inflammatory, and anticancer effects. This review article aims to comprehensively examine the diverse spectrum of nutrients contained in broccoli and explore its medicinal potential to promote human health.

The Involvement of Neutrophil in the Immune Dysfunction Associated with BVDV Infection.

Bovine viral diarrhea virus (BVDV) induces immune dysfunction that often results in a secondary bacterial infection in the infected animals. The underlying mechanism of BVDV-induced immune dysfunction is not well understood. The role of BVDV-infected macrophage-secreted factors was investigated. BVDV-infected monocyte-derived macrophage (MDM) supernatants down-regulated the expression of neutrophil L-selectin and CD18. Regardless of the biotype, phagocytic activity and oxidative burst were downregulated by BVDV-infected MDM supernatants. However, only supernatants from cytopathic (cp) BVDV down-regulated nitric oxide production and neutrophil extracellular traps (NET) induction. Our data suggested that BVDV-induced macrophage-secreted factors caused immune dysfunction in neutrophils. Unlike lymphocyte depletion, the negative impact on neutrophils seems to be specific to cp BVDV biotype. Interestingly the majority of modified live BVDV vaccines are based on cp strain of BVDV.

The effect of neonatal vaccination for bovine respiratory disease in the face of a dual challenge with bovine viral diarrhea virus and Mannheimia hemolytica.

Bovine respiratory disease is the greatest threat to calf health. In this study, colostrum-fed dairy X beef calves were vaccinated at ∼30 days of age with an adjuvanted parenteral vaccine containing modified live bovine viral diarrhea virus (BVDV) type 1 and type 2, bovine herpesvirus 1 (BHV-1), bovine parainfluenza type 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV) andM. haemolyticatoxoid (Group 1), or intranasal temperature-sensitive BHV-1, BRSV and PI3V concurrently witha parenteral vaccine containing modified live BVDV type 1 and type 2 andM. haemolyticatoxoid (Group 2) or a placebo (Group 3). The calves were challenged ∼150 days post vaccination intranasally with BVDV 1b and then 7 days later intratracheally withM. haemolytica. The calves wereeuthanized 6 days after theM. haemolyticachallenge. Clinical signs following BVDV infection were similar in all groups. There was increased rectal temperatures in the Groups 2 and 3 on day 3 and in Group 3 on days 8-13. Group 1 animals had a slight leukopenia following BVDV infection while Groups 2 and 3 had greater leukopenia. BVDV type 1 and 2 serum titers increased in Group 1 following vaccination while these titers waned in Groups 2 and 3. There were higher levels of BVDV in the buffy coats and nasal samples in Group 2 and Group 3 versus Group 1 (p < 0.01). Interferon-gamma response was higher (p < 0.01) in Group 1 animals than Groups 2 and 3. Group 1 had the lowest percent pneumonic tissue (1.6%) while Group 2 vaccinates had 3.7% and the control Group 3 was 5.3%. Vaccination in the face of maternal antibody with a parenteral adjuvanted vaccine resulted in better protection than the regimen of an intranasal vaccine anda parenteral adjuvanted BVDV andM haemolyticacombination vaccine in a BVDV-M. haemolyticadual challenge.

Efficacy of oleandrin and PBI-05204 against bovine viruses of importance to commercial cattle health.

BACKGROUND: Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV). and bovine coronavirus (BCV) threaten the productivity of cattle worldwide. Development of therapeutics that can control the spread of these viruses is an unmet need. The present research was designed to explore the in vitro antiviral activity of the Nerium oleander derived cardiac glycoside oleandrin and a defined N. oleander plant extract (PBI-05204) containing oleandrin. METHODS: Madin Darby Bovine Kidney (MDBK) cells, Bovine Turbinate (BT) cells, and Human Rectal Tumor-18 (HRT-18) cells were used as in vitro culture systems for BVDV, BRSV and BCV, respectively. Cytotoxicity was established using serial dilutions of oleandrin or PBI-05204. Noncytotoxic concentrations of each drug were used either prior to or at 12 h and 24 h following virus exposure to corresponding viruses. Infectious virus titers were determined following each treatment. RESULTS: Both oleandrin as well as PBI-05204 demonstrated strong antiviral activity against BVDV, BRSV, and BCV, in a dose-dependent manner, when added prior to or following infection of host cells. Determination of viral loads by PCR demonstrated a concentration dependent decline in virus replication. Importantly, the relative ability of virus produced from treated cultures to infect new host cells was reduced by as much as 10,000-fold at noncytotoxic concentrations of oleandrin or PBI-05204. CONCLUSIONS: The research demonstrates the potency of oleandrin and PBI-05204 to inhibit infectivity of three important enveloped bovine viruses in vitro. These data showing non-toxic concentrations of oleandrin inhibiting infectivity of three bovine viruses support further investigation of in vivo antiviral efficacy.

Mosaic Bovine Viral Diarrhea Virus Antigens Elicit Cross-Protective Immunity in Calves.

Bovine Viral Diarrhea Virus (BVDV) is an important pathogen that plays a significant role in initiating Bovine Respiratory Disease Complex (BRDC) in cattle. The disease causes multi-billion dollar losses globally due to high calf mortality and increased morbidity leading to heavy use of antibiotics. Current commercial vaccines provide limited cross-protection with several drawbacks such as safety, immunosuppression, potential reversion to virulence, and induction of neonatal pancytopenia. This study evaluates two prototype vaccines containing multiple rationally designed recombinant mosaic BVDV antigens for their potential to confer cross-protection against diverse BVDV strains. Genes encoding three novel mosaic antigens, designated E2123, NS2-31, and NS2-32, were designed in silico and expressed in mammalian cells for the formulation of a prototype protein-based vaccine. The mosaic antigens contain highly conserved protective epitopes from BVDV-1a, -1b, and -2, and included unique neutralizing epitopes from disparate strains to broaden coverage. We tested immunogenicity and protective efficacy of Expi293TM-expressed mosaic antigens (293F-E2123, 293F-NS2-31, and 293F-NS2-32), and baculovirus-expressed E2123 (Bac-E2123) mosaic antigen in calves. The Expi293TM-expressed antigen cocktail induced robust BVDV-specific cross-reactive IFN-γ responses, broadly neutralizing antibodies, and following challenge with a BVDV-1b strain, the calves had significantly (p < 0.05) reduced viremia and clinical BVD disease compared to the calves vaccinated with a commercial killed vaccine. The Bac-E2123 antigen was not as effective as the Expi293TM-expressed antigen cocktail, but it protected calves from BVD disease better than the commercial killed vaccine. The findings support feasibility for development of a broadly protective subunit BVDV vaccine for safe and effective management of BRD.

Bovine viral diarrhea virus compromises Neutrophil's functions in strain dependent manner.

Bovine viral diarrhea virus (BVDV) infection is a major problem that results in economically important diseases of the cattle industry worldwide. The two major consequences of this disease are persistent infection and immune dysfunction. A number of studies have been done to determine the underline mechanisms of BVDV-induced immune dysfunction, in particular targeting antigen-presenting cells, T- and B- cells and cytokine gene expression. However, little research has focused Eon the effect of BVDV on neutrophils. Neutrophils are one of the predominant leukocytes circulating in blood and are considered the first line of defense in the innate immune system along with macrophages. Neutrophils not only eliminate the invading bacteria but also activate innate as well as adaptive immune responses. Therefore, compromised neutrophil function would affect both arms of immune system and caused immune suppression. In the current study, we used virus strains from both BVDV-1 and BVDV-2 species. Including a highly virulent non-cytopathic type 2a BVDV (ncp BVDV2a-1373), moderately virulent non-cytopathic type 2a (ncp BVDV2a 28508-5), and a pair of non-cytopathic type 1b BVDV (ncp BVDV1b TGAN) and cytopathic type 1b BVDV (cp BVDV1b TGAC) strain isolated from a case of mucosal disease. The highly virulent ncp BVDV2a-1373 significantly increased neutrophil apoptosis. However, none of the other BVDV strains affected neutrophil viability. All BVDV strains used significantly reduced CD18 and L-selectin expression on neutrophils as well as their oxidative burst and neutrophil extracellular traps (NET) activity. Cp BVDV significantly reduced neutrophil's phagocytic activity but ncp BVDV did not have any effect on it. On the other hand, ncp BVDV significantly increased neutrophil's CD14 expression and chemotactic activity while cp BVDV did not show any effect either on neutrophil's CD14 expression or on chemotactic activity. In conclusion, BVDV affected neutrophils variability and functional activity in strain dependent manner. Results of the current study will further help in understanding the pathophysiology of different BVDV strains.

The Effect of Bovine Viral Diarrhea Virus (BVDV) Strains and the Corresponding Infected-Macrophages' Supernatant on Macrophage Inflammatory Function and Lymphocyte Apoptosis.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

Evaluation of essential oils and a prebiotic for newborn dairy calves.

A blend of essential oils (EO) and a prebiotic were combined (EOC) to formulate a colostrum-based liquid birth supplement and a separate feeding supplement (Start Strong and Stay Strong, Ralco Inc., Marshall, MN). These products were designed to promote immunity and stimulate appetite to diminish health challenges and stresses experienced by newborn calves. The hypothesis was that calves supplemented with an oral dose of liquid EOC at birth (10-mL aliquot at birth and 10 mL at 12 h of age) when fed the EOC feeding supplement would result in improved growth performance, health, and immunity. The objective was to determine if an additional feeding of liquid EOC at birth in combination with EOC in the milk replacer (MR) would allow calves to demonstrate improved growth, health, and immunity compare with calves only offered EO in MR. Sixty-one Holstein calves (18 males and 43 females) from a commercial dairy operation were blocked by birth date and randomly assigned to 1 of 3 treatments. Treatments were 1) Control (CON): a 24% crude protein (CP):20% fat (as-fed basis) MR; 2) EP: a 24:20 MR with EOC mixed at 1.25 g/d; or 3) EPC: a 24:20 MR with EOC mixed at 1.25 g/d in addition to calves receiving one 10-mL oral dose of liquid EOC at birth and 10 mL again at 12 h. The 24:20 MR was fed via bucket 2 times per day at a rate of 0.57 kg/calf daily for 14 d, increased to 0.85 kg/calf at 2 times per day until 35 d and was reduced to 0.43 kg at 1 time per day at 36 d to facilitate weaning after 42 d. Decoquinate was added to the MR at 41.6 mg/kg for coccidiosis control. Calves were housed in individual hutches bedded with straw with ad libitum access to a 20% CP-pelleted calf starter and water. All data were analyzed using PROC MIXED as a randomized complete block design. Calves in this study had similar (P > 0.10) average daily gains, body weight, and growth measurements. Calves fed EPC had significantly (P < 0.05) higher IgA titers on day 0 of the trial compared with calves fed EP or CON, which was expected as calves were supplemented with liquid EOC at birth and 12 h later demonstrating an increase in immune response. The use of a liquid EOC product being administrated after birth can improve IgA titers to improve the immune status of the new born calf to fight off potential diseases and pathogens. A formulation error resulted in the EOC being fed at half the rate of the previous experiment of 2.5 g/d, which appears to be below an efficacious dosage.

Protection against bovine respiratory syncytial virus in calves vaccinated with adjuvanted modified live vaccine administered in the face of maternal antibody.

Bovine respiratory syncytial virus (BRSV) is major viral contributor to bovine respiratory disease (BRD). BRD is a major cause of morbidity and mortality in all classes of cattle but particularly young beef and dairy calves. Passive antibodies not only help protect the calf against infection, but may interfere with the immune responses following vaccination. The purpose of this study was to evaluate the efficacy of an adjuvanted modified live virus (MLV) vaccine in the presence of well-defined maternal passive immunity. Calves were vaccinated at approximately 1 month of age and challenged ~90 days later when BRSV systemic antibodies were ≤1:4. Body temperature was lower at 6 and 7 days post challenge and other clinical signs were also lower in the vaccinates. Nasal viral shed was 3-4 times lower in the vaccinated animals as measured by virus isolation and polymerase chain reaction (PCR) and peaked 5 days post challenge compared to the controls (who peaked at days 6 and 7). On day 8 following challenge, animals were necropsied, and lung lobes were scored and tested for virus by PCR and indirect fluorescent assay (IFA). There was a 25-fold reduction in PCR virus detection in vaccinates and two of the vaccinated calves' lungs were PCR negative. Only 29.4% of vaccinated calves were BRSV positive on IFA testing at necropsy, while 87.5% of control calves were BRSV positive. Vaccinated calves developed a mucosal BRSV IgA response with over 50% of the vaccinated calves having IgA prior to challenge and all vaccinated calves were positive following challenge. Additionally, vaccination stimulated the production of Interferon gamma (IFN-γ) in mononuclear cells to prime the immune system. This study established that an adjuvanted MLV vaccine could provide protection against BRSV as measured by clinical, virological, and pathological parameters while also activating both mucosal and systemic immunity.

Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate.

Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS: showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.

Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens.

Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a live vector in cattle, we showed that recombinant human adenovirus-5 was cleared within one week following intradermal inoculation.