Grape seed extract against Aichi virus infectivity in model foods and contact surfaces.

Aichi virus (AiV) is an enteric virus that affects humans and is prevalent in sewage waters. Effective strategies to control its spread need to be explored. This study evaluated grape seed extract (GSE) for: a) antiviral potential towards AiV infectivity at 37 °C and room temperature (RT); b) antiviral behavior in model foods (apple juice (AJ) and 2% fat milk) and also simulated gastric environments; and c) potential application as a wash solution on stainless steel surfaces. GSE at 0.5 mg/mL decreased AiV suspensions containing ~4.75 log PFU/mL to titer levels that were not detected after 30 s at both 37 °C and RT. Infectious AiV titers were not detected after 5 min treatment with 1 mg/mL GSE at 37 °C in AJ. GSE at 2 mg/mL and 4 mg/mL in 2% fat milk decreased AiV after 24 h by 1.18 and 1.57 log PFU/mL (4.75 log PFU/mL to 2.86 and 3.25 log PFU/mL), respectively. As a surface wash, GSE at 1 mg/mL after 30 s decreased AiV to undetectable levels under clean conditions. With organic load (mimicking unclean conditions), 2 and 4 mg/mL GSE reduced AiV after 5 min by 1.13 and 1.71 log PFU/mL, respectively. Overall, GSE seems to be a promising antiviral agent against AiV at low concentrations and short contact times.

Evaluation of the Effectiveness of Metabolites of Bacterial Strains Bacillus thuringiensis against Human Influenza Virus A/Aichi/2/68 (H3N2) In Vitro and In Vivo.

The morphological and physiological characteristics of Bacillus thuringiensis strains were analyzed and conditions for obtaining culture fluid with maximum yield of secreted RNases were determined. Zymographic analysis showed that culture fluid of B. thuringiensis strains along with low-molecular-weight (15-20 kDa) RNases contained enzymes with a molecular weight ~55 kDa and their content depended on the duration and conditions of culturing. Preparations based on B. thuringiensis culture fluid were effective against human influenza virus A/Aichi/2/68 (H3N2). In experiments on mice infected with 10 LD50 influenza virus strain A/Aichi/2/68 (H3N2), we selected effective variants of preparations based on culture fluid of B. thuringiensi strains for preventive administration that provided reliable protection of infected animals (protection coefficient 50%), close to that of the reference drug Tamiflu.

Antiviral effects of blueberry proanthocyanidins against Aichi virus.

Blueberry polyphenols are known for their high antioxidant and antimicrobial potential. Aichi virus (AiV) is an emerging human enteric virus that causes gastroenteritis outbreaks worldwide. This study aimed to (1) determine the time- and dose-dependent effects of blueberry proanthocyanidins (B-PAC) against AiV over 24 h at 37 °C; (2) gain insights on their mode of action using pre- and post-treatment of host cells and Transmission Electron Microscopy; and (3) determine their anti-AiV effects in model foods and under simulated gastric conditions. AiV at ∼5 log PFU/ml was incubated with equal volumes of commercial blueberry juice (BJ, pH 2.8), neutralized BJ (pH 7.0), B-PAC (2, 4, and 10 mg/ml) prepared either in 10% ethanol, apple juice (AJ), 2% milk, simulated gastric fluid (SGF, pH 1.5) or simulated intestinal fluid (SIF, pH 7.5), and controls (malic acid (pH 3.0), phosphate buffered saline (pH 7.2), apple juice (pH 3.6) and 2% milk) over 24 h at 37 °C, followed by standard plaque assays. Each experiment was replicated thrice and data were statistically analyzed. Differences in AiV titers with 1 mg/ml B-PAC were 2.13 ±â€¯0.06 log PFU/ml lower after 24 h and ≥3 log PFU/ml (undetectable levels) lower with 2 and 5 mg/ml B-PAC compared to AiV titers in PBS after 24 h and 3 h, respectively. BJ at 37 °C resulted in titer differences (lower titers compared to PBS) of 0.17 ±â€¯0.06, 1.27 ±â€¯0.01, and 1.73 ±â€¯0.23 log PFU/ml after 1, 3, and 6 h and ≥3 log PFU/ml after 24 h. Pre- and post-treatment of host cells with 0.5 mg/ml B-PAC caused titer decreases of 0.62 ±â€¯0.33 and 0.30 ±â€¯0.06 log PFU/ml, respectively suggesting a moderate effect on viral-host cell binding. B-PAC at 2 mg/ml in AJ caused titer differences of ≥3 log PFU/ml after 0.5 h, while differences of 0.84 ±â€¯0.03 log PFU/ml with 5 mg/ml B-PAC in milk, and ≥3 log PFU/ml with B-PAC at 5 mg/ml in SIF after 30 min were obtained. This study shows the ability of BJ and B-PAC to decrease AiV titers to potentially prevent AiV-related illness and outbreaks.

Co-culture with Enterobacter cloacae does not Enhance Virus Resistance to Thermal and Chemical Treatments.

Human noroviruses (hNoV) are the primary cause of foodborne disease in the USA. Most studies on inactivation kinetics of hNoV and its surrogates are performed in monoculture, while the microbial ecosystem effect on virus inactivation remains limited. This study investigated the persistence of hNoV surrogates, murine norovirus (MNV) and Tulane virus (TuV), along with Aichi virus (AiV) under thermal and chemical inactivation in association with Gram-negative (Enterobacter cloacae) bacteria. Thermal inactivation of viruses in co-culture with E. cloacae revealed no protective effects of bacteria. At 56 °C, AiV with and without bacteria was completely inactivated by 10 min with decimal reduction values (D-values) of 41 and 43 s, respectively. Similar results were also observed for TuV. Conversely, MNV with bacteria was completely inactivated by 10 min while MNV alone remained stable up to 30 min at 56 °C. Both MNV and TuV were slightly more stable than AiV at 63 °C with TuV detection up to 2 min without bacteria. For chemical inactivation on stainless steel surfaces, viruses alone and in association with bacteria were treated with 1000 ppm sodium hypochlorite. Virus association with bacteria had no significant effect (p > 0.05) on virus resistance to bleach inactivation compared to virus alone. Specifically, exposure to 1000 ppm bleach for 5 min resulted in an average of 3.86, 2.14, and 0.94 log10 PFU/ml reductions for TuV, MNV, and AiV without bacteria, respectively. Reductions in TuV, MNV, and AiV were 3.50, 1.88, and 0.61 log10 PFU/ml when associated with E. cloacae, respectively.

Linear antimicrobial peptides with activity against herpes simplex virus 1 and Aichi virus.

Viruses are the major cause of disease and mortality worldwide. Nowadays there are treatments based on antivirals or prophylaxis with vaccines. However, the rising number of reports of viral resistance to current antivirals and the emergence of new types of virus has concerned the scientific community. In this scenario, the search for alternative treatments has led scientists to the discovery of antimicrobial peptides (AMPs) derived from many different sources. Since some of them have shown antiviral activities, here we challenged 10 synthetic peptides from different animal and plant sources against, herpes simplex virus 1 (HSV-1), and Aichi virus. Among them, the highlight was Pa-MAP from the polar fish Pleuronectes americanus, which caused around 90% of inhibition of the HSV with a selectivity index of 5 and a virucidal mechanism of action. Moreover, LL-37 from human neutrophils showed 96% of inhibition against the Aichi virus, showing a selectivity index of 3.4. The other evaluated peptides did not show significant antiviral activity. In conclusion, the present study demonstrated that Pa-MAP seems to be a reliable candidate for a possible alternative drug to treat HSV-1 infections. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 108: 1-6, 2017.

Aqueous Extracts of Hibiscus sabdariffa Calyces to Control Aichi Virus.

Aqueous Hibiscus sabdariffa extracts possess antimicrobial properties with limited information available on their antiviral effects. Aichi virus (AiV) is an emerging foodborne pathogen that causes gastroenteritis. Vaccines are currently unavailable to prevent their disease transmission. The objective of this study was to determine the antiviral effects of aqueous H. sabdariffa extracts against AiV. AiV at ~5 log PFU/ml was incubated with undiluted (200 mg/ml), 1:1 (100 mg/ml) or 1:5 (40 mg/ml) diluted aqueous hibiscus extract (pH 3.6), phosphate-buffered saline (pH 7.2 as control), or malic acid (pH 3.0, acid control) at 37 °C over 24 h. Treatments were stopped by serially diluting in cell-culture media containing fetal bovine serum and titers were determined using plaque assays on confluent Vero cells. Each treatment was replicated thrice and assayed in duplicate. AiV did not show any significant reduction with 1:1 (100 mg/ml) or 1:5 (40 mg/ml) diluted aqueous hibiscus extracts or malic acid after 0.5, 1, or 2 h at 37 °C. However, AiV titers were reduced to non-detectable levels after 24 h with all the three tested concentrations, while malic acid showed only 0.93 log PFU/ml reduction after 24 h. AiV was reduced by 0.5 and 0.9 log PFU/ml with undiluted extracts (200 mg/ml) after 2 and 6 h, respectively. AiV treated with 1:1 (100 mg/ml) and 1:5 (40 mg/ml) diluted extracts showed a minimal ~0.3 log PFU/ml reduction after 6 h. These extracts show promise to reduce AiV titers mainly through alteration of virus structure, though higher concentrations may have improved effects.

Comprehensive comparison of cultivable norovirus surrogates in response to different inactivation and disinfection treatments.

Human norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log10 units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log10 units by alcohols, in contrast to 2 and 3 log10 units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log10 units by 1,000 ppm chlorine, in contrast to 1 log10 unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.