Food and Environmental Virology最新文献

Aqueous extracts of Quillaja saponaria Molina are US FDA approved as food additives in beverages with known antiviral activity. Due to lack of commercially available vaccines against human noroviruses (HNoVs), alternate methods to prevent their spread and the subsequent emergence of variant strains are being researched. Furthermore, HNoVs are not yet culturable at high enough titers to determine inactivation, therefore surrogates continue to be used. This research analyzed the effect of aqueous Quillaja saponaria extracts (QE) against HNoV surrogates, Tulane virus (TV), murine norovirus (MNV-1), and feline calicivirus (FCV-F9) at room temperature (RT) and 37 °C. Viruses (~ 5 log PFU/mL) were individually treated with 1:1 or 1:5 (v/v) diluted QE (pH ~ 3.75), malic acid control (pH 3.0) or phosphate-buffered saline (pH 7.2, as control) at 37 °C or RT for up to 6 h. Individual treatments were replicated three times using duplicate plaque assays for each treatment. FCV-F9 at ~ 5 log PFU/mL was not detectable after 15 min by 1:1 QE at 37 °C and RT. At RT, 1:5 QE lowered FCV-F9 titers by 2.05, 2.14 and 2.74 log PFU/mL after 0.5 h, 1 h and 2 h, respectively. MNV-1 showed marginal reduction of < 1 log PFU/mL after 15 min with 1:1 or 1:5 QE at 37 °C without any significant reduction at RT, while TV titers decreased by 2.2 log PFU/mL after 30 min and were undetectable after 3 h at 37 °C. Longer incubation with higher QE concentrations may be required for improved antiviral activity against MNV-1 and TV.

SARS-CoV-2 contaminated items in the cold chain becomes a threat to public health, therefore the effective and safe sterilization method fit for the low temperature is needed. Ultraviolet is an effective sterilization method while its effect on SARS-CoV-2 under low-temperature environment is unclear. In this research, the sterilization effect of high-intensity ultraviolet-C (HIUVC) irradiation against SARS-CoV-2 and Staphylococcus aureus on different carriers at 4 °C and − 20 °C was investigated. The results showed that dose of 15.3 mJ/cm² achieved more than 3 log reduction of SARS-CoV-2 on gauze at 4 °C and − 20 °C. The vulnerability of coronavirus to HIUVC under − 20 °C was not significantly different than those under 4 °C. Four models including Weibull, biphasic, log-linear tail and log linear were used to fit the survival curves of SARS-CoV-2 and Staphylococcus aureus. The biphasic model fitted best with R² ranging from 0.9325 to 0.9878. Moreover, the HIUVC sterilization correlation between SARS-CoV-2 and Staphylococcus aureus was established. This paper provides data support for the employment of HIUVC under low-temperature environment. Also, it provides a method of using Staphylococcus aureus as a marker to evaluate the sterilization effect of cold chain sterilization equipment.

Graphical Abstract

Faecal shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent detection in wastewater turned the spotlight onto wastewater-based epidemiology (WBE) for monitoring the coronavirus-disease 2019 (COVID-19) pandemic. WBE for SARS-CoV-2 has been deployed in 70 countries, providing insights into disease prevalence, forecasting and the spatiotemporal tracking and emergence of SARS-CoV-2 variants. Wastewater, however, is a complex sample matrix containing numerous reverse transcription quantitative PCR (RT-qPCR) inhibitors whose concentration and diversity are influenced by factors including population size, surrounding industry and agriculture and climate. Such differences in the RT-qPCR inhibitor profile are likely to impact the quality of data produced by WBE and potentially produce erroneous results.

To help determine the possible impact of RT-qPCR assay on data quality, two assays employed by different laboratories within the UK’s SARS-CoV-2 wastewater monitoring programme were assessed in the Cefas laboratory in Weymouth, UK. The assays were based on Fast Virus (FV) and qScript (qS) chemistries using the same primers and probes, but at different concentrations and under different cycling conditions. Bovine serum albumin and MgSO₄ were also added to the FV assay reaction mixture. Two-hundred and eighty-six samples were analysed, and an external control RNA (EC RNA)-based method was used to measure RT-qPCR inhibition. Compared with qS, FV showed a 40.5% reduction in mean inhibition and a 57.0% reduction in inter-sample inhibition variability. A 4.1-fold increase in SARS-CoV-2 quantification was seen for FV relative to qS; partially due (1.5-fold) to differences in reverse transcription efficiency and the use of a dsDNA standard. Analytical variability was reduced by 51.2% using FV while qS increased the number of SARS-CoV-2 negative samples by 2.6-fold. This study indicates the importance of thorough method optimisation for RT-qPCR-based WBE which should be performed using a selection of samples which are representative of the physiochemical properties of wastewater. Furthermore, RT-qPCR inhibition, analytical variability and reverse transcription efficiency should be key considerations during assay optimisation. A standardised framework for the optimisation and validation of WBE procedures should be formed including concessions for emergency response situations that would allow flexibility in the process to address the difficult balance between the urgency of providing data and the availability of resources.

Indirect contact with contaminated surfaces is a potential transmission route for COVID-19. Therefore, it is necessary to investigate convenient and inexpensive surface sanitization methods, such as HOCl, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 surrogate, Phi6 (~ 7 log PFU/mL), was prepared in artificial saliva and tripartite matrices, spot inoculated on coupons of either stainless steel or vinyl, and allowed to dry. The coupons were sprayed with either 500 ppm or 1000 ppm HOCl, and remained on the surface for 0 s (control), 5 s, 30 s, or 60 s. Samples were enumerated via the double agar overlay assay. Statistical analysis was completed in R using a generalized linear model with Quasipoisson error approximations. Time, concentration, surface type, and inoculum matrix were all significant contributors to log reduction at P = 0.05. Significant three-way interactions were observed for 1000 ppm, vinyl, and 60 s (P = 0.03) and 1000 ppm, tripartite, and 60 s (P = 0.0121). A significant two-way interaction between vinyl and 60 s was also observed (P = 0.0168). Overall, increased HOCl concentration and exposure time led to increased Phi6 reduction. Notably, the highest estimated mean log reduction was 3.31 (95% CI 3.14, 3.49) for stainless steel at 60 s and 1000 ppm HOCl in artificial saliva, indicating that this method of sanitization may not adequately reduce enveloped viruses to below infective thresholds.

Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID₅₀ and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by < 0.5 log₁₀ after 14 days, while NoV reductions were already > 1.0 log₁₀ at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p > 0.05). TuV TCID₅₀ decreased steadily, and reductions were significantly different between the two temperatures (p < 0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3–1.7 log₁₀ higher than at 12 °C. After 3 weeks, reductions > 3.0 log₁₀ were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log₁₀. The length of depuration also had an influence on virus numbers. TuV reductions increased from < 1.0 log₁₀ after seven days to > 4.0 log₁₀ after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg²⁺ concentration (P < 0.05), in addition to primer quality. The optimal Mg²⁺ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 10⁰ PFU/15 g, 8.3 × 10¹ PFU/50 g, 8.3 × 10⁰ PFU/5 g, and 8.3 × 10⁰ PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

This study assessed the sources of contamination of water matrices in a rural area using detection of a host-specific virus (human adenovirus [HAdV], porcine adenovirus [PAdV] and bovine polyomaviruses [BoPyV]) as potential microbial source-tracking tool, and rotavirus A [RVA], given its epidemiological importance in Brazil. From July 2017 to June 2018, 92 samples were collected from eight points (P1-P8) of surface and raw waters in southeastern region of Brazil. Fifty-five (59.8%) were positive for HAdV, 41 (44.5%) for RVA, 10 (10.9%) for PAdV and four (4.3%) for BoPyV. HAdV and RVA were detected at all sites, and over the entire sampling period, PAdV was detected at a porcine breeding area and at Guarda River site, presenting high concentrations up to 2.6 × 10⁹ genome copies per liter [GC/L], and viral concentrations ranging from 9.6 × 10¹ to 7.1 × 10⁷, while BoPyV (1.5 × 10⁴ GC/L–9.2 × 10⁵ GC/L) was only detected in samples from the bovine breeding areas. The combination of human and animal virus circulation presents a potential impact in the environment due to raw sewage discharge from regional communities, as well as potential hazard to human and animal health.

The performance of dishwashers in removing live viruses is an important informative value in practical applications. Since foodborne viruses are present in contaminated food surfaces and water environments. Insufficient washing of dishes typically makes a carrier of foodborne viruses. Dishwashers have shown excellent performance in removing bacterial pathogens, but very limited reports related to eliminate foodborne viruses on contaminated dish surfaces. Here, murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and human coronavirus 229E (HCoV-229E) were experimentally inoculated on the dish surfaces (plate, rice bowl, and soup bowl). Plaque assay, 50% tissue culture infectious dose (TCID₅₀), and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to determine their removal efficiency of them through the general wash program of household dishwashers. Using titration assay, MNV-1 and HAV were reduced by 7.44 and 6.57 log10 PFU/dish, and HCoV-229E was reduced by 6.43 log10 TCID₅₀/dish through the general wash program, achieving a ≥ 99.999% reduction, respectively. Additionally, RT-qPCR results revealed that viral RNA of MNV-1 and HCoV-229E reduced 5.02 and 4.54 log10 genome copies/dish; in contrast, HAV was not detected on any dish surfaces. This study confirmed the performance of household dishwashers in removing pathogenic live viruses through the general wash program. However, residual viral RNA was not sufficiently removed. Further studies are needed to determine whether the viral RNA can be sufficiently removed using combination programs in household dishwashers.

Graphical Abstract

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes—nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

With the widespread availability of 3D food printing systems for purchase, users can customize their food in new ways. Manufacturer recommendations for cleaning these machines remain untested with regard to the prevention of foodborne pathogen transmission. This study aimed to determine if manufacturer cleaning recommendations for food ink capsules utilized in 3D food printers are adequate to control human norovirus (HuNoV). A HuNoV surrogate, Tulane virus (TuV; ~ 6 log₁₀ PFU/mL), was inoculated onto the interior surface of stainless steel food ink capsules. Capsules were either unsoiled or soiled with one of the following: butter, protein powder solution, powdered sugar solution, or a mixture containing all three food components. The capsules were allowed to dry and then one of three hygienic protocols was applied: manual washing (MW), a dishwasher speed cycle (DSC), or a dishwasher heavy cycle (DHC). The interaction effect between DSC and pure butter was a significant predictor of log reduction (P = 0.0067), with the pure butter and DSC combination achieving an estimated mean log reduction of 4.83 (95% CI 4.13, 5.59). The DSC was the least effective method of cleaning when compared with MW and the DHC. The 3-way interaction effects between wash type, soil, and capsule position were a significant predictor of log reduction (P = 0.00341). Capsules with butter in the DSC achieved an estimated mean log reduction of 2.81 (95% CI 2.80, 2.83) for the front-most position versus 6.35 (95% CI 6.33, 6.37) for the back-most position. Soil matrix, cleaning protocol, and capsule position all significantly impact capsule cleanability and potential food safety risk. The DHC is recommended for all capsules, and the corners should be avoided when placing capsules into the dishwasher. The current study seeks to provide recommendations for users of additive manufacturing and 3D food printing including consumers, restaurants, industry, and regulatory industries.