Occurrence and Accumulation of Human Enteric Viruses and Phages in Process Water from the Fresh Produce Industry

The virological quality of process water (PW) used by the produce industry has received limited attention. As a first step to overcoming technical limitations in monitoring viruses in PW, the analytical performance of ultrafiltration was assessed to concentrate viral particles from 20 L of spiked PW. The selected method used for sample concentration of PW was carefully validated, thus enabling the accurate quantification and estimation of viral titers of human enteric viruses and phages. PW from the produce industry was collected periodically from the washing tanks of commercial facilities. The analysis of coliphages was performed by plaque assay, while the occurrence of enteric viruses and crAssphage was determined by molecular techniques. Significant differences in the physicochemical composition of PW, mostly due to the different nature of fresh produce types and differences in the sanitizer used in commercial operation, were observed. Accumulation of crAssphage and coliphages was observed in PW, but correlation with human enteric viruses was not possible due to the low prevalence of these pathogens in the PW analyzed. The obtained results showed that depending on the type of product washed, the product/water ratio and the residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly.

Quantitative RT-PCR Detection of Human Noroviruses and Hepatitis A Virus in Surface Water and Fresh Produce

Vegetables can be considered as a vehicle for the transmission of human enteric viruses such as noroviruses (genogroups I and II) and hepatitis A virus (HAV) when irrigated with contaminated irrigation or when prepared by infected food handlers. In the current study, we investigated the presence of hepatitis A virus (HAV) and human noroviruses (genogroups I and II) in fresh produce and surface water used in cultivation of this produce using real-time PCR. Samples were collected from six different points in the Mansoura and Giza regions, Egypt. Our analysis showed that at least one virus was found in 41.6% (30/72) of surface water samples and 27% (13/48) of fresh produce samples. HAV (23/72) with a mean viral concentration = 4 × 106 genome copies/litter (GC/L) was the most frequently identified virus in surface irrigation water samples, followed by human norovirus genogroup II (HNoV GII) (15/72, with a mean concentration = 1.2 × 106 GC/L, and human noroviruses genogroup I (HNoV GI) (12/72, with a mean concentration = 1.4 × 104 GC/L). Additionally, HAV (10/48) with a mean concentration = 5.2 × 105 genome copies/gram (GC/g) was also the most frequently detected virus in the fresh produce samples, followed by HNoV GII (8/48, with a mean concentration = 1.7 × 104 GC/g), meanwhile HNoV GI (6/48) was less detected virus with a mean concentration = 3 × 103 GC/g. This work suggests a wide prevalence of human enteric viruses in surface irrigation waters and fresh produce, which is of concern when the fresh produce is eaten raw. Thus, Additional monitoring for viral pathogens in irrigation water and food is needed to increase produce safely.

Wastewater-Based Epidemiology of Enteric Viruses and Surveillance of Acute Gastrointestinal Illness Outbreaks in a Resource-Limited Region

Acute gastrointestinal illness (AGI) continues to be an important cause of morbidity and mortality among all ages. This study applied the principles of wastewater-based epidemiology for the preventive identification of potential outbreaks of acute viral gastroenteritis and hepatitis A by analyzing the presence of human enteric viruses in influents of small municipal wastewater treatment plants (WWTPs) handling domestic sewage, together with public health reports of acute diarrheal and hepatitis A disease in Costa Rica during 2013. Raw wastewater samples were collected during four seasonal periods with different rainfall levels. The presence of five human enteric viruses (rotavirus A, norovirus GI, norovirus GII, enterovirus, and hepatitis A virus) was studied by endpoint and real-time polymerase chain reaction in influents of five WWTPs. Cases of AGI were analyzed using historical public health reports of endemic levels and quartile ranges for each administrative and territorial area where the WWTPs are located and for its surrounding counties. A tendency for a higher rate of positive viral tests was present 1 week before an increase of AGI cases. Epidemiological weeks categorized as Outbreak (above the 75th percentile) and Success (below the 25th percentile) showed statistically significant differences in terms of positive viral test rates (Wilcoxon test, P = 0.05). Virological monitoring of wastewater in small WWTPs is an appropriate model for epidemiological surveillance of diarrheal and hepatitis A diseases in low- and middle-resource countries.

Norovirus Persistence in Oysters to Prolonged Commercial Purification

Depuration is generally the main treatment employed for bivalve mollusks harvested from contaminated sites. Commercial depuration has demonstrated to be effective for removal of bacterial pathogens, although it probably provides only limited efficacy against human enteric viruses. We evaluated the quantitative reduction of norovirus (NoV) genogroups I and II in naturally contaminated oysters after 1, 4, and 9 days of depuration. The process was conducted in an authorized depuration plant, and NoV concentration was determined by RT-qPCR according to ISO 15216-1:2017 method. Regardless of the NoV genogroup, our results showed no significant reduction in NoV concentration after 1 day of depuration. Higher mean reduction (68%) was obtained after 4 days of treatment, while no further increase was observed after 9 days. Overall, reduction was highly variable, and none of the trials showed statistically significant reduction in NoV RNA concentration at the end of each depuration period. Indeed, NoV concentration remained high in 70% of samples even after 9 days of depuration, with values ranging between 4.0 × 102 and 2.3 × 104 g.c./g. These results indicate that an extension of commercial depuration time does not appear to be effective for reducing or eliminating NoV in oysters.

Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

Effects of Contamination of Freshwater Habitat With Common Heavy Metals and Anions on the Prevalence of Human Adenoviruses and Enteroviruses

The occurrence and survival of enteric viruses in open surface waters can be impacted by a host of factors including fecal emission levels, seasonal variations, virus stability and the physicochemical parameters. In this research, we aimed to document the association between contaminations of water samples with human enteric viruses (adenoviruses and enteroviruses) from a freshwater lake with variations in chemical contaminants. We collected 216 water samples from October 2010 to April 2012, from a 4 km stretch along Lake Victoria (LV) basin in Homa Bay town located in the western region of Kenya. The samples were analyzed for the existence of human adenoviruses (HAdV) and human enteroviruses (HEV), using the nested PCR (nPCR). We also assessed in the water samples the levels of twelve chemical contaminants consisting of six heavy metal elements and six anions. About 8.3 % of the samples were found to be contaminated with the enteric viruses. The concentrations of the 12 chemical contaminants were found to be largely within the WHO suggested limits. Most of the chemical contaminants were not related to the detection rates of the viruses from the statistical analysis. However, some positive and negative associations between the viral genome's detection and the chemical concentrations were established for only three metals (Fe, Pb, Cd) and the PO43− Radical. Cd had a weak positive significant relationship with HAdV (rho = 0.146, p = 0.032) while Pb and Fe had a weak positive significant relationship with HEV genome detection (rho = 0.156, p = 0.022) and (rho = 0.148 and p = 0.029) respectively. There was a modest negative relationship between phosphate ions and HEV (rho = −0.174, p = 0.010). The results of our study do not provide support for the hypothesis of an association between the presence of human enteric viruses and the levels of twelve chemical contaminants.