Tracking enteric viruses in green vegetables from ...

Science of the Total Environment 637–638 (2018) 665–671

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Short Communication

Tracking enteric viruses in green vegetables from central Argentina: potential association with viral contamination of irrigation waters V.E. Prez a,b,⁎, L.C. Martínez a, M. Victoria c, M.O. Giordano a, G. Masachessi a,b, V.E. Ré a,b, J.V. Pavan a, R. Colina c, P.A. Barril b,d, S.V. Nates a a

Instituto de Virología “Dr. J. M. Vanella”, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Enfermera Gordillo Gómez s/n − Ciudad Universitaria, CP 5000 Córdoba, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas – CONICET, Argentina Laboratorio de Virología Molecular, CENUR Litoral Norte, Centro Universitario de Salto, Universidad de la República, Rivera 1350, Salto, Uruguay d Laboratorio de Microbiología de los Alimentos, Centro de Investigación y Asistencia Técnica a la Industria (CIATI A.C.), Expedicionarios del Desierto 1310, CP 8309 Centenario, Neuquén, Argentina b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Enteric virus was frequently detected in vegetables, irrigation waters and sewage. • Irrigation water is a possible source of viral contamination in raw vegetables. • Viral variants in the community were similar in vegetables and irrigation waters.

a r t i c l e

i n f o

Article history: Received 2 March 2018 Received in revised form 3 May 2018 Accepted 4 May 2018 Available online xxxx Editor: D. Barcelo Keywords: Food−borne viruses Sewage Enterovirus Norovirus Rotavirus Astrovirus

a b s t r a c t Consumption of green vegetable products is commonly viewed as a potential risk factor for infection with enteric viruses. The link between vegetable crops and fecally contaminated irrigation water establishes an environmental scenario that can result in a risk to human health. The aim of this work was to analyze the enteric viral quality in leafy green vegetables from Córdoba (Argentina) and its potential association with viral contamination of irrigation waters. During July–December 2012, vegetables were collected from peri−urban green farms (n = 19) and its corresponding urban river irrigation waters (n = 12). Also, urban sewage samples (n = 6) were collected to analyze the viral variants circulating in the community. Viruses were eluted and concentrated by polyethylene glycol precipitation and then were subject to Reverse Transcription Polymerase Chain Reaction to assess the genome presence of norovirus, rotavirus and human astrovirus. The concentrates were also inoculated in HEp−2 (Human Epidermoid carcinoma strain #2) cells to monitor the occurrence of infective enterovirus. The frequency of detection of the viral groups in sewage, irrigation water and crops was: norovirus 100%, 67% and 58%, rotavirus 100%, 75% and 5%, astrovirus 83%, 75% and 32% and infective enterovirus 50%, 33% and 79%, respectively. A similar profile in sewage, irrigation water and green vegetables was observed for norovirus genogroups (I and II) distribution as well as for rotavirus and astrovirus G−types. These results provide the first data for Argentina pointing out that green leafy vegetables are contaminated with a broad range of enteric viruses and that the irrigation water would be a source of contamination. The presence of viral genomes and infective particles in food that in general suffer minimal treatment before consumption underlines that green crops can act as potential sources of enteric virus transmission. Public intervention in the use of the river waters as irrigation source is needed. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Instituto de Virología “Dr. J. M. Vanella”, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Enfermera Gordillo Gómez s/n − Ciudad Universitaria, CP 5000 Córdoba, Argentina. E-mail address: [email protected] (V.E. Prez).

https://doi.org/10.1016/j.scitotenv.2018.05.044 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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V.E. Prez et al. / Science of the Total Environment 637–638 (2018) 665–671

1. Introduction Leafy green vegetables are important components of the current human diets but are one of the main foods involved in the transmission of human enteric viruses since they are eaten raw and usually without any further washing/decontamination procedures (Little and Gillespie, 2008). Enteric viruses can be accidentally introduced at different steps in vegetable chain production, being crop irrigation with fecally contaminated water the main critical vehicle of contamination (Beuchat, 2002; Hirneisen and Kniel, 2013; Miura et al., 2018). It should be noted that viruses can enter into environmental waters through the discharge of treated, insufficiently treated or untreated sewage. In recent years it has been reported a clear epidemiological evidence of the link between consumption of wastewater irrigated crops and viral community illness (Pavione et al., 2013; Mok et al., 2014). In Argentina, a high occurrence of enteric viruses, such as norovirus (NoV), rotavirus (RV), human astrovirus (HAstV), hepatitis A (HAV) and E viruses (HEV) and human enterovirus (EV) has recently been reported in a variety of water environments, some of which are used for irrigation purpose (Blanco Fernandez et al., 2012; Fernandez et al., 2012; Martinez Wassaf et al., 2014; Yanez et al., 2014; Barril et al., 2015; Ferreyra et al., 2015; Prez et al., 2015; Giordano et al., 2016; Farías et al., 2018). This underlines that raw vegetables could potentially be contaminated with these viruses through the irrigation water, but at present no such studies have been conducted in our country. Over 100 types of viruses, collectively known as enteric viruses and many of them recognized as pathogenic agents, are excreted in high concentration in the feces of infected humans and non−humans. They are highly stable in the environment because they lack the lipid envelope, being able to persist for long time in waters. Despite the relatively low concentration of viruses in fecal impacted waters, its presence carry health risks since they have very low infectious doses (10–100 virions) and therefore even a few viral particles in water can infect a person (La Rosa et al., 2012). Among the enteric viruses, RV and NoV are the most common etiological agents of gastroenteritis in humans. RV is widely known as one of the most important childhood diarrheal pathogens worldwide and it is estimated that causes approximately N600,000 children deaths annually all over the world. In Argentina, it is responsible for 40% of hospital admissions of acute diarrhea, and it is estimated that may cause between 30 and 50 deaths annually in the country (Bok et al., 2001). In January 2015 the monovalent rotavirus vaccine (G1[P8], GSK®) was introduced in the Argentine National Immunization Program. It has been reported that this vaccine has an estimated efficiency of 94.5% against severe disease and death (Ruiz−Palacios et al., 2006), but it does not prevent mild symptomatic infections in secondary contacts with the virus nor viral transmission to susceptible hosts. It must also be noted that RV has been implicated in waterborne and food−borne outbreaks worldwide (CDC, 2000; Mizukoshi et al., 2014). NoV is a major cause of acute viral gastroenteritis, affecting people of all age groups worldwide. Outbreaks of NoV gastroenteritis can be seasonal or sporadic cases that occur throughout the year (Glass et al., 2009). CDC estimates that each year NoV causes 19 to 21 million illnesses, 56,000 to 71,000 hospitalizations and 570 to 800 deaths. They are associated with outbreaks due to contact with infected persons or ingestion of contaminated food or water (Moore et al., 2015). Therefore, studying this viral group and its potential sources of infection in a region with scarce data, acquires great relevance. HAstV has been associated to endemic diarrheal episodes and outbreaks of gastroenteritis in industrialized and non−industrialized countries and has been detected in sewage and surface waters worldwide (Nadan et al., 2003; Jones et al., 2017). Human EVs comprise a large genus within the Picornaviridae family. They affect millions of people worldwide each year and are often found in respiratory secretions and in stool of infected persons. EV usually

cause subclinical infections, but sometimes they are associated with serious diseases, such as acute flaccid paralysis, aseptic meningitis and encephalitis, acute myocarditis, acute haemorrhagic conjunctivitis, and hand, foot, and mouth disease (Pallansch and Roos, 2007). Because most of the human EV can replicate in cell cultures, they are good indicators to confirm the presence of viable and infectious viruses in environmental samples. Moreover, EV infections have been linked to outbreaks of food−borne and waterborne diseases. The aim of this work was to determine the presence of NoV, RV, HAstV and infective EV (iEV) in fresh leafy green vegetables and to establish whether river water used for irrigation is a possible source of viral contamination in fresh vegetables. This study also focuses in the viral analysis of urban sewage waters as a mirror of the viral agents circulating in the community (van Zyl et al., 2006; Barril et al., 2010; Ruggeri et al., 2015). The findings of this work provide the first data for Argentina of the viral quality of green leafy vegetables and its link with the viral quality of the irrigation water. 2. Materials and methods 2.1. Background Córdoba city is the capital of the province of Córdoba, located in the central region of Argentina and has approximately 1,317,298 inhabitants with a population density of 2308 habitants/km2 (INDEC, 2010) (Fig. 1A). The Suquía River traverses Córdoba city from west to east. Its water flow is 10 m3/s, subject to a seasonal fluctuation. In its path through the city, the Suquía River receives the discharge of untreated or poorly treated industrial effluents and sewage and after leaving the city it receives the treated discharges of the main wastewater treatment plant (WWTP) named “Bajo Grande” (Fig. 1B). This sewerage system covers 61% of the population and no industrial wastewater is treated in this facility. The treatment at the WWTP involves the following steps: 1) grates that filter and extract solid waste, 2) grit chambers with buckets to extract finer trash from the liquid, 3) primary sedimentation tanks with surface sweeper and pumps that absorb sediment and pump it to a mud concentrator, 4) primary percolators, with air inlet through basal orifices and filled with ridge rolled (aerobic process), 5) secondary percolators, 6) secondary sedimentation tanks and 7) labyrinth of disinfection of the liquid with sodium hypochlorite. A system of irrigation canals is derived from the Suquía River, which is used to irrigate the vegetables of the farms located in a green belt area called “Chacra La Merced”. The vegetables harvested in this area are sold in a wholesale market. One of the main irrigation canal rises at the San Jose Bridge of the Suquía River, located 200 m downstream from the WWTP “Bajo Grande” (Fig. 1B). 2.2. Sample collection A total of 37 samples were collected during July to December 2012 in the city of Córdoba corresponding to waters or vegetables from three different points: raw sewage from the WWTP Bajo Grande (n = 6), superficial water from the San Jose Bridge (n = 12), and green leafy vegetables (n = 19) from a farm located at the green area Chacra La Merced (Fig. 1B). Raw sewage samples (1.5 L) were monthly collected from the inlet channel of the municipal WWTP. Irrigation waters (1.5 L) were collected twice a month at the San Jose Bridge, located approximately at the entrance of Chacra La Merced area (Fig. 1B). Water samples were taken on weekday mornings in sterile bottles. The vegetable sampling was conducted three times a month, collecting five types of green leafy vegetables that can be consumed uncooked, like spinach (Spinacia oleracea), lettuce (Lactuca sutiva), arugula (Eruca sativa), chicory (Cichorium intybus) and silver beet (Beta vulgaris var. cicla). The green vegetables were acquired according to the availability of recently green leafy vegetables harvested (approximately 700 g each). All sewage, irrigation water and vegetable samples were kept in individual


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Fig. 1. Sampling location sites in Córdoba, Argentina A) Sampling geographic area indicated in the map of the province of Córdoba, Argentina. B) Thick line in black indicates the Suquía River; black circles depict the monitoring stations: WWTP and San Jose Bridge; gray box shows Chacra La Merced area.

sterile containers at 4–8 °C until delivered to the laboratory, where they were processed for virus concentration within 24 h after collection. 2.3. Concentration of water samples The concentration of viruses in sewage and irrigation water samples was performed using the method of polyethylene glycol (PEG) precipitation (Lewis and Metcalf, 1988). Briefly, the 1.5 L water samples were concentrated 100−fold to 15 mL by high−speed centrifugation (two centrifugation steps, each of 8300 rpm for 20–25 min), elution (two steps at room temperature for 1 h) and PEG precipitation (10% PEG 6000, Anedra®, San Fernando, Mexico/2% NaCl Anedra®, San Fernando, Mexico, overnight at 4 °C). As a negative control distilled water autoclaved twice was concentrated in parallel. 2.4. Elution and concentration of viruses from vegetables The viral elution and concentration in vegetables was carried out according to Guevremont et al. (Guevremont et al., 2006), with minor modifications. As a negative control of the concentration method, a chicory sample was decontaminated by washing the leaves with sodium hypochlorite (1 mg chlorine/L) for 30 min with agitation, followed by several washes with distilled water to eliminate the chlorine. The presence of residual chlorine in the sample was evaluated with orthotoluidine and finally, the leaves were irradiated 40 min (20 min on each side) with UV light in a biological safety cabinet. Each vegetable sample (700 g) was coarsely chopped into pieces of approximately 2.5 cm × 2.5 cm and placed in a sterile polypropylene container. A total of 1.5 L of 2.9% (w/v) beef extract with 6% (w/v) glycine (pH 9.5) broth was added to elute viruses from the surface of the sample and gently mixed for 2 h at room temperature. Then, a solution of 10% (w/ v) PEG 6000 with 2% (w/v) NaCl was added to allow precipitation of the viral particles at 4 °C overnight. Viruses were concentrated by centrifugation at 12,000 ×g for 30 min at 4 °C and then the pellet was suspended on 15 mL of sterile PBS (Prez et al., 2016). The viral concentrate was stored at −80 °C. 2.5. Infective enterovirus detection Enterovirus infectivity was evaluated by cell culture in HEp−2 cell line. Prior to spiking the concentrated into the culture flasks, the

samples were quickly thawed at 37 °C and treated twice with chloroform (1:1); antibiotics and antimycotics were then added (penicillin 100,000 IU/mL; streptomycin 2.5% and amphotericin B 250 μg/mL). Then, each viral concentrate (1 mL/flask) was analyzed twice in HEp −2 cell flasks using standard operating procedures (WHO, 2004). After inoculation they were kept at 36 °C under 5% CO2 atmosphere for 5–7 days and cytopathogenic effects (CPE) were examined by inverted microscope every day. iEV was confirmed by indirect immunofluorescence assay. Monoclonal antibody blend used for EV detection consisted of coxsackievirus type A9, coxsackievirus type B (B1, B2, B3, B4, B5 and B6), echovirus (serotypes 4, 6, 9, 11, 30 and 34); poliovirus (serotypes 1, 2 and 3) and enterovirus (serotypes 60, 71 and Cox A16). The monoclonal antibody reagents were commercially prepared and were purchased from Chemicon International (Temecula, CA). As a negative control 1 mL PBS was inoculated in a HEp−2 cell flask and processed in parallel with the concentrated virus samples. The limit of detection of iEV by cell culture amplification coupled to indirect immunofluorescence assay was previously determined at 500 TCID50 (data not shown). According to Schiff et al. (Schiff et al., 1984) between 10 and 100 iEV particles are enough to initiate a viral infection in the host exposed. Therefore, in the present study every sample detected as “iEV positive” is a potential source of infection. 2.6. Virus genome detection and characterization 2.6.1. Nucleic acids extraction and cDNA synthesis For each sample (water and vegetable), 140 μL of the final concentrates were used to extract viral RNA with the commercial QIAmp Viral RNA kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. The final elution of RNA was carried out using 30 μL of elution buffer. Extracted RNA was reverse transcribed into cDNA using random hexamer primers and M−MLV reverse transcriptase (Invitrogen, CA, USA). Positive (stool sample positive for RV) and negative (distilled water autoclaved twice) controls were included. 2.6.2. Virus genome amplification and characterization 2.6.2.1. Norovirus. This assay was performed using the primers and conditions described by Vennema et al. and Boxman et al. (Vennema et al., 2002; Boxman et al., 2006). The detection was done using a heminested PCR that targets the region A of the viral RNA−dependent RNA

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polymerase (RdRp) gene. In the first round the primers NV1 and NV2 amplified a specific region for both GI and GII genogroups and gave an amplicon of 327 bp; then, in two separate second rounds, amplicons of 187 bp for GI (primers NV3 and NV2) and 236 bp for GII (NV1 and NV4) were generated. 2.6.2.2. Rotavirus. The amplification of the VP7 region was performed with the Beg9/End9 pair of primers described by Gouvea et al. (Gouvea et al., 1990). Then, multiplex heminested PCR with genotype −specific primers for VP7 (G genotypes) was used for detection and viral genotype characterization. The specific primers used in the multiplex heminested PCR were aBT1 (G1), aCT2 (G2), aET3 (G3), aDT4 (G4), aAT8 (G8) and aFT9 (G9) in combination with the antisense primer End9 (Gouvea et al., 1990). 2.6.2.3. Human astrovirus. The molecular detection was performed by nested PCR directed to the ORF2 region C. The first round was carried out with the primers PreCap1 and 12GR and the second round was a multiplex nested PCR with the serotype−specific primers HAstV−1 to HAstV−8 and End as described by Sakamoto et al. (Sakamoto et al., 2000). 2.6.3. Virus genome detection All the PCR products were resolved on 10% polyacrylamide gel electrophoresis (Laemmli, 1970) followed by silver staining (Herring et al., 1982), to achieve high resolution of the products obtained. Positive and negative controls were included in all PCR runs. 3. Results 3.1. Enteric virus detection At least one of the enteric viruses studied was found in all the sewage, irrigation water and leafy green vegetable samples. The virological results are summarized in Table 1. NoV, RV and HAstV genomes were detected in almost all the sewage and irrigation waters, however iEV was isolated from ≤50% of these samples, being intermittently detected. In green vegetables NoV and/ or RV and/or HAstV genomes were detected in 14/19 (73.6%) samples studied.

Table 1 Enteric viruses detection in sewage, irrigation water and green leafy crops samples. Samples (n)

Sewage (6) Irrigation water (12) Green leafy crops (19) Lettuce1 Lettuce2 Lettuce3 Lettuce4 Lettuce5 Lettuce6 Lettuce7 Spinach1 Spinach2 Spinach3 Chicory1 Chicory2 Arugula1 Arugula2 Silver beet1 Silver beet2 Silver beet3 Silver beet4 Silver beet5

Virus detection n (%) NoV

RV

HAstV

iEV

6 (100) 8 (67) 11 (58) + − − + − − + − + − − + + + + + − + +

6 (100) 9 (75) 1 (5) − − − − − − + − − − − − − − − − − − −

5 (83) 9 (75) 6 (32) − − + + − + − − − + − + − − + − − − −

3 (50) 4 (33) 15 (79) + + + + + − + + − + + + − + + − + + +

Note: the symbols “+” and “−” indicate positive and negative samples, respectively.

The temporal detection of the enteric viruses in the river waters used for irrigation and in the green leafy vegetables is shown in Fig. 2. NoV and HAstV were detected from July–December 2012 in at least one irrigation water sample per month analyzed, except in November. RV was also detected at least in one irrigation water sample per month analyzed, but not in September. iEV was only detected in 1 of the 2 irrigation water samples collected in July–September and December and was undetected in October and November. In the green leafy vegetables, NoV and iEV were detected during the whole studied period in at least one sample per month analyzed. HAstV was almost always detected in the green leafy crops but not in July, and RV was only detected in a lettuce sample collected in November. 3.2. Viral molecular characterization Multiple viral genotypes/genogroups were detected in the sewage samples, depicting the co−circulating variants in the community. Also, many water samples from river and some vegetable samples showed multiple genotypes/genogroups in one sample as a mixture of viruses in the water or vegetable analyzed. The molecular characterization of the viruses detected is shown in Table 2. NoV GI was by far the most frequent genogroup identified in the sewage and green leafy vegetables, meanwhile GII was the most frequently detected in the irrigation waters. RV VP7−gene was successfully characterized in all rotavirus positive samples. It was observed a similar pattern of G−type rotavirus distribution in sewage and irrigation waters. The only green vegetable sample (lettuce) that was positive for RV, was characterized as G3 type, being this G−type one of the most prevalent in sewage and irrigation water. Also, in green leafy vegetables, out of the six positive HAstV samples, HAstV genotypes were characterized in five, being G5 and G7 the most frequent types detected in sewage, irrigation waters and green vegetables. Also other HAstV genotypes were detected in the different matrixes (i.e. G2, G3, G6 and G8). Overall, the results obtained reflect the broad spectrum of rotavirus and astrovirus species circulating within the population (Table 2). 4. Discussion Food can be viral contaminated at the post and/or at the pre−harrvest stages. In the post−harvest stage, the main source of viral contamination can be attributed to infected food handlers who could be involved as the virus contaminated source during harvesting, packaging, and/or preparation of food (Daniels et al., 2000). Indeed, Bidawid et al. (Bidawid et al., 2000) showed that 9.2% of infectious virus particles on contaminated hands can be transferred to lettuce during its handling. In the pre−harvest stage, food can become contaminated in the farm during the growing stage by contact with contaminated fertilizers, sewage or the use of fecal contaminated irrigation water (Directorate, 2002). This is particularly relevant when food is green leafy vegetables that are eaten uncooked (Cheong et al., 2009). Fecal bacteria indicators are among the most used indicators for water microbial quality, despite the fact that the majority of the evidence indicates that direct correlation with pathogenic viruses is not observed and should not be expected because enteric viruses are generally more resistant than bacteria to sewage treatment procedures (Scandura and Sobsey, 1997; Pusch et al., 2005; Blatchley 3rd et al., 2007; Lambertini et al., 2011; Payment and Locas, 2011). In turn, enteric viruses can enter the environmental waters through a direct route of discharge of treated sewage. Currently there is not regulation that determines the control of enteric viruses in aqueous matrices in Argentina. To further contribute in the process of gathering information on viral contamination in raw vegetables, in this study qualitative molecular methods were applied to detect and characterize enteric viruses in irrigation water and associated selected raw vegetable samples in Córdoba,


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Fig. 2. Enteric viruses detection frequency in A) irrigation waters and B) green leafy vegetables collected at Córdoba, Argentina.

Argentina. A major drawback of the RT−PCR assay used in the detection of viruses is its inability to determine the viability and infectivity of the viruses detected. In this way, molecular techniques may pick up both infectious and non−infectious types. Non−infectious types in a sample could be advocated to natural factors such as sunlight, temperature and humidity that might decrease or inactivate the infectivity of enteric viruses. Thus, the presence of viral nucleic acids does not necessarily indicate the presence of infectious viruses (Hamza et al., 2009). Therefore, the molecular detection of enteric viruses cannot confirm the role of vegetables as transmitting vehicles of infectious enteric viruses. In order to exceed this limitation, in the present study, data are provided on infective enterovirus to assess the infectivity of viral particles present in irrigation water and vegetables associated. According to the limit of detection of iEV by cell culture amplification coupled to indirect

immunofluorescence assay previously determined, every sample detected as “iEV positive” is a potential source of infection. The evidence of EV viability could indicate adequate matrix conditions for maintaining the infectivity of the other viruses analyzed by genomic detection. The results revealed evidence of frequent detection of NoV, RV, HAstV and iEV in the irrigation water and also in the green leafy vegetables analyzed. This depicts that the contamination of these matrices occurred all−round the studied period, involving cold and warm months. Sewage waters revealed the continuous circulation in the community of NoV, RV and HAstV, but iEV was detected in 50% of the samples, which could be a consequence of the presence of inhibitory substances in these dirty samples that impede the amplification of the viable EV particles in the cell cultures. NoV GI and GII, RV G1−G4 and G9, and HAstV G5 and G7 were frequently detected in sewage, depicting a

Table 2 Proportional distribution of viral strains detected in sewage, irrigation waters and leafy green vegetables. Sample

NoV genogroup (%)

RV genotype (%)

HAstV genotype (%)

Sewage Irrigation waters Leafy green vegetables

GI(62.5), GII(37.5) GI(11), GII(89) GI(92), GII(8)

G1(14), G2(27), G3(23), G4(18), G9(18) G1(12), G2(16), G3(24), G4(16), G8(8), G9(24) G3(100)

G2(8), G5(38), G6(8), G7(31), G8(15) G1(31), G2(8), G5(38), G6(8), G7(15) G3(29), G5(43), G8(29)

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pattern of viral specimens circulating in the community similar to that observed in the contaminated irrigation waters. This situation suggests an involvement of the discharge of untreated or poorly treated local sewage into the environmental waters. Unfortunately, this situation seems to be a common occurrence in different regions of the world. Published data reported the occurrence of viruses in different water environments, following the route of contamination from raw sewage to the surface waters receiving wastewater discharges (Iaconelli et al., 2015; Rusinol et al., 2015). In addition, the results obtained indicate that the genotypes circulating in the community are also detected in leafy green vegetables, suggesting that contaminated irrigation water by fecal pollution could be a source of vegetable contamination with viral particles. It must be pointed out that iEV was detected in higher frequency in green vegetables (79%) as compared to the frequency detected in sewage (50%) and irrigation water (33%) samples, which could be related to the attachment of enterovirus to plants (Vega et al., 2005; Deboosere et al., 2012). Another explanation could be that vegetables are sprayed with the same irrigation water before being placed on the market. Perhaps the combination of both factors cited above could contribute to a greater contamination of the vegetables with infective viruses. The lack of quantitative data on viral particles detected in leafy green vegetables represents a limitation to figure out health risks associated with exposure to food−borne pathogens. However, the present study is the first in Argentina that conducts a comprehensive assessment of the prevalence of enteric viruses in fresh vegetables using cell culture and molecular methods. Stimulated by the results obtained, the genomic viral quantification directly on the plant surface will be carry out in a near future in order to estimate the human infection risk caused by RV and NoV in food. It is important to highlight that according to previously reported results the rate of virus recovery from water and vegetables with the concentration and molecular methods used in this study is in the range between 5 and 35% (Prez et al., 2016); therefore it could be possible that the results of the present paper are underestimated. In the last years, other authors have reported the presence of viruses in irrigation waters and associated raw vegetables (van Zyl et al., 2006; Cheong et al., 2009). However, the published papers reported limitations to establish the link between the viral contamination of irrigation waters and vegetable samples. These limitations refer to the lack of concurrent viral detection or the lack of sequencing of viral strains in the irrigation water and in the associated plant samples. Recent published studies reported RV detection in 11/134 (8%) of frozen vegetables in Mexico City (Parada−Fabian et al., 2016), and in 9/63 (14%) irrigation water and 1/61 (1.7%) of corresponding raw vegetables in three regions of southern Africa (van Zyl et al., 2006). Also, a study conducted in South Korea, which monitors the occurrence of enteric viruses in groundwater samples and associated raw vegetables, detected enteric viruses at frequencies of 17% and 10% respectively (Cheong et al., 2009). In that study, adenovirus was the most frequently detected virus in four groundwater and three vegetable samples; EV and NoV were detected in only one groundwater sample and one spinach sample, respectively and RV was undetected. The broad range of enteric viruses reported in the papers cited above is similar to that found in the present study and confirms the role of vegetables as transmitting vehicles of enteric viruses. Based on the concurrence of enteric viruses detection and the genotype profiles identified in water and in the associated vegetables samples, our results allow to hypothesize that the viral contamination of the green vegetables could partially be originated during the production phase of the farm−to−fork continuum. Although highly unlikely it is not possible to rule out post−harvesting contamination with enteric viruses, since no source tracking work was conducted. These results provide the first data for Argentina pointing out that green leafy vegetables are contaminated with a broad range of enteric viruses and that the irrigation water would be a source of

contamination. The presence of viral genomes and infective particles in food that in general suffer no treatment before consumption underlines that green crops can act as potential sources of enteric virus transmission. This preliminary study has highlighted the need for further in −depth surveillance of enteric viruses in water environments and the link to vegetables contamination in Argentina. Our findings reveal the need of public intervention in the use of the river waters as vegetables irrigation source. This would be particularly relevant to reduce the risk of food−borne viruses due to the consumption of raw green leafy vegetables.

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