Hepatitis A and E Viruses in Mussels from Cherrat Estuary in Morocco: Detection by Real-Time Reverse Transcription PCR Analysis

The aim of the present study was to evaluate hepatitis A virus (HAV) and hepatitis E virus (HEV) contamination in mussels (Mytilus galloprovincialis) from Cherrat estuary (Moroccan Atlantic Coast), Morocco. In total, 52 samples (n = 12 mussels/each) were collected at four sites in the estuary, monthly, between March 2019 and March 2020. HAV and HEV were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) according to the ISO/TS 15216 method. HAV was detected in 46.15% of analyzed samples. Conversely, HEV was not detected in any sample. Moreover, the HAV detection rate was significantly associated with seasonal rainfall variations. This qualitative study on HAV and HEV contamination highlights the interest of studying mussel samples from wild areas. As HAV presence in mussels represents a potential health risk, viral contamination surveillance of mussels is necessary to protect consumers. HAV shellfish contamination must be monitored at Cherrat estuary because of the role played by shellfish as HAV reservoirs and/or vehicles in fecal-oral HAV transmission.


Introduction
Shellfsh is a major component of the global aquatic food [1]. Tis term describes crustaceans and mollusks. Mollusks are subdivided into cephalopods, gastropods, and bivalves. Commercially important bivalve mollusks are mussels, oysters, clams, and scallops [2]. Mussels are organisms that feed by fltering water from the aquatic environment [3]. Terefore, they can concentrate microbial contaminants [3][4][5], including enteric viruses [6][7][8]. Moreover, mussels can live on sand/gravel sediments where enteric viruses persist in large quantities in the surface layer in contact with water [9,10] and can remain infectious for several months [11,12]. In the majority of cases of infection, diferent viral strains have been detected in the implicated shellfsh, suggesting contamination in their original environment by wastewater [13][14][15][16][17]. Indeed, entropic activities near the coast have a major impact on shellfsh quality [18]. Pollution of coastal waters can lead to contamination of bivalve mollusks [4], and their consumption (raw or undercooked) is associated with human viral diseases [19,20]. Importantly, enteric viruses are often identifed during enteric disease outbreaks caused by the consumption of shellfsh that meets the bacteriological standards [16,[21][22][23][24].
HAV is 28-30 nm in diameter, nonenveloped, 7.5 kb single-stranded, linear, positive-sense RNA virus, classifed in the genus Hepatovirus within the Picornaviridae family [35]. Tis virus is classifed into six genotypes (I-VI), of which genotypes I-III can infect humans [36]. HAV causes a self-limiting disease, without serious complications in most cases, and does not lead to chronic infection or chronic liver infection. However, fulminant hepatitis and death have been reported [37]. HAV is present worldwide, and the seroprevalence rate depends on hygiene conditions [38]. In developing countries (i.e., Asia, Africa, Latin, and Central America), HAV infection is highly endemic, and infected people are often asymptomatic [36].
HEV is a non-enveloped, 7.2 kb single-stranded, positivesense RNA virus belonging to the genus Orthohepevirus and the Hepeviridae family [39][40][41]. Each year, ∼20 million people are infected by HEV worldwide [7,42], mainly in developing countries [43]. HEV causes self-limiting acute hepatitis; however, extrahepatic manifestations (neurological and renal damage) and chronic infection, especially in immunocompromised patients and organ transplant recipients, are increasing, thus representing an important clinical problem [42]. Moreover, pregnant women can develop fulminant hepatic failure, with a mortality rate of up to 30% [44]. Although HEV has only one serotype, it shows great genetic diversity with four major genotypes (from 1 to 4) in the Orthohepevirus A species [39,40,45]. Genotypes 1 and 2 have been exclusively isolated in humans and are predominant in Asia, Africa, and Central America [41,46,47]. Tey have been implicated in major human epidemics due to consumption of HEV-contaminated water or food in countries with poor sanitation [39].
Morocco has put in place a national strategy for the protection and sustainable development of the environment [48]. In the framework of this strategy, Oued Cherrat is categorized as one of the priority sites of biological and ecological interest due to its ecological, social, and economic importance and also the strong pressure by human exploitation that is causing important alterations. Within this strategy, a protection program has been put in place to accompany this site development through the rational use of resources (including shellfsh) and ecotourism [49]. In this context, this article presents the frst environmental and virological study carried out at Cherrat estuary (Casa-Settat region in Morocco) to monitor viral contamination of mussels and the associated risk factors for a year.
Te study objectives were as follows: (i) To determine the contamination rate by HAV and HEV in mussels harvested at diferent sites at Cherrat estuary (ii) To study the correlation between seasonal factors and the mussel contamination rate by HAV and HEV

Study Area.
Cherrat estuary (33°49′52.71″°N -7°07′23.33″°W) is located on the Moroccan Atlantic coast in the province of Benslimane (Casa-Settat region), 53 km north of Casablanca. It is a transition area between the Cherrat beach and Oued Cherrat, a site of biological and ecological interest [49]. Te beach is classifed as category A for bathing water [50]: less than 100/100 mL amount of fecal coliforms (Escherichia coli) and enterococci (fecal streptococci), according to the relevant national standard (NM 03.7.200), transposed from the European Directive (76/160/ EEC) and the Directives WHO/UNEP, applicable to the health surveillance of marine bathing waters. Water quality in this area is afected by watershed runof, urban wastewater management malfunctions, and overfows of nearby sewerage systems.
Mussels were collected at four sites (S1, S2, S3, and S4) at Cherrat estuary ( Figure 1): (i) S1 and S2, located on the right rocky side of Cherrat estuary (with a distance step of 100 m) (ii) S3 and S4, located on the rocky left side of Cherrat estuary (with a distance step of 100 m) Tese sites correspond to an overexploited wild mussel harvesting area. Tese mussels are sold locally in an informal and traditional way by the local population, without any sanitary control.

Mussel
Collection. Naturally growing mussels (Mytilus galloprovincialis) were collected monthly at the four sites at Cherrat estuary over a 13-month period (from March 2019 to March 2020). In total, 52 samples (n � 12 mussels/sample) were collected. All samples were shipped to the laboratory in a refrigerated box within 24 h after collection.

Sample Processing and RNA Extraction.
Mussel samples were processed following the ISO/TS 15216-1:2017 (Microbiology of the food chain-horizontal method for determination of HAV and norovirus using real-time RT-PCR-part 1: method for quantifcation) protocol for HAV detection in food samples.
Mussels were rapidly rinsed, shucked, and dissected. Te hepatopancreas was removed and pooled to have a fnal weight of (2 ± 0.2) g for each sample. Digestive tissues were mixed with 10 μL of mengovirus (CeeramTOOLS ® ) as a nucleic acid extraction control. Tis was followed by incubation with proteinase K solution (2.0 ± 0.2 mL) at (37 ± 1.0)°C with shaking at 320 rpm for (60 ± 5) min to digest the tissues and a second incubation at (60 ± 2.0)°C for (15 ± 1) min in a waterbath. Ten, samples were centrifuged at 3000g at room temperature for (5.0 ± 0.5) min, and the supernatants were collected in clean tubes.  Table 1.
Te amplifcation protocol was as follows: reverse transcription reaction at 55°C for 10 min, initial denaturation at 95°C for 1 min, followed by 45 cycles of denaturation at 95°C for 10 s, and extension at 60°C for 1 min. Viral RNA extracted from each sample was amplifed in duplicate using 4 μL undiluted and ten-fold diluted sample to evaluate the presence of inhibitors, using the mengovirus analysis according to the manufacturer's instructions, by comparing the Ct values of the undiluted and diluted RNA samples. A Ct value diference <3.3 indicated the presence of inhibitors.
For each run, a mengovirus standard curve was generated using a ten-fold serial dilution. Extraction efciency was evaluated by comparing the Ct value of the mengovirus RNA extracted from the samples to this standard curve. Results ≥1% were considered valid. A sample was considered positive when the Ct value was ≤33 in at least two replicates, without evidence of amplifcation in the negative controls.
RNA extracted from the HAV HM 175 strain was used as a positive control. Two negative controls were included in each RT-PCR series: negative control of the extraction with the PCR mixture and negative control of the amplifcation (nuclease-free water with the PCR mixture).

HEV Detection. Te genesig real-time PCR detection
kit from Primerdesign ™ Ltd. supplied by oasig lyophilized One-Step RT-qPCR MasterMix was used for HEV detection (based on the detection of the ORF2 capsid proteinencoding gene). 5 μL of each RNA sample was amplifed in 20 μL of reaction mix that contained 1X reaction mix, 10 μL of oasig ™ lyophilized One-Step 2X RT-qPCR Master Mix, 1 μL of HEV primer/probe mix, 1 μL of internal extraction control primer/probe mix, and 3 μL of RNase/DNase-free water. Te primers have been described by the manufacturer in the genesig ® Advanced Kit handbook. Te amplifcation protocol was as follows: reverse transcription at 55°C for 10 min, enzyme activation at 95°C for 2 min, followed by 50 cycles of amplifcation with denaturation at 95°C for 10 s.
Each sample was amplifed in duplicate (undiluted and 1/10 diluted). Two negative controls were included in each RT-PCR run: negative control of the extraction with the PCR mixture and negative control of the amplifcation (RNase/ DNase-free water with the PCR mixture). Te positive control should have a Cq value between 16 and 23.
Five standards (from 2 × 10 4 copy numbers/μL to 2 copy numbers/μL) were prepared by dilution of the positive control (2 × 10 5 copy numbers/μL) for quantitative analysis. Te absence of PCR inhibitors was tested using the internal RNA extraction control from the RT-qPCR kit. Briefy, 4 μL of the internal extraction control RNA was added to each RNA sample, and the Ct values were compared with the Ct value of the negative control Advances in Virology containing the internal control. Te internal control was detected through the VIC channel and gave a Cq value of 28 ± 3.
All RT-PCR tests to detect HAV and HEV were performed on an AriaMx (Agilent Technologies, Santa Clara, CA) 96-well plate real-time PCR instrument. Fluorogenic data were collected at 60°C for 60 s through the FAM and VIC channels, using the Agilent Aria software v 1.71.

Statistical Analysis.
Spearman's rank correlation analysis was used to correlate the results of positive samples pooled by month and pluviometry. Tese correlations were performed with the total accumulated rain of the previous month (obtained from the Bouregreg and Chaouia hydraulic basin agency) assuming that each mussel harvesting area was mainly afected by the rain of the preceding month. All statistical analyses were performed using the statistical package SPSS Statistics 17.0. Statistical signifcance was determined by a P value <0.05.
Fisher's exact test showed signifcant diferences in contamination rates between the dry period (May to September) and the rainy period (October to April) (P < 0.0001). A signifcant positive correlation between the number of positive samples and the average rainfall was observed.
In this study, only HAV, but not HEV, was found in the collected mussel samples. Te diference in contamination rates among the four sites may be explained by their diferent location and the infuence of currents or other hydrographic variables. However, more studies are needed to test these hypotheses. Our results are in agreement with previous studies [58][59][60] reporting HAV presence in mussels collected at local markets, a culture farm, and during environmental monitoring.
HAV presence in mussels at Cherrat estuary is explained by the strong persistence of the virus in the environment [61] or by a continuous supply of viruses from runof from the watershed of the Oued Cherrat, urban wastewater, malfunctions, and overfows from the sewage system that is not   Advances in Virology up to standard [50]. Tis can induce difuse pollution and afect the estuarine environment where mussels naturally develop [62].
Regarding HEV, some studies on shellfsh did not detect HEV in mussels marketed in France [63], Italy [64], Denmark, [65], and Tailand [66]. On the other hand, several studies reported HEV presence in mussels grown naturally in wild areas and commercially available in Scotland [28], Spain [7,67,68], and Italy [4,69]. Similarly, there is evidence that HEV is circulating in the Moroccan population [70]. Te absence of detection at Cherrat estuary is explained by the number of HEV particles discharged in the environment is too low to be detected or the virus may have a very short period of persistence in human waste. More work on HEV persistence in human waste is needed to better evaluate this point.

Conclusion
Our study showed that HAV contamination rate in mussels from Cherrat estuary was quite high and signifcantly associated with seasonal variations. Terefore, updating the existing legislation to include viral hazards in the monitoring of bivalve mollusk harvesting areas is an urgent matter for the competent authorities. In addition, illegal harvesting of mussels from this area must be better controlled due to the threat to public health caused by the risk of viral contamination. Monitoring the presence of enteric viruses in bivalve mollusks may contribute to the prevention of viral food poisoning and the promotion of public health.

Data Availability
Te data used to support this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.