Detection and evaluation of rotavirus surveillance methods as viral indicator in the aquatic environments

Abstract

Group A rotaviruses (RVAs) have been introduced as the most important causative agents of acute gastroenteritis in the young children. One of every 260 children born globally will die due to rotavirus (RV) before 5 years old. The RV is widely known as a viral indicator for health (fecal contamination) because this pathogen has a high treatment resistance nature, which has been listed as a relevant waterborne pathogen by the World Health Organization (WHO). Therefore, monitoring of environmental is important, and RV is one of the best-known indicators for monitoring. It has been proved that common standards for microbiological water quality do not guarantee the absence of viruses. On the other hand, in order to recover and determine RV quantity within water, standard methods are scarce. Therefore, dependable prediction of RV quantities in water sample is crucial to be able to improve supervision efficiency of the treatment procedure, precise quantitative evaluation of the microbial risks as well as microbiological water safety. Hence, this study aimed to introduce approaches to detecting and controlling RV in environmental waters, and discussed the challenges faced to enable a clear perception on the ubiquity of the RV within different types of water across the world.

Introduction

The quality of environmental waters has a key role in human disease. Protozoa, viruses, and bacteria are recognized as the main origins of the waterborne infections [1] (Table 1). Despite recent improvements, diarrhea is the fourth main cause of death in children under the age of 5 and still accounts for an estimated nearly 500,000 children deaths every year especially in developing countries [2]. Research demonstrated that Rotavirus and Escherichia coli are the two main causes of moderate-to-acute diarrhea, and 38–67% of children with diarrhea admitted to hospitals were infected with group A rotavirus (RVA). Rotavirus (RV) is one of the leading causes of death in children aged under 5 years causing 146,000 deaths in 2015 [3]. Moreover, viral gastroenteritis outbursts are linked to water pollution originating from wastewater consisting of the viral particles that are released within surface waters [4, 5].

Table 1.

Indicator microorganisms, sources, and related diseases in environmental water [1, 6]

Category	Waterborne pathogens	Typical sources	Common pathways	Prevalent disease
Bacteria	Salmonella spp.	Humans and animal’s feces	Sewage	Salmonellosis, typhoid fever
	Vibrio cholerae	Aquatic environment	Sewage	Diarrhea
	Legionella	Biofilms in water systems, urine of animals	Sewage, water environment	Legionnaires disease
	Escherichia coli O157:H7	Human and Cattle feces	Sewage	Hemorrhagic diarrhea
	Fecal Streptococci	Human and animal feces contamination	Sewage, water environment	Gastroenteritis, diarrhea
	Shigella spp.	Intestinal tract of humans and animals	Sewage	Shigellosis
	Leptospira interrogans	Urine and tissues of animals	Sewage, fresh water	Leptospirosis
	Campylobacter spp.	Human and animal feces	Sewage	Gastroenteritis
	Klebsiella spp.	Pulp and paper mill waste	Urban and industrial sewage	Gastroenteritis
Viruses	Enteroviruses	Human feces	Sewage	Gastroenteritis, meningitis, diarrhea, poliomyelitis
	Hepatitis A, E	Human and animal waste	Sewage	Infectious hepatitis
	Astrovirus	Human feces	Sewage	Gastroenteritis
	Adenovirus (40/41)	Human feces	Sewage	Gastroenteritis
	Adenovirus (C)	Human feces	Sewage	Respiratory disease
	Rotavirus	Human feces	Sewage	Gastroenteritis, diarrhea
	Norovirus	Feces or vomit of contaminated patients	Sewage	Gastroenteritis
Protozoa	Cryptosporidium oocysts	Human and animal feces	Sewage, runoff	Cholecystitis, hepatitis, respiratory disease
	Acanthamoeba castellamii	Free-living Amoeba, environment	Sewage, marine sediments	Amoebic encephalitis
	Giardia lamblia (cysts)	Human and animal waste	Sewage, runoff	Giardiasis

In comparison with protozoa and bacteria, waterborne human enteric viruses have high infectivity with an increased risk of transmission [7]. Different species of enteric viruses (over 140 strains) have been isolated from the aquatic environments, which are responsible for diseases like gastroenteritis, hepatitis, fever, meningitis, conjunctivitis, diabetes, and rash [8, 9]. Some of these viral indicators transmitted through direct contact or fecal-oral route are rotavirus, norovirus, adenovirus, astrovirus, and enterovirus, which are mainly established in the host gastrointestinal tract [10, 11]. Rotaviruses (RVSs) have been introduced as the commonest cause of diarrhea among young children and infants [12–15]. However, the prevalence of RVA infection in children hospitalized with diarrhea is similar worldwide (~ 30–50%); > 90% of children with fatal rotavirus infections live in low-income countries, which is likely because of limited access to health care, lack of available hydration therapy, and a greater prevalence of malnutrition and some other factors [16]. In the infected person, RVs are excreted in very large quantities (> 10¹¹ particles of RVA per gram of stool) to be counted. The RVs are released into the surface water, such as rivers and lakes, because of stability to typical wastewater treatment process (WWTP) and effluent used for irrigation and recreation purposes [6, 17]. The outbreak of RV serotype has been reported in many countries during 2010–2018 (Table 2).

Table 2.

The RVA serotypes detected in previous studies

Year	Country	Source	Percent Positive	Genotypes	Reference
2013	Egypt	Raw sewage	16/7	NR	[18]
2013	Egypt	Drinking water	12/5	NR	[18]
2013	Egypt	Raw water	29/2	NR	[18]
2010	China	Raw water	34/6	NR	[19]
2010	China	Drinking water	22/4	NR	[19]
2014	China	Raw sewage	34/4	G9, P [10]	[19]
2011	Canada	Raw sewage	24	NR	[20]
2010	Brazil (Porto Alegre)	Raw sewage	28/6	NR	[21]
2011	South Africa (King William Town)	Surface water	13/9	NR	[22]
2017	South Africa	WWTP	41/7	NR	[23]
2015	Nigeria	WWTP	14/2	G1, G8, G9, G3, G4, G2	[24]
2011	Italy (Naples, Bari, Palermo)	Raw sewage	60/4	G1, G2, G3, G4, G6, G9, G26, P [4], P [25], P [10], P [11], P [26], P [27]	[28]
2011	China	Raw sewage	67	NR	[25]
2016	Pakistan	Drinking water	35	NR	[29]
2015	Pakistan	Drinking water	9/4	NR	[30]
2013	Brazil	Raw sewage	45/8	G3, G4, G9, P [25], P[10]	[31]
2013	Brazil	Drinking water	16/4	NR	[32]
2012	Brazil	Surface water	23/9	NR	[33]
2018	Brazil	WWTP	33/3	NR	[34]
2012	Egypt	Drinking water	16/6	NR	[35]
2012	Egypt	Raw water	4/1	NR	[35]
2010	Bangladesh	Ground water	40	NR	[36]
2010	Kenya (Karen)	Raw sewage	69/2	G1, G4, G5, G8, G9, G10, G11, G12, P [4], P [10]	[36]
2011	Iran	WWTP	25	G1, G1/G4	[37]
2012	USA (Arizona)	Raw water	58/3	NR	[38]
2014	China	WWTP	93/5	G9, P [10]	[39]
2012	Argentina (Cordobe)	WWTP	91/4	G1, G2, G3, G4, G8, G9,P [4], P [10]	[40]
2014	Uruguay (Melo, Treinta, Bella Union)	Raw water	52/6	G1, G2, G3, G12, P [3], P [4], P [10]	[41]
2010	Thailand	Surface water	18/2	G1, G2, G3, G9	[42]

Serogroup II (RAV categorization on the basis of VP6 evaluation)

WWTP wastewater treatment plant, NR not reported

Rotavirus structure and their persistence in water environments

The RV is a family member of Reoviridae with the genus Rotavirus. RV is non-enveloped with a diameter of 70-nm 18.5-kb genome length. The genome contains 11 segments of the double-stranded RNA encoding 6 structural proteins (VP1–4, 6, and 7) as well as 6 non-structural proteins (NSP1–6) (Fig. 1) [43, 44]. An icosahedral symmetry of virus capsid has 3 layers involving an outer layer containing VP7 protein, an interior VP6 glycoprotein layer, the VP2-covered core shell, and VP4 protein spikes [45]. The importance of RV as a viral indicator for quality of water is related to (1) stabilization, insistent, and high diffusion in various water resources, including wastewater, surface water, and drinking water; (2) continuous identification of the virus in sewage during all seasons in untreated wastewaters; (3) high resistance to treatment processes and disinfectants such as chlorine and ozone; and (4) human host specificity (in particular fecal contamination of human beings) in addition to the loss of duplication outside of the host [26, 46, 47].

Fig. 1.

Human rotavirus: structure and morphology. Human rotaviruses are miniature RNA viruses of three double-stranded layers pertaining to the Reoviridae family that are specified through their wheel-type architecture (in Latin rota = wheel)

Global distribution and predominating genotypes

The International Committee of Taxonomy of Viruses (ICTV) has recognized 10 species of RV, referred to as A, B, C, D, E, F, G, H, I, and J. Moreover, RV strains have a binary taxonomy system according to the VP4 and the VP7 proteins [48, 49]. The VP7 protein is responsible for G (glycoprotein), and VP4 protein contributes to P (protease sensitive) serotype/genotype of RV. Results showed G1P [10], G2P [4], G3P [10], and G4P [10] as the most pervasive genotypes in the humans’ infection. Researchers currently proposed a classification with the use of 11 segments of the genome. To date, they recognized not less than 51 P types and 36 G types in animals and humans [47, 50, 51].

In spite of wide distribution throughout the world, the outbreak rate of RV depends on the geographical regions.

Based on the epidemiology of RV infections, there are a minimum of 3 P genotypes (P [4], P [10], and P [25]) and 6 G genotypes (G1–G4, G9, and more recently G12) circulating throughout the world to generate substantial efficacy on general health [52, 53]. The five major genotypes of the RV in America, Australia, and Europe including G1P [10], G2P [4], G3P [10], G4P [10], and G9P [10] have been considered as the cause of above 90% of the RV gastrointestinal diseases in the mentioned countries. However, G1P [10] is the predominant genotype in those regions in the prolonged time. Nonetheless, epidemiologic situations are not constant due to the natural variations. The genotype G9P [10] of RV has been reported to take especially local predominance for single seasons in the 2000s. While reports indicate greater variability and the number of the circulating RV genotypes in Asia and Africa, it has been demonstrated that G1P [10] and G9P [10] are widely the dominant RV types (Table 2) [46].

Reduction and recovery rotaviruses by treatment process

Log reduction value (LRV) test determines the removal efficiency of a treatment process unit. An LRV evaluates the competency of the treatment procedures in eliminating pathogenic microorganisms that are identified by calculating the pathogen concentration ratio logarithm within the influent and sewage water pertaining to the treatment procedure. However, the virus removal efficiency in these units depends on the conditions for the treatment process, indicating significantly various performances during the same reactor for the treatment. Moreover, RV emission can be 2 × 10⁸ viral particles during the wastewater treatment [10, 27].

For most pathogenic groups (bacteria, viruses, and protozoa), the most resistant pathogen must be used as the basis for calculating log₁₀ reductions for each treatment process unit. The effective techniques to eliminate the pathogen particles in the treatment plants are filtration, flocculation, coagulation, and sedimentation, as well as disinfection techniques such as ozonation and chlorination. This is usually evinced in terms of “log₁₀ reduction values” or LRVs, where a “one log₁₀ reduction” balances to 90% reduction of a pathogen, a “two-log₁₀ reduction” balances to 99% reduction, a “three-log₁₀ reduction” balances to 99.9% reduction, and so on (VIC Guidelines, 2013). Sano et al. [54] reported 0.87 removal efficiency of RVA in wastewater treatment units by the membrane bioreactor (MBR) as well as the conventional activated sludge (CAS) procedures. The conventional water treatment processes have shown high abundance for HAdV (human adenovirus) and RV in untreated surface water and treatment water, while no bacterial indicator has been found in tap water, specifying standard for drinking water bacterial quality. This is due to the feature to be processes conventional treatment is not effective in completely eliminating these pathogens [27, 54].

Rotavirus concentration methods

Concentration technique is chosen by water and sewage properties and analysis of the sampling volumes. Moreover, the impact of virus features should be evaluated [55, 56]. A good method concentrating on the water specimens and sewage must adhere to these factors: (1) appropriate viral recovery trend with good accuracy, (2) avoidance of false negative and positive results by excluding the preventative or cross reacting materials extensively found on the water surface, (3) final concentration to apply in viral culture without intermediate processing, (4) the same recovery method for DNA and RNA viruses, (5) consideration of diverse environmental water matrices, such as treated water and sewage, and (6) simple and cost-effective techniques for a majority of samples or water quality laboratories. Therefore, primary concentration stage could be used to decrease the volume of water sample [20, 57, 58]. Amidst the processes of the large volume of water specimens (10–100 L), disc/cartridge filters or ultrafiltration (HFUF) is typically implemented for primary concentration. The sample volume is reduced greatly using a secondary concentration step in all methods of concentration, so that the sample can be concentrated significantly to improve the remaining sample and accuracy of the estimation of the virus counts. Pang et al. [20] reported an average of 31% RV recovery from 1000 L of tap water by filtration, elution, and flocculation in filter cartridge method. Generally, the used electropositive filter included 1 MDS and Nanocream disk or cartridge filters. The advantages of most electropositive filters have been considered to be their simplistic utilization with no preliminary sample preparation of the water samples as well as cartridge format, enabling the filtration of larger volumes of water (> 1000 L) at the increased filtration rates without blocking in most cases (Table 3) [59]. In 1996, US Environmental Protection Agency (USEPA) introduced the elution technique with the use of 1.5% beef extract for viral concentration procedure and electropositive 1MDS filter for drinking water. Utilization of the 1MDS cartridge filters has been accompanied by variable recovery (≥ 50%) of RV from waste water and water samples. Verheyen et al. [60] reported lower RV recovery (2.1%) by 1 MDS almost from different types of water, while Ikner et al. [22] obtained an average of 80% RV recovery from 20 L of tap water using 1 MDS cartridge filters. However, Virosorb 1 MDS has consistently displayed effective virus adsorption from different types of water for both small and large volumes; it is costly. PEG/dextran precipitation, ultracentrifugation, and organic flocculation have been proposed to be usually utilized as the primary concentration procedures for the sewage sample analyses. Kargar et al. [61, 62] used two-phase method for sewage sample concentration in numerous studies. According to the method, the samples have been mixed by dextran and PEG and remained in a separation funnel for 24 h. Ultracentrifugation has been also used. Zhou et al. [39] showed that viruses have been concentrated from water sample extracted by ultracentrifugation at very high rates (180,000 to 210,000 g) for 10–20 L water samples. He recovered 93.5% of RV from 1-l sample in WWTP. In addition, two types of filter are available to absorb the virus, including electronegative and electropositive filters (Fig. 2) [18, 39, 63]. The PEG precipitation for RV in the sample seeded into beef extract showed about 40% recovery [64]. Finally, Ruggeri et al. [28] reported an average of 60.4% of RV recovery from 1 L of sample using ultracentrifugation.

Table 3.

Optimal concentration method for RVA using different types of filter in water samples

Filter	Sample (L)	Water type	RV recovery (%)	Final volume (mL)	Reference
Nanocream vs2.5-5	100	DW	21	5	[11]
Nanocream cartridge filter	10	RW	31	1	[65]
Nanocream electropositive 90 mm	10	TW	19–21	30	[57]
1 MDS	10	DW	2/1	1	[60]
Electronegative membrane 45 mm	20	DW	NR	10	[66]
Nanocream 45 mm	1	RS, GW	88	4	[20]

DW drinking water, RW raw water, TW tap water, RS raw sewage, G ground water

Fig. 2.

Flow chart of RVA concentration methods in water and sewage: electropositive filter and two-phase methods for water and sewage

Detection methods of rotaviruses in water and wastewater

Cell culture

There are some cell culture-based techniques, such as plaque assay for several enteric viruses, but they are time-consuming and improper ways to ensure a significant reduction in viral infectivity in wastewater reclamation systems [67]. Moreover, experts in the field introduced constraints like lack of correlation between the detected viral genome and its infection level that shows the necessity for applying other methods like cell culture to improve detection. The cell culture is still available as the standard method for viruses, but numerous cell lines would be necessary for various cultivable viruses. Moreover, viral culture demands days to weeks prior to the outputs’ verification. In addition, researchers have not yet established in vitro culture system substantially for many enteric viruses. Recent advances in the molecular detection methods provide high accuracy and more sensitive non-culture-based methods such as nucleic acid extraction followed by reverse transcription polymerase chain reaction (RT-PCR) [68, 69].

Immunological detection

The most widely performed methods for RV detection are based on identifying the protein antigens on the virus surface (VP6, VP7) in all concentrated water and wastewater samples. Therefore, the most appropriate antigen detection method for most monitoring studies is an EIA (enzyme immuno assay) that uses RV special antibodies to capture antigen onto the wells of plates in proprietary kits [70, 71]. In a study conducted by Soltan et al. [26], more than 85% of the samples were detected with ELISA (enzyme-linked immunosorbent assay). The EIA is a dominant in vitro detection method due to its highly sensitive, specific, and adaptability features to large sample volumes, without need for specialized equipment. In addition, serological methods are often utilized, and ELISA is the commonest method. Nevertheless, one of the prominent drawbacks is to pretreat the limit-requiring sample for concentrating the viral contents [37, 69, 72].

Immunomagnetic particles

A quantitative, rapid, and sensitive method for capturing, separating, concentrating, and detecting infectious RV in water has been used to amino pink magnetic microparticles. Villamizar-Gallardo [69] showed that magnetic features of microparticles enable the concentration and separation of the viral particles from water (Fig. 3). In case of the use of the above technique, the ground would be provided for eliminating the background interference such PCR suppressors. Moreover, utilization of an antibody as the molecular receptor would ensure that the captured antigen is corresponding to the intact infectious particles [72]. Researchers have reported the immunomagnetic separation (IMS) technique as an efficient one for determination and concentration of multiple virus types, like RVA and HAdV [73].

Fig. 3.

Concentration of RV with magneto fluorescent microparticles conjugated using antigen-antibody reaction for RV detection [69]

Microscopic characterization

Electron microscopy (EM) has been proposed as an important method for virus detection. EM is popular for simplicity, speed, good preservation of the three-dimensional structure of virus structure and high resolution [74]. It is possible to employ the transmission electron microscopy (TEM) and atomic force microscopy for confirming the presence of anti-RV antibody connected to the magnetic microparticle surfaces and the surface of the bond viruses. This technique can be combined with immuno methods to improve viral detection or to localize viral antigens. The disadvantages of EM were that it was not ideal for quantifying virus and that the technique was time-consuming and expensive. Due to the difficulty associated with the use of EM however, rotavirus molecular detection methods have been developed [69, 75, 76].

Molecular methods

When compared with the cell culture procedures, molecular RV detection techniques enjoy benefits like quickness and reliability. They also enable the quantitative determination of viruses and measure the viral particles. In addition, quantitative PCR has been introduced as the preferred molecular technique for RV counting characterized by high sensitivity and potentially detection of viruses that unable to be cultured [19, 77–79]. The sensitivity of enteric virus detection has promoted using the real-time-PCR (qPCR), which is mostly used in monoplex mode, in which only a single virus type can be quantified per assay [17]. Most of qPCR methods available for genogrouping of the VP6 gene target the VP6 gene such as the TaqMan and the SYBR green assay. For detection and quantification of RV, studies indicated that qPCR is preferable in sensitivity with electron microscopy or immunological methods [80–82].

Immunomagnetic separation combined with quantitative reverse transcription polymerase chain reaction

Immunomagnetic separation combined with quantitative reverse transcription polymerase chain reaction (IMS-RT-qPCR) has been developed to detect RV in the water samples [83]. It has been found that IMS enhanced detection sensitivity by nearly one order of magnitude from the clean water in comparison when compared with the extracting RNA with the use of commercial kits. The IMS-RT-qPCR has been shown to be successful in purifying and detecting RV seeded in 10³-fold concentrated wastewater influents and water samples. Research revealed rapidity, sensitivity, and reliability of the IMS-RT-qPCR to detect RV in the complicated water contexts. IMS-RT-qPCR in 2004 has been developed for viruses as a highly sensitive and specific assay. For example, Yang et al. [73] showed higher reliability of IMS-PCR in detecting the potentially infectious viruses and presented proper and worthwhile outputs for evaluating the health risks, in particular, for the viruses, which can be hardly cultured in vitro.

Integrated cell culture RT-PCR

For resolving the restrictions considered for detection techniques of RV in the environmental specimens like the prolonged interval with the conventional cell culture–based plaque assays, inabilities for detecting infectivity with RT-PCR-based molecular techniques as well as the declined sensitivity with ELISA tests, experts in the field proposed an integrated cell culture and reverse transcription quantitative PCR (ICC-RT-qPCR) assay for detecting the infectious RV with regard viral RNA detection in the course of replication in the cells [84, 85]. It is notable that the cell culturing step prior to the qPCR enables replication and detection of the infectious RV because such RVs have been considered to be the merely ones, which may infect the cells and generate RNA. In a study conducted by Li et al. [25], more than 42% of the RVs have been detected with ICC-RT-PCR while only 21% and 12% of samples were positive with plaque counting or the RT-qPCR method, respectively. Moreover, the sensitivity of ICC-RT-PCR method to detect RV less than 0.01 has been evaluated. Finally, ICC-RT-qPCR has been considered to be a fast and quantitative procedure to determine the infectious RV in the environmental waters, which obviously differentiate them from the inactivated RV [25].

Next-generation sequencing

The term next-generation sequencing refers to a number of different techniques developed in the last 15–20 years. Next-generation sequencing (NGS) is a new technology used for DNA and RNA sequencing and variant/mutation detection that can sequence hundreds and thousands of genes or whole genome in a short period of time [86]. It is widely accepted that the NGS for much parallel sequencing had unique advances in microbial ecology. NGS methods are high-throughput technologies with capabilities of sequencing large numbers of different DNA (massively parallel) sequences at once. NGS can sequence hundreds and thousands of genes or whole genome in a short period of time While a Sanger reaction returns a single DNA sequence, a typical NGS run can yield up to a billion unique reads, which is made possible by running several samples parallel. Also, transcripts can be identified without prior knowledge of a particular gene and taking alternative splicing and sequence variation into account at the same time [2, 3].

The major advance offered by NGS technologies is the ability to produce, in some cases, in excess of one billion short reads per instrument run, which makes them useful for many biological applications. NGS offers an almost unlimited opportunity to examine samples but on the contrary also produces massive quantities of data, which makes data analysis procedures compulsory. Sequencing procedures designed developed for typing RV serotypes from clinical samples have been used to describe VP4 as well as VP7 genes from the environment. The RV strains have been typed via the semi-nested RT-PCR procedures with the primers special for encoding VP4 (P-type) and VP7 (G-type) proteins [39, 45, 53]. One of the major disadvantages of the NGS method is that it is an expensive and time-consuming sample preparation protocol. The most important advantages of this method are (1) massively parallel sequencing capability, (2) single input of DNA/RNA, (3) simultaneous screening of multiple genes in multiple samples, (4) quantitative and sensitive detection of genomic aberrations, and (5) decreased sequencing costs per gene [87].

Conclusions

It has been found that the viral gastroenteritis prevalence is related to the environmental contamination caused by the wastewater involving the viral particles released in surface waters and rivers. RV is globally a key infectious agent for acute pediatric gastroenteritis. It is found in the aquatic environments because of the contaminations by raw sewage, even when there are no indicator bacteria that have been regarded as the prominent indicators in the case of the microbial evaluation of the water quality. Due to problems and diseases associated with RV for the children health around the world, the epidemiological study of RV-related gastroenteritis is very momentous in different countries. According to an analytical perspective, detection of viruses is difficult because of their lower concentration in water (1 to 10³ viral particles in each liter). Hence, sensitive techniques would be crucial for viruses to be detected. Researchers have been attracted to investigate new methods of concentration, detection, and discovery of the water resources pathogenic viruses. Moreover, identification of RV in the wastewater revealed possible risks for human health, especially in the case of untreated wastewater. According to this review, for RV concentration, detection can be carried out using a lot of methods that have their own advantages and different efficiency. However, further studies and investigations are needed to address such an important condition.

Now, some recommendation will be presented to estimate the number of RA in different kinds of water:

1. Experts in the field especially proposed effective recovery of RV using disk and cartridge filters for water resources, and such procedures may be assessed for types of aquatic environments.

2. Molecular methods based on the standard qPCR are widely recommended for viral genome detection in the water samples.

3. The recent research based on the molecular methods, that is, PCR-based techniques such as qPCR, RT-PCR, and real-time PCR, has been reported as a number of the most often utilized methods due to their specific higher sensitivity.

4. In many studies, RV genetic variation has been carried out in water samples. Lately, developed NGS methods have been used successfully for viruses, such as RV; therefore, it is suggested for further research.

5. Since the number of RV in sewage is high and causes the spread of RV diseases through the transfer of sewage to peripheral waters, it is recommended to monitor sewage in different regions of the world.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.