key: cord-0005554-jb9kg3h0
authors: Betancourt, Walter Q.; Gerba, Charles P.
title: Rethinking the Significance of Reovirus in Water and Wastewater
date: 2016-06-18
journal: Food Environ Virol
DOI: 10.1007/s12560-016-9250-8
sha: b579dd066bd55d6171418e03834661e96f90ae67
doc_id: 5554
cord_uid: jb9kg3h0

The genus Orthoreovirus contains nonenveloped viruses with double-stranded gene segments encased in a double-layered icosahedral capsid shell. These features constitute major determinants of virion stability in the environment and virion resistance against physical and chemical agents. Reovirus (ReoV) is the general term most commonly used for all virus strains that infect humans and nonhuman animals. Several studies have demonstrated the frequent occurrence of ReoV in wastewaters and natural waters, including surface and ground waters from different geographical areas. Most of these studies have reported higher concentrations of ReoV than any other enteric virus analyzed. They are more commonly isolated in chlorine-disinfected wastewaters than other enteric viruses, and appear to survive longer in water. The ability of ReoV to form large aggregates, even with different types of enteric viruses (e.g., poliovirus) and their ability to undergo mechanisms of gene segment reassortment among different serotypes may also explain their greater stability. Different approaches have been applied for concentration of ReoV from water; however, the recovery efficiency of the filtration methods has not been fully evaluated. Recently, molecular methods for identification of ReoV strains and quantification of virus genome have been developed. Studies have shown that the overall detection sensitivity of ReoV RNA is enhanced through initial replication of infectious virions in cell culture. More studies are needed to specifically address unresolved issues about the fate and distribution of ReoV in the environment since this virus is not commonly included in virological investigations.

Reovirus (acronym for respiratory enteric orphan virus) belongs to a highly diverse group of nonenveloped and double-stranded RNA viruses (dsRNA) classified within the Reoviridae family. Orthoreovirus is the genus name (the true ''reovirus'') for one of the 15 genera of the Reoviridae viruses that infect mammals, birds, and reptiles. This designation distinguishes the orthoreoviruses from other members of the family that infect plants, fish, mollusks, fungi, and insects (Clarke and Tyler 2010; Day 2009; Dermody et al. 2013) . The Mammalian orthoreovirus (MRV, prototypical member of the Reoviridae) is the type species of the Orthoreovirus genus that was initially associated with a subgroup of respiratory and enteric viruses present in healthy humans with minimal or no symptoms of respiratory or enteric disease; therefore, the virus was considered an ''orphan'' (Sabin 1959; Jackson and Muldoon 1973; Norman and Lee 2000; Day 2009; Dermody et al. 2013) . Reovirus (ReoV) is a general term that refers to the MRV and is most frequently used throughout this review. Current studies indicate that ReoV may be the most common enteric virus found in raw and treated wastewater; however, the concentration efficiency of ReoV in these matrices including natural waters has not been thoroughly evaluated. This review summarizes previous and recent findings about the occurrence of ReoV in water. Recent progress in concentration and molecularbased detection methods combined with traditional cell & Walter Q. Betancourt wbetancourt@email.arizona.edu culture techniques will undoubtedly lead to more effective approaches to uncover the significance of ReoV in water and wastewater.

The Orthoreovirus genus is divided into two distinct phylogenetic subgroups: the fusogenic orthoreoviruses (i.e., induce fusion of infected cells through formation of large, multinucleated syncytia) which infect mammals, birds, and reptiles; and the nonfusogenic mammalian orthoreoviruses which infect virtually all mammals, including humans (Day 2009; Dermody et al. 2013) .

The Reoviridae are further divided into two subfamilies based upon virion morphology: the Sedoreovirinae subfamily, which includes genera containing ''smooth'' viruses almost spherical in appearance; and the Spinareovirinae subfamily which contains viruses with large ''spikes'' or ''turrets'' at the 12 icosahedral vertices of the core particle. The Orthoreovirus genus is included within the Spinareovirinae subfamily (Day 2009; Dermody et al. 2013) .

Three morphologically similar groups of ReoV have been described based on genetic divergence and antigenic properties among serotypes as described later in this review. Based on available sequence data, the current taxonomic organization of the orthoreoviruses comprises five species groupings (I-V): (I) the prototypical mammalian ReoV, plus Ndelle virus which correspond to nonfusogenic viruses; (II) avian reoviruses; (III) Nelson Bay virus and related bat-origin reoviruses such as Paulu virus (PuIV) and Melaka virus (MeIV); (IV) baboon reovirus; and (V) the reptilian reoviruses. Species group II, III, IV, and V belong to the fusogenic viruses (Clarke and Tyler 2010; Day 2009; Dermody et al. 2013) .

ReoVs are ubiquitous in their geographical distribution, and virtually all mammals, including humans, serve as host for infection (Scott 1971; Mochizuki et al. 1992; Decaro et al. 2005; Dermody et al. 2013) . ReoVs infect their hosts by the fecal-oral and respiratory routes. Infectious virions enter the body by ingestion of contaminated food or inhalation of virus-containing aerosols. At both portals of entry, ReoVs infect epithelial cells and disseminate to peripheral sites where they cause disease (Boehme et al. 2013) . Studies indicate that ReoVs are neurotropic viruses that use both bloodborne and neural pathways to spread systemically within their hosts to cause disease (Boehme et al. 2013) . Most ReoV infections in humans occur in infancy or early childhood; therefore, full seroconversion against all three major serotypes is attained by late childhood, (around 5 years of age) (Norman and Lee 2000; Day 2009; Dermody et al. 2013) . Human ReoVs are rarely associated with disease, except in the very young. They are known to cause either asymptomatic or mild symptoms of upper respiratory and intestinal infections, in some cases febrile exanthema with fever (Jackson and Muldoon 1973; Clarke and Tyler 2010; Dermody et al.2013) . ReoVs have been occasionally found in fecal specimens associated with childhood diarrhea (Giordano et al. 2005) . More recently novel ReoVs from nonhuman animals have been identified with some infecting humans and thus raising concerns about their zoonotic potential (Chua et al. 2007; Zhang et al. 2011; Kohl et al. 2012; Dermody et al. 2013; Yamanaka et al. 2014; Kawagishi et al. 2016 ).

Fine structural analysis by cryoelectron microscopy (cryo-EM) and three-dimensional image reconstruction techniques has revealed that the ReoV virions are spherical, nonenveloped particles approximately 85 nm in diameter (Day 2009; Dermody et al. 2013) . The virions have a particle mass of approximately 1.3 9 10 8 Daltons of which about 15 % is RNA, while the remainder is protein. In cesium chloride gradients, virion particles band at a buoyant density of 1.36-1.38 g/cm 3 (Silverstein et al. 1976) . Table 1 provides general features of reovirus particles.

All members of the Orthoreovirus genus contain 10 linear segments of double-stranded RNA with collinear and complementary [?] and [-] strands encased in a doublelayered icosahedral capsid shell ). These features constitute major determinants of virion stability in the environment and virion resistance against physical and chemical agents (Gomatos and Tamm 1963; Harris et al. 1987; Drayna and Fields 1982a, b; Norman and Lee 2000) . The inner-capsid shell or core particle has an internal diameter of approximately 50-60 nm and encloses the genome. In addition, the inner capsid possesses 12 prominent projections or spikes situated as though on vertices of a regular icosahedron. The outercapsid shell is 38 nm in diameter and surrounds the core particle . The 10 dsRNA genome segments are divided into three size classes based upon their migration in polyacrylamide gels: three large (L: L1, L2, L3), three medium (M: M1, M2, M3), and four small (S: S1, S2, S3, S4) segments. These segments encode mRNAs for synthesis of 12 viral proteins that execute the ReoV replication program in a variety of cell types within diverse animal hosts. Eight of these segments encode a single protein, while two encode two proteins each. Eight of the 12 proteins are structural components of virions (k1, k2, k3, l1, l2, r1, r2, and r3) and four are nonstructural (lNS, lNSC, r1S, and rNS). The function of these virus-encoded proteins has been described in detail elsewhere (Schiff 1998; Dermody et al. 2013) . Cell attachment r1 protein is the most type-specific reovirus protein. It is also the reovirus-hemagglutinin and is a major determinant of host-range/tissue specificity and of the nature of interaction of reovirus with cells of the immune system (Schiff 1998; Dermody et al. 2013 ). Interestingly, studies have shown that proteins r3 and l1 interact extensively within the outer capsid and the extensive contacts between r3 and l1 molecules have been suggested to stabilize outer-capsid structure and thereby facilitate survival of virions during transmission of ReoV between hosts (Nibert et al. 1995; Schiff 1998) . Furthermore, outer-capsid proteins r3, l1, and r1 have each been associated with the stability of ReoV particles against particular physicochemical agents (Drayna and Fields 1982a, b) .

Three morphologically similar groups of ReoV have been described based on genetic divergence and antigenic properties among serotypes. These occur mainly in the gene coding for cell attachment r1 protein that gives rise to antibodies with strong, completely type-specific hemagglutination inhibition (HI) activity; strong, almost completely type-specific neutralization activity; and ability to precipitate ReoV particles (Gaillard and Joklik 1982; Cashdollar et al. 1985; Douville et al. 2008; Dermody et al. 2013) . The three classically recognized serotypes represent the prototype strains: type 1 Lang (T1L), type 2 Jones (T2J), type 3 Dearing (T3D), and type 3 Abney (T3A). A putative fourth serotype strain isolated from a mouse in the Cameroon has been identified and named Ndelle (T4N) (Clarke and Tyler 2010; Day 2009; Kohl et al. 2012; Dermody et al. 2013 ). These three serotypes currently circulate in humans and other mammals (Boehme et al. 2013) . They are closely related in sequence and can exchange their ten genomic dsRNA segments by genetic recombination which occurs primarily through reassortment. This process generates some progeny (reassortants) that stably behaves as the wild type that can be grown under nonpermissive temperature conditions (Shiff 1998). It is known that T1L and T3D share approximately 25 % identity in their r1 protein, while other outer capsid and viral core proteins are highly conserved with 90-98 % identity (Douville et al. 2008) . Serotypes 1, 2, and 3 differ in their response to a variety of chemical and physical inactivating agents as discussed below.

ReoV replication and assembly occurs in the cytoplasm of host cells within cytoplasmic structures called viral factories (VFs) or viral inclusion bodies (VIBs). Following virus replication, different morphotypes of reovirus particles can be found in infected cells, including genomecontaining virions, infectious or intermediate subvirion particles (ISVPs), core particles, and genome-lacking particles .

The size of the ReoV genome is approximately 23,500 base pairs (Day 2009; Dermody et al. 2013) . Genomes of the prototype strains and several fusogenic orthoreovirus isolates have been completely sequenced (National Center for Biotechnology Information) including novel reassortant Isoelectric point measured for serotype 3 (T3D) varies between 3.8 (electrophoretic mobility using light microscopy for detection) and 3.9

(isoelectric focusing using dense aqueous solution) Reovirus genome Genome consists of 10 linear double-stranded RNA (dsRNA) gene segments that enables reassortment and viral evolution Gene segments of three size classes:

Three large segment (L1, L2, L3), three medium size segments (M1, M2, M3), and four small segments (S1, S2, S3, and S4) Genome encased in a double concentric icosahedral capsid shell (inner shell or core and outer shell) Gene segments are transcribed into full-length mRNAs Gene segments encode one or two proteins: k, l, and r size class proteins Food Environ Virol (2016) 8: 161-173 163 MRV (Anbalagan et al. 2014; Lelli et al. 2015) . Studies have indicated that 75 % of the RNA (15 9 10 6 Daltons) in the virion is contained in these 10 dsRNA segments, while about 25 % of the RNA in the virions ( *4.5 9 10 6 Daltons) is composed of short single-stranded adenylic-rich oligonucleotides. Around 70 % of these oligonucleotides are 2--9-residue products of abortive transcription and terminate with 5 0 -GC(U)(A). The remainder ( *30 %) are oligoadenylates that range in length from 2 to 20 residues (Silverstein et al. 1976; Dermody et al. 2013 ).

The frequent occurrence of ReoV in natural waters including surface and ground waters from different geographical areas has been documented in previous studies either as presence/absence-qualitative results (i.e., typing with conventional neutralization tests, distinctive cytopathogenic effects, immunofluorescence with specific antibodies, or molecular methods)-or as virus titers using different quantitative assays of infectivity in cultured cells.

Most of the published work on ReoVs in ambient waters and wastewaters, with some exceptions (Dahling et al. 1989; Symonds et al. 2009 ) reported higher concentrations of these viruses than any other enteric virus analyzed. Table 2 summarizes selected studies on the occurrence of ReoVs in natural waters including streams, source waters (groundwater and surface waters) used for drinking water supply, and seawater. Between the late 1960s and the mid-1980s, several studies demonstrated the frequent occurrence of ReoVs in wastewater (Adams et al. 1982; England et al. 1967; England 1972; Berg 1972; Sattar and Westwood 1978; Sellwood et al. 1981; Ridinger et al. 1982; Payment et al. 1986 ). Adams et al. (1982) using antibody immunofluorescence in cell culture was able to detect up to 210,000 ReoV per liter of untreated wastewater. Overall these studies documented higher levels of ReoV than any other enteric virus co-occurring in raw sewage and treated wastewater (Table 3) . Surprisingly, ReoV occurrence in wastewaters was less frequently reported in subsequent years. This may be due to the types of cell lines used and the incubation time. The most commonly used cell line for testing for viruses in water is the BGM cell line. While ReoV will grow in this cell line, it usually requires 14 days or longer incubation for the observation of cytopathogenic effects.

More recent investigations confirmed the frequent occurrence of ReoV in raw sewage and treated effluent in the United States (Sedmak et al. 2005) , the Netherlands (Lodder and de Roda Husman 2005) , and Taiwan (Lim et al. 2015) . The comparative reductions of ReoV and other human enteric viruses after wastewater treatment have been documented in previous studies and also summarized in Table 3 . Sedmak et al. (2005) conducted the longest term study of the relative occurrence of ReoV. They used different cell lines for detection of infectious virus in raw sewage, activated sludge-treated disinfected effluent, and the intake to a drinking water treatment plant. ReoVs were the most commonly isolated viruses followed by enteroviruses and adenoviruses. All of their isolates were from BGM cells which had been maintained for 14 days. The occurrence of ReoV relative to the other enteric viruses after disinfection increased suggesting greater resistance of the ReoV to removal by activated sludge treatment and/or chlorine disinfection.

Recently Qiu et al. (2015) conducted a study on the removal of enteric viruses by activated sludge treatment, disinfection, and ultrafiltration at a full-scale wastewater treatment facility in Canada. Virus removal was assessed by both qPCR and integrated cell culture PCR (ICC-PCR). Interestingly ReoV, in contrast to the other enteric viruses, was rarely detected in the primary effluent and after other steps of the treatment process. However, it was the most commonly isolated virus by the ICC-PCR infectivity assay after secondary treatment, UV light disinfection, chlorination, and ultrafiltration. It was the only infective virus isolated after disinfection and ultrafiltration.

ReoV appears to be common in sewage sludge and have been detected in raw, anaerobically, aerobically digested, and lagoon sewage sludge by infectivity assays (Wellings et al. 1976; Sattar and Westwood 1978; Gallagher and Margolin 2007) . The relative concentration to other enteric viruses is difficult to judge because of different methods for recovery from the sludge and cell lines used for assay in the different studies. Also, quantitative assessment of removal of naturally occurring ReoV is difficult because extracting data on the operation of the processes is often not provided in enough detail. Using a primary cell line from a primate Cliver (1975) isolated ReoV in anaerobically digested sludge more commonly and in greater numbers than enteroviruses. Also, using a primary cell line Subrahamanyan (1983) reported the almost exclusive isolation of ReoV. It was also the most common isolate before sludge digestion. Laboratory studies suggest that ReoV may be more resistant to thermal inactivation than poliovirus. Brewster et al. (2005) found that ReoV was more resistant than poliovirus 1 at thermophilic (50-55°C) and mesophilic (30-35°C) temperatures used in anaerobic sludge treatment. However, Coxsackie and polio virus were shown to have similar inactivation rates during drying of sewage ReoV was detected in every type of water. Constant electrophoretic patterns were associated with reovirus type 1 and 3.

Milde et al.

Coastal waters (estuary and seawater) of the middle northern Adriatic Sea, Italy

Prefiltration of water samples through polypropylene membranes followed by tangential flow filtration using polysulfone membrane with a 10,000 molecular weight exclusion size, elution in 3 % beef extract, and a final concentration step using a polysulfone membrane.

BGM and Hep-2cells used for isolation of viruses. Cell culture homogenous lysates tested for EVs by RT-PCR and ReoV dsRNA using nondenaturing polyacrylamide gel electrophoresis.

ReoV found with higher frequency than EV in all samples. 

Seawater samples from two areas of the Adriatic Sea Prefiltration of water samples through polypropylene membranes followed by tangential flow filtration using polysulfone membrane with a 10,000 molecular weight exclusion size, elution in 3 % beef extract, and a final concentration step using a polysulfone membrane. sludge (Ward and Ashley 1977) . The mechanisms of ReoV inactivation in sewage sludge are complex depending on temperature, pH, ammonia, and the presence of detergents. Inactivation of ReoV results from the breakdown of the viral particles (Ward 1983 ). ReoV appears to show similar resistance to enteroviruses and MS2 phage to lime treatment of sludges (Ward 1983; Brewster et al. 2005; Katz and Margolin 2007) . In lime-treated fecal sludge, from latrines, the reduction of ReoV type 3 was found to be similar to adenovirus type 1 and related to ammonia concentrations above pH 9 (Magri et al. 2015) . The existing data suggest that ReoV may be more abundant than ReoV detected at a higher frequency (89 %) than AdV and EV (between 15 and 21 %) after testing CPE-positive cell culture using reovirus-specific primers Lee and Jeong (2004) Source water for drinking water production at three locations in the Netherlands enteroviruses in sewage sludge and may be more resistant to digestion than enteroviruses. ReoV has been suggested as a better model for sludge treatment than enteroviruses (Brewster et al. 2005) .

Different approaches have been applied for concentration of ReoV from sewage including cotton gauze swab, precipitation by protamine sulfate treatment (salmine) and isoelectric coagulation (i.e., flocculation). Early studies demonstrated that protamine sulfate treatment provided an efficient method for concentrating ReoV and adenoviruses which tended to be overgrown by more rapid replicating enteroviruses in cell culture (England 1972) . The efficiency of the protamine sulfate method with some modifications for the recovery of ReoV from waste waters and sewage was later confirmed by another study in the U.S, (Adams et al. 1982) . In this study, infectious ReoV and potentially infectious ReoV particles (particles with complete outer coat) were concentrated from water and sewage by protamine sulfate precipitation followed by addition of 0.25 % fetal bovine serum and 0.005 % protamine sulfate for the first precipitation of the sample plus 0.0025 % for the second. The study reported virus recoveries between 80 and 100 % with levels of infectious ReoV greater than 10 5 per liter of untreated sewage, which suggested that the modified protamine precipitation method when combined with fluorescent-antibody staining was useful in determining the potential roles of the infectious virus and ReoV particles in water quality testing.

Another study (Bottiger 1973) investigated the viral content of sewage collected with gauze swabs at the inlet of three major sewage treatment plants of the Stockholm area. Echovirus type 1 and ReoV were found throughout the entire period of the study using primary monkey kidney cells. Later, a comprehensive study from the United Kingdom (Sellwood et al. 1981 ) conducted over a 3-year period at a local sewage works (biological oxidation beds) found that multiple cell lines were necessary for efficient evaluation of the virus content of raw sewage and treated effluent. In this study, Vero and primary monkey kidney cells yielded the most Coxsackievirus and ReoV isolates, which were frequently isolated along with echovirus from the plant inlet (raw sewage) and outlet swabs. In a similar study in the United States, the sensitivities to ReoV (serotypes 1, 2, and 3) of 12 continuous cell lines was compared with sewage isolates precipitated by protamine sulfate ). Among the cell lines evaluated, Madin-Darby bovine kidney (MDBK) was the most sensitive for isolation of ReoV, and when combined with an immunofluorescent cell count assay, the detection rate gave a ReoV titer in excess of 10 4 /liter of raw sewage.

Adsorption/elution methods (e.g., membrane, cartridge, and glass wool filtration) and entrapment or size exclusion techniques which involve ultrafiltration systems (e.g., tangential flow filtration) have been applied for processing large volumes of water including treated sewage and surface waters (Milde et al. 1995; Muscillo et al. 1997; Fout et al. 2003; Lodder and de Roda Husman 2005; Sedmak et al. 2005) . Following primary concentration, different secondary concentration procedures have been used to further isolate ReoV and other enteric viruses. Two-phase separation with dextran and polyethylene glycol (PEG)-or PEG and NaCl-beef extract elution, and PEG (1996) Activated sludge with nutrient removal followed by advanced treatment: ultraviolet (UV) disinfection, ultrafiltration (UF) and chlorine disinfection Edmonton, Canada

Virus concentration by NanoCeram 90-mm laminated disc filter and organic flocculation. Detection by ICC-qPCR using BGM cells Infectious ReoV was the only virus detected by ICC-qPCR after chlorine disinfection (12 %, 3 of 25) and ultrafiltration (8 %, 1 of 13) of the final effluent Qiu et al. (2015) precipitation have been most commonly used for the secondary concentration step (Wyn-Jones 2007). Very few studies have evaluated the recovery efficiency of ReoV from water. It is very important since the sorption behavior of virus particles is governed by electrostatic interactions which are given by the virus isoelectric point (IEP); this is a characteristic parameter of the virion in equilibrium with its environmental water chemistry (Michen and Graule 2010) . In this situation, the sorption behavior of a particular virus may substantially differ from the most common human viruses (i.e., poliovirus) or viral surrogates (i.e., bacteriophages) used in recovery efficiency experiments. For instance, reported isoelectric points for poliovirus strains range from 4.0 to 7.4, MS2 coliphage 2.2-4.0, and ReoV type 3 from 3.8 to 3.9 (Michen and Graule 2010) . The detection of ReoV in water concentrates has been accomplished through different assay methods. As previously mentioned, virus replication in continuous cell lines of monkey kidneys was initially applied for isolating ReoV from water; however, they have the ability to infect many continuous mammalian cell lines maintained in clinical laboratories . The identification of ReoV in water based on observation of typical cythopathic effect (CPE) has been applied simultaneously with quantitative assays such as hemagglutination or immunological methods which quantify the presence of infectious and noninfectious virus particles (Condit 2013) . ReoV produces a very characteristic cytopathogenic effect (CPE) in monkey kidney cell lines. The cells become granular and do not slough off as readily as with enteroviruses. Often they remain fasten to the glass by a single process and flutter in the medium as the flask/tube is agitated during microscopic examination. The addition of the proteolytic enzyme can be used to enhance infectivity in cell culture (Spendlove and Schaffer 1965) . Cell culture assay methods have been concomitantly applied for quantification of ReoV infectivity such as most probable number assays (MPN) which derive MPN titers (i.e., virus titer) in replicates of cell monolayers inoculated with different equivalent volumes of a water concentrate (Lee and Jeong 2004; Sedmak et al. 2005) . Studies have also shown that multiple blind passages on BGM cells may increase the presence of virus particles for subsequent detection of ReoV RNA by RT-PCR or polyacrylamide gel electrophoresis (PAGE) (Muscillo et al. 2001) . Interestingly, these studies have also demonstrated that the simultaneous presence of ReoV and enteroviruses can have a masking effect by hindering enterovirus amplification and detection in environmental samples highly contaminated with ReoV (Carducci et al. 2002) .

ReoV RNA obtained from cell culture homogenous lysates has been used to study dsRNA segment patterns separated on a nondenaturing polyacrylamide gel (Milde et al. 1995; Muscillo et al. 1997) . Further combination of cell culture with molecular amplification of viral RNA sequences (ICC-PCR) added another feasible and relatively more sensitive approach applicable to ReoV detection in water (Spinner and DiGiovanni 2001; Lee and Jeong 2004) .

Genome-based molecular techniques offer rapid detection and identification of ReoV. These assays involve reverse transcriptase polymerase chain reaction (RT-PCR) with primers designed to amplify specific viral RNA sequences. Genomic sequencing and phylogenetic analysis are subsequently used to identify specific genotypes and/or to confirm the isolate serotype as well as to reveal virus diversity. Most studies included initial replication of the virus in cell culture to increase viral genomes available for molecular identification and typing assays (Spinner and DiGiovanni 2001; Muscillo et al. 2001) .

Primers that target conserved regions of the ReoV L1, L3, and S2 genes have been developed and applied for molecular detection of ReoV RNA in water (Muscillo et al. 2001; Spinner and DiGiovanni 2001; Leary et al. 2002) . The sensitivity of amplification of ReoV RNA with these primers was evaluated differently. The detection sensitivity with primers against the L1 gene was evaluated with serial dilutions of the purified PCR-positive control. The lowest concentration in which a band could be visualized defined the sensitivity of the assay which corresponded to 100 targets per reaction (Symonds et al. 2009 ). Spinner and DiGiovanni (2001) used serially diluted purified nucleic acids from ReoV types 1, 2, and 3 to determine the sensitivity of the ReoV primers against the L3 gene. The detection sensitivities corresponded to approximately 3, 30, and 0.3 PFU per reaction mixture, respectively. The sensitivity of the RT-PCR reactions with primers against the S2 region was tested on the RNA extracted from serial dilutions of virus stock ranging from 10 5 tissue culture infectious dose (TCID 50 ) to 0.1-fold. In this case, the S2 reactions were positive on the reference strains with a sensitivity of less than 10 2 -10 TCID 50 viral particles (Muscillo et al. 2001) .

Additionally, reverse transcription quantitative real-time PCR (RT-qPCR) methods have been developed for sensitive, rapid, and specific detection of ReoV RNA in wastewater samples (Gallagher and Margolin 2007; Qiu et al. 2015) . These assays have not been tested for detection of ReoV in other water matrices.

Studies have shown that ReoV is very environmentally stable; its high stability outside the host enables this virus to remain viable for up to a year at 4°C (Berard and Food Environ Virol (2016) 8:161-173 169 Coombs 2009; Nibert et al. 1995) . Additional studies conducted under controlled laboratory conditions using distilled water and wastewater demonstrated that among different enteric viruses evaluated ReoV and rotavirus survived more than 30 days at 26°C and longer than 1 year at 8°C (McDaniels et al. 1983) . Similarly, studies which involved the incubation of ReoV and poliovirus in a buffered solution (29 Saline Sodium Citrate: 300 mM NaCl, 30 mM sodium citrate, pH 7.0) at different temperatures (22, 37, 39.5, 42, 45, and 50°C) indicated that survival of ReoV as determined by plaque assay was greater than for poliovirus. This study provided good evidence to support ReoV being more resistant to temperature-induced inactivation than PV (Brewster et al. 2005) .

ReoVs have been shown to be resistant to high temperature (55°C) for short periods of time (McVey et al. 2013) .

ReoV is very stable in aerosols in relative humidity above 75 % or below 40 % and may be expected to survive spray irrigation with wastewater (Spendlove and Fannin 1982; Sattar and Springthorpe 1999) .

Several studies have demonstrated that ReoV are more resistant to Ultraviolet (UV) light inactivation than singlestranded human RNA viruses (Chevrefils et al. 2006; Lytle and Saripanti 2005; Harris et al. 1987; Hill et al. 1970 ). Doses of UV light for ReoV type 1 (Lang) have been observed to be almost double of poliovirus type 1 for a 99.9 % reduction in titer (24 vs. 46 mW/sec/cm 2 ) (Harris et al. 1987) . McClain and Spendlove (1966) observed differences in UV light sensitivity of ReoV 1-3 at higher doses of UV, with ReoV type 3 being the most resistant. They also observed a nonlinear rate of inactivation and attributed to it experimentally to multiplicity of reactivation, i.e., two or more viruses infecting the same cell with different regions of damaged genome. They also observed that multiplicity of reactivation occurred between the different ReoV types, i.e., ReoV 1 could serve to compliment ReoV 2 infecting the same cell. It has also been suggested that the greater resistance of ReoV to UV light is due to its double-stranded segmented genome, a thicker protein coat than the single-stranded enteroviruses, and greater ability to aggregate (Harris et al. 1987) . In a study of resistance of naturally occurring coliphages and ReoV to UV light in secondary effluent, the rate of ReoV inactivation was found to be almost identical to F-specific coliphages (Nieuwstand et al. 1991) . Liu et al. (1971) reported that highly purified ReoV types 1-3 were more sensitive to chlorine in buffered water and Potomac River water than various types of adenovirus, poliovirus, echovirus, and Coxsackievirus. The most detailed studies on ReoV inactivation by disinfectants was conducted by Sharp et al. (1975) . They found that inactivation of ReoV and enteroviruses was highly dependent on the size and number of viral aggregates. The viruses could be made to aggregate and deaggregate by changing the pH, salt concentration, and the types of salts in solution (Floyd and Sharp 1977; 1978a; 1978b) . Their studies also suggested that the observed tailing effect during bromine disinfection was due to aggregates larger than 1,000 virions. These ''super sized'' aggregates were too rare to be seen under the electron microscope, but by sonication they could break some of these resulting in an increase in titer. They suggested that these ''super'' aggregates were formed in cell culture and in addition to aggregated virus might also contain cell debris. Another phenomenon observed was the ability of ReoV and poliovirus type 1 to form aggregates together (Floyd 1979) . Thus, dissimilar viruses could form aggregates. Since they found that 10 5 -10 6 virions were needed to form aggregates, they concluded this was not a significant factor in viral resistant in wastewater. However, with modern molecular methods such as qPCR for quantifying genome copies of viruses, concentrations of 10 5 -10 6 per mL of adenoviruses, Aichi viruses, and pepper mild mottle virus (PMMoV) are known to occur in wastewater ). In addition, the formation of disinfectant-resistant virial aggregates may be possible, especially considering that the total concentration of all human, plant, and bacterial viruses likely exceeds 10 7 per mL in domestic wastewater .

Reovirus exhibits greater resistance to inactivation by preformed chloramines and ozone than poliovirus, but is more sensitive to inactivation by chlorine and chlorine dioxide, 95 % ethanol and quaternary ammonium disinfectants, such as Roccal ROCCAL Ò -D Plus used in veterinary practice based on laboratory studies of purified virus (Drulak et al. 1984; Liu et al. 1971; McVey et al. 2013 ).

ReoV has not been studied as much in the environment, as enteroviruses and adenoviruses, especially using qPCR. Existing data suggest that they may be among the most common enteric viruses in wastewater, if not the most common. Studies on disinfected wastewater suggest that they may be more sensitive to chlorine than enteroviruses. However, they are more resistant to ultraviolet light disinfection than enteroviruses because of their dsRNA genome. ReoV also appears to be more environmentally stable than enteroviruses. Fout et al. (1996) found ReoV to be the most common viruses in groundwater in the United States. Studies at managed aquifer sites also suggest that they may travel further than other enteric viruses in the subsurface (Betancourt et al. 2014 ). However, their role in human disease is unclear, but given the role of bats in emerging zoonotic diseases such as SARS, Ebola, and MERS and zoonotic potential (including bats) of ReoV, additional studies on routes of human exposure are needed.

Moreover, the ubiquitous presence of ReoV in mammals, frequent occurrence in sewage and relative resistance to ambient conditions suggest that these viruses may be used to determine the vulnerability of watersheds, coastal areas, and groundwater sites to fecal pollution and consequently as an indication of enteric virus contamination (Muscillo et al. 1997; Spinner and DiGiovanni 2001; Fout et al. 2003; Shoeib et al. 2009 ). This review suggests that environmental transmission of ReoV clearly needs more study both to better assess its potential for transmission and its usefulness of an indicator of enteric viruses in the environment.

Protamine precipitation of two reovirus particle types from polluted waters

Genome sequence of the novel reassortant mammalian orthoreovirus strain MRV00304/13, isolated from a calf with diarrhea from the United States

Enteric viruses in a wastewater treatment plant in Rome. Water Air Soil Pollution

Mammalian reoviruses: propagation, quantification and storage

Reassessment of the virus problem in sewage and in surface and renovated waters

Assessment of virus removal by managed aquifer recharge at three full-scale operations

Microbiological assessment of ambient waters and proposed water sources for restoration of a Florida wetland

Mechanisms of reovirus bloodstream dissemination

Experiences from investigations of virus isolations from sewage over a two-year period with special regard to polioviruses

Enteric virus indicators: reovirus versus poliovirus

Interference between enterovirus and reovirus as a limiting factor in environmental virus detection

Sequences of the S1 genes of the three serotypes of reovirus

UV light dose required to be achieved incremental log inactivation of bacteria, protozoa and viruses

A previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans

Reoviruses: General Features

Virus association with wastewater solids

Principles of Virology

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities

The diversity of the orthoreoviruses: molecular taxonomy and phylogenetic divides

Virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in Italy

Detection of human enteric viruses in stream water with RT-PCR and cell culture

Orthoreoviruses

Reovirus serotypes elicit distinctive patterns of recall immunity in humans

Biochemical studies on the mechanism of chemical and physical inactivation of reovirus

Genetic studies on the mechanism of chemical and physical inactivation of reovirus

The effectiveness of six disinfectants in inactivation of Reovirus 3

Concentration of reovirus and adenovirus from sewage and effluents by protamine sulfate (salmine) treatment

Virologic assessment of sewage treatment at

Viral aggregation: mixed suspensions of poliovirus and reovirus

Aggregation of poliovirus and reovirus by dilution in water

Viral aggregation: quantitation and kinetics of the aggregation of poliovirus and reovirus

Viral aggregation: effects of salts on the aggregation of poliovirus and reovirus at low pH

A multiplex reverse transcription-PCR method for detection of human enteric viruses in groundwater

ICR microbial laboratory manual

Quantitation of the relatedness of reovirus serotypes 1, 2, and 3 at the gene level

Development of an integrated cell culture-real-time RT-PCR assay for detection of reovirus in biosolids

Discrepancies in viral gastroenteritis diagnosis: an unusual dual reovirusadenovirus infection case

Animal and plant viruses with double-helical RNA

Ultraviolet inactivation of selected bacteria and viruses with photoreactivation of the bacteria

Ultraviolet devitalization of eight selected enteric viruses in estuarine water

1-year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated-sludge purification plant

Viruses causing common respiratory infection in man. IV. Reoviruses and adenoviruses

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

Reverse genetics for fusogenic bat-borne Orthoreovirus associated with acute respiratory tract infections in humans: role of outer capsid protein rC in viral replication and pathogenesis

Relative abundance and treatment reduction of viruses during wastewater treatment processes-Identification of potential viral indicators

Isolation and characterization of three mammalian orthoreoviruses from European bats

Detection of mammalian reovirus rna by using reverse transcription-pcr: sequence diversity within the k3-encoding l1 gene

Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliability of waterborne virus detection

Detection and characterization of a novel reassortant Mammalian Orthoreovirus in Bats in Europe

High incidence of mammalian Orthoreovirus identified by environmental surveillance in Taiwan

Relative resistance of twenty human enteric viruses to free chlorine in Potomac water

Presence of noroviruses and other enteric viruses in sewage and surface waters in The Netherlands

Presence of enteric viruses in source waters for drinking water production in the Netherlands

Predicted inactivation of viruses of relevance to biodefense by solar radiation

Inactivation of adenovirus, reovirus and bacteriophages in fecal sludge by pH and ammonia

Multiplicity reactivation of reovirus particles after exposure to ultraviolet light

Rotavirus and reovirus stability in microorganism-free distilled and wastewaters

Reoviridae

Isoelectric point of viruses

Occurrence of reoviruses in environmental water samples

Serotype 2 reoviruses from the feces of cats with and without diarrhea

Enteric virus detection in Adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis

A new RT-PCR method for identification of reoviruses in seawater samples

Reoviruses and their replication

Hydraulic and microbiological characterization of reactors for ultraviolet disinfection of secondary wastewater effluent

Reovirus as a novel oncolytic agent

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

Assessment of human virus removal during municipal wastewater treatment in

Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage

Reoviruses: a new group of respiratory and enteric viruses formerly classified as ECHO type 10 is described

Reoviruses, In Waterborne Pathogens

Viral pollution of surface waters due to chlorinated primary effluents

Reovirus Capsid Proteins cd and J11: Interactions That Influence Viral Entry, Assembly, and Translational Control

Feline reovirus

9-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a wastewater treatment plant in

Viruses in sewage as an indicator of their presence in the community

Nature of the surviving plaque-forming unit of reovirus in water containing bromine

Comparative assessment of mammalian reoviruses versus enteroviruses as indicator for viral water pollution

The reovirus replicative cycle

Methods for characterization of viruses in aerosols

Enzymatic enhancement of infectivity of reovirus

Detection and identification of mammalian reoviruses in surface water by combined cell culture and reverse transcription-PCR

Virological hazards associated with sewage and sludge: Investigation in southern Ontario

Eukaryotic viruses in wastewater samples from the United States

Seasonal distribution of adenoviruses, enteroviruses and reoviruses in urban river water

Destruction of viruses in sludges by treatment processes

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation

Demonstration of solids-associated virus in wastewater and sludge

The detection of waterborne viruses

Imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus

A potentially novel reovirus isolated from swine in northeastern China