- double-stranded RNA virus with wheel- ("rota-") shape
- most infective of all the enteric viruses: estimated to cause >500,000 deaths per year
- more than 85% of these deaths occur in low-income countries in Africa and Asia
- children under five years of age (especially those between 6 months and two years), the elderly, and the immunocompromised are most vulnerable to the disease
- common in domestic wastewater and focally polluted surface waters/well water
- may be present in large numbers in the environment of infected communities
- exhibits greater resistance to common disinfection strategies than most other enteric viruses
Rotaviruses are double-stranded RNA viruses that cause disease in humans and animals. The primary mode of transmission is fecal-oral ( Kapikian and Chanock 2001
), although some have reported low titers of virus in respiratory tract secretions and other body fluids ( Zheng et al. 1990
) and is frequently associated with co-infection by respiratory syncytial virus (RSV) ( Fournel et al. 2008
). Because the virus is stable in the environment, transmission can occur through ingestion of contaminated water or food and contact with contaminated surfaces. They are an important cause of severe diarrhea among infants and young children in developed countries and a major cause of morbidity and mortality in developing countries in the same age group. According to the WHO, rotaviruses are estimated to be responsible for approximately 527,000 deaths each year (CDC), with more than 85% of these deaths occurring in low-income countries in Africa and Asia, and over two million are hospitalized each year with pronounced dehydration. Children under five years of age, especially those between 6 months and two years are most vulnerable to the disease.
It is estimated that by the age of 2 years old more than 96% of children in Mexico are infected with rotavirus (Velázquez et al. 1996). Interestingly, this same study revealed that natural infection confers protection against subsequent infections and that protection increases with each new infection and reduces the severity of the diarrhea. Among the 7 rotavirus species (named A to G), rotavirus A, B, and C can cause disease in humans with rotavirus A being the most common. All 7 species can cause infections in animals. Simian rotavirus SA-11, which closely resembles rotavirus of human origin is used as a surrogate of human rotaviruses in many water-relevant studies. Group A, which contains 9 serotypes, is endemic worldwide, is the most studied and has been documented as a cause of waterborne outbreaks in humans. Infections are referred to as infantile diarrhea, winter diarrhea, acute non-bacterial infectious gastroenteritis and acute viral gastroenteritis. Children 6 months to 2 years of age, premature infants, the elderly and the immunocompromised are particularly prone to more severe symptoms caused by infection with group A rotavirus. It is the leading cause of severe diarrhea among infants and children and accounts for about half of the cases requiring hospitalization. In the United States, approximately 3.5 million cases occur each year ( AWWA 2006). Outbreaks caused by Group B rotavirus, also called adult diarrhea rotavirus, have also been reported in the elderly and adults, although infection in the latter is very uncommon and usually subclinical. Group C rotavirus has been associated with rare and sporadic cases of diarrhea in children in many countries.
Rotaviruses can be found in all water types such as fresh, saline and sewage, with water being an important vehicle of transmission for this virus. Rotaviruses are excreted in very large quantities in the feces of infected subjects, at a rate of up to 1011
virus particles per gram in the acute phase ( Gratacap-Cavallier et al. 2000
). The stability of these viruses with regards to several physical conditions such as pH, temperature and moisture in conjunction with their resistance to commercially available disinfectants ( Harakeh and Butler 1984
) or wastewater treatments, contribute significantly to their persistence in the environment. The survival characteristics explain the presence of large amounts of infectious particles in wastewater ( Ansari et al. 1991
; Dubois et al. 1997
) and generally in environmental waters ( Abad et al. 1998
). Enteric viruses can also move from sources of contamination, such as broken sewage pipes and septic tanks, into groundwater aquifers ( Fout et al. 2003
). Several studies have mentioned the presence of rotaviruses in drinking water ( Deetz et al. 1984
; Keswick et al. 1985b
). In parallel with interhuman contamination, drinking water might thus play a role in the occurrence of sporadic cases. Considering the low infectious dose of these viruses in the range between 10 to 100 viral particles, only a small amount, present in the contaminated food, is usually sufficient to infect a human host, which in turn will shed a large number of viral particles in the acute phase of the disease, thus increasing the risk of contaminating the environment through fecal contamination.
Examples of reported rotavirus concentrations in aqueous environments:
- Lodder and Husman 2005 detected between 57 - 5,386 PCR detectable units (PDU) per liter in river water in two large rivers in The Netherlands. Although the high virus concentrations determined by PCR may in part be explained by the detection of virus RNA instead of infectious particles, the counts were nevertheless alarmingly high. Ojeh et al. 1995 demonstrated that virus RNA remained amplifiable after 24 hours of drying, although infectivity was reduced 1000-fold when compared with controls.
- A study conducted in southern Africa on the occurrence of rotavirus in raw and treated drinking water supplies, irrigation water and irrigated raw vegetables revealed the presence of rotavirus at several points ( van Zyl et al. 2006).
- Group A rotaviruses were detected in 11.8% of partially treated and 1.7% of finally treated drinking water samples and in 14% of irrigation water samples and 1.7% of corresponding raw vegetable samples. Although the study was not quantitative, the potential risk of waterborne transmission was highlighted. Furthermore, the seven final treated drinking water samples in which rotaviruses were detected met the World Health Organization and the South African Bureau of Standards microbial specifications for drinking water quality ( van Zyl et al. 2006). Considering the low infectious dose of rotavirus, these results underline shortcomings in quality specifications.
- Rotavirus was also demonstrated to be the most frequently detected gastroenteritis virus occurring naturally in the stream waters of Manaus, Brazil, an area heavily impacted by disordered urbanization. Around 44% of all positive water samples were found to be infected with this virus, and viral load was primarily shown to be linked to recreational activities such as bathing ( Miagostovich et al. 2008). As previously indicated in a study by Baggi et al. 2001, the presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.
In agreement with their tolerance to a wide range of environmental conditions and their high resistance to disinfection, Rotavirus has been shown to survive secondary sewage treatment. Further to the study by Lodder and Husman 2005
mentioned previously, sewage treatment appeared to be ineffective at removing rotavirus from effluent, showing log10
removals of only 0.9, 1.1 and 0.03 on three occasions and even an apparent increase through the treatment plant on two occasions of -0.1 and -1.8 log10
removals. Furthermore, although numerous technologies exist for the disinfection of wastewater prior to discharge back into the environment, rotavirus has demonstrated a tenacious resistance to many of these, whether physical or chemical. As with most other enteric viruses, rotavirus is resistant to inactivation at both low (3.5) and high (10) pH ( Health Canada 2004
; AWWA 2006
). Recently Caballero has demonstrated that a system of recombinant rotavirus surrogates opens new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced ( Caballero et al. 2004
). This should improve our understanding of effective disinfection practices for rotavirus in situ.
To kill or inactivate rotavirus from contaminated well water, CDC recommends bring water to a rolling boil for one minute (at elevations above 6,500 feet three minutes are recommended; see http://www.cdc.gov/healthywater/drinking/private/wells/disease/rotavirus.html). The water should then be allowed to cool and should be stored in a clean sanitized container with a tight cover, and refrigerated. Filters do not remove the virus from the water due to the small size fo the virus.
Selected studies on disinfection are summarized in the following:
- During a study of the persistence of viral pathogens and bacteriophages during sewage treatment, Baggi et al. showed that while most forms of secondary treatment were effective in reducing the numbers of bacteria (as determined by traditional fecal indicators), only those with a final sand filtation step were somewhat effective at reducing the viral load of the wastewater, prior to discharge to open rivers ( Baggi et al. 2001).
- One study subjected rotavirus to UV irradiation (253.7 nm) for 2.5 h at an intensity of 200 ~μW / cm2 ( Ojeh et al. 1995). The authors also tested other formulations including: 6% hydrogen peroxide-0-85% phosphoric acid (Endo-Spor, Globe Medical, Largo, FL, USA); 79% ethanol-0.1% orthophenylphenol (Lysol Disinfectant Spray, L&F Products, Montvale, NJ, USA); 80% (w/w) ethanol; and household bleach diluted to achieve a final concentration of approximately 2,500 ppm of available chlorine. Infectious virus was eliminated by exposure to all the aforementioned treatments. When these samples were evaluated for elimination of target sequences of RNA, as detected by RT-PCR, the researchers found that treatment with 6% H202, 2,500ppm chlorine, an ethanol-phenolic disinfectant, ultraviolet light and autoclaving eliminated amplifiable virion RNA, but 80% ethanol was not sufficient to prevent virus RNA amplificaition. However another study by Nasser et al. 2006 demonstrated that using a low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3 log10 inactivation of rotavirus even when the turbidity of the water was <5 NTU.
- Vaughn et al. 1987 showed that rotavirus was rapidly inactivated by ozone concentrations of 0.25 mg/L or greater at all pH levels tested. Comparison of the virucidal activity of ozone with that of chlorine in identical experiments indicated little significant difference in rotavirus-inactivating efficiencies when the disinfectants were used at concentrations of 0.25 mg/liter or greater.
- In a comparative study, Harakeh and Butler 1984 found that human rotavirus was the most resistant enteric virus (out of coxsackie, poliovirus, f2 coliphage, echovirus and simian rotavirus) to chlorine, chlorine dioxide, peracetic acid and ozone. In this study, concentrations of chlorine and peracetic acid capable of inactivating at least 99.9 % of simian rotavirus SA11 within 5 min permitted about 50% of the human rotavirus to survive. The differential observed with chlorine dioxide was almost as great, while in ozone SA11 was about 100-fold less sensitive than human rotavirus.
- Several recreational and commercial water treatment systems rely on the use of electrolytically generated copper and silver ions as a relatively safe and odourless alternative method for water disinfection. In a comparative study by Abad et al. 1994, the effectiveness of chlorine alone versus the combination of chlorine with copper and silver ions was studied to evaluate the effects on disinfection of rotavirus. This study demonstrated that addition of 700 µg of copper and 70 µg of silver per liter of water did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of free chlorine per liter.
Although viruses cannot replicate outside their host's tissues and therefore cannot multiply in the environment, they can survive for several months in fresh water and for shorter periods in marine water. Rotavirus can survive in water for days to weeks, depending on conditions of water quality and temperature. Increased microbial activity and sunlight intensity do have a detrimental effect on survival ( Conroy et al. 1996
; McGuigan et al. 1999
). Rotaviruses have the ability to attach to surfaces and to adhere to sediments via adsorption and dependant on autochthonous flora which might be beneficial for their survival and which has to be considered in sampling procedures ( Butot et al. 2007
). Infectivity of rotaviruses in seawater was shown to be prolonged in the presence of sediments.
Examples of studies on rotavirus persistence are given in the following:
- Rotavirus infectivity and genome persistence in ground- and surface water were investigated by molecular methods (reverse transcriptase qPCR) and conventional virology (focus forming units in cell cultures) as well as investigating the tolerance of the viruses to chlorine disinfection ( Espinosa et al. 2008). This study demonstrated that rotavirus infectivity was affected by enterobacterial content of the water as well as extrinsic factors such as light and temperature. These factors also affected genome stability, but to a lesser degree. The study also demonstrated that infectivity of rotavirus is tolerant to residual chlorine concentrations higher than those recommended by WHO for water used for human consumption. This observation, together with the fact that infectious rotavirus is able to persist for months in groundwater, indicates that protection of water sources from viral contamination, adequate disinfection procedures, specific viral indicators, and microbiological water quality monitoring are important issues to consider.
- Despite heated debate in the media, Butot et al. 2007 demonstrated through the development of an effective elution protocol, that attachment of enteric viruses (including rotavirus) to PET and glass water bottles is unlikely in the absence of autochthonous flora and that the bottling of uncontaminated water is not a likely vehicle for viral disease. This evidence is supported by very early work conducted by LaBelle and Gerba 1979 and Rao et al. 1984 when these authors demonstrated that the adsorption of enteric viruses to estuarine sediments played a major role in their hydrotransportation and survival, with rotavirus retaining its infectivity for up to 19 days when associated with sediments versus only 9 days when freely suspended in the same water.
- In a review of rotavirus survival and spread in the natural and manmade environments, Ansari et al. 1991 wrote that in temperate regions nosocomial outbreaks of the disease occurred mainly in cold dry weather, whereas in tropical settings the seasonality was less well defined. They indicated that although waterborne outbreaks of rotavirus gastroenteritis are most often recorded, that air, hands, fomites and food may also act as vehicles for this infection. They went on to state that rotaviruses can survive for weeks in potable and recreational waters and for at least 4 hours on human hands. In air and on nonporous inanimate surfaces, the survival of rotaviruses is favored by a relative humidity of <50% and viral infectivity can be retained for several days.
- Pancorbo et al. (1987) highlighted the survival of rotavirus in freshwater sources, identifying pH as significantly affecting the aggregation of viral particles and subsequently their susceptibility to chlorine and other virucidal factors of the fresh and treated water. They also demonstrated that in all case studied, infectivity decreased at a faster rate than antigenicity.
- Raphael et al. 1985 made an interesting supporting discovery that in fact treating (chlorinating) water may enhance the survival of rotavirus by removing potentially predatory autochthonous flora from the environment. They showed that the loss of virus infectivity was most rapid in raw water held at 20°C, taking about 10 days for a 99.0% reduction in the plaque titre of the virus versus municipally treated tap water held at 4 °C, where there was no significant drop in the virus titre even after 64 days, and at 20°C the titre in this same water was reduced by only 2 log10 over the same period.
It is estimated that the infectious dose is around 10-100 viral particles ( Health Canada 2004
). Viral particles are considered to be inactivated by gastric juice. In a study administering different numbers of infectious porcine rotaviruses to highly susceptible (colostrum deprived, cesarean derived, and given sodium biocarbonate to prevent gastric inactivation of viruses) newborn miniature swine piglets, the lowest dose of rotavirus to induce clinical illness was 1 PFU ( Graham et al. 1987
Enteric viruses are typically detected in contaminated waters by concentrating virus particles from large volumes. Viruses present in the concentrate often are detected with plaque or quantal cell culture assays ( Beller et al. 1997
). These cell culture analytical methods are labor intensive, requiring about 1 week to more than 6 weeks ( Fout et al. 1996
). Rotavirus has routinely been cultivated and detected using plaque assays in cell cultures of MA104 monolayers ( Rao et al. 1988
As for other viruses, molecular methods are commonly applied for rotavirus detection. The majority of molecular methods used for the detection of rotavirus have, however, been largely qualitative, ignoring quantification because of the low infectious dose and supposing that simply the presence of an infectious viral particle constitutes a hazard. Molecular methods are typically applied in combination with ultrafiltration to concentrate viral particles from water samples.
Selected molecular detection methods are summarized in the following:
- Flow Cytometry: A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) was developed by ( Abad et al. 1998). The efficiency of the procedure was compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM). This method admittedly required a sample size of 500L of river water to be concentrated in order to achieve the detection results obtained. The limit of sensitivity for the detection of the cell-adapted strain Itor P13 rotavirus, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively. The IIF-OM detection method, although efficient, is cumbersome and requires well-trained personnel, while IIF-FC is an automatable procedure ( Bosch et al. 2004).
- RT-PCR: Soule et al. showed that RT-PCR yielded the same sensitivity as cell culture (the compendial method of detection), detecting experimentally contaminated samples as low as 1 TCID50%/L ( Soule et al. 2000). In the same study, 1 tap water sample tested positive using the RT-PCR assay while the culture assay was unable to detect the virus.
- RT-PCR and IAC: RT-PCR provides a means to rapidly detect low levels of human enteric viruses, but it is sensitive to inhibitors that are present in water samples. This problem requires that adequate controls be used to distinguish true from potentially false-negative results. In a study by Parshionikar et al. 2004 researchers developed homologous viral internal controls several enteric viruses including rotavirus. There are three possible types of RT-PCR results that can be obtained when testing environmental water samples seeded with internal control RNA for presence of virus: (1) the internal control and virus do not amplify (2) the internal control amplifies, but not the virus, or (3) the virus amplifies, but not the internal control. The first type indicates the presence of PCR inhibitors. This false-negative result cannot be interpreted as the absence of virus in the sample. The second type of result should indicate the absence of virus in the sample. However, it may also be obtained if the internal control is in excess over the viral RNA, such that it suppresses viral amplification. Using a minimum amount of internal control RNA can prevent this problem. In addition, suppression of virus by internal control RNA can be detected by using a "virus alone" control. The third type can happen if the virus is present in great excess over the internal control. This result is still a true positive for the presence of virus and does not interfere with the purpose of the test, which is to determine the presence or absence of virus. Laboratories may want to test the effects of co-amplification when the virus is in excess over the internal control. Another suggested use of internal controls is the quantitation of single stranded viruses by competitive amplification ( Atmar et al. 1995). The number of viral particles can be calculated from the dilution of virus at which the signal intensity of the virus derived amplicon is equal to that of the transcript derived amplicon. In the case of rotavirus however, it may not be accurate to compare signal intensities for quantitation purposes as rotavirus has double stranded RNA while the internal control (ROTAIC) developed in this study was single stranded RNA. This may make the thermodynamics of the two amplification reactions unequal ( Parshionikar et al. 2004).
- Nested & Seminested RT-PCR: Rotavirus genomic segments have been shown to possess unique sequences both at 3' and at 5' ends, which are highly conserved among all strains. The majority of PCR primers have been designed to amplify regions within the gene encoding for the vp7 glycoprotein. Gratacap-Cavallier et al. 2000 were able to detect rotaviruses in drinking water using seminested PCR. In the first round of PCR, primers from a variety of authors were used to amplify a full length fragment of the vp7 glycoprotein gene using reverse transcriptase. Second round conventional PCR was carried out to amplify a nested sequence of 341bp. The sensitivity of the whole procedure had been standardized using a bovine strain of rotavirus to 1 TCID50%/L. In their experiment, 4/56 drinking water samples (2L) taken from homes where children had been confirmed as being infected with rotavirus tested positive using this seminested RT-PCR method.
In the nested PCR method ( Kittigul et al. 2005) the detection limit was 1.67 PFU per assay alone and 1.46 PFU per assay if a concentration method was applied to 1L seeded tap water. Of 120 water samples, rotavirus RNA was detected in 20 samples (16.7%): 2/10 (20%) of the river samples, 8/30 (26.7%) of the canal samples, and 10/40 (25%) of the sewage samples but was not found in any tap water samples (0/40). Only three water samples were positive for rotavirus antigen determined using a commercial ELISA.
In another study, to amplify part of the VP7 rotavirus gene segment, a nested RT-PCR was carried out. Primers RoV5 and RoV5a (sense) and primer RoV6 (antisense) were used in the first step. Subsequent nested PCR mix contained a mixture of the antisense primers RoV8a-c and the sense primer RoV7. VP7 primers, their genome positions and reaction conditions are according to ( Oh et al. 2003).
Using nested RT-PCR, six water samples from a river in Switzerland revealed rotavirus in 100% of the sites ( Gilgen et al. 1997). The rotavirus amplicons from the nested PCR were subsequently typed by RFLP analysis. All six samples revealed serotype 1, the most prevalent serotype isolated from children as well as environmental samples.
- Multiplex RT-PCR: Monoplex and multiplex RT-PCR were also optimized for the detection of Hepatitis A virus and Rotavirus ( Brassard et al. 2005). The analytical sensitivity of each method was evaluated by using known titers of HAV and rotaviruses in artificially inoculated bottled spring water samples. Compared to the multiplex RT-PCR, the analytical sensitivity of the RT-PCR performed in the monoplex reaction was found to be at least a hundredfold more sensitive for rotaviruses (10-3 TCID50%/ml) and at least ten-fold more sensitive for HAV (10-1 TCID50%/ml). However, the multiplex RT-PCR offers a simultaneous detection of both viruses, from a single amplification step, with analytical sensitivities of at least 1 TCID50%/mL for HAV and at least 0.1 TCID50%/mL for rotaviruses. This shows three orders of magnitude increase in sensitivity over the RT-PCR developed just a few years earlier by ( Gratacap-Cavallier et al. 2000) due to advance in effective concentration methods.
- NASBA-ELISA: Both monoplex and multiplex NASBA were optimized for the detection of Hepatitis A virus and Rotavirus, with a subsequent ELISA assay included to confirm specificity of the NASBA ( Jean et al. 2002a; Jean et al. 2002b). Amplicons from the NASBA reaction were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA-RNA hybrids were colorimetrically detected by the addition of streptavidin-peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4x101 PFU/mL) and 15 PFU (3x103 PFU/mL) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. Compared to a similar study using HAV, the detection limit for rotavirus was shown to be 10 times more sensitive ( Jean et al. 2002a).
- Microarray detection and genotyping: Honma et al. have developed a DNA Microarray for the detection and genotyping of 5 clinically relevant human rotavirus VP4 (P) genotypes and five additional human rotavirus VP7 (G) genotypes on one chip ( Honma et al. 2007). In their study, 128 rotavirus-positive human stool samples were collected in three countries (Brazil, Ghana, and the United States) to validate the chip. Interestingly 100% of the specimens were typeable from the US (although 1 P genotype was discordant with the microarray) while the Brazilian and Ghanan samples showed high levels of being nontypeable or discordant. Despite current technological hinderances and the need to develop more thorough type libraries for identification, the microarray shows promise for the rapid identification of several of the major strains of human relevant rotavirus.
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
Transmission electron micrograph of intact rotavirus particles, double-shelled. Distinctive rim of radiating capsomeres.
Photo ID: 273
Content provider(s): Centers for Disease Control and Prevention/ Dr. Erskine Palmer
This electron micrograph reveals a number of RNA rotavirus virions, and a number of unknown, 29nm virion particles.
A rotavirus has a characteristic wheel-like appearance when viewed by electron microscopy, i.e., rotavirus is derived from the Latin rota, meaning "wheel". Rotaviruses are nonenveloped, double-shelled viruses, making the virus stable in the environment.
Photo ID: 5620
Content provider(s): Centers for Disease Control and Prevention/ E. L. Palmer