Home Viruses Calicivirus
Caliciviruses PDF Print E-mail
Thursday, 18 March 2010 00:00

  • Caliciviruses include norovirus, sapovirus, lagovirus, and vesivirus
  • Norovirus is the genus of most concern in terms of impact on human health
  • Norovirus is well-known from outbreaks on cruise ships and is sometimes referred to as 'cruise ship virus'
  • transmitted via the fecal-oral route
  • survival favored by colder temperatures
  • outbreaks caused by contaminated water occur predominantly in the colder months of the year
  • relatively high resistance to chlorine compared with other enteric indicator viruses
Caliciviruses contain positive-sense, single stranded RNA genomes of app. 7.6 kb and have been grouped into the genera norovirus, sapovirus, lagovirus and vesivirus ( Ando et al. 2000; Green et al. 2001; Hansman et al. 2007a). Only the first two genera comprise human pathogens. Noroviruses are the genus of most concern in terms of impact on human health. Five different Norovirus genogroups (GGI to GGV) are distinguished, based on the sequence of the RNA-dependent RNA polymerase (RdRp). Noroviruses belonging to the genogroup I and II comprise mostly human pathogens ( Rutjes et al. 2006). To a lesser extent, also genogroup IV are associated with disease ( Trujillo et al. 2006). Their importance derives from the fact that caliciviruses are estimated to be responsible for more than 90% of acute viral gastroenteritis worldwide and for a large number of the viral waterborne outbreaks ( Green KY 1997). People of all age groups are susceptible ( Koopmans and Duizer 2004). Both outbreaks originating from drinking water and from recreational water contaminated with feces have been reported ( Boccia et al. 2002; Hafliger et al. 2000; Hoebe et al. 2004; Kukkula et al. 1999; Sartorius et al. 2007). Outbreaks on cruise ships tend to receive much public attention ( Dahl E 2006; Depoortere et al. 2006). Finland could provide valuable data since the implementation of an improved outbreak surveillance system. A study associated 18 of 41 waterborne outbreaks from 1998 to 2003 with noroviruses as the causative agent ( Maunula et al. 2005). With the exception of 2001 several norovirus outbreaks occurred every year. The largest epidemics in 2000 were caused by contaminated groundwater with more than 10,000 people exposed resulting in 2,000-5,500 illnesses ( Maunula et al. 2005). In an outbreak in Sweden associated with recreational activity in a lake, swimming less than 20 m from the shore and having water in the mouth in the main swimming area were considered risk factors ( Sartorius et al. 2007). Also contaminated food is a common reason for outbreaks ( De Wit et al. 2007; Schmid D 2007). Noroviruses were formerly called small round-structured viruses (SRSVs) or Norwalk-like viruses. Norwalk virus is a reference strain of genotype I noroviruses, while Snow Mountain virus is a prototype strain for genogroup II. Norwalk virus was isolated during an outbreak of gastroenteritis in 1968 in a school in Norwalk, Ohio. Sapporo virus is the prototype strain for the genus sapovirus. Like noroviruses, sapoviruses are also classified into 5 genogroups (GI – GV), among which all but GIII can infect humans ( Hansman et al. 2007a). Infections are mainly known to affect young children and normally occur by the age of 5 years. Although the knowledge about sapoviruses is limited, they have been detected in water samples including treated and untreated wastewater and river water ( Hansman et al. 2007b).
Typically water gets contaminated by infected people. Individuals release viral particles in high numbers both via vomitus and feces ( Westrell et al. 2006). During the symptomatic phase, shedding numbers as high as 108 (vomits) and 109 (faeces) virus particles per ml have been estimated using electron microscopy ( Westrell et al. 2006). Contamination of water with noroviruses leads to numerous outbreaks every year due to the low infectious dose (Centers for Disease Control and Prevention 2003). Whereas norovirus transmission can occur during the entire year, outbreaks caused by contaminated water show a seasonal pattern and occur predominantly in the colder months of the year ( Mounts et al. 2000). In the before mentioned Finish study, 13 of 18 norovirus outbreaks between 1998 and 2003 occurred in winter and spring (December to May). The remaining 5 outbreaks were reported in July and August affecting a smaller number of people who drank water from wells. Whereas this seasonality is striking, norovirus concentrations can also reach levels that can cause disease and outbreaks in summer months as shown by infection in recreational water or drinking water ( Hoebe et al. 2004; Boccia et al. 2002). Interestingly, the study in Finland related most epidemics to contaminated groundwater systems, which are most commonly used as drinking water source in Finland. Only in three cases, surface, lake, or river water was the cause of the outbreaks ( Maunula et al. 2005). While this example shows the vulnerability of groundwater to viral contamination, in general surface waters are considered more prone to pathogen contamination. Especially surface waters impacted by untreated sewage can have elevated viral concentrations ( Lodder and de Roda Husman 2005). Untreated sewage contamination can for example be caused by heavy rainfalls overwhelming sewage treatment systems.

Selected publications about prevalence in water are summarized in the following:

  • Quantification of noroviruses in river water using RT-PCR provided concentrations ranging from 108 to 4,303 PDU/liter. The mean concentration was 1,241 NDU/liter. Concentrations quantified by qNASBA were slightly higher with a mean of 1,405 NDU/liter (27 – 5,498 NDU/liter). The concentration method of choice was filtration on negatively charged membranes, followed by ultrafiltration and RNA isolation with magnetic silica beads ( Rutjes et al. 2006) A study examining Norovirus concentrations in another moderately polluted and highly eutrophicated river in The Netherlands by RT-PCR, revealed a strong seasonality. Several peaks of varying duration and magnitude were observed in the winter with maximum concentrations of app. 240 PDU/liter in January. Samples between mid-February and beginning of October tested negative except on positive samples in May (2.5 PDU/liter) ( Westrell et al. 2006). In the following winter viral concentrations were app. 10-fold higher. The higher levels might have been caused by a significant increase of norovirus outbreaks in the watershed during this time. Heavy rainfall causing sewer overflows was reported as a possible reason. Peaks did not coincide with concentrations of enteroviruses, F-specific bacteriophages or turbidity ( Westrell et al. 2006).
  • Another study in The Netherlands examined concentration ranges for norviruses in different water samples in winter months. Enumeration was performed using RT-PCR. Estimated concentrations (in PDU per liter) were 4 - 4,900 for river water, 896 – 7,499 for treated sewage, and 5,111 – 850,000 for raw sewage. The average norovirus concentrations in two large Dutch rivers (Maas and Waal) impacted by raw and untreated sewage were 200 PDU/liter (Maas river) and 2000 PDU/liter (Waal river). The greater catchment area of rainwater in the case of the Waal river was given as a possible explanation for the higher viral numbers as more surface area might allow collecting more microbial contamination from wastewater sources. The authors pointed out that the high virus concentrations determined by PCR may partly be explained by detection of naked RNA instead of infectious particles. Comparing values before and after sewage treatment, an average removal rate of 1.8 log10 units by treatment was calculated. This number compared to average removal rates of 1.4 for enteroviruses, 1.3 for reoviruses, 1.6 for F-specific phages, 1.1 for somatic phages, and 0.2 for rotaviruses. Sequencing data indicated the presence of 10 different genotypes in the sewage and surface samples ( Lodder and de Roda Husman 2005).
  • An earlier study by the same group reported high noroviral loads in sewage samples that were taken close to the source of an outbreak. Levels as high as 107 PDU per liter were found. Sequences of RT-PCR amplicons were identical with sequences in stool samples of patients ( Lodder et al. 1999). 
  • In a study examining four French rivers monthly or semimonthly over one year, Norwalk-like viruses genogroup II were detected in 1.5% (n=68) samples using RT-PCR, whereas no Norwalk viruses genogroups I were found. The sample volume was 20 liters ( Hot et al. 2003).
  • A 14-month survey in Tokyo, Japan, detected noroviruses of genotypes I and II in 4.1 and 7.1% of tap water samples (n=98; 100 to 532 liters) ( Haramoto et al. 2004). More recently, the same group found norovirus RNA concentrations in raw sewage between 1.7-2,600 genome copies/liter (determined by qPCR) for genogroup I and 24-19,00 copies/liter for genogroup II ( Haramoto et al. 2006). Higher values were observed in winter months.
  • In a study examining three different brands of European bottled mineral water (‘groundwater’), 33% of samples (n=159) tested positive for caliciviruses in a 1-year monitoring program ( Beuret et al. 2002). Percentages of positive results were very similar for all three brands in the range between 30 and 36%. In positive samples, viral titers were estimated to be in the range between 10 to 100 viral particles per liter as determined by RT-PCR.
Caliciviruses can be highly resistant to disinfection conditions encountered in water treatment, especially to chlorine. It was reported that discharged sewage can contain high viral levels and can significantly increase viral concentrations in surface water. Lodder and de Roda Husman point out that primary and secondary sewage treatment processes do not efficiently eliminate viruses, whereas tertiary treatment reduces the concentrations ( Lodder and de Roda Husman 2005). The removal efficiency of noroviruses during sewage treatment in a study in The Netherlands varied between 0.7 and 2.1 log10 units. Another study in Sweden reported removal by 0.9 log10 units comparing inlet and outlet from secondary WWTPs ( Ottoson et al. 2006). There was not correlation between removal of pathogens and fecal indicators. More efficient removal of 2.3 and 3.7 log10 units were reported for genogroups I and II in a Japanese WWTP ( Haramoto et al. 2006). Again, bacteria were found to be unreliable indicators of norovirus concentrations in the final effluent.

Chlorine treatment: Noroviruses are not inactivated by exposure to chlorine at a concentration 3.75 mg/liter (yielding a residual free chlorine of app. 0.5 to 1 ppm) as determined by a human volunteer experiment ( Keswick et al. 1985). This chlorine concentration range is typical for residual free chlorine in distribution systems meaning normal chlorination in water supply systems might not be sufficient to inactivate noroviruses. Interestingly, an outbreak was reported that was linked to contaminated water containing 0.7 to 1 mg/liter of residual iodine ( Keswick et al. 1985). Infectivity was lost only in water with 10 mg/liter chlorine (yielding a free residual chlorine level of 5-6 mg/l). The Norwalk virus strain in this study was found to be more resistant than poliovirus type 1, human and simian rotavirus and f2 bacteriophage.  Due to the high resistance to chlorine compared with other enteric indicator viruses and the lack of an infectivity model, the effectiveness of water treatment procedures is unclear. It was suggested that routine chlorination alone is probably not reliable for norovirus inactivation ( Keswick et al. 1985). High chlorine resistance was also reported by Doultree et al. 1999 for feline caliciviruses which are considered a norovirus surrogate for which culture infectivity tests exist. Treatment with 100 ppm hypochlorite only resulted in a reduction of 1.75 log10 units. Also Duizer et al. reported that hypochlorite solutions were necessary (>300 ppm) to inactivate canine and feline caliciviruses ( Duizer et al. 2004). As these studies were, however, performed with viral suspensions containing considerable amounts of organic matter (which might have caused a loss of chlorine’s virucidal activity), chlorine sensitivity was examined with partially purified feline caliciviruses ( Urakami et al. 2007). Only 0.3 mg/liter chlorine was required for an inactivation of more than 4.6 log10 units (measured in a cell culture infectivity test) suggesting that noroviruses might not be as resistant to free chlorine as suggested earlier. Although the results obtained with feline caliciviruses might not translate entirely to noroviruses leaving room for uncertainty, the findings would correlate with the observation that a norovirus outbreak in a swimming pool (caused by a chlorination failure) could be successfully terminated by hyperchlorination ( Podewils et al. 2007). Hyperchlorination was performed by elevating the concentration to 3.5 mg/l and again adding several cups of 65% chlorine granules after half a day. It can be said for sure that the inactivation efficiency will greatly depend on the chlorine demand of the water the virus is suspended in. Other disinfectants whose efficiency does not depend to this extent on the presence of organic material, yielded mixed results ( Doultree et al. 1999).

Other disinfection methods: Glutaraldehyde (0.5%) and iodine (0.8%) solutions effectively inactivated feline caliciviruses, whereas a quaternary ammonium compound (‘Pinoclean’, diluted 1:10) failed to inactivate the virus. Ethanol (75%) and an anionic detergent yielded maximum reduction of 1.25 and 0.5 log10 units, respectively. Concentrated virus stocks were exposed to the disinfectants for 1 min before diluting out the disinfectant and harvesting the viral particles. Heating at 56°C for 1 or 3 min did not inactivate the viruses, whereas 1 hour at 56°C or 5 min at 70°C resulted in complete inactivation ( Doultree et al. 1999). Duizer et al. observed that the time for a 3 log10 unit inactivation decreased from 24 h to 8 min, when exposing feline and canine caliciviruses to 56°C compared to 37°C. At 20°C it took one week to see the same reduction in infectivity ( Duizer et al. 2004). The same 3 log inactivation was achieved by exposure to 34 mJ/cm2 UV-B radiation or to 70% ethanol for 30 min. The viruses were also inactivated at low or high pH, However, as judged by RNA stability noroviruses appeared more stable at low pH than their animal surrogates (for details see Duizer et al. 2004). Studies with human volunteers have shown that noroviruses remained infective after exposure to pH 2.7 for 18 hours or incubation at 60°C for 30 min ( AWWA 2006). A study monitoring survival of dry feline calicivirus samples on surfaces found that ozonation can reduce the concentration of infectious viruses by a factor of more than 103 with an ozone concentration of 20-25 ppm which was maintained for 20 min ( Hudson et al. 2007). Ozone also effectively reduces viral titers in water: A reduction of Norwalk virus by >3 log10 units within a contact time of 10 sec was reported with an ozone dose of 0.37 mg per liter (pH 7, 5°C) ( Shin and Sobsey 2003).

In general, low temperatures in winter months seem to be beneficial for norovirus survival as indicated by higher concentrations in water and higher number of outbreaks ( Mounts et al. 2000; Haramoto et al. 2006).

Examples of survival in different water types are given in the following:

  • In a reactor fed with tap water containing 0.15 ppm chlorine, the number of seeded canine caliciviruses (as a surrogate for human caliciviruses) in preconditioned drinking water-associated biofilms (grown under high-shear turbulent flow) decreased only 30% over a 4 week period as analyzed by PCR (Lehtola MJ 2007). In the outflowing water viral concentrations decreased more quickly but slower than the expected washout rate. These results indicate the viruses get trapped in the biofilms and are slowly released.
  • Survival of feline caliciviruses in dechlorinated tap water was examined at different temperatures. Feline caliciviruses can serve as a norovirus surrogate, their infectivity can be assayed in cell culture. D values representing the number of days needed for a 90% reduction in titer (determined in a in-vitro infectivity test) increased with decreasing storage temperatures. Mean values were 2.0 (at 37°C), 5.2 (at 25°C) and 7.3 (at 4°C). They were comparable with the onces obtained with E. coli under the same conditions using plate counts, whereas F-specific coliphages (MS2) survived better with D-values of 2.7, 18.7 and 25.7 at the three temperatures ( Allwood et al. 2003). The observation that colder temperatures prolong survival, correlate with the observation that norovirus outbreaks are more prevalent in colder months ( Mounts et al. 2000).
  • Caliciviruses in bottled drinking water were still found after 6 months in all 10 samples examined, when bottles were stored in the dark at room temperature. After 12 months, 9 of the 10 samples still tested positive. ( Beuret et al. 2002). Although infectivity after storage could not be determined, the authors considered survival of naked RNA from degraded viral particles in mineral water for this time period improbable. Infectivity could thus not be excluded ( Beuret et al. 2002).
The minimum infectious dose is estimated to be in the range between 10 to 100 viral particles ( Dreier et al. 2006; Trujillo et al. 2006). A viral dose of 10 to <104 PCR-detectable units (PDUs) led to gastrointestinal disease in two-thirds of volunteers ( Lindesmith et al. 2003). The fact that RT-PCR cannot distinguish naked RNA from RNA in infectious viruses, contributes to the difficulty of risk assessment.
Due to the lack of appropriate host cells, cell culture methods (including cell culture-PCR which is a powerful tool for determining infectivity) cannot be applied to detecting caliciviruses. Also animal models are not available for studies of infectivity impeding the study of these noroviruses. Molecular RNA-based tools are the methods of choice. Electron microscopy (EM) and immunological assays cannot be used for water samples due to limited sensitivity, but they are valuable for stool samples with higher virus concentrations ( Dreier et al. 2006; Hymas et al. 2007). The challenge for nucleic acid-based assays is that sequence heterogeneity among caliciviruses makes the design of primers and probes difficult causing conflicting results depending on the genetic target and assay design ( Hymas et al. 2007). Novel Eclipse hybridization probes that bind to minor grooves were recently published that allow the use of shorter oligonucleotides while maintaining high melting temperatures and specificity ( Hymas et al. 2007). Methodology regarding sample collection, concentration, and processing, virus detection and and confirmation and assessment of infectivity was discussed in a workshop in 2002, outcomes were summarized by Karim et al. 2004b.

Examples of molecular detection methods:

  • RT-PCR - reverse line blot hybridization has been used for detection and genotyping. A region of the highly conserved pol gene (coding for RNA polymerase) was amplified by RT-PCR and biotin-labeled amplicons were hybridized to multiple oligonucleotide genotype-specific probes. The detection limit was reported to be between 3-30 RNA-containing particles per reaction. Ninety four percent of studied strains (n=132) could be  correctly typed ( Vinjé and Koopmans 2000).
  • RT-PCR-AGE: Lodder and de Roda Husman 2005 applied a RT-PCR assay originally developed for norovirus detection in stool samples ( Vinjé and Koopmans 1996; Vinjé et al.1997) to detection in water. Primers target the pol gene. PCR amplicons were visualized on stained agarose gels. The detection limit of the assay was 10 to 100 RNA-containing particles. Specificity was confirmed using Southern hybridization ( Lodder et al. 1999). The generic primers published by Vinjé and Koopmans ( Vinjé and Koopmans 1996; Vinjé and Koopmans 2000) were modified by Vennema et al. 2002, the novel primers tested favorable when validated with stool samples. These primers together with newly developed primers were also used in a seminested assay together with an IAC for detecting norovirus genogroups I and II in different water types including seawater, estuarine water, and WWTP effluents.  The new assay produced longer products containing more phylogenetic information ( La Rosa et al. 2007).
  • Bead capture-RT-PCR: A protocol was developed using magnetic beads coated with oligonucleotides for norovirus RNA capture. The same oligonucleotide used for capture of viral RNA served as a primer for reverse transcription of the captured RNA. Different primers targeting the pol gene were used for PCR amplification. Amplification products were visualized on a polyacrylamide gel. Alternatively, amplicons were dotted onto a nylon membrane and hybridized with different probes. The bead capture increased detection sensitivity compared to classic RT-PCR without bead capture when validating the methods with sewage samples. The binding capacity of beads was found to be the most important parameter for sensitivity. Moreover, inhibitors were efficiently removed by bead capture ( Loisy et al. 2000).
  • qRT-PCR targeting the conserved ORF1-ORF2 junction. The TaqMan assay presented by Kageyama et al. can distinguish between genogroups I and II. The degenerate primers target the ORF1-ORF2 region. Amplification was performed on a ABI Prism 7700 sequence detector. The detection limit was reported to be 10 copies of cDNA per reaction. The dynamic range was from 10 to 107 cDNA copies ( Kageyama et al. 2003). The assay with some modifications (different RNA extraction kit and reverse transcription using random hexamers instead of specific primers) was applied in a study examining noroviruses in tap water in Japan on a ABI Prism 7200 platform ( Haramoto et al. 2004). This TaqMan assay was further optimized, expanded and validated with clinical and water samples by Trujillo et al. on a LightCycler platform. Detection limits were estimated to be <10 RNA copies per reaction for the genogroup II assay and < 100 copies for the genogroup I assay. In addition primers for genotype IV were presented also targeting the ORF1-ORF2 junction. The assay succeeded in detecting norovirus RNA in filtered potable water from a cruise ship whereas conventional RT-PCR failed (Trujillo et al. 2006). Another recent qRT-PCR assay by Wolf et al. allowed the simultaneous detection and differentiation of genogroups I, II, and III (Wolf et al. 2007). Genogroup III comprises bovine noroviruses. The multiplex assay is based on three reasonably degenerate primer-probe pairs also targeting the ORF1-ORF2 region. It could detect less than 10 plasmid copies with target cRNA sequences for all three genogroups. A qRT-PCR assay presented by Logan et al. could detect norovirus genogroup IV in addition to I and II in independent reactions (the primers targeted the pol gene). Moreover, primers and probes were presented for detection of sapoviruses (genogroups GI, II, and IV) targeting the gene coding for a polyprotein. Reliable detection of 100 cRNA copies per reaction was achieved for each genogroups of norovirus and sapovirus (Logan et al. 2007).
  • Eclipse qRT-PCR assay: A one step quantitative RT-PCR assay was presented with very good correlation to the alternative TaqMan RT-PCR assay ( Trujillo et al. 2006). The use of Eclipse probes containing a minor groove binder and structurally modified bases allowed the design of shorter probes. As Eclipse probes are not hydrolyzed during the reaction, they remain available for melting curve analysis. An internal RNA control was incorporated to monitor RNA extraction and amplification inhibition. Two different primer and probe sets allowed the selective detection and typing of genogroups I and II (targeting the ORF1-ORF2 region) with detection limits of app. 50 copies per reaction ( Hymas et al. 2007).
  • Real-time NASBA: targeting RdRp gene coding for the RNA-dependent RNA polymerase. Viruses from surface waters were concentrated by filtration on a negatively charged membrane, followed by two-phase separation or ultrafiltration. NASBA was shown to be more sensitive and less susceptible to inhibition than RT-PCR ( Rutjes et al. 2006).
  • One step qRT-LAMP: A loop-mediated isothermal amplification assay was developed for detection of norovirues and tested with clinical specimens. The specificity of LAMP-based assays is very high as six primers are used which recognize eight distinct regions of the targeted sequence. Primer sets were developed for genogroups I and II targeting the ORF1-ORF2 region. Very good correlation was found with the qRT-PCR TaqMan assay published by Kageyama et al. 2003. Moreover, the sensitivity of the assay was reported to be equal or better than that of a commercial LAMP end-point detection kit as tested for multiple norovirus genotypes (Yoda et al. 2007). The commercial test was based on qRT-LAMP assay published by Fukuda et al. 2006. This group presented recently another qRT-LAMP assay allowing the simultaneous detection of norovirus genogroups I and II in a single tube. Primers targeted the pol gene region. The test was based on the previous, but included some changes in the reaction mixture and three new primers. The detection limits were reported to be in the range between 102 to 104 copies per tube depending on the genotype ( Fukuda et al. 2007). The genogroup-specific assay ( Fukuda et al. 2006) was about one log10 unit more sensitive.
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.


Figure 1:

                                         Caliciviruess_photo 1                                               

This transmission electron micrograph (TEM) revealed some of the ultrastructural morphology displayed by norovirus virions, or virus particles.

Source: http://phil.cdc.gov/phil/details.asp
Photo ID: 10708
Content provider(s): Centers for Disease Control and Prevention/ Charles D. Humphrey

Figure 2:

                                         Caliciviruses_photo 2                                                 

This transmission electron micrograph (TEM) revealed some of the ultrastructural morphology displayed by norovirus virions, or virus particles.

Source: http://www.cdc.gov/media/subtopic/library/diseases.htm
Photo ID: 10709
Content provider(s): Centers for Disease Control and Prevention/ Charles D. Humphrey

Figure 3:

                                          Caliciviruses_photo 3                                                  

This electron micrograph reveals the morphologic traits exhibited by the feline calicivirus (FCV), a Caliciviridae family member.

Source: http://phil.cdc.gov/phil/details.asp
Photo ID: 198
Content provider(s): Centers for Disease Control and Prevention/ Dr. Erskine Palmer 

Will pull out specific links from main web links section.Links to useful external websites are provided in the following.

Center for Disease Control:

Online Biology Degree:


Last Updated on Wednesday, 05 December 2012 11:15


Hepatitis A
Hepatitis E


Copyright © 2017 Waterborne Pathogens. All Rights Reserved. Powered by SuSanA