Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log₁₀ reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log₁₀ gc/liter; 95% credible interval [CI], 3.5, 4.3 log₁₀ gc/liter) is larger than the value for NoV GI (1.5 log₁₀ gc/liter; 95% CI, 0.4, 2.4 log₁₀ gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log₁₀ reductions were −2.4 log₁₀ gc/liter (95% CI, −3.9, −1.1 log₁₀ gc/liter) for NoV GI, −2.7 log₁₀ gc/liter (95% CI, −3.6, −1.9 log₁₀ gc/liter) for NoV GII, and −2.9 log₁₀ PFU per liter (95% CI, −3.4, −2.4 log₁₀ PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were −1.4 log₁₀ gc/liter (95% CI, −3.3, 0.5 log₁₀ gc/liter) for NoV GI, −1.7 log₁₀ gc/liter (95% CI, −3.1, −0.3 log₁₀ gc/liter) for NoV GII, and −3.6 log₁₀ PFU per liter (95% CI, −4.8, −2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log₁₀ reduction in NoV GII and the mean log₁₀ reduction in MSCs.     

Human norovirus occurrence and diversity in the Llobregat river catchment, Spain

Human noroviruses (NoV) were quantified and characterized in an 18 month survey conducted along the Llobregat river catchment in Spain. Sample types included freshwater, untreated and treated wastewater and drinking water. High NoV genome copy numbers were reported, reaching up to 10(6) l(-1) and 10(9) l(-1) in freshwater and raw sewage respectively. In both types of samples, GII NoV genome copies outnumbered those of GI, although without significance. All samples of semi-treated and treated drinking water were negative for NoV. A clear seasonality of NoV occurrence was observed both in river water and sewage samples, with significantly higher genome copy numbers in the cold than in the warm months period. Mean NoV log reduction rates after biological treatment of sewage were 2.2 and 3.1 for GI and GII respectively. A total of 77 NoV strains isolated in the Llobregat river catchment could be phylogenetically characterized, 44 belonging to GI and 33 to GII. The most prevalent genotype was GI.4, followed by GII.4 and GII.21. Several variants of the pandemic GII.4 strain were detected in the environment, corroborating their circulation among the population.     

Quantification and Genotyping of Torque Teno Virus at a Wastewater Treatment Plant in Japan

Torque teno virus (TTV) DNA was quantitatively detected in influent and effluent samples collected from a wastewater treatment plant in Japan, with the highest concentration being 4.8 × 10⁴ copies/liter. Genogroup-specific nested PCR demonstrated that TTV of genogroup 3 was the most abundant in wastewater among the five genogroups tested.     

Deciphering the Diversities of Astroviruses and Noroviruses in Wastewater Treatment Plant Effluents by a High-Throughput Sequencing Method

Although clinical epidemiology lists human enteric viruses to be among the primary causes of acute gastroenteritis in the human population, their circulation in the environment remains poorly investigated. These viruses are excreted by the human population into sewers and may be released into rivers through the effluents of wastewater treatment plants (WWTPs). In order to evaluate the viral diversity and loads in WWTP effluents of the Paris, France, urban area, which includes about 9 million inhabitants (approximately 15% of the French population), the seasonal occurrence of astroviruses and noroviruses in 100 WWTP effluent samples was investigated over 1 year. The coupling of these measurements with a high-throughput sequencing approach allowed the specific estimation of the diversity of human astroviruses (human astrovirus genotype 1 [HAstV-1], HAstV-2, HAstV-5, and HAstV-6), 7 genotypes of noroviruses (NoVs) of genogroup I (NoV GI.1 to NoV GI.6 and NoV GI.8), and 16 genotypes of NoVs of genogroup II (NoV GII.1 to NoV GII.7, NoV GII.9, NoV GII.12 to NoV GII.17, NoV GII.20, and NoV GII.21) in effluent samples. Comparison of the viral diversity in WWTP effluents to the viral diversity found by analysis of clinical data obtained throughout France underlined the consistency between the identified genotypes. However, some genotypes were locally present in effluents and were not found in the analysis of the clinical data. These findings could highlight an underestimation of the diversity of enteric viruses circulating in the human population. Consequently, analysis of WWTP effluents could allow the exploration of viral diversity not only in environmental waters but also in a human population linked to a sewerage network in order to better comprehend viral epidemiology and to forecast seasonal outbreaks.     

Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability

Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal αfucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical αfucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.     

Detection of genogroup IV norovirus in wastewater and river water in Japan

Aims:  To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV).
Methods and Results:  Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption–elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6·9 × 10⁴ and 1·5 × 10⁴ copies l⁻¹, respectively.
Conclusions:  Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks.
Significance and Impact of the Study:  This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.     

Vomiting as a Symptom and Transmission Risk in Norovirus Illness: Evidence from Human Challenge Studies

BackgroundIn the US, noroviruses are estimated to cause 21 million cases annually with economic losses reaching $2 billion. Outbreak investigations frequently implicate vomiting as a major transmission risk. However, little is known about the characteristics of vomiting as a symptom or the amount of virus present in emesis.

Methodology and Principal Findings

Emesis samples and symptomology data were obtained from previous norovirus human challenge studies with GI.1 Norwalk virus, GII.2 Snow Mountain virus, and a pilot study with GII.1 Hawaii virus. Viral titers in emesis were determined using strain-specific quantitative RT-PCR. In all four studies, vomiting was common with 40–100% of infected subjects vomiting at least once. However, only 45% of subjects with vomiting also had diarrhea. Most of the emesis samples had detectable virus and the mean viral titers were 8.0 x 10⁵ and 3.9 x 10⁴ genomic equivalent copies (GEC)/ml for GI and GII viruses, respectively (p = 0.02). Sample pH was correlated with GII.2 Snow Mountain virus detection.

Conclusions and Significance

Half of all subjects with symptomatic infection experienced vomiting and the average subject shed 1.7 x 10⁸ GEC in emesis. Unlike shedding through stool, vomiting is more likely to result in significant environmental contamination, leading to transmission through fomites and airborne droplets. This quantitative data will be critical for risk assessment studies to further understand norovirus transmission and develop effective control measures. The correlation between sample pH and virus detection is consistent with a single site of virus replication in the small intestine and stomach contents becoming contaminated by intestinal reflux. Additionally, the frequency of vomiting without concurrent diarrhea suggests that epidemiology studies that enroll subjects based on the presence of diarrhea may be significantly underestimating the true burden of norovirus disease.     

Characterization of norovirus infections in Seoul,Korea

The present study has determined the detection rate of norovirus (NoV) with acute gastroenteritis (AGE) in hospitalized children and describes the molecular epidemiology of NoV circulating in Seoul, Korea. Six hundred and eighty‐three (9.8%) of samples were positive for NoV. Of these, the NoV GII genogroup was the most commonly found, with a prevalence of 96.2% (683 of 710). Only 27 samples were positive for the NoV GI genogroup. Ten kinds of GI genotype (GI/1, GI/2, GI/3, GI/4, GI/5, GI/6, GI/7, GI/9, GI/12, and GI/13) and eight kinds of GII genotype (GII/2, GII/3, GII/4, GII/8, GII/14, GII/15, GII/16, and GII/17) were identified in children with AGE during the years 2008–2011.     

Environmental surveillance and molecular characterization of human enteric viruses in tropical urban wastewaters

Aims: To study the prevalence and genotypes of waterborne pathogenic viruses in urban wastewaters in the tropical region. Methods and Results: Viruses in wastewaters collected at three water reclamation plants in Singapore were studied by molecular methods. Over a 6‐month sampling period, adenoviruses, astroviruses and both norovirus genogroups I (GI) and II (GII) were detected in 100% of the sewage and secondary effluent. Enteroviruses and hepatitis A viruses (HAV) were found in 94 and 78% of sewage, and 89 and 28% of secondary effluent, respectively. By using quantitative real‐time PCR, estimated concentrations of astrovirus in the sewage were 1–2 orders of magnitude higher than those for adenovirus, noroviruses GI and GII. Genotyping of environmental isolates revealed multiple genotypes of GI and GII noroviruses. Coxsackieviruses A, astrovirus type 1 and adenovirus type 41 were prevalent. Norovirus GII/4 and coxsackievirus A24 isolates in wastewaters were closely related to respective outbreak strains isolated previously in Singapore. Conclusions: This study showed the widespread occurrence of all tested enteric virus groups in urban wastewaters. Genetic diversity of astroviruses, enteroviruses and noroviruses in the tropical region was observed. Significance and Impact of the Study: The high prevalence and great genetic diversity of human enteric viruses in urban wastewaters strongly supports the need of further comprehensive studies for evaluating the public health risk associated with viral pathogens in water environments.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Molecular epidemiology of norovirus in asymptomatic food handlers in Busan,Korea, and emergence of genotype GII.17

The molecular epidemiology of norovirus infections was studied in food handlers without any symptoms from January to December 2015 in Busan city, Korea. A total of 2,174 fecal specimens from asymptomatic food handlers were analyzed, and 2.3% (49/2,174) were norovirus-positive. Fourteen of 335 samples (4.2%) were positive in January; fifteen of 299 samples (5.0%) in February, and seven of 189 samples (3.7%) in December. However, norovirus was rarely detected in other months. From sequencing analysis, 11 genotypes (five GI and six GII genotypes) were detected. Among the 42 capid gene sequences identified, 14 were from the GI genogroup, while 28 were from the GII genogroup. The most commonly detected genotype was GII.17, comprising 15 (35.7%) of positive samples. From January 2012 to December 2015, 5,138 samples were collected from gastroenteritis patients and outbreaks in Busan. The most detected genotype in 2012, 2013, and 2014 was GII.4 (121, 24, and 12 cases, respectively), but in 2015, GII.17 (25 cases) was the most common. The GII.4 genotype was the major cause of acute gastroenteritis from 2012 to 2014, but the GII.17 genotype became the most prevalent cause in 2015. Continued epidemiological surveillance of GII.17 is needed, together with assessment of the risk of norovirus infection.