Detection and Molecular Characterization of Gemycircularvirus from Environmental Samples in Brazil

Gemycircularvirus (GemyCV) is a group of viruses which has been recently proposed as a new viral genus detected in fecal and environmental samples around the world. GemyCVs have been detected in human blood, brain tissue, cerebrospinal fluid, and stool sample. In the present study, we demonstrate for the first time, through molecular detection and characterization, the presence of GemyCVs in environmental samples from Brazil. Our results show a percentage of positivity ranging from 69 (25/36) to 97 % (35/36) in river water samples collected in Manaus, Amazon region, and wastewater from a wastewater treatment plant located in Rio de Janeiro, respectively, revealing GemyCVs as an important environmental contaminant.     

GIV Noroviruses in Wastewaters and in Stool Specimens from Hospitalized Patients

Noroviruses (NoVs) are important human pathogens associated with foodborne and waterborne gastroenteritis. These viruses are genetically highly heterogeneous, with more than forty genotypes within three genogroups (GI, GII, and GIV) identified in humans. However, the vast majority of human infections are associated with variants of a unique genotype, GII.4. Aside from these NoV strains of epidemiological relevance, NoV strains of genogroup GIV (Alphatron-like) are reported in a sporadic fashion and their overall prevalence in the community is unknown and this likely reflects the lack of specific diagnostic tools. We analyzed raw sewages collected from 32 wastewater treatment plants distributed throughout Italy (307 samples) and stool specimens collected from hospitalized patients with clinical signs of diarrhea of unknown etiology (285 samples). By using specific qualitative and quantitative RT-PCR assays, 21.8 % of the sewage samples and 3.2 % of the stool specimens tested positive for GIV NoVs. The number of genome copies in fecal samples ranged from 5.08 × 10⁴ to 1.73× 10⁶/g of feces. Sequence analysis showed limited genetic variability in human GIV viruses. The presence of GIV NoV both in sewage and in clinical samples confirms that not only GI and GII NoVs but also GIV strains are circulating in humans. Monitoring of GIV NoV is recommended in order to understand the dynamics of circulation in human populations, environmental contamination, and potential health risks.     

Distribution of Naturally Occurring Norovirus Genogroups I,II, and IV in Oyster Tissues

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10² RNA copies/g of digestive tissues, 2.62 × 10² RNA copies/g of gills, and 1.61 × 10³ RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.     

Occurrence of Norovirus GIV in Environmental Water Samples from Belém City,Amazon Region,Brazil

Noroviruses are the major cause of non-bacterial acute gastroenteritis outbreaks in humans, with few reports about the occurrence of the norovirus GIV strain. We investigated the presence of norovirus GIV in surface water (river, bay, and stream) and untreated sewage, and we determined a positivity rate of 9.4 % (9/96). The strains genotyped were GIV.1. To our knowledge, this is the first report of GIV in Brazil.     

Oyster Contamination with Human Noroviruses Impacted by Urban Drainage and Seasonal Flooding in Vietnam

This study investigated the level of norovirus contamination in oysters collected at a lagoon receiving urban drainage from Hue City for 17 months (August 2015–December 2016). We also investigated the genetic diversity of norovirus GI and GII in oyster and wastewater samples by using pyrosequencing to evaluate the effect of urban drainage on norovirus contamination of oysters. A total of 34 oyster samples were collected at two sampling sites (stations A and B) in a lagoon. Norovirus GI was more frequently detected than GII (positive rate 79 vs. 41%). Maximum concentrations of GI and GII were 2.4 × 10⁵ and 2.3 × 10⁴ copies/g, respectively. Co-contamination with GI and GII was observed in 35% of samples. Norovirus GII concentration was higher at station A in the flood season than in the dry season (P = 0.04, Wilcoxon signed-rank test). Six genotypes (GI.2, GI.3, GI.5, GII.2, GII.3, and GII.4) were identified in both wastewater and oyster samples, and genetically similar or identical sequences were obtained from the two types of samples. These observations suggest that urban drainage and seasonal flooding contribute to norovirus contamination of oysters in the study area.     

Eco-industrial park development in Rio de Janeiro,Brazil: a tool for sustainable development

The development of eco-industrial parks (EIPs) is an emerging concept that is being spread worldwide as a new industrial model that can reconcile the three dimensions of sustainability: social, economic and environmental. In Rio de Janeiro, Brazil, EIPs were launched through formal legislation as a means to foster sustainable development and to ameliorate the distress caused by unplanned urban and industrial development. The objective of this study is to present and analyze the development of EIPs in Rio de Janeiro, focusing on two of the initiatives launched. We argue that Rio de Janeiro has the potential for developing a sustainable industrial system through EIP implementation, but the continuity of EIPs will only be successful if there is convergence of interests among the actors involved.     

Human Enteric Viruses Occurrence in Shellfish from European Markets

Different sources were consulted to obtain information on the occurrence of viruses in bivalve molluscs on the European market. Twenty-six peer-reviewed articles were identified reporting on the molecular detection of viral RNA in 4,260 samples in total. The data obtained will be presented geographically on virus types detected, the origin and treatment of the shellfish, and the detection technique applied. The data demonstrate that viral RNA can be detected in shellfish from polluted areas, in depurated shellfish as well as those for human consumption. The European Rapid Alert System for Food and Feed (RASFF) database was consulted as another source. Twenty-eight notifications were identified on the presence of hepatitis A virus or norovirus in shellfish on the European market. The most recent report of the European laboratory network was referred to, to gain insight into the laboratory capability at present for the analyses of shellfish on the presence of viruses. Approximately 67% of 27 participating laboratories obtained intended results for all samples, consisting of lenticules loaded with 10³ copies norovirus (genogroup I (GGI) and/or genogroup II (GGII)) and/or 1 × 10⁵–8 × 10⁴ copies of hepatitis A virus. From 1993, there has been a continuous development of molecular detection techniques and tools have been described to ensure quality assurance. End product testing will, however, not be achievable. As depuration has been shown not to be effective for the complete elimination of viruses, shellfish should not be in contact with faecal contaminated water in order to minimise the risk of shellfish-transmittable viral diseases.     

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP’s outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75 % and AsV in 67 %. HAV were not detected in wastewater. Overall, 92 % of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92 % of samples and at concentrations up to 7.7 × 10³ genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66 % of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.     

Environmental Surveillance of SARS-CoV-2 RNA in Wastewater and Groundwater in Quintana Roo,Mexico

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 10³ to 7.5 × 10³ genome copies per liter (GC l⁻¹), using the N1 RT-qPCR assay, and from 2.4 × 10² to 5.9 × 10³ GC l⁻¹ using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area.

     

Effects of Bacterial, Chemical, Physical and Meteorological Variables on Virus Removal by a Wastewater Treatment Plant

The purpose of wastewater treatment is to minimize chemical and microbial contamination of recipient waters. The present study evaluated the impacts of meteorological variables, such as temperature and rainfall, on the removal of human viruses and indicators by a wastewater treatment plant servicing Pisa, Italy. Data were obtained during four sampling campaigns from 2007 to 2010. Wastewater sewage samples were analyzed for human adenovirus (HAdV) and norovirus using quantitative molecular techniques. In parallel, Escherichia coli, enterococci and somatic coliphages were measured, and meteorological and chemical data were recorded. We detected a continuous presence of HAdV in both influent and effluent samples with an average removal rate of 2.01 log₁₀ Genomic Copies/l. An association between meteorological parameters and viral removal rates was detected only for rainfall and HAdV removal during a specific sampling campaign. No correlation was found between viral data and microbial, chemical and physical ones. Viral removal rates were not strongly influenced by meteorological conditions and were unrelated to other process indicators routinely monitored. Our results suggest that HAdV is a suitable parameter to assess the viral removal efficiency of wastewater treatment plants, particularly in the case of heavy rainfall.     

Surveillance of Enteric Viruses and Thermotolerant Coliforms in Surface Water and Bivalves from a Mangrove Estuary in Southeastern Brazil

This study was conducted to evaluate the microbiological quality of a mangrove estuary in the Vitória Bay region, Espírito Santo, Brazil. We analyzed the presence and concentration of enteric viruses and thermotolerant coliforms in water, mussels (Mytella charruana and Mytella guyanensis), and oysters (Crassostrea rhizophorae), collected over a 13-month period. Human adenovirus, rotavirus A (RVA), and norovirus genogroup II were analyzed by quantitative PCR. The highest viral load was found in RVA-positive samples with a concentration of 3.0 × 10⁴ genome copies (GC) L⁻¹ in water samples and 1.3 × 10⁵ GC g⁻¹ in bivalves. RVA was the most prevalent virus in all matrices. Thermotolerant coliforms were quantified as colony-forming units (CFU) by the membrane filtration method. The concentration of these bacteria in water was in accordance with the Brazilian standard for recreational waters (< 250 CFU 100 mL⁻¹) during most of the monitoring period (12 out of 13 months). However, thermotolerant coliform concentrations of 3.0, 3.1, and 2.6 log CFU 100 g⁻¹ were detected in M. charruana, M. guyanensis, and C. rhizophorae, respectively. The presence of human-specific viruses in water and bivalves reflects the strong anthropogenic impact on the mangrove and serves as an early warning of waterborne and foodborne disease outbreaks resulting from the consumption of shellfish and the practice of water recreational activities in the region.

     

A One Year Study on the Concentrations of Norovirus and Enteric Adenoviruses in Wastewater and A Surface Drinking Water Source in Norway

Enteric viruses transmitted via the faecal-oral route occur in high concentrations in wastewater and may contaminate drinking water sources and cause disease. In order to quantify enteric adenovirus and norovirus genotypes I and II (GI and GII) impacting a drinking source in Norway, samples of surface water (52), wastewater inlet (64) and outlet (59) were collected between January 2011 and April 2012. Samples were concentrated in two steps, using an electropositive disc filter and polyethylene glycol precipitation, followed by nucleic acid extraction and analysis by quantitative polymerase chain reaction. Virus was detected in 47/52 (90.4 %) of surface water, 59/64 (92 %) of wastewater inlet and 55/59 (93 %) of wastewater outlet samples. Norovirus GI occurred in the highest concentrations in surface water (2.51e + 04) and adenovirus in wastewater (2.15e + 07). While adenovirus was the most frequently detected in all matrices, norovirus GI was more frequently detected in surface water and norovirus GII in wastewater. This study is the first in Norway to monitor both sewage and a drinking water source in parallel, and confirms the year-round presence of norovirus and adenovirus in a Norwegian drinking water source.     

Environmental Surveillance of Polioviruses in Rio de Janeiro,Brazil, in Support to the Activities of Global Polio Eradication Initiative

Wild polioviruses still remain endemic in three countries (Afghanistan, Pakistan, and Nigeria) and re-emergency of wild polio has been reported in previously polio-free countries. Environmental surveillance has been used as a supplementary tool in monitoring the circulation of wild poliovirus (PVs) and/or vaccine-derived PVs even in the absence of acute flaccid paralysis cases. This study aimed to monitor the presence of polioviruses in wastewater samples collected at one wastewater treatment plant located in the municipality of Rio de Janeiro, Brazil. From December 2011 to June 2012 and from September to December 2012, 31 samples were collected and processed. RD and L20B cell cultures were able to isolate PVs and non-polio enteroviruses in 27/31 samples. Polioviruses were isolated in eight samples (type 1 Sabin = 1, type 2 Sabin = 5, and type 3 Sabin = 2). Vaccine-derived polioviruses were not detected nor evidence of recombination with other PVs or non-polio enterovirus serotypes were observed among the isolates. The Sabin-related serotypes 2 and 3 presented nucleotide substitutions in positions associated with the neurovirulent phenotype at the 5′-UTR. Changes in important Amino acid residues at VP1 were also observed in the serotypes 2 and 3. Environmental surveillance has been used successfully in monitoring the circulation of PVs and non-polio enteroviruses and it is of crucial importance in the final stages of the WHO global polio eradication initiative. Our results show the continuous circulation of Sabin-like PVs and non-polio enteroviruses in the analyzed area during the study period.     

Detection and Characterization of Hepatitis A Virus and Norovirus in Mussels from Galicia (NW Spain)

Shellfish are recognized as a potential vehicle of viral disease and despite the control measures for shellfish safety there is periodic emergence of viral outbreaks associated with shellfish consumption. In this study a total of 81 mussel samples from Ría do Burgo, A Coruña (NW Spain) were analysed. Samples were collected in seven different harvesting areas with the aim to establish a correlation between the prevalence of norovirus (NoV) and hepatitis A virus (HAV) in mussel samples and the water quality. In addition, the genogroup of the detected HAV and NoV strains was also determined. The HAV presence was detected in 18.5 % of the samples. Contamination levels for this virus ranged from 1.1 × 10² to 4.1 × 10⁶ RNA copies/g digestive tissue. NoV were detected in 49.4 % of the cases reaching contamination levels from 5.9 × 10³ to 1.6 × 10⁹ RNA copies/g digestive tissue for NoV GI and from 6.1 × 10³ to 5.4 × 10⁶ RNA copies/g digestive tissue for NoV GII. The χ²-test showed no statistical correlation between the number of positive samples and the classification of molluscan harvesting area based on the E. coli number. All the detected HAV strains belong to genogroup IB. NoV strains were assigned to genotype I.4, II.4 and II.6.     

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

城市污水中人类星状病毒的检测及变化规律

人类星状病毒是典型的水媒病毒,可引起腹泻等疾病. 为了明确污水中人类星状病毒的分布及去除规律,利用常规PCR和定量PCR方法对西安市3个污水处理厂的进水和出水进行了为期1 a的监测;采用特异性引物,确立了人类星状病毒在环境水体中的定性PCR和荧光定量PCR的检测方法. 结果表明,人类星状病毒在进水和未消毒二级出水中呈较高的阳性率,分别为91.5%和36.6%. 经过核苷酸测序鉴定,人类星状病毒的主要血清型为HAst V-1和HAst V-4. 人类星状病毒在进水和未消毒二级出水中的平均浓度分别为1.47×104和1.03×103 copies/mL,污水处理厂对人类星状病毒的去除率为93.15%. 污水处理厂进水中人类星状病毒浓度呈季节性变化,春季和冬季较高,夏季和秋季较低,这一分布规律与该地区历年人类星状病毒的流行规律相似.