Rotavirus Surveillance in Tap Water,Recycled Water,and Sewage Sludge in Thailand: A Longitudinal Study, 2007–2018

The objective of this study was to describe the epidemiological and molecular surveillance of rotaviruses in tap water, recycled water, and sewage sludge in Thailand from 2007 to 2018. Three hundred and seventy tap water, 202 recycled water, and 72 sewage sludge samples were collected and processed to detect the rotavirus VP7 gene using RT-nested PCR. Rotavirus G genotypes were identified by DNA sequencing and phylogenetic analysis. The frequency of rotavirus detection was 0.54% of the tap water samples, 30.2% of the recycled water samples, and 50.0% of the sewage sludge samples. During the 12-year surveillance, G1 was prevalent most years and constantly predominant in recycled water and sewage sludge. G2 was identified in a tap water sample and in recycled water samples. G3 and G9 were observed in both recycled water and sewage sludge samples. The uncommon G6 rotavirus strain was identified in one recycled water sample. The rotavirus VP4 gene was detected in rotavirus strains with an identified G genotype using RT-multiplex nested PCR. The unusual P[6] genotype was the most frequently detected, followed by mixed P[6]/[4] and P[4] genotypes. Phylogenetic analysis of both G and P genotypes showed a close genetic relationship with sequences of human rotavirus strains. The high nucleotide identity of the rotavirus strains found in this study to human rotavirus strains suggests that the rotaviruses are derived from human source. These results represent useful epidemiological and molecular information for evaluating rotavirus distribution in water for consumption and irrigation, and in biosolids for agricultural application.

     

Prevalence of Rotaviruses Groups A and C in Egyptian Children and Aquatic Environment

The objective of this study is to compare the prevalence of rotaviruses groups A and C in Egyptian children and aquatic environment. From 110 stool specimens of children with acute diarrhea and using RT-PCR, 35 samples (31.8 %) were positive for human rotavirus group A and 15 samples (13.6 %) were positive for human rotavirus group C. From 96 samples collected from Zenin wastewater treatment plant over a 2-year period (November 2009–October 2011) and using RT-PCR, rotavirus group A was detected in (4/24) 16.7 %, (5/24) 20.8 %, (4/24) 16.7 %, and (4/24) 16.7 %, while rotavirus group C was detected in (2/24) 8.3 %, (3/24) 12.5 %, (3/24) 12.5 %, and (0/24) 0 % in raw sewage, after primary sedimentation, after secondary sedimentation, and after final chlorination, respectively. Moreover, from 96 samples collected from El-Giza water treatment plant over a 2-year period (November 2009–October 2011), rotavirus group A was detected in (7/24) 29.2 %, (6/24) 25 %, (5/24) 20.8 %, and (3/24) 12.5 %, while rotavirus group C was detected in (3/24) 12.5 %, (1/24) 4.2 %, (1/24) 4.2 %, and (0/24) 0 % in raw Nile water, after sedimentation, after sand filtration, and after final chlorination, respectively. Using SYBR Green real-time RT-PCR, the number of human rotavirus group A genome or infectious units was higher than rotavirus group C. VP6 sequence analysis of the RT-PCR positive rotavirus group C samples revealed that four clinical specimens and three environmental samples showed similar sequences clustered with Moduganari/Human Nigerian strain AF 325806 with 98 % homology, and two clinical specimens and one environmental sample showed similar sequences clustered with Dhaka CB/Human Bangladesh strain AY 754826 with 97 % homology.     

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.     

Assessment of Gastroenteric Viruses from Wastewater Directly Discharged into Uruguay River,Uruguay

The aim of this study was to assess the viral contamination of group A rotavirus (RVA), norovirus (NoV), and human astrovirus (HAstV) in sewage directly discharged into Uruguay River and to characterize RVA genotypes circulating in Uruguay. For this purpose, sewage samples (n = 96) were collected biweekly from March 2011 to February 2012 in four Uruguayan cities: Bella Unión, Salto, Paysandú, and Fray Bentos. Each sample was concentrated by ultracentrifugation method. Qualitative and quantitative RT-PCR for RVA, NoV, and HAstV were performed. A wide dissemination of gastroenteric viruses was observed in the sewage samples analyzed with 80 % of positivity, being NoV (51 %) the most frequently detected followed by RVA with a frequency of 49 % and HAstV with 45 %. Genotypes of RVA were typed using multiplex semi-nested RT-PCR as follows: P[8] (n = 15), P[4] (n = 8), P[10] (n = 1), P[11] (n = 1), G2 (n = 29), and G3 (n = 2). The viral load ranged from 10³ to 10⁷ genomic copies/liter, and they were detected roughly with the same frequency in all participant cities. A peak of RVA and HAstV detection was observed in colder months (June to September), whereas no seasonality was observed for NoV. This study demonstrates for the first time, the high degree of gastroenteric viral contamination in the country; highlighting the importance of developing these analyses as a tool to determine the viral contamination in this hydrographic boundary region used by the local populations for recreation and consumption, establishing an elevated risk of gastroenteric diseases for human health.     

江苏省某市部分农村河道水体和污水处理厂主要工艺环节病毒污染状况调查

为研究江苏省某市农村河道水体和污水处理厂主要工艺环节病毒的污染状况,分析常见理化因素与病毒污染之间的相关性,对江苏省某市部分农村河道水体和污水处理厂主要工艺环节7个水样、1个污泥和2个沉积物样点进行为期9个月的病毒(包括诺如病毒G Ⅰ、诺如病毒G Ⅱ、轮状病毒、札如病毒、腺病毒、星状病毒、肠道病毒)污染状况监测采样(共90份样本),通过阴离子膜吸附-洗脱法进行富集,荧光定量RT-PCR检测法对病毒进行检测,分析病毒污染状况;同时,监测水温、pH、COD(化学耗氧量)、TP(总磷)、TN(总氮)、EC(电导率)和DO(溶解氧)等7个理化指标的变化情况,探讨这些常见理化因素与病毒污染之间的相关性. 结果表明:90份样本中,诺如病毒G Ⅱ检出率为36.67%,肠道病毒检出率为30%,其他病毒检出率均低于诺如病毒G Ⅱ和肠道病毒. 污水处理后出水中有诺如病毒G Ⅱ、肠道病毒和轮状病毒检出,相关性分析显示,水温与诺如病毒G Ⅱ检出率呈显著负相关(P < 0.05). 研究显示,江苏省某市农村河道水体和污水处理厂主要工艺环节存在病毒污染,诺如病毒G Ⅱ为优势毒株;目前的污水消毒处理工艺并不能完全去除病毒,人群在接触外环境水体时,有潜在病毒暴露风险.      

Frequent Detection and Genetic Diversity of Human Bocavirus in Urban Sewage Samples

The prevalence and genetic diversity of human bocaviruses (HBoVs) in sewage water samples are largely unknown. In this study, 134 raw sewage samples from 25 wastewater treatment plants (WTPs) in Italy were analyzed by nested PCR and sequencing using species-specific primer pairs and broad-range primer pairs targeting the capsid proteins VP1/VP2. A large number of samples (106, 79.1 %) were positive for HBoV. Out of these, 49 were classified as HBoV species 2, and 27 as species 3. For the remaining 30 samples, sequencing results showed mixed electropherograms. By cloning PCR amplicons and sequencing, we confirmed the copresence of species 2 and 3 in 29 samples and species 2 and 4 in only one sample. A real-time PCR assay was also performed, using a newly designed TaqMan assay, for quantification of HBoVs in sewage water samples. Viral load quantification ranged from 5.51E+03 to 1.84E+05 GC/L (mean value 4.70E+04 GC/L) for bocavirus 2 and from 1.89E+03 to 1.02E+05 GC/L (mean value 2.27E+04 GC/L) for bocavirus 3. The wide distribution of HBoV in sewages suggests that this virus is common in the population, and the most prevalent are the species 2 and 3. HBoV-4 was also found, representing the first detection of this species in Italy. Although there is no indication of waterborne transmission for HBoV, the significant presence in sewage waters suggests that HBoV may spread to other water environments, and therefore, a potential role of water in the HBoV transmission should not be neglected.     

Molecular Detection of human Noroviruses in Influent and Effluent Samples From Two Biological Sewage Treatment Plants in the Region of Monastir,Tunisia

Noroviruses (NoVs) are responsible for numerous cases of waterborne and foodborne gastroenteritis every year. They are released in the sewage and their detection in this environment can reflect the epidemiology of the viral strains circulating in the community. A three-year (2007–2010) survey was conducted in order to evaluate the presence of human NoVs using RT-PCR in 518 sewage samples collected at the entrance and exit of two biological sewage treatment plants located in Monastir region, Tunisia. In this study, we aimed to genetically characterize the most prevalent GI and GII NoV strains, in order to obtain a rough estimate of the efficacy of disinfection treatments and to compare the results with clinical data documented in the same area during the same period. This work confirms the wide circulation and the genetic diversity of NoVs in Tunisia and the widespread distribution of NoV variants in both raw and treated wastewater. Indeed, NoV was detected in 192 (37.1 %) sewage samples, among them mixed infections with group A rotavirus were detected in 125 (65.1 %) cases. The genotypes of the GI NoVs were GI.1, GI.2, GI.4, GI.5, and GI of unassigned genotype (GI.UA), and the genotypes of the GII NoVs were all GII.12. This study enhances the currently poor environmental virological data gathered in Tunisia, demonstrates the benefit of environmental surveillance as a tool to determine the epidemiology of NoVs circulating in a given community, and underlines the need for the design and support of similar long-term studies in our country, in order to compensate for the absence of a national surveillance system for gastroenteric viruses.     

Enteric Viruses in a Large Waterborne Outbreak of Acute Gastroenteritis in Finland

In Nokia city about 450,000 l of treated sewage water was for 2 days allowed to run into the drinking water supplies of the city due to a personal error of one employee. Within the next 5 weeks about 1,000 people sought care at the municipal health centre or regional hospital because of gastroenteritis. Here we report the results of viral analyses performed by gene amplification assays from the earliest water and sewage samples as well as from close to 300 patient samples. The contaminating treated sewage was shown to harbour several enteric viruses known to cause acute gastroenteritis. Likewise, the drinking water sample was positive for noro-, astro-, rota-, entero- and adenoviruses. Noroviruses were also found in 29.8% of stool samples from affected patients, while astro-, adeno-, rota- and enteroviruses were detected in 19.7, 18.2, 7.5 and 3.7% of the specimens, respectively. An erratum to this article can be found at     

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 10⁶) > treated sewage (2.9 × 10⁵) > river waters (6.1 × 10²) > estuarine waters (4.8 × 10²) > bathing waters (8.5 × 10¹) > groundwater (5.9 × 10¹). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

     

The First Detection of Human Bocavirus Species 2 and 3 in Raw Sewage and Mussels in South Africa

Human bocavirus (HBoV) has a global distribution and is associated with respiratory and enteric infections, particularly in the paediatric population. In this study, raw sewage and mussel samples were analysed for the presence of HBoV using nested PCR with primers targeting the VP1/VP2 junction. Amplification and sequencing of the 382 bp region followed by phylogenetic analysis indicated the presence of HBoV 2 in mussel samples and HBoV 3 in sewage samples. This is the first report describing the presence of enteric-associated HBoV in environmental samples from South Africa and in mussel samples from the African continent. The results signify the need for further studies examining the potential risk of foodborne transmission of HBoV and highlight the importance of continued screening to determine the prevalence and epidemiology of HBoV in South Africa.     

Detection of Rotavirus Strains in Freshwater Clams in Japan

Bivalve molluscan shellfish like clams and oysters, etc., are capable to bioaccumulate surrounding contaminants from waters into their digestive systems and posing serious threats of food poisoning. Detection of rotaviruses (RVs) in shellfish is of particular importance because RVs are prone to genome reassortment resulting in the emergence of new RV variants that may compromise vaccine safety. Herein, we have detected the wild-type RVs and Rotarix/RotaTeq vaccine strains in freshwater clams collected on the riverside, Kawasaki city, from July 2019 to January 2020 and correlated the detected genotypes with that of gastroenteritis cases of nearby clinics to understand the transmission of RVs in the environment. The wild-type RVs were detected in 62 (64.6%) out of 96 freshwater clams in every study month: July, September, November, and January that are considered as off-season for RV infections. The most frequent genotypes were G2 (42.9%), G8 (28.6%), G3 (14.3%), G1 (7.1%), and G10 (7.1%), which remained comparable with genotypic distribution found in the clinical samples over the last few years indicating that these RVs may accumulate in clams since a long time. However, G10 genotype was detected in clam but not in clinical samples suggesting the presence of asymptomatic infection or RVs could be carried out from a long distance. Importantly, vaccine strains, RotaTeq (1%) but not Rotarix (0%), were also detected in a clam. Attention must be paid to monitoring the potential transmission of wild-type and vaccine RV strains in the environment to prevent the emergence of new variants generated from genome reassortment with vaccine strains.

     

水中轮状病毒实时定量PCR外标准品的构建

采用细胞培养和T-A克隆技术,在轮状病毒主要结构蛋白VP7基因序列上设计合成引物,经PCR扩增后将特异性产物连接入pGEM-T-easy载体中,经过酶切鉴定和测序分析获得轮状病毒cDNA标准品.利用常规PCR和实时定量PCR方法对所获得的cDNA标准品进行特异性、稳定性和重复性指标的检验.结果表明,利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系(斜率为-3.353,R2=0.995);实时定量PCR熔解曲线分析表明,温度在81℃±0.5℃的PCR产物是轮状病毒VP7序列上的特异性产物,表明此标准品是轮状病毒特异性的;同时,所制备的标准品在实时定量PCR检测中具有较大的线性范围(9×100～9×1011 copies/μL,每个反应最低可以检测到9个拷贝数的轮状病毒cDNA,表明利用该标准品进行实时定量PCR分析时具有较高的检测灵敏性;此外,该标准品具有较高的稳定性和重复性(3次独立实验CV值在0.2%～0.9%之间),可长期稳定保存.构建的标准品可用作检测水环境中轮状病毒的实时荧光定量PCR的外标准品.     

Detection and Molecular Characterization of Human Noroviruses in Korean Groundwater Between 2008 and 2010

RT-PCR, nucleotide sequencing, and phylogenetic analysis were performed for genotyping and molecular characterization of noroviruses isolated from Korean groundwater. Among 160 samples collected from 80 sites between 2008 and 2010, 14 samples (8.7?%) from 12 sites were positive for noroviruses (NoVs). The percentages of NoV-positive samples in 2008, 2009, and 2010 were 22.2, 3.2, and 0?%, respectively, representing a yearly decrease. GII-positive samples (n?=?9, 5.6?%) outnumbered GI-positive samples (n?=?5, 3.1?%). The genotypes of the GI NoVs were GI.2, GI.5, and GI.6, and the genotypes of the GII NoVs were all GII.4. One sample, HM623465, was very similar to CUK-3 and CBNU2 and two GII.4 sequences isolated from the stool of Korean gastroenteritis patients. A BLASTN search revealed several nucleotide sequences highly similar to those of NoVs isolated in this study. The original isolation sources for these similar NoVs were mostly stool (n?=?731, 80.0?%) and groundwater (n?=?135, 14.8?%), and all the countries from which they were isolated were almost in Asia (96.0?%); specifically, China (n?=?192, 21.0?%), Japan (n?=?383, 41.9?%), Korea (n?=?296, 32.4?%), and other Asian countries (n?=?6, 0.7?%). These results suggest that Korean groundwater might be contaminated with NoVs from the stool of infected patients and that these NoVs in turn cause new cases of gastroenteritis through a typical fecal-oral route with region-specific circulation. Therefore, it is important to properly treat sewage, which may include waterborne viruses and manage point sources in groundwater for national health and sanitation. In addition, continuous molecular surveillance remains important for understanding circulating NoVs.     

Coxsackievirus B4 as a Causative Agent of Diabetes Mellitus Type 1: Is There a Role of Inefficiently Treated Drinking Water and Sewage in Virus Spreading?

This study proposed to detect the enterovirus (EV) infection in children with type 1 diabetes mellitus (T1D) and to assess the role of insufficiently treated water and sewage as sources of viral spreading. Three hundred and eighty-two serum specimens of children with T1D, one hundred serum specimens of children who did not suffer from T1D as control, and forty-eight water and sewage samples were screened for EV RNA using nested RT-PCR. The number of genome copies and infectious units of EVs in raw and treated sewage and water samples were investigated using real-time (RT)-PCR and plaque assay, respectively. T1D markers [Fasting blood glucose (FBG), HbA1c, and C-peptide], in addition to anti-Coxsackie A & B viruses (CVs A & B) IgG, were measured in control, T1D-negative EV (T1D–EV^?), and T1D-positive EV (T1D–EV⁺) children specimens. The prevalence of EV genome was significantly higher in diabetic children (26.2%, 100 out of 382) than the control children (0%, 0 out of 100). FBG and HbA1c in T1D–EV^? and T1D–EV⁺ children specimens were significantly higher than those in the control group, while c-peptide in T1D–EV^? and T1D–EV⁺ children specimens was significantly lower than that in the control (n = 100; p < 0.001). Positivity of anti-CVs A & B IgG was 70.7, 6.7, and 22.9% in T1D–EV⁺, T1D–EV^?, and control children specimens, respectively. The prevalence of EV genome in drinking water and treated sewage samples was 25 and 33.3%, respectively. The prevalence of EV infectious units in drinking water and treated sewage samples was 8.5 and 25%, respectively. Quantification assays were performed to assess the capabilities of both wastewater treatment plants (WWTPs) and water treatment plants (WTPs) to remove EV. The reduction of EV genome in Zenin WWTP ranged from 2 to 4 log₁₀, while the reduction of EV infectious units ranged from 1 to 4 log₁₀. The reduction of EV genome in El-Giza WTP ranged from 1 to 3 log₁₀, while the reduction of EV infectious units ranged from 1 to 2 log₁₀. This capability of reduction did not prevent the appearance of infectious EV in treated sewage and drinking water. Plaque purification was performed for isolation of separate EV isolates from treated and untreated water and sewage samples. Characterization of the EV amplicons by RT-PCR followed by sequencing of these isolates revealed high homology (97%) with human coxsackievirus B4 (CV B4) in 60% of the isolates, while the rest of the isolates belonged to poliovirus type 1 and type 2 vaccine strains. On the other hand, characterization of the EV amplicons by RT-PCR followed by sequencing for T1D–EV⁺ children specimens indicated that all samples contained CV B4 with the same sequence characterized in the environmental samples. CV B4-contaminated drinking water or treated sewage may play a role as a causative agent of T1D in children.     

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters,Downstream River Waters,and Drinking Waters

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.     

Occurrence and Genetic Diversity of Human Cosavirus in Sewage in Italy

Human Cosavirus (HCoSV) is a newly discovered virus whose role in human enteric diseases is still unknown. In Italy, the prevalence and genetic diversity of HCoSV are unexplored. One hundred forty-one raw sewage samples collected throughout Italy were screened for HCoSV by RT-nested PCR. HCoSV was detected in 25.5% of samples. Species A, C, and D, and a potentially new species were detected. Our results show a significant circulation and heterogeneity of HCoSV in Italy.