Relative Abundance of Human Bocaviruses in Urban Sewage in Greater Cairo,Egypt

Human bocavirus (HBoV) is predominantly found in the respiratory tract infections and in the stool of patients with gastroenteritis symptoms. However, data on the prevalence of HBoV genotypes in environmental samples are limited. Here we addressed the prevalence of HBoV in sewage collected from three different wastewater treatment plants in Egypt. HBoV-1, HBoV-2, and HBoV-3 were detected, whereas none of the samples were positive for HBoV-4. The median concentration of HBoV in influent samples was 8.5 × 10³ GC/l for HBoV-1, 3.0 × 10⁴ GC/l for HBoV-2, and 2.5 × 10⁴ GC/l for HBoV-3. The concentration was reduced but not completely removed in the effluent samples. The median concentration in the outlet samples was 2.9 × 10³ GC/l for HBoV-1, 4.1 × 10³ GC/l for HBoV-2, and 2.1 × 10³ GC/l for HBoV-3. Moreover, no seasonality pattern of HBoVs was found. The high incidence of HBoV in sewage samples provided an evidence of its circulation in the local population. Although the role of HBoV in respiratory or gastro-intestinal infections still remains to be fully elucidated, the risk of infection via contaminated water should be taken into consideration.     

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.     

Human Bocavirus: Detection,Quantification and Molecular Characterization in Sewage and Surface Waters in Uruguay

Human bocavirus (HBoV) infections are related to respiratory and gastroenteric diseases. The aim of this study was to investigate the presence of HBoV in both sewage and surface waters in Uruguay. Sixty-eight sewage samples from the cities of Salto, Paysandú, Bella Unión, Fray Bentos, Treinta y Tres and Melo and 36 surface water samples from the cities of Salto, Florida and Santa Lucía were studied. HBoV was screened by multiplex qPCR for the detection of the four subtypes, followed by monoplex qPCRs for the independent quantification of each subtype. A qualitative PCR followed by DNA sequencing and phylogenetic analysis was carried out for molecular characterization of HBoV strains. HBoV was present in a high frequency (69%) in sewage and only one positive sample (3%) was found in surface water. Concerning sewage samples, HBoV1 was detected in 11 (23%) out of the 47 positives samples, with a mean concentration of 8.2 × 10⁴ genomic copies/Liter (gc/L), HBoV3 was detected in 35 (74%) of the positive samples with a mean concentration of 4.1 × 10⁶ gc/L and subtypes 2 and/or 4 were detected in 39 (83%) of the positive samples with a mean concentration of 7.8 × 10⁶ gc/L. After the phylogenetic analysis performed by a Bayesian approach, the four HBoV subtypes were confirmed. This is the first study determining a high frequency of HBoV and the presence of the four HBoV subtypes in aquatic matrices in Latin America, mainly in sewage. Although HBoV was scarcely detected in surface water, a waterborne transmission is likely to occur if people enter in contact with polluted surface waters for recreational activities such as fishing or swimming since an elevated frequency of HBoV was detected in raw sewage which is usually directly discharged into surface waters.     

Isolation and Identification of Enteroviruses from Sewage and Sewage-Contaminated Water in Lagos,Nigeria

Studies have confirmed silent circulation of enteroviruses in the environment even in the absence of associated clinical conditions in the community. In this light, 26 samples of sewage and sewage-contaminated water serving selected high-risk communities in Lagos Nigeria were examined between June and September 2010. To concentrate virus particles in the sample, 480 μL of each sample was centrifuged at 3,000 rpm for 1 h at 4 °C. Subsequently, pellets were pooled, chloroform treated and further centrifuged at 1,500 rpm for 20 min at 4 °C. The water phase (concentrate) was then collected and stored at ?20 °C. The concentrates were subsequently inoculated into RD and L20B cell lines. Recovered isolates were identified by real-time RT-PCR (rRT-PCR), serotyping, VP1 amplification, sequencing and phylogenetic analysis. Overall, 9 (34.6 %) of the samples showed characteristic enterovirus cytopathic effect in RD cell line and were subsequently confirmed by pan-enterovirus rRT-PCR. The isolates were further identified by serotyping to include three E7, one E11 and one E13 isolates whilst four isolates were untypable. Further characterisation by VP1 sequencing confirmed the results of serotyping and rRT-PCR for all but isolate E13. Also, the four previously untypable isolates were identified to include two E19, one E20 and one E7 by VP1 sequencing. Results of the study confirmed circulation of Sub-Saharan Africa-specific enterovirus clades in the region, provide information on their molecular epidemiology and emphasise the need to combine methods of identification to enhance enterovirus surveillance.     

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.     

Quantification and Genotyping of Aichi Virus 1 in Water Samples in the Kathmandu Valley,Nepal

Aichi virus 1 genomes were detected by quantitative PCR in groundwater from shallow dug (10/22) and tube wells (1/15), river water (14/14), and sewage (1/1), with the maximum concentration of 4.0 × 10⁹ copies/l. Nucleotide sequencing analysis demonstrated the prevalence of genotype B in the virus positive samples.     

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.

     

DNA Heat Treatment for Improving qPCR Analysis of Human Adenovirus in Wastewater

PCR inhibitory substances in complex sample matrices can cause false negatives or under-estimation of target concentration. This study assessed DNA heat treatment for reducing inhibition during qPCR analysis of human adenovirus (HAdV) in wastewater samples. Inhibition was reduced by heat treating DNA, where mean HAdV concentration was increased by 0.71 log₁₀ GC/L (and up to 3.04 log₁₀ GC/L in one case), and replicate variability and false negatives were reduced. DNA heat treatment should be further investigated for improving reliability of HAdV concentration estimates in water, which can support more accurate assessment of health risks associated with viral pathogen exposure.     

Occurrence of <Emphasis Type="Italic">Pepper Mild Mottle Virus</Emphasis> (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula,Mexico

The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels <40 MPN/100 ml. The highest average concentrations of somatic and male F+ specific coliphages were 920 and 330 plaque forming units per 100 ml, respectively, detected in freshwater during the rainy season. PMMoV RNA was detected in 85% of the samples with gene sequences sharing 99–100% of nucleotide identity with PMMoV sequences available in GenBank. Quantification of PMMoV genome copies (GC) by quantitative real-time PCR indicated concentrations ranging from 1.7 × 10¹ to 1.0 × 10⁴ GC/L, with the highest number of GC detected during the rainy season. No significant correlation was observed between PMMoV occurrence by season or water type (p > 0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.     

北京市城市污水雌激素活性的研究

采用重组酵母雌激素筛检法(YES法)对北京市3个城市污水处理厂工艺流程污水样品的雌激素活性进行了评价,并采用GC/MS分析了样品中8种内分泌干扰物(EDCs),以进一步阐明城市污水雌激素活性的变化.结果表明,污水处理厂能较好地降低污水雌激素活性,降低率为82.2%～97.0%,但出水仍具有一定的雌激素活性,雌二醇(E2)当量浓度(EEQ)为2.6～16.0 ng/L.GC/MS检测显示,污水处理厂并不能完全去除目标化合物,出水中平均浓度最低的是17α-E2,为13.5 ng/L,最高的是BPA,为106.4 ng/L;出水的雌激素活性主要来自类固醇雌激素.污水厂的出水排放具有潜在的环境风险,在污水处理过程中,应特别关注对类固醇雌激素的去除.     

Presence of Torque Teno Virus (TTV) in Tap Water in Public Schools from Southern Brazil

Torque teno virus (TTV) was surveyed in tap water collected in schools from three municipalities located in the south of Brazil. TTV genomes were found in 11.7 % (4/34) of the samples. TTV DNA was detected in 10.5 % (2/19) of the samples collected at the city of Caxias do Sul and in 25 % (2/8) of the samples from Pelotas. Those cities have a low rate of sewage treatment. All samples from Santa Cruz do Sul, which has nearly 92 % of its sewage treated, were negative. These results suggest that the amount of sewage treated may have an effect on the detection rates of TTV DNA in drinking water in a given urban area, showing a mild negative correlation (r = ?0.76), when comparing the percentage of sewage treatment to the detection of TTV genomes. The detection rate of TTV was also compared with Escherichia coli, showing a strong correlation (r = 0.97), indicating that TTV may be a suitable marker of fecal contamination.     

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.     

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.     

Detection of Norovirus GII.17 Kawasaki 2014 in Shellfish,Marine Water and Underwater Sewage Discharges in Italy

Norovirus (NoV) is a major cause of non-bacterial acute gastroenteritis worldwide, and the variants of genotype GII.4 are currently the predominant human strains. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been reported as the cause of gastroenteritis outbreaks in Asia, replacing the pandemic strain GII.4 Sydney 2012. The GII.17 Kawasaki 2014 variant has also been reported sporadically in patients with gastroenteritis outside of Asia, including Italy. In this study, 384 shellfish samples were subjected to screening for human NoVs using real-time PCR and 259 (67.4%) tested positive for Genogroup II (GII) NoV. Of these, 52 samples, selected as representative of different areas and sampling dates, were further amplified by conventional PCR targeting the capsid gene, using broad-range primers. Forty shellfish samples were characterized by amplicon sequencing as GII.4 (n = 29), GII.2 (n = 4), GII.6 (n = 2), GII.12 (n = 2), and GII.17 (n = 3). Sixty-eight water samples (39 seawater samples from the corresponding shellfish production areas and 29 water samples from nearby underwater sewage discharge points) were also tested using the above broad-range assay: eight NoV-positive samples were characterized as GII.1 (n = 3), GII.2 (n = 1), GII.4 (n = 2), and GII.6 (n = 2). Based on full genome sequences available in public databases, a novel RT-PCR nested assay specific for GII.17 NoVs was designed and used to re-test the characterized shellfish (40) and water (8) samples. In this second screening, the RNA of GII.17 NoV was identified in 17 additional shellfish samples and in one water sample. Upon phylogenetic analysis, these GII.17 NoV isolates were closely related to the novel GII.17 Kawasaki 2014. Interestingly, our findings chronologically matched the emergence of the Kawasaki 2014 variant in the Italian population (early 2015), as reported by hospital-based NoV surveillance. These results, showing GII.17 NoV strains to be widespread in shellfish samples collected in 2015 in Italy, provide indirect evidence that this strain has started circulating in the Italian population. Notably, using a specific assay, we were able to detect many more samples positive for GII.17 NoV, indicating that, in food and water matrices, broad-range assays for NoV may grossly underestimate the prevalence of some, less common, NoVs. The detection of the GII.17 strain Kawasaki 2014 in clinical, water and food samples in Italy highlights the need for more systematic surveillance for future disease control and prevention.     

Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011–2016) Revealed by Next-Generation and Sanger Sequencing

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013–2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.     

基于吡啶甲基萘酰亚胺的水中痕量铁检测方法研究

合成了一种新型的荧光分子探针N-(2-吡啶甲基)-1,8-萘酰亚胺(PMN),研究了其在环境水样中痕量铁离子的检测应用.结果表明,该方法选择性好,灵敏度高,操作简便,且常见金属离子不干扰测定.Fe3+浓度在1×10-7~8×10-5mol/L范围内,荧光强度与其浓度呈良好的线性关系.方法用于自来水、井水、生活污水中痕量铁的测定,回收率在95.78%~104.02 %之间.     

对水中有机磷农药样品保存方法的优化

优化了对水中11种有机磷农药检测的样品保存方法。加标水样在4℃下保存一定时间后,通过液液萃取,气相色谱-质谱（选择离子模式）法测定有机磷农药的回收率。在不加入稳定剂的情况下,水样的保存时间不宜超过16h。为延长样品的保存时间,向水样中加入有机溶剂和pH调节剂作为稳定剂。实验结果表明,当在1000ml样品中加入10ml正己烷和10mlpH=3．6的醋酸-醋酸钠缓冲溶液时,可以将样品保存时间延长至48h。为验证方法的有效性,以地表水作为实际水样,加标浓度水平在0．2μg／L和0．04μg／L时,48h后的平均加标回收率在76．2％-95．0％之间,RSD在2．4％-7．2％之间。     

Quantification of Human Adenoviruses in European Recreational Waters

The presence of human adenoviruses (HAdV) in recreational water might cause disease in the population upon exposure. HAdV detected by PCR could also serve as indicators of the virological water quality. In order to assess the applicability of HAdV to the evaluation of the faecal contamination in European bathing waters, a real-time quantitative PCR assay was used for the quantification of HAdV in 132 samples collected from 24 different recreational marine and freshwater sites in nine European countries. Selected samples presenting positive nested PCR results for HAdV were analyzed using quantitative PCR and 80 samples from a total of 132 produced quantitative results with mean values of 3.2 × 10² per 100 ml of water, being human adenovirus 41 the most prevalent serotype between the samples where adenovirus was typified. HAdV were quantified in samples from all 15 surveillance laboratories. Statistical analysis showed no homogeneous linear relation between HAdV and E. coli, intestinal enterococci or somatic coliphages concentrations in the tested samples when considering all the data together. Significant correlations between HAdV and at least one of the other indicators were observed only when data from individual laboratories were considered. The quantification of HAdV may provide complementary information in relation to the use of bacterial standards in the control of water quality in bathing water.