Assessment of adenovirus,hepatitis A virus and rotavirus presence in environmental samples in Florianopolis,South Brazil

Aims: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV‐A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. Methods and Results: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources‐with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT‐PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture‐PCR (ICC‐PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV‐A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV‐A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV‐A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 10⁴ gc g^?1 (oyster farm south) and 1·5 × 10⁵ gc g^?1 (oyster farm north) and in waters ranging from 2·16 × 10⁶ (lagoon water) to 1·33 × 10⁷ gc l^?1 (untreated drinking water). Conclusions: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. Significance and Impact of the Study: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.     

First outbreak of norovirus in Albania

Aims: Noroviruses (NoVs) represent the most important enteric viruses responsible for acute gastroenteritis world‐wide. This study objective is to characterize the first outbreak of NoV that occurred in Ballsh, a small city in Albania. Methods and Results: Stool specimens were collected from people attending to the hospital. Samples were also collected from the aqueduct for bacteriological and virological tests. Overall 33 stools and five drinking water samples were collected, respectively, from the hospital in Ballsh and from the municipal aqueduct. No water samples were scored positive whereas ten stool samples (30·3%) were scored GGII NoV positive. All the GGII isolates were identified as GGII·4 genotype, and no GGI was identified. The alignment and protein analysis were performed using, respectively, Clustal V and the mega 4 software. Conclusions: This is the first report of NoV GGII·4 in Albania causing an outbreak. The genetic analysis showed several point mutations and amino acid substitutions with respect to the international strains. Significance and Impact of Study: Over the last decades, Albania has suffered from different outbreaks as cholera, poliomyelitis, hepatitis A and now, for the first time, it has been documented an outbreak of NoV.     

Diagnosis of HEV infection by serological and realtime PCR assays: a study on acute non-A-C hepatitis collected from 2004 to 2010 in Italy

ABSTRACT: BACKGROUND: The impact of hepatitis E in developed countries, like Italy, still requires a clear definition. In the present study, we evaluated HEV infection in patients with acute non-A-C hepatitis by an approach comparing data from Real-time PCR and serological assays. METHODS: In a first analysis, sera from 52 patients hospitalized with a diagnosis of acute viral non-A-C hepatitis in Italy were tested by in-house Real-Time PCR assay for identification of Hepatitis E Virus (HEV) RNA and by anti-HEV IgM and IgG assays. In a subsequent analysis, selected samples were evaluated by additional IgM tests to confirm diagnosis. RESULTS: Among the 52 samples, 21 showed positive results for all three markers (IgM, IgG and HEV RNA). One patient showed HEV RNA as single marker. Uncertain results were found in 8 samples while the remaining 22 were negative for all markers. Further analysis of the 8 undefined samples by additional IgM tests confirmed HEV infection in 1 patient. Overall, acute HEV infections were reliably identified in 23 (44.2%) out of 52 patients. CONCLUSIONS: In the present paper, we performed a study evaluating HEV infection in 52 sporadic non-A-C acute hepatitis cases. All samples were collected from 2004 to 2010 in Italy. By a diagnostic strategy based on genomic and serological assays we identified HEV infections in 23 out of 52 patients (44.2%), a percentage higher than previous estimates. Thus, the actual impact of HEV infections in Italy needs to be further evaluated on a national scale by a diagnostic strategy based on multiple and last generation assays.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa

AIMS: Human adenoviruses (HAds), of which there are 51 serotypes, are associated with gastrointestinal, respiratory, urinary tract and eye infections. The importance of water in the transmission of HAds and the potential health risks constituted by HAds in these environments are widely recognized. Adenoviruses have not previously been quantified in river and treated drinking water samples. In this study, HAds in river water and treated drinking water sources in South Africa were detected, quantified and typed. METHODS AND RESULTS: Adenoviruses were recovered from the water samples using a glass wool adsorption-elution method followed by polyethylene glycol/NaCl precipitation for secondary concentration. The sensitivity and specificity of two nested PCR methods were compared for detection of HAds in the water samples. Over a 1-year period (June 2002 to July 2003), HAds were detected in 5.32% (10/188) of the treated drinking water and 22.22% (10/45) of river water samples using the conventional nested PCR method. The HAds detected in the water samples were quantified using a real-time PCR method. The original treated drinking water and river water samples had an estimate of less than one copy per litre of HAd DNA present. The hexon-PCR products used for typing HAds were directly sequenced or cloned into plasmids before sequencing. In treated drinking water samples, species D HAds predominated. In addition, adenovirus serotypes 2, 40 and 41 were each detected in three different treated drinking water samples. Most (70%) of the HAds detected in river water samples analysed were enteric HAds (serotypes 40 and 41). One HAd serotype 2 and two species D HAds were detected in the river water. CONCLUSIONS: Adenoviruses detected in river and treated drinking water samples were successfully quantified and typed. The detection of HAds in drinking water supplies treated and disinfected by internationally recommended methods, and which conform to quality limits for indicator bacteria, warrants an investigation of the risk of infection constituted by these viruses. The risk of infection may have implications for the management of drinking water quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique as it is the first report on the quantification and typing of HAds in treated drinking water and river water. This baseline data is necessary for the meaningful assessment of the potential risk of infection constituted by these viruses.     

Detection and molecular characterization of enteric viruses in environmental samples in Monastir,Tunisia between January 2003 and April 2007

Aims: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. Methods and Results: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). Conclusion: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. Significance and Impact of the Study: Our results address the potential health risks associated with transmission of human enteric viruses through water‐related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.     

Real-Time PCR Detection of Enteric Viruses in Source Water and Treated Drinking Water in Wuhan, China

A total of 48 water samples were collected from six water treatment plants in Wuhan and analyzed by real-time PCR assay for viral identification of enterovirus (EV), rotavirus group A (RVA), human adenovirus (HAdV) as well as human adenovirus subgroup F (HAdVF) during the period from December 2010 to October 2011. HAdV, HAdVF, and RVA were all positively detected in the samples of source water and treated drinking water. EV could be found in 46 % (11/24) of all the source water samples, but only 21 % (5/24) positive in treated drinking water. The concentrations of these three kinds of enteric viruses detected were as follows: HAdV > RVA > EV. The highest removal rate was EV (97 %), followed by RVA (82 %), HAdV (73 %), and HAdVF (72 %). HAdV and RVA have been abundant in untreated river water and finished water after conventional processes of water treatment plants, while bacterial indicators could not be detected in tap water, which met the standard of China for drinking water bacterial quality. Some factors that could affect the accuracy of qPCR detection are also discussed in this study.     

Enteric viruses in renovated water in Manitoba

Using various concentration procedures (including the gauze pad method, ultrafiltration, and adsorption-elution), enteric viruses, mainly vaccine strains of polioviruses, were isolated from 61.8% of sewage, 20.5% of effluents, 3.0% of river water, and 6.7% of drinking water samples collected from a waterway system in Manitoba. The presence of polioviruses in the drinking water of the town utilizing this waterway was not correlated with an increase in the number of infectious diseases reported.     

新疆奶牛戊型肝炎病毒RNA检测及序列分析

从新疆两个奶牛场的戊型肝炎病毒(Hepatitis E virus,HEV)抗体检测阳性的奶牛中分别采集牛粪便或肛拭子样品,利用反转录套式聚合酶链反应(RT-nPCR)检测所采集样品中的HEV RNA,结果来自第一个奶牛场的7份牛粪便样品为阳性,阳性率11.67%,来自第二奶牛场的1份肛拭子样品为阳性,阳性率为3.23%。将PCR扩增产物克隆、测序并进行序列分析,结果表明,对应于HEV ORF2 189bp核苷酸序列的8个牛源HEV基因组扩增片段的同源性为96.3%~100.0%,应当属于相同的基因型;它们与HEV 1、2、3和4型ORF2 189bp核苷酸平均同源性分别为78.5%~86.4%,81.7%~83.8%,79.1%~85.3%和84.3%~95.8%,与4型的同源性最高达93.2%~95.8%。基于这些已测序核酸片段绘制的基因进化树显示:本研究中的8株牛源HEV ORF2 189bp核苷酸序列与HEV 4型人源C5株、猪源swC3与swXJ株位于同一进化枝上,同属HEV基因4型,提示新疆奶牛中可能存在HEV感染,并且奶牛可能是人类HEV传染源中除猪之外的新宿主。     

Quantitative detection of sapoviruses in wastewater and river water in Japan

Aims:  To detect sapoviruses at a wastewater treatment plant (WWTP) and in a river in Japan, quantitatively. Methods and Results:  Influent and effluent samples at a WWTP and river water samples were collected monthly for 1 year. The water samples were subjected to virus concentration using an HA electronegative filter, followed by quantification of sapoviruses using real‐time PCR. The concentration of sapoviruses in influent ranged from 2·8 × 10³ to 1·3 × 10⁵ copies per litre, showing a higher value in winter. Seven (58%) of 12 effluent samples were positive for sapoviruses, as were 23 (64%) of 36 river water samples collected from three sites along the Tamagawa River. Conclusions:  Sapoviruses were abundant in the influent even in the nonepidemic period, suggesting that sporadic and asymptomatic infections occur throughout the year. Increasing concentration of sapoviruses was discharged into the river during the epidemic period winter. Significance and Impact of the Study:  This is the first study demonstrating the quantitative detection of sapoviruses in aquatic environments.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viral pollution of the rivers in Toyama City

Viral pollution of the river water in Toyama City was surveyed during the two-year period from July 1979 to July 1981, and the ecology of viruses in the river water is discussed. Virus isolation from the river water samples, or from the water squeezed from cotton pads that were immersed in the stream for 3 days, was carried out by the "filter adsorption/elution" method. River waters were found to be contaminated with various species of enteric viruses, that is, poliovirus, echovirus, coxsackievirus, adenovirus, and reovirus. Poliovirus was isolated during the period immediately after the oral administration of polio vaccine, and coxsackie B virus was frequently isolated all year around. The enterovirus concentration in the river water was significantly high with a maximum of five plaque-forming units of coxsackie B2 virus per 250 ml. The species and type distribution of enteroviruses isolated from the river water coincided well with that of viruses isolated from inhabitants of Toyama Prefecture, with the exception of reovirus which was the largest population of virus species in the river water.     

Optimization of sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees

Aims: To develop fast and reliable sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4‐mm‐diameter, 43‐cm‐long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1–4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56·6% for the scheme based on a single drill to 96·8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0·05) from those based on two and three drillings, whose efficiency was 72·6% and 83·9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of the Study: Fast and reliable sampling procedures make DNA‐based diagnosis more suitable for tree inspection procedures.