Genetically distinct genogroup IV norovirus strains identified in wastewater.

We investigated the prevalence and genetic diversity of genogroup IV norovirus (GIV NoV) strains in wastewater in Arizona, United States, over a 13-month period. Among 50 wastewater samples tested, GIV NoVs were identified in 13 (26 %) of the samples. A total of 47 different GIV NoV strains were identified, which were classified into two genetically distinct clusters: the GIV.1 human cluster and a unique genetic cluster closely related to strains previously identified in Japanese wastewater. The results provide additional evidence of the considerable genetic diversity among GIV NoV strains through the analysis of wastewater containing virus strains shed from all populations.

Occurrence and genetic diversity of human cosavirus in influent and effluent of wastewater treatment plants in Arizona, United States.

Human cosavirus (HCoSV) is a novel member of the family Picornaviridae. We investigated the prevalence and genetic diversity of HCoSV in influent and effluent wastewater in Arizona over a 12-month period, from August 2011 to July 2012. HCoSV sequences were identified in six (25%) influent samples and one (4%) effluent sample, with the highest concentration of 3.24 × 10(5) and 1.54 × 10(3) copies/liter in influent and effluent, respectively. The strains were characterized based on their 5' untranslated region and classified into species A and D, demonstrating that genetically heterogeneous HCoSV were circulating with a clear temporal shift of predominant strains in the study area.

Norovirus Outbreaks Among Colorado River Rafters in the Grand Canyon, Summer 2012.

OBJECTIVE: To investigate the incidence and causative agent of the recurrent outbreaks of acute gastrointestinal illness (AGI) among different rafting groups on the Colorado River in the Grand Canyon National Park during the 2012 summer season. METHODS: Confidential illness reports were completed by all individuals with symptoms of AGI, and samples of fecal matter and vomitus, surface swabs of rafting equipment, and environmental swabs at stops along the hiking corridor were collected and tested for the presence of norovirus using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: During the active outbreak period between May 9 and July 9, 2012, 97 rafters (1.4%) from 10 trips (2.9% of all trips) declared AGI symptoms. AGI incidence within the 10 infected trips varied from 6% to 88%. Outbreaks occurred in 3 distinct temporal clusters that involved 2 different genogroups of norovirus. All available toilet fecal samples (5 samples) were positive for norovirus RNA: 1 with genogroup I (GI) and 4 with GII. The vomitus sample tested positive for GI. None of the fomite samples from rafting equipment or from the hiking corridors were confirmed for norovirus. CONCLUSIONS: The results suggest that norovirus may have been introduced by ill or asymptomatic individuals actively shedding the virus in their vomitus or feces, and spread within, or between, river trips by different modes of transmission. This study reinforces the importance of appropriate guidance and practice regarding norovirus prevention and the necessity of postoutbreak containment in relatively isolated groups of individuals.

Microbial atrazine breakdown in a karst groundwater system and its effect on ecosystem energetics.

In the absence of sunlight energy, microbial community survival in subterranean aquifers depends on integrated mechanisms of energy and nutrient scavenging. Because karst aquifers are particularly sensitive to agricultural land use impacts due to rapid and direct hydrologic connections for pollutants to enter the groundwater, we examined the fate of an exogenous pesticide (atrazine) into such an aquifer and its impact on microbial ecosystem function. Atrazine and its degradation product deethylatrazine (DEA) were detected in a fast-flowing karst aquifer underlying atrazine-impacted agricultural land. By establishing microbial cultures with sediments from a cave conduit within this aquifer, we observed two distinct pathways of microbial atrazine degradation: (i) in cave sediments previously affected by atrazine, apparent surface-derived catabolic genes allowed the microbial communities to rapidly degrade atrazine via hydroxyatrazine, to cyanuric acid, and (ii) in low-impact sediments not previously exposed to this pesticide, atrazine was also degraded by microbial activity at a much slower rate, with DEA as the primary degradation product. In sediments from both locations, atrazine affected nitrogen cycling by altering the abundance of nitrogen dissimulatory species able to use nitrogenous compounds for energy. The sum of these effects was that the presence of atrazine altered the natural microbial processes in these cave sediments, leading to an accumulation of nitrate. Such changes in microbial ecosystem dynamics can alter the ability of DEA to serve as a proxy for atrazine contamination and can negatively affect ecosystem health and water quality in karst aquifers.

Genetic Analysis of Norovirus Strains that Caused Gastroenteritis Outbreaks Among River Rafters in the Grand Canyon, Arizona.

Toilet solid waste samples collected from five outbreaks among rafters in the Grand Canyon were subjected to sequencing analysis of norovirus partial capsid gene. The results revealed that a GI.3 strain was associated with one outbreak, whereas the other outbreaks were caused by GII.5 whose sequences shared >98.9% homology.

Relative abundance and treatment reduction of viruses during wastewater treatment processes--identification of potential viral indicators.

Waterborne pathogenic viruses discharged from wastewater treatment plants (WWTPs) pose potential public health risks. In the present study, we investigated the relative abundance, occurrence, and reduction of eleven different viruses at two WWTPs in southern Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent samples from the two WWTPs were collected monthly. Viruses were concentrated using an electronegative filter method and quantified using TaqMan-based quantitative PCR (qPCR) assays for each of the virus types (i.e., genogroup I, II and IV noroviruses, sapovirus, enterovirus, group A rotavirus, Aichi virus, pepper mild mottle virus, adenovirus, and JC and BK polyomaviruses), with murine norovirus internal control for the monitoring of extraction-RT-qPCR efficiencies. The pepper mild mottle virus, a plant virus, was found to be the most prevalent virus in both influent and effluent wastewater (annual mean concentration of 3.7-4.4×10(6) copies/L and 4.6-6.3×10(5) copies/L in influent and effluent wastewater, respectively), showing a low reduction by the treatment processes (0.76-0.99 annual mean log10 reduction), and no significant seasonal change in concentration. Aichi virus, a human enteric virus, was also found in greater abundance, and showed lower reduction during wastewater treatment than other human enteric viruses. Our results suggest that these viruses could be used as potential indicators of wastewater reclamation system performance, with respect to virus occurrence and removal.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

Quantification and genetic analysis of salivirus/klassevirus in wastewater in Arizona, USA.

Salivirus/klassevirus sequences were identified in 7 (15%) wastewater samples collected in Arizona monthly for a year, with the highest concentration of 2.28 × 10(5) and 2.46 × 10(4) copies/L in influent and effluent, respectively. This is the first report of quantification and genetic analysis of salivirus/klassevirus in water samples in the United States.

Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples.

The extraction and purification of nucleic acids is a critical step in the molecular detection of enteric viruses from environmental or fecal samples. In the present study, the performance of three commercially available kits was assessed: the MO BIO PowerViral Environmental DNA/RNA Isolation kit, the Qiagen QIAamp Viral RNA Mini kit, and the Zymo ZR Virus DNA/RNA Extraction kit. Viral particles of adenovirus 2 (AdV), murine norovirus (MNV), and poliovirus type 1 (PV1) were spiked in molecular grade water and three different types of sample matrices (i.e., biosolids, feces, and surface water concentrates), extracted with the kits, and the yields of the nucleic acids were determined by quantitative PCR (qPCR). The MO BIO kit performed the best with the biosolids, which were considered to contain the highest level of inhibitors and provided the most consistent detection of spiked virus from all of the samples. A qPCR inhibition test using an internal control plasmid DNA and a nucleic acid purity test using an absorbance at 230 nm for the nucleic acid extracts demonstrated that the MO BIO kit was able to remove qPCR inhibitors more effectively than the Qiagen and Zymo kits. These results suggest that the MO BIO kit is appropriate for the extraction and purification of viral nucleic acids from environmental and clinical samples that contain high levels of inhibitors.