Akihiko Hata

Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples.

ABSTRACT Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

Occurrence and reduction of human viruses, F-specific RNA coliphage genogroups and microbial indicators at a full-scale wastewater treatment plant in Japan

To evaluate and compare the reductions of human viruses and F‐specific coliphages in a full‐scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays.

Effects of rainfall events on the occurrence and detection efficiency of viruses in river water impacted by combined sewer overflows

Rainfall events can introduce large amount of microbial contaminants including human enteric viruses into surface water by intermittent discharges from combined sewer overflows (CSOs). The present study aimed to investigate the effect of rainfall events on viral loads in surface waters impacted by CSO and the reliability of molecular methods for detection of enteric viruses. The reliability of virus detection in the samples was assessed by using process controls for virus concentration, nucleic acid extraction and reverse transcription (RT)-quantitative PCR (qPCR) steps, which allowed accurate estimation of virus detection efficiencies. Recovery efficiencies of poliovirus in river water samples collected during rainfall events (<10%) were lower than those during dry weather conditions (>10%). The log10-transformed virus concentration efficiency was negatively correlated with suspended solid concentration (r(2)=0.86) that increased significantly during rainfall events. Efficiencies of DNA extraction and qPCR steps determined with adenovirus type 5 and a primer sharing control, respectively, were lower in dry weather. However, no clear relationship was observed between organic water quality parameters and efficiencies of these two steps. Observed concentrations of indigenous enteric adenoviruses, GII-noroviruses, enteroviruses, and Aichi viruses increased during rainfall events even though the virus concentration efficiency was presumed to be lower than in dry weather. The present study highlights the importance of using appropriate process controls to evaluate accurately the concentration of water borne enteric viruses in natural waters impacted by wastewater discharge, stormwater, and CSOs.

Risk management of viral infectious diseases in wastewater reclamation and reuse: Review

Abstract Inappropriate usage of reclaimed wastewater has caused outbreaks of viral infectious diseases worldwide. International and domestic guidelines for wastewater reuse stipulate that virus infection risks are to be regulated by the multiple-barrier system, in which a wastewater treatment process composed of sequential treatment units is designed based on the pre-determined virus removal efficiency of each unit. The objectives of this review were to calculate representative values of virus removal efficiency in wastewater treatment units based on published datasets, and to identify research topics that should be further addressed for improving implementation of the multiple-barrier system. The removal efficiencies of human noroviruses, rotaviruses and enteroviruses in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes were obtained by a systematic review protocol and a meta-analysis approach. The log10 reduction (LR) of norovirus GII and enterovirus in MBR were 3.35 (95% confidence interval: 2.39, 4.30) and 2.71 (1.52, 3.89), respectively. The LR values of rotavirus, norovirus GI and GII in CAS processes were 0.87 (0.20, 1.53), 1.48 (0.96, 2.00) and 1.35 (0.52, 2.18), respectively. The systematic review process eliminated a substantial number of articles about virus removal in wastewater treatment because of the lack of information required for the meta-analysis. It is recommended that future publications should explicitly describe their treatment of left-censored datasets. Indicators, surrogates and methodologies appropriate for validating virus removal performance during daily operation of wastewater reclamation systems also need to be identified.

Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples

ABSTRACT Aichi viruses (AiVs) have been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. In the present study, we developed a TaqMan minor groove binder (MGB)-based reverse transcription-quantitative PCR (RT-qPCR) system that is able to quantify AiVs and differentiate between genotypes A and B. This system consists of two assays, an AiV universal assay utilizing a universal primer pair and a universal probe and a duplex genotype-specific assay utilizing the same primer pair and two genotype-specific probes. The primers and probes were designed based on multiple alignments of the 21 available AiV genome sequences containing the capsid gene. Using a 10-fold dilution of plasmid DNA containing the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0 × 101 to 1.0 × 107 copies/reaction, and the genotype-specific assay reacts specifically to each genotype. To validate the newly developed assays, 30 clinical stool specimens were subsequently examined with the assays, and the AiV RNA loads were determined to be 1.4 × 104 to 6.6 × 109 copies/g stool. We also examined 12 influent and 12 effluent wastewater samples collected monthly for a 1-year period to validate the applicability of the assays for detection of AiVs in environmental samples. The AiV RNA concentrations in influent and effluent wastewater were determined to be up to 2.2 × 107 and 1.8 × 104 copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples.

Wastewater Analysis Indicates that Genetically Diverse Astroviruses, Including Strains Belonging to Novel Clades MLB and VA, Are Circulating within Japanese Populations

ABSTRACT Human astroviruses (HAstVs) are a common etiological agent of infantile gastroenteritis. Recent studies revealed that novel astrovirus (AstV) strains of the MLB clade (MLB-AstVs) and VA clade (VA-AstVs), which are genetically distinct from the classic HAstVs, are circulating in the human population. In the present study, we quantified classic HAstVs as well as carried out a genetic analysis of classic and novel HAstVs in wastewater in Japan. The concentration of classic HAstVs in the influent water samples ranged from 104 to 105 copies per liter, and the amount removed by wastewater treatment was determined to be 2.4 ± 0.3 log10. Four types of classic HAstV strains (HAstV types 1, 2, 5, and 4/8) as well as novel AstV strains belonging to the MLB-2, VA-1, and VA-2 clades were identified using reverse transcription-PCR (RT-PCR) assays, including assays newly developed for the detection of strains of the MLB and VA clades, followed by cloning and nucleotide sequencing. Our results suggest that genetically diverse AstV strains are circulating among the human population in Japan. The newly developed (semi)nested RT-PCR assays for these novel AstV clades are useful to identify and characterize the novel AstVs in environmental waters.

Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

ABSTRACT Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances.

A review on recent progress in the detection methods and prevalence of human enteric viruses in water

Waterborne human enteric viruses, such as noroviruses and adenoviruses, are excreted in the feces of infected individuals and transmitted via the fecal-oral route including contaminated food and water. Since viruses are normally present at low concentrations in aquatic environments, they should be concentrated into smaller volumes prior to downstream molecular biological applications, such as quantitative polymerase chain reaction (qPCR). This review describes recent progress made in the development of concentration and detection methods of human enteric viruses in water, and discusses their applications for providing a better understanding of the prevalence of the viruses in various types of water worldwide. Maximum concentrations of human enteric viruses in water that have been reported in previous studies are summarized to assess viral abundances in aquatic environments. Some descriptions are also available on recent applications of sequencing analyses used to determine the genetic diversity of viral genomes in water samples, including those of novel viruses. Furthermore, the importance and significance of utilizing appropriate process controls during viral analyses are discussed, and three types of process controls are considered: whole process controls, molecular process controls, and (reverse transcription (RT)-)qPCR controls. Although no standards have been established for acceptable values of virus recovery and/or extraction-(RT-)qPCR efficiency, use of at least one of these appropriate control types is highly recommended for more accurate interpretation of observed data.

Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters

ABSTRACT F-specific RNA phages (FRNAPHs) are considered potential viral indicators of water pollution due to their occurrence and stability in water environments. However, their suitability as viral indicators is not fully elucidated because the characteristics of FRNAPHs are variable depending on the genotype. In this study, for the characterization of infectious FRNAPH genotypes, integrated culture reverse transcription-PCR coupled with the most probable number approach was applied to surface water samples. Further, to recover low concentrations of FRNAPH genotypes, an FRNAPH recovery method was developed. The novel FRNAPH recovery method using a noncharged microfiltration membrane could effectively recover FRNAPH strains without inactivation, while a method using an electronegative microfiltration membrane resulted in the inactivation of some strains. Infectious FRNAPH genotypes in surface water samples were successfully quantified with an efficiency comparable to that of the conventional plaque assay. Genotype I (GI) and GII FRNAPHs tended to be predominant at locations impacted by treated and untreated municipal wastewater, respectively. The numbers and proportions of infectious FRNAPHs tended to be higher during the winter season when water temperature decreased. IMPORTANCE Properties of FRNAPHs are highly variable depending on their genotypes. Previous typing methods for FRNAPHs are not quantitative and/or are based on molecular assays, which cannot differentiate infective strains from inactive strains. Due to the reasons mentioned above, the utility of FRNAPHs as viral indicators of water pollution has not been fully validated. In this study, a quantitative genotyping method for infectious FRNAPHs was developed and applied to surface water samples. The method enabled characterization of infectious FRNAPH genotypes in terms of their occurrence and seasonality. Moreover, comparison of the method to a conventional molecular assay (reverse transcription-quantitative PCR) enabled characterization of their stability. Our approach can provide novel findings for further validation of FRNAPHs as viral indicators of water pollution.

Development of a high resolution melting analysis for detection and differentiation of human astroviruses.

Human astroviruses (AstVs), the common causes of viral gastroenteritis, consist of 8 different sero- or genotypes in which a variety of subtypes have been found. In the present study, a rapid and high-throughput method for detection and sequence-discrimination of AstVs by high resolution melting (HRM) analysis was developed. A newly designed primer set for the assay targeting ORF1b-ORF2 junction region of AstVs successfully reacted with all 8 serotypes of AstVs and allowed genotyping using their amplicons. The HRM assay consists of intercalating dye based real time quantitative PCR (qPCR) and melting curve analysis. The qPCR assay was sensitive enough to detect 1.0×10(1) copies/reaction of AstV serotypes. However, 1.0×10(3) copies/reaction of AstVs gene was required to obtain a sequence-specific difference curve, indicating that pre-amplification is necessary to apply the assay to samples containing low numbers of AstVs. AstVs in clinical specimens were subjected to the HRM assay after pre-amplification. The strains possessing same nucleotide sequences at the target region showed an identical difference curve and those possessing different nucleotide sequences showed a distinguishable difference curve. The newly developed HRM assay is an effective technique for screening of AstVs to quantify and discriminate the strains.