Detection and Genetic Analysis of Human Sapoviruses in River Water in Japan

Abstract

We investigated the prevalence of sapoviruses (SaVs) in the Tamagawa River in Japan from April 2003 to March 2004 and performed genetic analysis of the SaV genes identified in river water. A total of 60 river water samples were collected from five sites along the river, and 500 ml was concentrated using the cation-coated filter method. By use of a real-time reverse transcription (RT)-PCR assay, 12 (20%) of the 60 samples were positive for SaV. SaV sequences were obtained from 15 (25%) samples, and a total of 30 SaV strains were identified using six RT-PCR assays followed by cloning and sequence analysis. A newly developed nested RT-PCR assay utilizing a broadly reactive forward primer showed the highest detection efficiency and amplified more diverse SaV genomes in the samples. SaV sequences were frequently detected from November to March, whereas none were obtained in April, July, September, or October. No SaV sequences were detected in the upstream portion of the river, whereas the midstream portion showed high positive rates. Based on phylogenetic analysis, SaV strains identified in the river water samples were classified into nine genotypes, namely, GI/1, GI/2, GI/3, GI/5, GI/untyped, GII/1, GII/2, GII/3, and GV/1. To our knowledge, this is the first study describing seasonal and spatial distributions and genetic diversity of SaVs in river water. A combination of real-time RT-PCR assay and newly developed nested RT-PCR assay is useful for identifying and characterizing SaV strains in a water environment.

Diverse sapovirus genotypes identified in children hospitalised with gastroenteritis in selected regions of South Africa

“…Strains from SA shared even higher nucleotide identity (99-100%) with SaVs from other countries, particularly recent strains reported in Asia (2012) [18] and Japan (2003) [5]. In GII.5, the most closely-related SaV (93-95% nucleotide identity) was identified from wastewater in Japan (2005) [22] and in GII.6 GenBank matches with < 90% nucleotide identity over the genotyped region included SaVs detected in stool from Burkina Faso [14], Thailand [37] and Japan and river water in Spain [23], from 2004 to 2009. In GII.7, closely-related SaVs from other countries included recent strains from the US (2010) and China (2011) [38].…”

“…The region was amplified by nested polymerase chain reaction (PCR) using published primers [22,23] as previously described [24], with minor adjustments. Briefly, in the first round of PCR 5 µl cDNA was added to a 50 µl reaction with 0.4 µM of each primer (SV-F13, SV-F14, SV-DS3 and SV-DS4) and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), with cycling parameters as previously described [24].…”

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

“…Since cell culture assays for classic HAstVs are time-consuming, less sensitive than RT-PCR, and not specific, RT-PCR-based assays are more effective for determination of the occurrences of classic HAstVs. In general, the amplification efficiency/sensitivity of PCR for environmental virus detection can be affected by several factors, such as the primer and probe sequences used and the sample matrix (34,35). Because environmental samples contain multiple virus strains, selection of a broadly reactive RT-PCR assay is important to obtain representative results.…”

Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

“…and environmental samples including river water (Kitajima et al 2010;Sano et al 2011), wastewater (Haramoto et al 2008;Kitajima et al 2011) and shellfish (Hansman et al 2007b;Iizuka et al 2010;Ueki et al 2010); predominantly in J a p a n a n d S p a i n . H o w e v e r , w h e n c o m p a r e d w i t h N o V s , t h e r e i s l i m i t e d information available on SaVs circulating in the environment worldwide.…”

“…Sapoviruses are most frequently detected in environmental samples using realtime reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Haramoto et al 2008;Kitajima et al 2010;Sano et al 2011). This molecular method allows for sensitive and specific detection and quantification of SaVs (Chan et al 2006) but its application can be hampered by the presence of inhibitory compounds in the sample.…”