Prevalence and Genetic Diversity of Klassevirus in Wastewater in Japan

Klassevirus is a novel virus belonging to the family Picornaviridae. This study examined the prevalence and genetic diversity of klassevirus in wastewater. Raw sewage (100 ml) and secondary-treated sewage (2 l) were collected monthly for 14 months between January 2011 and February 2012 from a wastewater treatment plant in Japan. Klassevirus in the sample was concentrated by the electronegative membrane-vortex method, followed by qualitative detection by means of three types of reverse transcription (RT)-nested polymerase chain reactions (PCRs). Klassevirus was detected in seven of the 14 raw sewage (50 %) and four of the 14 secondary-treated sewage (29 %) samples by the RT-nested PCRs targeting the 2C and/or 3D regions. In contrast, none of the samples tested positive for the virus by the RT-nested PCR targeting the VP0/VP3 region. Based on direct nucleotide sequence analysis of the klassevirus-positive nested PCR fragments, the tested samples showed high nucleotide sequence similarities of 94.7-100.0 % and 93.2-100.0 % in the 2C and 3D regions, respectively, indicating the presence of a single klassevirus strain. To our knowledge, this is the first study evaluating seasonal prevalence and genetic diversity of klassevirus in environmental waters.