Occurrence of pepper mild mottle virus in drinking water sources in Japan

Abstract

Pepper mild mottle virus (PMMoV) is a plant virus that has been recently proposed as a potential indicator of human fecal contamination of environmental waters; however, information on its geographical occurrence in surface water is still limited. We aimed to determine the seasonal and geographic occurrence of PMMoV in drinking water sources all over Japan. Between July 2008 and February 2011, 184 source water samples were collected from 30 drinking water treatment plants (DWTPs); viruses from 1 to 2 liters of each sample were concentrated by using an electronegative membrane, followed by RNA extraction and reverse transcription. Using quantitative PCR, PMMoV was detected in 140 (76%) samples, with a concentration ranging from 2.03×10(3) to 2.90×10(6) copies/liter. At least one of the samples from 27 DWTPs (n=4 or 8) was positive for PMMoV; samples from 10 of these DWTPs were always contaminated. There was a significant difference in the occurrence of PMMoV among geographical regions but not a seasonal difference. PMMoV was frequently detected in samples that were negative for human enteric virus or Escherichia coli. A phylogenetic analysis based on the partial nucleotide sequences of the PMMoV coat protein gene in 12 water samples from 9 DWTPs indicated that there are genetically diverse PMMoV strains present in drinking water sources in Japan. To our knowledge, this is the first study to demonstrate the occurrence of PMMoV in environmental waters across wide geographical regions.

Fig 1

Concentration of PMMoV in source water samples at 30 DWTPs in Japan. Lines within the boxes represent median values; the upper and lower lines of the boxes represent 25th and 75th percentiles, respectively, and the upper and lower bars outside the boxes represent the maximum and minimum values, respectively. Source water samples were collected 4 and 8 times from each DWTP, indicated with and without an asterisk, respectively.

Fig 2

Phylogenetic analysis of PMMoV sequences identified in source water samples. The tree was generated by using the neighbor-joining method with 1,000 bootstrap replicates based on the 431-nt coat protein sequences. The sequence of tobacco mosaic virus was used as an outgroup. The numbers on each branch indicate the bootstrap values, and the scale bar represents the number of nucleotide substitutions per position. PMMoV sequences obtained in this study are labeled with boldface type and italics, and they indicate the GenBank accession number, region name, DWTP identification, and month and year of sample collection. Reference sequences are indicated by the GenBank accession number and strain name.