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

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.

Expression of hyaluronan synthases and CD44 messenger RNAs in porcine cumulus-oocyte complexes during in vitro maturation.

The transient synthesis and accumulation of hyaluronan (HA), an extracellular matrix component of cumulus cells, brings about expansion of cumulus-oocyte complexes (COCs) in preovulatory mammalian follicles. In this study, we investigated the mRNA expressions of hyaluronan synthase 2 (has2), hyaluronan synthase 3 (has3), and CD44, as well as the responsiveness to eCG and porcine follicular fluid (pFF) of these genes, in porcine COCs, oocytectomized complexes (OXCs), and oocytes during in vitro maturation. Immunolocalization of CD44 was also analyzed in COCs. After 12 h of culture, the area of cumulus expansion in medium 199 supplemented with both 10 IU/ml eCG and 10% (v/v) pFF was significantly greater than that in the medium supplemented with eCG or pFF. Oocytectomy reduced the expansion area in the group supplemented with eCG. In reverse transcription-polymerase chain reaction analysis, all transcripts were identified in COCs, but has3 transcript was not found in OXCs. Only has3 mRNA was detectable in oocytes, indicating that cumulus cells express has2 and CD44 mRNAs, and oocytes express has3 mRNA. The expression levels of has2 and CD44 mRNAs in COCs and OXCs increased in the presence of eCG and pFF after 24 h of culture, suggesting that these genes have a positive dependency on eCG and pFF. In contrast, the high level of has3 mRNA was detected in COCs cultured in the medium alone. Oocytectomy slightly reduced the expression level of has2 mRNA. On immunostaining for CD44, CD44 was expressed apparently in COCs cultured with eCG and pFF for 24 h. The positive staining was distributed on cytoplasm along the perimembrane of cumulus cells and at the junctions between cumulus cells and oocytes. CD44 was also localized on cytoplasm of some oocytes. These results indicate that 1) porcine oocytes promote eCG-dependent cumulus expansion and the expression of has2 mRNA in cumulus cells, but these are not essential for expansion of cumulus cells and the expression of has2 mRNA; 2) HAS2 is involved in HA synthesis during cumulus expansion, and eCG and pFF up-regulate its expression; 3) the expression profile of the has3 mRNA that is transcribed in oocytes is different from those of has2 and CD44 mRNA; and 4) CD44 may participate in the interaction between cumulus cells and oocytes.