title: Quantification of cyprinid herpesvirus 3 in environmental water by using an external standard virus.
abstract: Cyprinid herpesvirus 3 (CyHV-3), a lethal DNA virus that spreads in natural lakes and rivers, infects common carp and koi. We established a quantification method for CyHV-3 that includes a viral concentration method and quantitative PCR combined with an external standard virus. Viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. The recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44%+/-19%, n=3; ultrafiltration method, 50%+/-3%, n=3); however, the former method was faster and more suitable for routine determinations. The recovery of seeded CyHV-3 based on the cation-coated filter method varied by more than 3 orders of magnitude among the water samples. The recovery yield of CyHV-3 was significantly correlated with that of the seeded lambda phage, and the average ratio of lambda to the CyHV-3 recovery yield was 1.4, indicating that lambda is useful as an external standard virus for determining the recovery yield of CyHV-3. Therefore, to quantify CyHV-3 in environmental water, a known amount of lambda was added as an external standard virus to each water sample. Using this method, CyHV-3 DNA was detected in 6 of the 10 (60%) types of environmental water tested; the highest concentration of CyHV-3 DNA was 2x10(5) copies liter(-1). The lowest recovery limit of CyHV-3 DNA was 60 copies liter(-1). This method is practical for monitoring CyHV-3 abundance in environmental water.
authors.affiliation: Research Institute for Humanity and Nature, 457-4 Motoyama Kamigamo, Kita-ku, Kyoto 603-8047, Japan. mhonjo@chikyu.ac.jp
authors.forename: Mie N
authors.initials: MN
authors.lastname: Honjo
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Minamoto
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Matsui
authors.forename: Kimiko
authors.initials: K
authors.lastname: Uchii
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Alata A
authors.initials: AA
authors.lastname: Suzuki
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Kohmatsu
authors.forename: Takaji
authors.initials: T
authors.lastname: Iida
authors.forename: Zen'ichiro
authors.initials: Z
authors.lastname: Kawabata
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000302:isolation & purification; D002347:Carps / Q000821:virology*; D005374:Filtration / Q000379:methods; D006564:Herpesviridae / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014462:Ultrafiltration / Q000379:methods; D019562:Viral Load; D014773:Virology / Q000379:methods*; D014871:Water Microbiology