Main content area

Quantification of Cyprinid Herpesvirus 3 in Environmental Water by Using an External Standard Virus

Honjo, Mie N., Minamoto, Toshifumi, Matsui, Kazuaki, Uchii, Kimiko, Yamanaka, Hiroki, Suzuki, Alata A., Kohmatsu, Yukihiro, Iida, Takaji, Kawabata, Zen'ichiro
Applied and environmental microbiology 2010 v.76 no.1 pp. 161-168
Cyprinid herpesvirus 3, Cyprinus carpio, DNA, bacteriophages, filters, koi, lakes, monitoring, polymerase chain reaction, quantitative analysis, rivers, ultrafiltration, virus-like particles
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 λ phage, and the average ratio of λ to the CyHV-3 recovery yield was 1.4, indicating that λ 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 λ 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 2 x 10⁵ 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.