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

Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects the common carp (Cyprinus carpio L.) and koi carp (C. carpio koi). The occurrence of the disease in the United Kingdom has been dated to 1996, following outbreaks in the United States, Israel, Europe, and South Asia (10), and has afflicted cultured ornamental and common carps, causing severe losses to fish breeders, retailers, and hobbyists (28). Therefore, the characterization and diagnosis of the disease have been the subject of intensive research (15). In recent years, the mortality of wild carp has been reported in natural freshwater environments (11, 18, 23). In Lake Biwa in Japan, 60 to 80% of the wild carp population (>100,000) died in 2004, presumably due to CyHV-3 infection (Shiga Prefectural Government, http://www.pref.shiga.jp/g/suisan-s/seika/files/seikah1711.pdf [in Japanese]) (18). The mass mortality of wild carp can directly and indirectly affect community composition and environmental ecosystems (18). Nevertheless, the occurrence of the disease and the means of transmission of CyHV-3 in the natural environment are still not well understood. CyHV-3 is present in several organs of infected fish, such as the intestines, kidneys (7), and gills (29). CyHV-3 is also detected in droppings (3); therefore, infected fish are suspected of releasing CyHV-3 into natural waters. Seasonal variation and the spatial distribution of CyHV-3 may be important for understanding the transmission routes and mechanisms by which CyHV-3 spreads. However, the lack of a reliable method for quantifying CyHV-3 in environmental water precludes our elucidation of how this disease spreads. In general, the concentration of a pathogen in environmental water is considerably lower than that found in host bodies. Therefore, a CyHV-3 concentration method is required to detect and quantify the virus in environmental water. Several methods have been developed for determining concentrations of viruses in water samples. Ultrafiltration can concentrate a pathogen from a large volume of water in