www.ksfp.org 한국어병학회지 제28권 제2호 (2015) pISSN 1226-0819, eISSN 2233-5412 J. Fish Pathol., 28(2) : 113~116 http://dx.doi.org/10.7847/jfp.2015.28.2.113 Aquaculture has grown rapidly over the past few decades. However, because of high culture densities and environmental pollution, various infectious dis- eases have appeared (Wolf, 1988; Austin and Austin, 1999; Woo and Bruno, 1999). In particular, viral dis- eases are a threat to aquaculture because of the lack of available treatments. Infectious haematopoietic ne- crosis (IHN), viral hemorrhagic septicemia, red sea bream iridovirus disease, and viral nervous necrosis are representative viral diseases (Wolf, 1988; Essba- uer and Ahne, 2001). A virological examination is conducted using a fish cell line to diagnose a viral disease in diseased fish (Oh et al., 2001; Kim et al., 2009; OIE, 2012). Gen- erally, fish samples are transported on ice, and in- ternal organs are aseptically collected and homogen- ized with nine volumes of Hanks’ balanced salt sol- ution (HBSS). After centrifugation (2,000－4,000 × g, 10－20 min, 4°C), the supernatant is filtered through a 0.45 µm membrane filter, and 100 µl is inoculated onto fish cell lines in 24-well plates. The cell cultures are incubated at 15－25°C and examined for the de- velopment of cytopathic effects. We determined that virus titers decreased dramati- cally after filtration of virus supernatants with 0.45 µm filters. Thus, using a filter may be a dangerous procedure for virus extraction from fish samples. Effect of virus infectivity titer following centrifugation and filtration during virus extraction from fish samples Wi-Sik Kim, Jong-Oh Kim and Myung-Joo Oh † Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu 59626, Korea A 0.45-µm membrane filter is generally used to remove bacterial contamination during virus ex- traction from fish samples. However, the number of fish viruses is drastically reduced after filtration with a 0.45 µm filter. In this study, we investigated the effect of filters on virus infectivity titer and the change in virus titer and bacterial number following different centrifugation conditions to determine a suitable procedure for virus extraction from fish samples. 10 4.05 and 10 5.05 TCID 50 /ml of infectious hematopoietic necrosis virus (IHNV) and 10 4.05 and 10 4.55 TCID 50 /ml of Oncorhynchus masou virus (OMV) were not detectable after filtration with two types of 0.45-µm filters, except the IHNV titer was reduced by about 10 fold after filter use (company A). No significant difference was found in the virus titer following centrifugation at 880 × g (30 min) or 3,500 × g (30 min), whereas IHNV and OMV titers were reduced by about 10 and 10–1000 fold by centrifugation at 14,000 × g (30 min) and 14,000 × g (10 and 30 min), respectively. A total of 97.7–99.9% Escherichia coli were eliminated by centrifugation at 880 × g (30 min) and 3,500 × g (30 min). These results show that fish viruses were affected by filtering, even though the effect differed by virus species and filter type. Therefore, centrifugation at 3,500 × g (30 min) and use of medium with antibiotics may be useful for virus extraction along with a reduction in bacteria. Key words: Fish virus, Centrifugation, 0.45 µm filter, Virological examination † Corresponding author: Myung-Joo Oh Tel: 82-61-659-7173; Fax: 82-61-659-7173 E-mail: ohmj@jnu.ac.kr Note