Preparation of an inoculum of white spot syndrome virus for challenge tests in Penaeus japonicus

An inoculum of white spot syndrome virus (WSSV), called penaeid rod-shaped DNA virus (PRDV) in Japan, was prepared for challenge tests in kuruma shrimp Penaeus japonicus. The hemolymph was drawn with PBS from moribund shrimp, which were intramuscularly (IM) inoculated three days before with a virus suspension prepared from naturally affected shrimp. The virus concentration in the hemolymph was quantified as 1.5 * 10E7 genome copies/micro L hemolymph by competitive PCR. The LD50 of the inoculum prepared from the hemolymph stored at -80degC for 40 days was calculated by IM challenges as 10E-4.2 micro L hemolymph/g shrimp or 9.5*10E2 genome copies/g shrimp. Virulence testes were also conducted through IM injections with the virus preparation stocked for 5 and 16 months. As a result, there was no significant difference in cumulative mortalities (p>0.05) among the 3 challenge tests made after preservation of the virus n the hemolymph at -80degC for 40 days, 5 or 16 months.