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White spot syndrome virus (WWSV) has become one of the most widespread causes of mortality in commercial shrimp farming. In the present study, we used PCR to determine the shrimp infectious dose 50% endpoint (SID50 ml-1) of a Chinese isolate of WSSV in 5 different sizes of pathogen-free Litopenaeus vannamei inoculated intramuscularly. The lethal dose 50% endpoint (LD50 ml-1) was also determined from the percentage of dead shrimp. The LD50 ml-1 for 2, 4, 6, 8, and 10 cm shrimp were 104.68, 105.7, 106.70, 107.75, and 108.81, respectively, and the SID50 ml-1 were 104.68, 105.70, 106.90, 107.75, and 108.94, respectively. There was no significant difference between the LD50 ml-1 and SID50 ml-1 for each shrimp size, which indicated that all infected shrimp died. The lethal and infectious titer decreased about 1 log10 as shrimp size decreased 1 grade. These data clearly indicate that adult shrimp were more susceptible to WSSV than juvenile shrimp. The horizontal comparison showed that the amount of virus in the shrimp organs increased over the experimental period. The vertical comparison showed that virus quantity was lowest in the organs of 10 cm shrimp and highest in 2 cm shrimp, which indicates that the smaller shrimp had higher levels of viral replication. Hence, the optimal size for WSSV challenge in shrimp inoculated intramuscularly was 2 cm. The determination of virus titers in different sizes of shrimp represents a step towards creating strategies to reduce the negative impacts of WSSV in the aquaculture industry.