Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea

Abstract

White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen with a broad host range. Among the hosts, though mud crab, Scylla olivacea is reported to be more susceptible to WSSV than S. serrata and S. paramamosain, a detailed study on the pathogenicity and genome stability of the virus after multiple passages has yet to be reported. Firstly, to test the pathogenicity of the virus, WSSV was intramuscularly injected into healthy shrimp, Penaeus vannamei. Experimentally infected P. vannamei showed the first mortality at 36 h post-injection (hpi), followed by 100 % cumulative mortality in 7 days post-injection (dpi). However, S. olivacea injected with the WSSV inoculum derived from infected shrimp showed the first mortality at 48 hpi and a cumulative mortality of 70 % at the end of the ten days experiment. Subsequently, WSSV was sequentially passaged five times in Scylla olivacea to find out any change in the virulence of the virus in each passage. S. olivacea groups injected with 1st, second, third and fourth passages derived from the crab recorded the first mortality between 48 and 56 hpi and the cumulative mortality of 60 to 70 % at the end of the ten days experiment. Injection of WSSV inoculum in P. vannamei derived from multiple passages in S. olivaceae revealed the retention of the pathogenicity of the virus. Shrimp groups injected with WSSV derived from different passages showed first mortality between 24 and 36 hpi and cumulative mortality of 100 % between 6 and 7 dpi. The average viral load in the shrimp groups injected with WSSV inoculum derived from shrimp was 3.6 × 108, whereas in shrimp injected with the inoculum derived from 1st, third and fifth passages from crab showed 4.0 × 108, 4.7 × 108 and 4.3 × 108 copies per 100 ng DNA. Histological examination of the gill and stomach tissue of shrimp injected with inoculum prepared from shrimp as well as the inoculum derived from 1st, third and fifth passages in S. olivacea revealed characteristic pathological manifestations of the WSSV infection in gill and stomach tissues such as hypertrophied nuclei, Cowdry A-type inclusions as well as massive basophilic intranuclear inclusions. Further, to study the genome stability, the primers targeting highly variable regions of the WSSV genome (ORF94, ORF125, ORF75, variable region (VR) 14/15 and VR 23/24) were used to amplify WSSV derived from different passages and the amplified PCR products were sequenced. The sequence analysis revealed the WSSV genome stability after multiple passages in mud crab, S. olivacea.