A pair of primers created from information of PmNOBⅢ genome DNA Sal I fragment produced a 355bp band by using Penaeus chinensis non occluded baculovirus (PcNOBV),the WSBV isolate from P.hinensis in China's Mainland,as the DNA template.The specific PCR product was cloned,sequenced and labeled with digoxigenin (DIG)DNA labeling kit(Boehringer Mannheim).The DIG labeled fragment was tested by dot blot hybridization for sensitivity and specificity with purified PcNOBV nucleocapsid,PcNOBV infected shrimp tissues and healthy shrimp tissues.The detection limit of the DNA probe is 6.8pg of purified PcNOBV DNA.No hybridization signals were observed using DNA from healthy shrimp as template.Healthy P.chinensis,artificially infected P.chinensis and pond reared adult P.chinensis were screened for PcNOBV infection by both PCR and the hybridization assay.The results showed a good relationship between PCR and the hybridization assay.These findings demonstrate that the DIG labeled probe can be used as a sensitive,specific and cost effective reagent for detection of PcNOBV.
In 1996,a serious epizootic called white spot syndrome caused high mortality of cultivated Penaeus chinensis in Qingdao from June to August.Electron microscopy and reinfection test showed that Penaeus chinensis non occluded baculovirus (PcNOBV),the WSBV isolate from P.chinensis in China's Mainland,is the major causative agent of the epizootic.Meanwhile,reovirus like particles were observed in some of the diseased P.chinensis from the following sample tissues:gill,stomach,foregut and ovary.The reovirus like particles could sometimes be found infecting the same shrimp or the same tissue and even the same cell with PcNOBV.The viral particles were spherical with double shelled structure characteristic of reovirus without envelope and located in the cytoplasm of infected tissue cells.The diameter of the particle varies in different tissues ranging from 80 110nm.The size of inner core is 60nm.Based on its morphologic characteristics,the virus is assumed to be a reovirus and named Penaeus chinensis reo like virus(PcREO)temporally.It’s not clear currently what roles does it play in the occurrence of the epizootic.However,the facts of systemic infection of the virus in P.chinensis and dual infection with PcNOBV strongly indicate that PcREO might contribute to the occurrence of the epizootic.Further research is required to determine its characteristics of pathogenicity,biology and molecular biology.
An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.
