POLYDNAVIRUS OF THE PARASITIC WASP CHELONUS-INANITUS (BRACONIDAE) - CHARACTERIZATION, GENOME ORGANIZATION AND TIME POINT OF REPLICATION

Ultrastructural analysis of the polydnavirus of the braconid wasp Chel onus inanitus revealed that virions consist of one cylindrical nucleoc apsid enveloped by a single unit membrane. Nucleocapsids have a consta nt diameter of 33.7 +/- 1.4nm and a variable length of between 8 and 4 6 nm. Spreading of viral DNA showed that the genome consists of circul ar dsDNA molecules of variable sizes and measurement of the contour le ngths indicated sizes of between 7 and 31 kbp. When virions were expos ed to osmotic shock conditions to release the DNA, only one circular m olecule was released per particle suggesting that the various DNA mole cules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate g enome size is in the order of 200 kbp. By partial digestion of viral D NA with HindIII or EcoRI in the presence of ethidium bromide and subse quent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfect ion into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clone s were obtained. Southern blots revealed that complete and unrearrange d segments were cloned with this approach, and restriction maps for fi ve segments were obtained. Part of a 16.8 kbp segment was sequenced, f ound to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the cou rse of pupal-adult development of the wasp was investigated and seen t o be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initi ate at a specific stage of pupal-adult development.