Attenuated marmoset herpesvirus isolated from recombinants of virulent marmoset herpesvirus and hybrid plasmids.

Marmoset herpesvirus (MarHV) deletion mutants in the thymidine kinase (TK) gene were isolated after infection of OMK cells with TK+ MarHV DNA and the hybrid plasmid, pMAR401, which lacks a 2.6-kb KpnI-M fragment in the coding region of the MarHV TK gene. After plaque purification in TK- HeLa(BU25) cells, the DNA's from five recombinant araT-resistant MarHV clones were analyzed with restriction nucleases to verify that the 2.6-kb KpnI-M fragment (and a 0.9-kb BglII-Q fragment) were deleted from the viral DNA's. Molecular hybridization experiments using 32P-labeled pMAR4 probes and viral DNA fragments also showed that the recombinant viral DNA's lacked the KpnI-M and BglII-Q fragments, that BamHI-I of parental virus was shortened, and that three new HindIII fragments replaced the parental virus HindIII-G fragment. The recombinants did not induce TK activity in LM (TK-) cells. To study the relative virulence of the recombinants, 3-week-old Swiss mice were injected intracerebrally (Ic) or subcutaneously (Sc) in the sacro-lumbar region with either parental or recombinant viruses. The LD50 for the parental virus was 3 p.f.u. (Ic) and 7,600 p.f.u. (Sc). The recombinant viruses were significantly less virulent than TK+ MarHV after Ic inoculation (LD50 of 62,000 and 32,000 p.f.u., respectively, for viruses 5D-6B and 5D-4B) and gave no fatalities after Sc inoculation. Mice surviving TK- MarHV infections were protected from challenge with TK+ parental MarHV. Recombinant TK- MarHV's may be useful as vectors for the expression of foreign genes in animal cells and as the starting material for the design of vaccines.