A strategy for cloning infectious molecular clones of retroviruses from serum or plasma

To enable biological characterisation of lentiviral variants which emerge d uring infection and development of AIDS, a method was developed to construc t molecular clones from circulating simian immunodeficiency virus (SIV) par ticles present in as little as 20 mu l of serum from infected rhesus monkey s. This technique uses a long distance RT-PCR method optimised for the ampl ification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones whi ch, after transfection, were able to replicate well in rhesus peripheral bl ood mononuclear cells (PBMCs) and in various human T-cell lines inducing sy ncytia. In addition to the study of molecular cloned virus quasispecies eme rging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clone s may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in th is way can be inserted quickly in new recombinant expression vectors and ma y then be applied for DNA vaccination approaches. (C) 2000 Elsevier Science B.V. All rights reserved.