A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.
To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.
Holterman
L
L
Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands.
Dubbes
R
R
Mullins
J
J
Haaijman
J
J
Heeney
J
J
D016915:AIDS Vaccines / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000235:genetics; D006678:HIV / Q000235:genetics; D006801:Humans; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D013601:T-Lymphocytes / Q000821:virology; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics; D014766:Viremia / Q000821:virology; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D016915:AIDS Vaccines; D004279:DNA, Viral; D019444:Vaccines, DNA