The presence of JCV in peripheral blood lymphocytes (PBLs) in both viral induced progressive multifocal leukoencephalopathy (PML) and non-PML patients has strengthened the concept that JCV is carried to the brain by a hematogenous route. At the same time, it has focused a part of our studies to develop a quantitative analysis of the level of JCV in these cells and other tissues. It may be important in helping to assess the risk of non-PML patients and to evaluate the affect nucleotide analogs for the treatment of PML. Noncompetitive and competitive or quantitative PCR (QPCR) analysis procedures are being developed to ascertain the level of JCV present in different tissues. Control JCV DNA templates are being constructed to serve as external or internal standards for the quantitation of the virus. These procedures utilize the same primer set used to detect JCV in patient samples by PCR analysis which eliminates the possible differential hybridization kinetics when using different primer sets. A rapid and sensitive chemiluminescent procedure is being used to analyze the PCR products in these studies. An examination of the nucleotide sequences present in the prototype (Mad1) and brain-type (Mad 8B) JCV is being made to assess the role of these sequences in the pathogenicity of JCV. One major difference is the presence of a 23bp sequence which is present in most strains of JCV isolated from brain biopsies from PML patients. This 23bp sequence has a putative binding site for the transcription factor SP1. However, we have found that SP1 binds to this sequence very weakly as seen by competitive binding and DNase 1 protection assays. In addition, construction of CAT reporter plasmids containing either the Mad1 or Mad8B regulatory region has shown that the Mad1 sequence is much more active that from Mad8B. Further studies are underway to understand the role of the 23-bp sequence in the expression of the Mad8B strain in different tissues.