Optimal Conditions forRecovery oftheHumanImmunodeficiency Virus fromPeripheral BloodMononuclear Cells

Optimal conditions fordemonstrating thepresence ofinfectious human immunodeficiency virusin peripheral bloodmononuclear cells (PMCs)fromseropositive individuals involved cocultivation ofinfected cells withphytohemagglutinin-stimulated PMCsfromseronegative donorsinthepresence of2,ug ofPolybrene perml.Thesize oftheculture vessel also influenced theresults; smaller numbersofinfected cells were detected underconditions ofincreased cell density. Inaddition, an increased normaldonor/patient PMC ratio was helpful. Thecocultivation approach permitted identification ofhumanimmunodeficiency virusin over 90% of seropositive individuals withdifferent clinical conditions. Moreover, reconstruction experiments indicated that this methodallows detection ofone productively infected CD4+cell ina population ofover 106PMCs. Current serological tests, suchastheimmunofluorescentantibody andWestern blot (immunoblot) assays, determine prior exposure tothehumanimmunodeficiency virus (HIV) (14,24).Theydonot,however, indicate thepresence of virus orits replication inthehost. Ultimately, thedefinitive test foranactive infection istherecovery ofHIVfromcells oftheinfected individual. TheHIVprovirus iscopied together withhostcell DNA witheveryroundofreplication, andwheninfected cells divide, theygenerally produce thelargest amounts ofvirus (16, 22). Forthis reason, phytohemagglutinin (PHA), which stimulates DNA replication andcellular proliferation ofT lymphocytes, hasbeenusedtoamplify thenumberof infected cells (presumed tobeprimarily CD4+helper T lymphocytes). Moreover, this mitogen stimulates therelease ofvirus bythecells sothat culture supernatants havelevels detectable bystandard reverse transcriptase (RT)assays (1, 2,5,22).