Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.
Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus. Similarly, spliced HIV-1 mRNA could be detected within 24 h of infection of CEM cells by HIV-1IIIB. This NASBA-based infection assay was shown to titer the neutralization of the HIV-1IIIB isolate by serum from an infected human and by a monoclonal antibody to gp120. Furthermore, the inhibitory effects of azidothymidine (AZT) and soluble CD4 on HIV-1IIIB infection were quantitated by this assay. The early detection of virus by NASBA minimizes the contribution of secondary infection, thereby permitting more accurate evaluation of antiviral agents and neutralizing antibodies. This assay may be useful for the study of infection of phenotypically distinct HIV-1 isolates, which differ in terms of their replication kinetics.
Romano
J W
JW
Advanced BioSciences Laboratories Inc., Kensington, MD 20895, USA.
Shurtliff
R N
RN
Sarngadharan
M G
MG
Pal
R
R
D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015704:CD4 Antigens / Q000494:pharmacology; D002478:Cells, Cultured; D017931:DNA Primers; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D021141:Nucleic Acid Amplification Techniques; D012326:RNA Splicing; D012367:RNA, Viral / Q000032:analysis*; D015215:Zidovudine / Q000494:pharmacology
D000914:Antibodies, Viral; D015704:CD4 Antigens; D017931:DNA Primers; D012367:RNA, Viral; D015215:Zidovudine