Journal of Virological Methods 115 (2004) 199–205 Viral growth assay to evaluate the replicative capacity of HIV-1 isolates Emanuele Nicastri b , Loredana Sarmati a , Luca Dori a , Marco Montano a , Gabriella d’Ettorre d , Anna Rita Buonomini a , Saverio G. Parisi c , Ercole Concia c , Vincenzo Vullo d , Massimo Andreoni a,∗ a Department of Public Health and Cellular Biology, University of Rome Tor Vergata, via di Tor Vergata 135, Rome 00133, Italy b National Institute of Infectious Diseases, IRCCS L. Spallanzani, Rome, Italy c Institute of Immunology and Infectious Diseases, University of Verona, Verona, Italy d Department of Infectious Diseases, University of Rome La Sapienza, Rome, Italy Received 12 June 2003; received in revised form 29 September 2003; accepted 29 September 2003 Abstract The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes. A cross sectional study was carried out on 10 HIV-infected na¨ ıve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 na¨ ıve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve. A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r = 0.63, P< 0.001 and r = 0.67, P< 0.001, respectively). Although all patients failing therapy had strains with impaired replicative capacity, a wide range of values (0.1–74.5%) was detected. All strains with a replicative capacity above 10% had non-nucleoside reverse transcriptase inhibitors related mutations. A viral assay to evaluate the HIV replicative capacity is described. The high variability of replicative capacity confirms the need to undertake replicative capacity assay using the whole virus. © 2003 Elsevier B.V. All rights reserved. Keywords: Viral growth assay; Replicative capacity; Drug resistance; HIV 1. Introduction The emergence of drug resistance is often associated with impaired viral replication compared to wild type viruses. The selection of drug resistant mutants during antiretroviral ther- apy reflects the effect of a mutation on the drug activity and on the viral replicative capacity. The genetic polymorphisms inside and outside the protease gene influence the replication competence of drug-resistant mutants by affecting the abil- ity to accommodate resistance mutations (Mammano et al., 1998; Nijhuis et al., 1999). Furthermore, in the clinical set- ting, the fitness of any one viral strain depends on the ac- cumulation of mutations in the protease and in the reverse ∗ Correspnding author. Tel.: +39-06-72596873; fax: +39-06-72596873. E-mail address: andreoni@uniroma2.it (M. Andreoni). transcriptase gene, as well as on any other selection (e.g., immunological) the virus is exposed to within the host en- vironment (Nijhuis et al., 2001). It has been reported that in patients failing highly active antiretroviral therapy, mu- tations of the p7/p1 and p1/p6 Gag protease cleavage sites, linked to protease inhibitor exposure, can influence viral protein incorporation and have effect on the viral replicative capacity (Maguire et al., 2002). Recent data suggest that the cleavage site mutation is necessary but not sufficient for full recovery of viral fitness and additional gag mutations are required (Myint et al., 2002). In patients failing highly active antiretroviral therapy (HAART) an impaired replicative capacity of HIV may provide a good explanation for the discordant clinical re- sponses, characterized by sustained CD4 cell increases despite virological failure (d’Ettorre et al., 2002). Although careful biochemical analysis of enzyme activity in the 0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.09.032