Human Immunodeficiency virus (HIV) Antiviral Services


HIV antiviral assays are run on a daily basis at our facilities. RetroVirox staff has decades of experience in the use of in vitro antiviral assays. Multiple cell-based assays to evaluate anti-HIV compounds are available, including replication assays with primary (PBMC cells) or transformed T cell lines, and use of reporter T cell lines to monitor virus infectivity. In parallel with antiviral assays, our services include evaluation of compound’s cytotoxic effects in several uninfected cells. We utilize a variety of reporter genes and readouts adapted to the specific needs of each client’s assays. In addition, we offer a number of customized assays to investigate the mechanism of action of candidate antivirals, such as the emergence of resistant strains and characterization of viral targets. We also perform screening of small molecule libraries with several cell-based assays developed in-house or following the client’s needs. All the antiviral assays include the presence of positive controls (known ARV inhibitors).


Anti-HIV efficacy in primary cells and T cell lines

Cell-based antiviral assays are performed with either transformed T cell lines (SupT1, H9, Molt4), primary blood-derived mononuclear cells (PBMCs) or macrophages. PBMC cells are prepared, stimulated and then infected with HIV laboratory-adapted or primary isolates utilizing different coreceptors.  Experimental readout of HIV replication can be performed by ELISA of p24 viral antigen, monitoring reverse transcriptase, reporter virus or by intracellular or surface staining of viral antigens (e.g. Gag protein or Env, respectively). Antiviral assays can be performed with a set of primary and laboratory adapted HIV isolates (displaying X4, R5 or dual tropism), and virus resistant to each family of ARVs.  Multi-drug resistant isolates are also available. In vitro antiviral assays can be performed at low and medium throughputs (up to 100,000 molecules).


Single-cycle infectivity assays

A variety of stable reporter cell lines expressing HIV receptor and coreceptors are used to evaluate infectivity of HIV or HIV-based pseudotypes using single cycle or quasi-single cycle assays. We also offer multi-tiered assays to evaluate the antiviral activity of late-stage antiviral compounds (e.g. maturation inhibitors). These latter assays require production of viral particles in compound treated cells and subsequent analysis of infectivity in permissive cells in the absence of compound.


In vitro models for HIV latency

Several assays are available to evaluate the ability of small molecules and biologics to reactivate viral replication from latently infected cells. Our assays utilize latent primary cells that upon treatment become reactivated and produce HIV virus. Unlike other latency assays, viruses recovered from these cells are functionally active and fully replication competent.


Mechanism of action studies

Studies to address the mechanism of action of anti-HIV compounds are available. Among these studies we offer “time of addition” assays in synchronized infections, evaluation of viral entry inhibition, determination of HIV reverse transcriptase activity, integrase assays and several assays to determine whether the inhibitor acts at late stages of the virus cycle. Experiments aimed at characterizing the molecular target of candidate inhibitors are also offered.


Resistance Selection and Characterization 

Selection of resistant variant virus by serial passage in the presence of escalating concentration of antiviral compound is routinely performed. Resistant selection may be carried out in either transformed or primary cells with a variety of viral isolates. Selections for multidrug resistant variants are also available using a combination of antiviral compounds including approved drugs likely to be used in combination therapy in the clinic.  Characterization of resistant variants includes determination of genotypic and phenotypic variations.


Combination therapy

Assays evaluating synergistic efficacy with candidate antiviral compounds in combination with approved or clinical stage compounds are available. Combination studies are performed with multiple classes of drug including nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, proteinase inhibitors, entry inhibitors and integrase inhibitors. Efficacy of combination antivirals is evaluated as synergistic, additive or antagonistic using software packages including Prichard and Shipman MacSynergy II and Chou and Talalay median dose effect (Calcusyn).


Pseudovirions to evaluate viral entry

Pseudotyping HIV virus with heterologous viral envelope proteins can be used as a surrogate assay to evaluate viral entry in a number of viruses. These studies have been used to identify and study candidate viral entry inhibitors against HCV, Lassa Fever, Dengue virus, West Nile virus, Ebola, Yellow Fever, highly pathogenic avian influenza and many other infectious agents for which entry assays are difficult to perform because they are not available, or when available, they require the use of highly infectious virus under strict Biosafety level conditions (BSL3 or BSL4).

Additional HIV assays

Other available assays include evaluation of HIV protease activity, entry assays, integration assays, antibody neutralization assays, coreceptor determination, HIV-induced downmodulation of CD4 and class I MHC, anti-HIV activity in chronically infected cells (with established HIV replication), evaluation of CPE effect in infected cells (syncytia formation, apoptosis, direct killing). These assays will help address the mechanism of action of lead compounds. RetroVirox also offers customized assays adapted to the specific needs of our clients. Additional information on these and other assays are available upon request. Our staff will work with you to develop additional assays appropriate to your needs.

For additional information about HIV antiviral services call us at (858) 677-9315 or contact us at

To see more information about antiviral assays offered against other viruses click on the following links:

Influenza and Parainfluenza

Human Respiratory Syncytial Virus (HRSV)

Dengue Virus (DENV)


Human Cytomegalovirus (HCMV)

Human Rhinovirus (HRV)

Hepatitis B Virus


Multi-virus panel

HTS campaigns with live virus