Determination of virus burst size in vivo using a single-cycle SIV in rhesus macaques

Abstract

A single-cycle simian immunodeficiency virus (scSIV) that undergoes only one round of infection and replication was constructed to calculate the total number of virons produced by an SIV-infected cell in vivo. Four Mamu-A*01 rhesus macaques were inoculated on two occasions 11 weeks apart with the scSIV by ex vivo infection and i.v. reinfusion of autologous cells. After each inoculation, plasma viral loads peaked between 1 and 2.5 days and then declined exponentially in one or two phases to below detection limits within 2 weeks. Although higher levels of SIV-specific cytotoxic T lymphocytes and modest increases in antibody responses were observed for each animal after the second inoculation, decay rates of the infected cells were only minimally affected. Analyzing the viral load data with a mathematical model, the in vivo viral burst size averaged 4.0 x 10(4) and 5.5 x 10(4) virions per cell for the first and second inoculations, respectively, with no significant difference between the two inoculations. This estimate, in conjunction with our prior understanding of other quantitative viral and cellular parameters during SIV and HIV infection, provides critical insights into the dynamic process of viral production and its interplay with the infected host in vivo.

Fig. 1.

Plasmid constructs used to generate scSIV. (a) SIVΔenv contains the SIVmac239 proviral genome with a 415-bp deletion at the 5′ end of the env gene. The black box indicates deleted sequences. (b) VSV-G contains the env gene of the VSV. Infectious scSIV were generated by cotransfecting 293T cells with the SIVΔenv and the VSV-G plasmids. Western blot analysis of the protein expression profile in both transfected cells and culture supernatants revealed comparable patterns to those of the wild-type SIVmac239 except for the loss of the envelope glycoprotein gp130 and the addition of the 67-kDa VSV-G glycoprotein (data not shown). In vitro measurements of the scSIV particles in transfected cell supernatants indicated that initial infectivity was conferred by the intact VSV-G on the virion surface. Subsequent rounds of infection were not detected because of the defective env within the virion genome (data not shown).

Fig. 2.

Plasma viral loads and specific immune responses after the first and second inoculations of scSIV-infected PBMCs. SIV RNA levels in the plasma were measured by real-time PCR using SIV gag-specific primers and a molecular beacon. The detection limit of the assay was ≈250 copies per ml. (a) SIV-specific CTL responses to the p11c epitope of SIV Gag were determined by tetramer staining. (b) Antibody responses to SIV p27 and gp130 were measured by ELISA.

Fig. 3.

Decay curves of scSIV-infected PBMCs. Plasma viral load data were fitted by using a nonlinear least-square method to determine the decay rate of productively infected cells. The best-fit theoretical curves to the viral load changes are presented. After the peak of viremia, viral load decays adhered to either a two-phase or a single-phase decay model as determined by the F test, a statistical test that compares the “goodness” of fit between the two models. The horizontal line through each graph indicates the detection limit of the real-time RT-PCR assay (≈250 copies per ml).