High specific infectivity of plasma virus from the pre-ramp-up and ramp-up stages of acute simian immunodeficiency virus infection

Abstract

To define the ratio of simian immunodeficiency virus (SIV) RNA molecules to infectious virions in plasma, a ramp-up-stage plasma pool was made from the earliest viral RNA (vRNA)-positive plasma samples (collected approximately 7 days after inoculation) from seven macaques, and a set-point-stage plasma pool was made from plasma samples collected 10 to 16 weeks after peak viremia from seven macaques; vRNA levels in these plasma pools were determined, and serial 10-fold dilutions containing 1 to 1,500 vRNA copies/ml were made. Intravenous (i.v.) inoculation of a 1-ml aliquot of diluted ramp-up-stage plasma containing 20 vRNA copies infected 2 of 2 rhesus macaques, while for the set-point-stage plasma, i.v. inoculation with 1,500 vRNA copies was needed to transmit infection. Further, when the heat-inactivated set-point-stage plasma pool was mixed with ramp-up-stage virions, infection of inoculated macaques was blocked. Notably, 2 of 2 animals inoculated with 85 ml of a pre-ramp-up plasma pool containing <3 SIV RNA copies/ml developed SIV infections characterized by high levels of viral replication, demonstrating that "vRNA-negative" plasma collected from macaques in the pre-ramp-up stage is infectious. Furthermore, there is a high ratio of infectious virions to total virions in ramp-up-stage plasma (between 1:1 and 1:10) and a lower ratio in set-point-stage plasma (between 1:75 and 1:750). Heat-inactivated chronic-stage plasma can "neutralize" the highly infectious ramp-up-stage virions. These findings have implications for the understanding of the natural history of SIV and human immunodeficiency virus infection and transmission.

FIG. 1.

Characteristics of the plasma samples, collected from donor animals negative for SIV RNA in plasma, that were used to produce the pre-ramp-up plasma pools. (A to D) Plasma vRNA levels at the time each sample was collected (circled time points) for use in the pool identified above each panel. Note that all the donor animals were vaginally inoculated weekly with 10³ TCID₅₀ of SIVmac251. The animals contributing plasma to pools A and B were necropsied prior to the onset of viremia, while the animals contributing plasma to pool C became viremic prior to necropsy. Plasma “pool” D was a single aliquot of plasma collected from a single animal during a blip in plasma vRNA (731 vRNA copies/ml plasma) that was detected 5 weeks prior to more-typical ramp-up viremia. (E to G) Detailed characterization of the samples and animals contributing plasma to pool C. (E) Relationship between the onset of ramp-up-stage viremia and the number of samples contributed to the pool. (F) Relationship between the onset of ramp-up-stage viremia and the volume of plasma contributed to the pool. (G) Relationship between the total volume of pool C and the volume of plasma contributed by each donor animal.

FIG. 2.

Plasma vRNA levels in SIV-naïve recipient animals after intravenous infusion of the pre-ramp-up-stage plasma pools. As indicated in each panel, relatively large volumes of plasma pools A to C were transferred to the naïve animals, and these plasma pools contained <3 copies of vRNA/ml, which is the limit of detection for our assay. However, plasma “pool” D consisted of only 2 ml of plasma, with 731 vRNA copies/ml, collected at one time from a single animal. Both animals that received 85 ml of vRNA⁻ pre-ramp-up-stage plasma pool C became infected and developed persistently high levels of vRNA in plasma.

FIG. 3.

vRNA⁺ plasma samples used to produce the ramp-up-stage plasma pool and outcome of challenge of recipient animals with the serially diluted ramp-up-stage plasma pool. (A) Plasma vRNA levels in donor animals that were vaginally inoculated twice in one day with 10⁵ TCID₅₀ of SIVmac251 or weekly from 0 to 13 weeks with 10³ TCID₅₀ of SIVmac251 until infection was detected. Each sample used to make up the ramp-up-stage pool is circled. (B) Plasma vRNA levels in SIV-naïve recipient animals after i.v. infusion of the ramp-up-stage plasma pool. While 1 animal inoculated i.v. with 2 SIV RNA copies (animal 32970) did not become infected, 2 of 2 animals inoculated i.v. with 20 SIV RNA copies (animals 33815 and 35036) did become infected. These two animals had a typical pattern of viremia after the plasma transfer.

FIG. 4.

vRNA⁺ plasma samples used to produce the set-point-stage plasma pool and outcome of challenge of recipient animals with the serially diluted set-point-stage plasma pool. (A) Plasma vRNA levels in donor animals that were vaginally inoculated weekly with 10³ TCID₅₀ of SIV mac251 at the time each sample was collected (circled time points) for use in the set-point-stage pool. (B) Plasma vRNA levels in SIV-naïve recipient animals after i.v. infusion of the set-point-stage plasma pool. Neither of two animals became infected after i.v. inoculation with 1.5 SIV RNA copies or 15 SIV RNA copies, and none of three animals inoculated i.v. with 150 SIV RNA copies became infected. However, all three animals (animals 33952, 34846, and 34373) became infected after i.v. inoculation with 1,500 SIV RNA copies. Two of the animals had a typical pattern of viremia, and one animal (animal 34846) had a delay to detection of plasma vRNA after the plasma transfer. (C) Kaplan-Meier survival analysis to compare the infection rates in macaques after inoculation with the serial 10-fold dilutions of the ramp-up-stage and set-point-stage plasma pools. Note that the number of SIV RNA molecules required for establishing infection is significantly higher for the set-point-stage plasma pool (P = 0.0027) than for the ramp-up-stage plasma pool.

FIG. 5.

Plasma vRNA levels in SIV-naïve recipient animals after i.v. infusion of 20 vRNA molecules from the pre-ramp-up-stage plasma pool mixed with the heat-inactivated set-point-stage plasma pool. One animal (animal 36068) became infected after i.v. inoculation with 20 vRNA copies from the ramp-up-stage pool mixed with 0.5 ml of heat-inactivated plasma from a SIV-naïve monkey, but of two animals inoculated i.v. with a mixture of 20 SIV RNA molecules from a ramp-up-stage pool and 0.5 ml of the heat-inactivated set-point-stage plasma pool (animals 33681 and 32350), both remained uninfected, and one animal (animal 32970) inoculated i.v. with only the heat-inactivated set-point-stage plasma pool remained uninfected.