Research ArticleAIDS/HIVVirology Open Access | 10.1172/jci.insight.183751

Kevin D. Raehtz,1 Cuiling Xu,1,2 Claire Deleage,3 Dongzhu Ma,2 Benjamin B. Policicchio,4 Egidio Brocca-Cofano,2 Daniele Piccolo,5 Kathryn Weaver,1 Brandon F. Keele,3 Jacob D. Estes,3,6 Cristian Apetrei,2,4 and Ivona Pandrea1,4

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Raehtz, K. in: JCI | PubMed | Google Scholar |

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Xu, C. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Deleage, C. in: JCI | PubMed | Google Scholar |

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Ma, D. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Policicchio, B. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Brocca-Cofano, E. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Piccolo, D. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Weaver, K. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Keele, B. in: JCI | PubMed | Google Scholar |

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Estes, J. in: JCI | PubMed | Google Scholar |

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Apetrei, C. in: JCI | PubMed | Google Scholar

1Department of Pathology and

2Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

3AIDS and Cancer Virus Program, Frederick National Laboratory of Cancer Research, Frederick, Maryland, USA.

4Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

5Università di Pavia, Udine, Italy.

6Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon, USA.

Address correspondence to: Cristian Apetrei, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, 843 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. USA. Phone: 412.383.1272; Email: apetreic@pitt.edu. Or to: Ivona Pandrea, Department of Pathology, University of Pittsburgh, 739 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA. Phone: 412.383.5834; Email: pandrea@pitt.edu

Authorship note: CA and IP contributed equally to this work.

Find articles by Pandrea, I. in: JCI | PubMed | Google Scholar

Authorship note: CA and IP contributed equally to this work.

Published December 6, 2024 - More info

Published in Volume 9, Issue 23 on December 6, 2024
JCI Insight. 2024;9(23):e183751. https://doi.org/10.1172/jci.insight.183751.
© 2024 Raehtz et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published December 6, 2024 - Version history
Received: June 10, 2024; Accepted: October 11, 2024 View PDF Abstract

African green monkeys (AGMs) are natural hosts of SIV whose infection does not progress to AIDS. Since early events of infection may be critical to pathogenesis in nonnatural hosts, we investigated early SIV infection in 29 adult male AGMs intrarectally inoculated with SIVsab92018 (SIVsab) and serially sacrificed throughout acute into early chronic infection to understand patterns of viral establishment, dissemination, and their effect on disease progression. Using this model, we showed that foci of virus replication could be detected at the site of inoculation and in the draining lymphatics as early as 1–3 days postinfection (dpi). Furthermore, testing with ultrasensitive assays showed rapid onset of viremia (2–4 dpi). After systemic spread, virus was detected in all tissues surveyed. Multiple transmitted/founder viruses were identified, confirming an optimal challenge dose, while demonstrating a moderate mucosal genetic bottleneck. Resident CD4+ T cells were the initial target cells; other immune cell populations were not significantly altered at the site of entry. Thus, intrarectal SIVsab infection is characterized by swift dissemination of the virus, a lack of major target cell recruitment, and no window of opportunity for interventions to prevent virus dissemination during the earliest stages of infection, similar to intrarectal transmission but different from vaginal transmission in macaques.

Introduction

African nonhuman primates (NHPs) are the natural hosts of SIVs, with over 40 different SIVs known to circulate among a variety of NHP species in the wild (1, 2). Unlike Asian macaques, which are not naturally infected by SIVs (3–6) and progress to AIDS after SIV infection, African NHPs rarely show signs of disease progression to AIDS (7), and they generally maintain the homeostasis of their immune cell populations and effective immune responses despite active viral replication (7–9). This lack of disease progression is not due to some inherent lack of virulence of the infecting SIV strains, since chronic, high levels of viral replication equal to or greater than the levels typically observed in persons living with HIV persist for the remaining lifespan of the African hosts (10–13). Instead, natural SIV hosts have evolved various adaptations (14–16) to better manage the detrimental effects of SIV infection, rather than direct control or clearance of the virus by exquisite host immune responses (2, 17–24).

Previous research has indicated that the mechanisms by which natural hosts might avoid disease progression occur very early on during SIV infection, probably within days following transmission (17, 24–29), at the mucosal sites of viral entry and the initial sites of viral replication. However, relatively little has been known about the earliest events of mucosal transmission in natural hosts, such as African green monkeys (AGMs). Most of our knowledge of the early events of SIV mucosal transmission was derived from studies of intravaginal transmission of SIVmac in rhesus macaques (RMs) (30–35). These studies show that the virus must first cross the epithelial barrier, which is a significant impediment to transmission (30, 34, 36–38). After crossing the epithelium, the virus enters the submucosa and infects a small number of target cells. This initial target cell population is composed of CD4+ T cells, which can be more numerous at mucosal sites than other resident immune cells types (39). However, the initial target cell density is generally still not high enough to sustain long-term viral replication (R0 < 1). Thus, more CD4+ T cells must be recruited to the initial foci of replication by the virus-induced innate and inflammatory immune responses (37). Local dissemination through lymphatic drainage leads to even greater viral expansion when the virus reaches the draining lymph nodes (LNs), where there is an extremely high concentration of densely packed cells that might be targeted by the virus. Lymphatic dissemination also allows the virus to move into the bloodstream through the thoracic duct and thereby enter systemic circulation (30, 31). Several studies of SIVmac intrarectal (IR) transmission in RMs indicate that the virus follows the same general pattern of dissemination after IR exposure, albeit with different timing (40, 41).

The macaque vaginal model of mucosal transmission also predicts that the combination of the epithelial barrier, the paucity of resident CD4+ T cells in many regions of the vaginal mucosa, and the antiviral immune responses imposes a severe “mucosal bottleneck” on the number of transmitted/founder (T/F) viral variants that can successfully establish infection (42). Indeed, among 80% of heterosexual transmissions of HIV, only a single T/F genotype was identified to establish systemic infection (43, 44). Similarly, only 1–2 SIV T/F variants are normally transmitted in either adult or juvenile AGMs (26). Likewise, only a single T/F virus was found to establish infection in multiple AGMs in the wild (45). This implies that the general features of mucosal transmission are similar in natural hosts (i.e., AGMs) and pathogenic hosts (i.e., humans and macaques).

Nevertheless, there are several important characteristics of natural hosts that set them apart from pathogenic hosts that can affect transmission. Most notably, AGMs maintain significantly lower levels of CCR5+CD4+ T cell targets in blood, LNs, and mucosal sites than seen in humans and RMs (22, 46). In wild AGMs, while this reduced mucosal target cell availability is not sufficient to prevent vaginal SIVsab92018 (SIVsab) transmission to adults, it appears to limit oral transmission to juveniles and infants; these groups have even lower target cell levels than adults and exceedingly rare instances of SIV transmission, even during frequent breastfeeding (26, 46, 47). The target cell availability at the mucosal sites determining transmission efficacy is further supported by the observation that pig-tailed macaques, which have higher levels of CCR5+CD4+ T cells at mucosal sites than AGMs, can be IR infected with a dose of SIVsab 1 log lower than those needed to infect the adult AGMs (26).

To better understand the barriers to SIV transmission and the immunological determinants of early SIVsab IR infection and disease progression, we performed a detailed characterization of the early events occurring at the site of virus entry following IR inoculation in Caribbean AGMs, employed as a model of natural SIV infection (48, 49). Our goal was to characterize both the virological and immunological events of the earliest stages of mucosal transmission in AGMs, particularly at the site of inoculation. To this end, we used quantitative PCR (qPCR) analysis of both plasma and tissue sites proximal and distal to the sites of entry to track viral dissemination throughout the body from the site of inoculation. We also performed a thorough dissection of the rectum and distal colon to identify: (a) the initial foci of viral replication; (b) the population of founder cells in the mucosa; (c) changes in immune activation, immune cell recruitment, or cell death at the mucosal site; and (d) the mucosal bottleneck of virus transmission, by enumerating the T/F viral variants. We report that, upon IR inoculation, despite the limited mucosal target cell availability, SIVsab became established and disseminated systemically almost immediately. Meanwhile, the immune response to viral infection remained minimal, both at the site of inoculation and at more distal sites. Taken together, these findings indicate that virus amplification and spread occurred virtually concomitantly upon IR SIVsab challenge of AGMs, with no feasible window of opportunity for interventions aimed at preventing systemic infection.

Results

Study design. To thoroughly characterize the very earliest stages of viral replication and dissemination, 29 adult male AGMs were IR challenged with 1 × 107 copies of SIVsab. The inoculum consisted of diluted plasma collected from an acutely infected AGM, which had been established to be effective in a preliminary study and was shown to contain T/F variants (26). Twenty-seven of the 29 SIV-challenged AGMs became infected. The remaining 2 AGMs (AGM137 and AGM16) were not infected and therefore were excluded from all analyses. Infected AGMs were euthanized at well-defined time points covering both acute and early chronic SIV infection. They were divided into 5 groups based on their predicted viral loads (VLs): (a) preinfection (baseline); (b) pre–ramp-up (1–3 days postinfection [dpi]); (c) ramp-up (4–6 dpi); (d) peak (9–12 dpi); and (e) set point (46–55 dpi). Four unchallenged AGMs were included as a control group (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.183751DS1).

At the time of each necropsy, numerous compartments were sampled from each AGM. The collected tissues were processed for qPCR, histology, and flow cytometry (blood, gut, and LNs only). As the site of inoculation, the entire rectum and distal colon were excised and dissected into small (1 × 1 cm2) segments for qPCR and histology. Using this overall strategy, we increased the likelihood of capturing a rare instance of early viral replication at the site of inoculation, while being able to monitor the presence of virus throughout the body.

Rapid onset of viremia upon IR SIVsab challenge of AGMs. We monitored the early dynamics of viremia with a single copy assay (SCA), which has a theoretical limit of 1 viral RNA (vRNA) copy/mL (cp/mL) (50). At 4 dpi, 2 of 3 AGMs had VLs above 1 cp/mL (Figure 1). Beyond 5 dpi, all AGMs were viremic. In addition, 2 AGMs were viremic at 2 and 3 dpi, but virus was only detectable by testing very large volumes of plasma (AGM122 at 3 dpi had 5.8 × 10–1 vRNA cp/mL and AGM124 at 2 dpi had 1.1 × 10–1 vRNA cp/mL, respectively). The average plasma VLs then peaked between 9 and 12 dpi at 1 × 105 to 1 × 108 (geometric mean [GM], 20 × 106) vRNA cp/mL. Finally, set point VLs were controlled to between 1 × 104 and 1 × 105 (GM, 3.6 × 104) vRNA cp/mL. Note that the set point group AGMs were also sampled at 12 dpi and tested within the same range as the peak group AGMs.

Figure 1

Plasma VLs in African green monkeys (AGMs) intrarectally (IR) infected with SIVsab. The plasma VLs are shown as log10 values. Each AGM that became infected after inoculation (n = 27) is represented by symbol with a unique color and shape combination as shown on the right. Blood samples from the chronically infected AGMs (yellow) were sampled both at peak infection and during early chronic infection, as indicated by the appearance of the symbols for those AGMs twice and the dotted lines connecting the same animal. Limits of detection for conventional and single-copy assays are shown by dotted horizontal lines, with the area below the SCA limit shaded in gray. The colors represent the different stages of infection, as defined by viral replication status: pre–ramp-up (green), ramp-up (blue), peak (red), and set point (yellow). The bars represent the geometric mean and SD of each group. AGMs that were negative for viremia are plotted directly on the x axis.

Cerebrospinal fluid (CSF) exhibits similar, but generally lower, VLs to plasma. Due to limited volume, CSF was assessed by conventional qPCR but not SCA, which showed VLs with GMs of 6.0 × 104 vRNA cp/mL at 9 dpi and 8.0 × 106 vRNA cp/mL at 12 dpi, respectively. The VLs of the set point samples ranged from 1 × 103 to 1 × 104 (GM 8 × 103) vRNA cp/mL. Two other CSF samples tested positive: AGM125 with 5 × 102 vRNA cp/mL at 4 dpi and AGM136 with 5.0 × 102 vRNA cp/mL at 5 dpi (Figure 2). Therefore, CSF testing supported the rapid systemic dissemination of the virus and further spread into the CSF, though it was unclear if the virus first entered the CSF through the blood or the lymphatics. Previous studies in macaques and more recent studies in humans have shown that, while VLs are typically lower in the CSF, the overall dynamics of viral burden in the CSF can mirror what was observed in the plasma in this study (51–54).

Figure 2

Cerebrospinal fluid (CSF) SIVsab viral loads in African green monkeys (AGMs) during acute and early chronic infection. The CSF viral loads are shown as log10 values. Each infected AGM (n = 27) is represented by a unique color and shape combination as shown on the right. The limit of detection for conventional qPCR (30 viral RNA copies/mL plasma) is indicated by a dotted line. The colors represent the different stages of infection as defined by viral replication status: pre–ramp-up (green), ramp-up (blue), peak (red), and set point (yellow). The bars represent the geometric mean and geometric SD of each group. AGMs that were negative for viremia are plotted directly on the x axis.

Rapid systemic dissemination of SIVsab from the site of inoculation. To examine the tissue replication kinetics, both within the site of inoculation and distally, vRNA (Figure 3A) and vDNA (Figure 3B) from multiple tissues were quantified via qPCR. In sections collected from AGMs in the pre–ramp-up group (1–3 dpi), the initial foci of replication yielded both detectable vRNA and vDNA, with the vRNA being more readily detectable (15 vRNA+ tissues versus 9 vDNA+ tissues). The pre–ramp-up group yielded multiple (AGM25, AGM13, AGM124, and AGM122) rectum or distal colon sections that contained either vRNA or vDNA, while AGM126 yielded only 1 vRNA+ section. Furthermore, in the pre–ramp-up AGMs, SIVsab vRNA was additionally detected in the draining colonic LNs (AGM15, AGM122), along with a sole instance of vRNA in the PBMCs (AGM123). We also detected vRNA in the colonic LNs of the ramp-up group (AGM125, AGM128) and the duodenum (AGM125), while vDNA was found solely at the site of inoculation. Taken as a whole, tissue VLs from the pre–ramp-up AGMs were low, with 1 × 100 to 1 × 101 (GM, 2.0 × 101) vRNA copies/1 × 106 cells (Figure 3A) and only 100 (GM, 5.0 × 100) vDNA copies/1 × 106 cells (Figure 3B).

Figure 3

Total viral RNA and DNA in tissues of SIVsab-infected African green monkeys (AGMs). (A and B) The total viral RNA (A) and DNA (B) from each of the 38 tissues collected from the infected AGMs tested (n = 27) are shown, with dotted lines delineating each individual tissue. Viral loads are shown on a logarithmic scale and represent the total number of SIV genome copies per 1 × 106 cells. The names of the tissues are listed below the x axis. The data are shown as box-and-whisker plots displaying the median, 1st and 3rd quartiles, and the minimum/maximum outliers. The colors represent the different stages of infection as defined by viral replication status: pre–ramp-up (green), ramp-up (blue), peak (red), and set point (yellow). For the rectum and distal colon, S1-S6 indicates from which strip of tissue the section was taken.

After the pre–ramp-up stage, while viremia was detected in the majority of the AGMs (4–6 dpi), of the 38 tissue types tested, 37 had detectable but highly variable levels of vRNA (1 × 10–1 to 1 × 105 vRNA copies/1 × 106 cells) and 36 had detectable levels of vDNA 1 × (10–1 to 1 × 104 vDNA copies/1 × 106 cells). Even immune-privileged sites like the testes and the brain tested positive for both vRNA and vDNA, although they had the lowest VLs on average. By the peak of infection, vRNA and vDNA were detectable in all tissues, at 1 to 1 × 107 vRNA copies/ 1 × 106 cells and 1 to 1 × 104 vDNA copies/1 × 106 cells, respectively. When viral replication reached its set point during the early chronic stage of infection, VLs in all tissues fell to 1 × 100 to 1 × 106 vRNA copies/1 × 106 cells and 1 × 100 to 1 × 104 vDNA copies/1 × 106 cells (Figure 3), respectively.

Multiple T/F viruses established infection in each AGM, though a mucosal bottleneck still occurred. All AGMs were challenged with a single dose of plasma taken from an acutely infected AGM, which was diluted to 1 × 107 viral copies (26). We utilized single genome amplification (SGA) and phylogenetic analyses to enumerate the T/F viral variants that established infection in viremic AGMs to ensure the AGMs were not overdosed. We detected between 3–10 T/F variants in each AGM, consistent with a moderate dose that was likely to infect all AGMs after a single challenge (Figure 4). Phylogenetic analysis showed that these T/F viruses were genetically distinct from each other, clustering randomly in relation to the different viral lineages found within the inoculum (Figure 4).

Figure 4

Single genome amplification of SIVsab transmitted/founder (T/F) viruses. The totality of T/F viruses from AGMs (n = 14) listed on the left are shown as a circular phylogenetic tree. The 14 AGMs shown here represent AGMs for which viremia was reliably confirmed by conventional qPCR. The color of the variant name corresponds to the color of the AGM name, with the total number of T/F variants per AGM listed to the right of the name. Viruses indicated by the red arrows represent the viral species found in the original inoculum used to infect AGMs. The set-point AGMs (AGM129-AGM132) represent viral diversity from blood draws at 12 dpi. All sequences were aligned using MUSCLE Alignment implemented in Geneious (https://www.geneious.com/) and then manually inspected and optimized. Phylogenetic trees were based on nucleotide sequences and constructed using the neighbor-joining method with Tamura-Nei distance model.

vRNA was found in the lamina propria and lymphoid aggregates of the rectum and distal colon following inoculation. We confirmed the qPCR detection of vRNA in the lamina propria of the rectum and distal colon and the colonic and distal LNs by RNAScope in situ hybridization (ISH) and histology (Figure 5). vRNA+ cells were detected in multiple pre–ramp-up AGMs, but these cells were rare and only in the lamina propria and the T cell zone of the colonic LNs. At this early stage of infection, these vRNA+ cells were typically single, isolated cells and not clustered foci of vRNA+ cells. During ramp-up, vRNA+ cells were found not only within the colonic lamina propria, but also in the colonic lymphoid aggregates in both the T cell zone and B cell follicle. Additionally, in these AGMs, vRNA+ cells were now found not only in the colonic LNs in the T cell zone and B cell follicles, but also in the more distal iliac LNs. The viral foci were still extremely rare, though, with the virus being detected less frequently in the lamina propria and lymphoid aggregates of the distal colon than in both the colonic and iliac LNs. In the samples tested, the virus was localized mostly to the T cell zone but also could be observed occasionally within the B cell follicles (Figure 5).

Figure 5

RNAScope for SIVsab RNA at the site of inoculation and in the draining lymphatics. RNAScope was performed on multiple sections of the rectum and the distal colon, which represent the site of inoculation. Only images from the distal colon are shown for consistency. Viral RNA was stained red, with the surrounding tissue counterstained purple. The red arrows point to foci of viral replication, especially at the earlier time points, when the virus is still rare. Each column represents a different tissue type, and each row represents a different time group. The BCFs in the LNs are all outlined with a dashed black line. All images were captured at 200× magnification with an Olympus FV10i confocal microscope. The time groups are shown on the left side of the figure, reflecting the status of viral replication, with: pre–ramp-up (1–3 dpi), ramp-up (4–6 dpi), peak (9–12 dpi), and set point (42 dpi). LP, lamina propria; LA, lymphoid aggregate; TCZ, T cell zone; BCF, B cell follicle.

In AGMs necropsied at later time points of the acute infection, SIVsab was frequently found in both the lamina propria and lymphoid aggregates of the colon. We also observed large amounts of vRNA+ cells within the T cell zones of the LNs and abundant trapping of the virus by follicular DCs in the germinal centers (Figure 5). By the set point (>42 dpi), virus was detected rarely in the lamina propria and the lymphoid aggregates of the rectum and distal colon. In the LNs, large numbers of virions were trapped by follicular DCs, with little to no vRNA+ cells in the T cell zones (Figure 5). Overall, SIVsab detection by RNAScope supported both the virus-forming isolated foci of replication at the site of inoculation and the qPCR results showing early and rapid viral dissemination.

SIVsab target cells at the site of inoculation are CD4+ T cells and myeloid cells. The primary targets of both HIV and SIV are CCR5+CD4+ T cells (1, 22, 55–58). However, SIVsab is multitropic, as it can use multiple coreceptors, including CCR5, CXCR4, and the alternative coreceptor CXCR6 (7, 12, 59). To establish the identity of the target cell population that supported initial infection at the site of inoculation, rectum and distal colon sections were tested with a combined RNAScope and immunofluorescence assay specific for SIVsab vRNA, and they were costained for CD4+ T cells and myeloid lineage cells (CD68+ CD163+ HAM56+) in all AGMs (Figure 6). In all the tissues tested, vRNA+ cells always colocalized with CD4+ T cells but not with myeloid lineage cells. In the very early stages of infection (2 dpi), only rare single SIV-infected CD4+ T cells could be identified, though they were found to be present in both the rectum and the distal colon. Furthermore, the samples collected later in infection, when cells were infected through either direct local transmission or through systemic seeding, also showed that vRNA+ cells were consistently only CD4+ T cells. This strongly indicates that the T/F viruses are preferentially infecting CD4+ T cells resident to lamina propria of the rectum and distal colon, as was also reported for the HIV T/F strains (60–62).

Figure 6

RNAScope for SIVsab RNA combined with immunofluorescence for CD4+ T cells and myeloid lineage cells. Combined RNAScope with immunofluorescence was used to identify with what cells the SIVsab RNA genomes colocalize. Viral RNA copies are shown in red, CD4+ T cells are shown in blue, and myeloid lineage cells (CD68+CD163+HAM56+) are shown in green. The tissue type and dpi are shown in white in the lower right corner of each image. All images were captured at 600× magnification with an Olympus FV10i confocal microscope using a 60× phase contrast oil-immersion objective and by imaging using sequential mode to separately capture the fluorescence from the different fluorochromes. For the colonic LN, both the B cell follicle (BCF) and T cell zone (TCZ) are shown.

No significant alterations in the number of target cells at the site of inoculation. Having established that CD4+ T cells at the site of inoculation are the primary target cells of SIV, CD4+ T cells isolated from the rectum at the time of necropsy were analyzed by flow cytometry to assess the changes occurring in response to SIV infection.