Infection and clinical characterization of RM cohorts
Three cohorts of RMs were used for this study (Fig 1). The first cohort consisted of two adult females and one adult male. Animals were infected with 1x104, 1x105, or 1x106 focus forming units (ffu) of ZIKV (PRVABC59). The infectious dose was divided over 10 subcutaneous injections over bilateral hands and arms. Blood and urine were sampled daily through 10 dpi, as well as on 14, 21, and 28 dpi. Euthanasia was performed at 28 dpi and tissues collected at necropsy for analysis of viral loads. A second cohort, consisting of two adult RM (one male, one female) was infected with 1x105 ffu, followed by daily sampling of blood and urine through 7 dpi, at which time animals were euthanized as above. The third cohort, consisting of two adult male RM, was infected with 1x105 ffu followed by daily sampling of blood and urine through 35 dpi. All animals developed a transient fever, rash on the arms and upper torso, as well as lymphadenopathy of their axillary lymph nodes. Additionally, 3 of 7 animals developed conjunctivitis lasting 3–5 days. None of the infected animals experienced weight loss or signs of clinical disease other than those described above. Analysis of blood chemistry revealed no significant changes following ZIKV infection (S2 Fig).
ZIKV viral loads and tissue tropism
All infected animals developed plasma viremia as detected by RT-qPCR of viral genomes that typically peaked at 2 dpi and was detectable out to 5–7 dpi (Fig 2A and 2B). We were unable to titer virus directly from plasma samples. However, infectious virus, detected by co-culture of plasma with C6/36 cells, was observed in indicated cases between 2 to 4 dpi (Fig 2A, stars). Viral RNA in the urine was detected from 3–10 dpi with peak levels at 5 dpi (Fig 2A and 2B). We detected viral RNA positive urine samples outside of the initial 3–10 dpi window, which is consistent with other reports of ZIKV infections of NHP . This finding indicates that ZIKV infection in NHP is dynamic and remains persistent.
Fig 2. Viral loads in plasma and urine from ZIKV-infected rhesus macaques.
One-step qRT-PCR was used to measure ZIKV RNA loads in the plasma (top panels) and urine (bottom panels) from each animal at indicated days pi and represented as copies per milliliter of fluid. (A) Cohort 1 and 3: Animal 24961(1x104 ffu)-red lines; 25147 (1x105 ffu)-blue lines; 25421 (1x106 ffu)-black lines; 26021 (1x105 ffu)-green lines; 26023 (1x105 ffu)-purple lines. (B) Cohort 2: Animal 24504 (1x105 ffu)-red lines; and Animal 27679 (1x105 ffu)-blue lines. 1/10th of total RNA extracted from 100 μl plasma or urine was used in each reaction. Approximate limit of detection at 1e4 genomes/ml is based on a detection limit of ~100 genomes in each reaction (S1 Fig) as indicated by the dotted line. Asterisks indicate infectious virus co-cultured from plasma harvested on day shown.
Following euthanasia and necropsy, RNA was isolated from individual tissues and the viral genomes were quantified by qRT-PCR (viral loads of positive tissues in Fig 3A and 3B; complete list of tissues samples in S1 Table). At 7 dpi (cohort 2, 1x105 ffu), viral RNA was detected in multiple tissues: lymphoid tissue—including lymph nodes distributed throughout the body as well as the spleen; joints—most prominently joints near the site of inoculation but in some instances more distal joint tissue as well; peripheral nervous tissue, specifically the sciatic nerve, brachial plexus and trigeminal ganglion. Additionally, viral RNA was found associated with the spinal cord (cervical, lumbar and thoracic), but not in CSF or in the brain, at this time point. This may indicate neurological tropism, but an inability to effect retrograde transport of infectious virus into the CNS or that infection of the CNS requires additional time. Viral RNA was detected in the kidney and bladder of the male animal (27679), although not in the testes or prostate. Viral RNA was found in the uterus of the female (24504).
Fig 3. Viral loads in the tissues following necropsy of ZIKV-infected rhesus macaques.
One-step qRT-PCR was used to measure ZIKV RNA loads in the tissues of animals in Cohort 2 (A), Cohort 1 (B), and Cohort 3 (C). Total RNA was generated using the Trizol method on precleared samples following bead beating. Approximately 80 different tissues were assessed for the presence of viral RNA. Shown are the tissues with positive detection in at least one of the animals per cohort. Arrows indicate samples in which virus was successfully co-cultured from tissue homogenate. Approximate limit of detection at 1e4 genomes/ml is based on a detection limit of ~100 genomes in each reaction (S1 Fig) as indicated by the horizontal line. (D) Paraffin sections of sciatic nerve cut in cross section were hybridized with ZIKV specific chromogenic probe (red) and counterstained with hematoxylin (blue). Nerve fibers (NF) show a normal distribution within the endoneurium surrounded by perineurium (PN) and perineurial adventia (PA). Hybridization for ZIKV was robust but limited to the PA region. Original magnification was 50X.
We were able to co-culture infectious ZIKV in C6/36 insect cells from homogenates of the axillary and inguinal lymph nodes, finger joints, kidney and bladder derived from the male monkey (27679) (Fig 3A, black arrows). Together these data indicate that ZIKV quickly disseminates to many tissues throughout the body including lymph nodes/spleen, peripheral nerves, and skin as well as the genital/urinary tract.
At 28 dpi, (cohort 1, inoculated with 1x104 1x105 and 1x106 ffu) viral RNA was still detected in both lymphoid and joint tissues in all animals (Fig 3B). In general, vRNA tissue distribution was greatest for the 1x105 and 1x106 ffu infected animals. The axillary (draining) lymph nodes and spleen showed the highest level of viral RNA in all three animals, while other lymph nodes were positive for at least 2 of 3 animals. Joint tissues close to the site of inoculation (wrist and finger) were also positive in all three animals 28 dpi. Additional joints, muscles of the arms and legs, and heart were positive for ZIKV RNA in subsets of animals. In animal 25421 (female, 1x106 ffu) viral RNA was detected in the reproductive tissues (uterus and vagina) suggesting that the virus can infect these tissues and persist there for at least 4 weeks post infection. This finding may have important implications for viral transmission and fetal infections during pregnancy. Viral RNA was also detected in the sciatic nerve and eyes from this subject. Interestingly, ZIKV RNA was detected in the cerebellum of animal 24561 (female, 1x104 ffu), indicating penetration to the CNS. Co-culture of homogenates from tissues collected at 28 dpi with C6/36 cells did not amplify infectious virus.
At 35 dpi, (cohort 3, inoculated with 1x105 ffu) positive viral RNA detection occurred in neuronal tissues, lymph nodes, and joint/muscle tissues (Fig 3C). Animal 26023 displayed extensive neuronal tissue involvement with viral RNA detected in the occipital and parietal lobes of the brain, lumbar region of the spinal cord, dorsal root ganglia, brachial plexus, and eye. In Animal 26021, ZIKV RNA was not detected in the brain but was present in the trigeminal ganglia, as well as cervical, lumbar and thoracic regions of the spinal cord and peripheral nerves (brachial plexus and sciatic nerve). Interestingly, in situ hybridization on cross sections of sciatic nerve using ZIKV-specific chromogenic probes detected robust virus RNA levels in the perineurial adventitial space from animal 26023 (Fig 3D). Virus was not detected in the nerve fibers. Both animals had viral RNA in their axillary lymph nodes. These results combined with the viral detection data from the day 28 animals confirm the long-term persistence of ZIKV RNA in neuronal, lymph node and joint/muscle tissues.
Histologic examination of sections taken from tissues of infected RM found few specific abnormalities, although several areas of inflammation were observed (S3 Fig). An uncharacteristic prostatitis characterized by interstitial neutrophilic and lymphoplasmacytic cellular infiltrates and glandular microabscesses were noted 7dpi in animal 27679 infected with 1x105 ffu (S3A Fig). Minimal perivascular lymphocytic or lymphoplasmacytic inflammatory cell infiltrates were present in sections of skin from the upper torso affected with a rash for both animals examined 7dpi (S3B Fig). Viral RNA was also detected in this area of skin in this animal. Similarly, variable perivascular inflammatory infiltrates composed of lymphocytes, eosinophils and plasma cells were observed in the joints and muscles of animal 24504 (S3C & S3D Fig). Focal lymphohistiocytic inflammation was associated with a meningeal vessel in the cerebrum of the high dose (1x106 ffu) animal 25421 at 28 dpi suggestive of an ongoing infection of the brain vasculature (S3E Fig). Animal 25147 (28 d pi, 1x105 ffu) had focal lymphocytic infiltration of the dorsal root ganglion of the cervical spinal cord (S3F Fig). The lack of correlation of detection of viral RNA with sites of inflammation in the prostate, brain, and DRG may indicate highly focal areas of infection, or clearance of virus prior to resolution of inflammation.
In order to determine which cell types within lymphoid tissues were positive for viral RNA, we sorted macrophage, dendritic cell, B-cells and T-cells from the splenocytes and axillary lymphocytes by positive magnetic bead selection (S4 Fig). RNA was isolated from each cell population and ZIKV RNA quantified by qRT-PCR. As shown, at 28 dpi RNA is primarily found in the macrophage and B cell subsets with reduced levels in DC subsets but rarely present in the T cell fractions (Fig 4A). In situ hybridization with ZIKV- and Influenza-specific probes detected ZIKV but not Flu RNA in multiple axillary lymph node follicles from animal #25421 (Fig 4B), confirming the presence of the ZIKV RNA in the macrophage, B cell and DC rich regions of the germinal center. Overall, this data indicates that ZIKV spreads to multiple tissue types and the infection of many of these tissues persists in macrophages, as well as other cell types for at least 4–5 weeks post infection.
Fig 4. Infected cell types in spleen and axillary lymph nodes of ZIKV-infected rhesus macaques.
Cell subpopulations were isolated by positive selection magnetic bead separation from lymphocytes isolated from the spleen and axillary lymph nodes at 28 dpi. For macrophage and T cell isolation, CD14-microbeads were used to isolate macrophages from total spleen and axillary lymph node lymphocytes, and then anti-CD3 was used to isolate T cells from macrophage depleted flow-through. For B cell and DC isolation, total splenocytes or axillary lymph node lymphocytes were first positively selected for CD20+ B cells and the depleted fraction was bound to CD1c microbeads to isolate DCs. To increase purity of the isolated cell populations all positively selected samples were eluted after primary selection and then re-bound to a second fresh column. Depiction of isolated cell populations as characterized by flow cytometry is shown in S4 Fig. Total RNA was isolated from the positively selected cell fractions and quantified. One-step qRT-PCR was used to measure ZIKV RNA loads in each of the cell fractions isolated from animals at 28dpi (A). Viral RNA loads were highest in the macrophage and B cell fractions, with consistently less vRNA detected in DCs and rarely in the T cells. (B) Serial paraffin sections of axillary lymph node tissue from animal #25421 were hybridized with ZIKV specific chromogenic probe (red) or Influenza specific chromogenic probe (red) and counterstained with hematoxylin (blue) showed strong positive staining for ZIKV in the lymphoid follicles. Original magnification was 50X.
Immune activation following ZIKV infection
We performed a detailed phenotypic analysis of immune cell subsets by flow cytometry to characterize activation of innate immune cells (monocyte/macrophage/DC/NK cells) as well as adaptive immune cell proliferative responses (T and B cells). We also characterized cytokines and antibodies present in the sera of infected RM. Within 1–2 days pi, all of the animals showed innate immune cell activation, as demonstrated by the presence of CD169+ staining (Fig 5). RM 24961 (1x104 ffu) displayed a more protracted innate immune response, compared to 25421 (1x106 ffu), 25147, 26021 and 26023 (1x105 ffu). While all animals showed an increase in CD169+ monocytes and DCs at 2–4 dpi, the number of activated cells waned between day 8–10 pi in animals infected with 1x105 or 1x106 ffu, while the number of activated cells in the animal infected with 1x104 ffu did not return to baseline levels until 14–21 dpi.
Fig 5. ZIKV-infection induces robust innate immune cell activation.
Total peripheral blood mononuclear cells from all time points were stained with fluoroflore-conjugated antibodies directed against the cellular markers CD3, CD8, CD11c, CD14, CD16, CD169 and HLA-DR in order to assess changes in the activation of A) monocyte/macrophages; B) myeloid dendritic cells; C) other dendritic cells; and D) NK cells. Multi-color flow cytometry was used to visualize the stained cells. The percentage of activated cells (CD169+) was calculated using FlowJo and the data was graphed in GraphPad Prism v6 software.
Cytokine expression in the plasma largely did not change following infection. However, expression of 4 cytokines (IL-1RA, MCP-1-CCL2, IP-10-CXCL10, and I-TAC-CXCL11) was induced over background levels in the plasma (Fig 6A–6D). Expression of these cytokines was elevated within the first several days post infection but returned to baseline levels by 10 dpi. Low levels of cytokine activation in vivo may be an indirect effect of routine ketamine treatment  or a result of direct inhibition by ZIKV of innate immune pathways that direct synthesis and secretion of pro-inflammatory cytokines. To examine the latter possibility we employed a reporter assay for which the readout is luciferase expression that responds to NF-κB or JAK/STAT pathway (type I IFN) activation [32,33]. As shown in Fig 6E and 6F, rhesus fibroblasts infected with ZIKV for 48h at 5 FFU/cell showed significantly diminished LUC signal relative to uninfected cells following treatment with either poly(I:C) or human IL-1β. These represent distinct NF-κB-terminal signaling pathways with poly(I:C) induction resulting from activation of the TLR3 pattern recognition receptor and TRIF adaptor protein as well as IL-1β triggering the IL1 receptor and associated MyD88 adaptor protein. Similarly, activation of JAK/STAT signaling by IFNβ1 treatment was also repressed by ZIKV infection (Fig 6G). These results agree with previous observations that ZIKV infection promotes degradation of STAT2 and subsequent inhibition of type I IFN signaling . As such we hypothesize that ZIKV exhibits an inhibitory phenotype that operates downstream of the convergence of these pathways, likely targeting activation of NF-κB itself but delimiting the mechanisms associated with this point will require further experimentation.
Fig 6. Rhesus cytokine and chemokine production in response to ZIKV infection and block of NF-kB signaling in Rhesus fibroblasts.
A 29-plex-cytokine/chemokine/growth factor magnetic bead assay was performed on plasma from rhesus monkeys at all time points post infection. Cytokine analysis revealed changes in only A) IL-RA; B) MCP-1-CCL2; C) IP-10-CXCL10; and D) ITAC-CXCL11. Reporter assay showing induction of NF-κB-dependent (E, F) or interferon stimulated response element (ISRE)-dependent (G) LUC expression in fibroblasts infected for 56h with ZIKV at MOI = 5ffu/cell. Luminescence was measured 8h after treatment with 60μg/mL poly(I:C) (E) 100ng/mL human IL-1β (F) or 5,000 units/ml IFNβ1 (G). Values displayed are average fold changes (three replicates) of stimulated versus untreated cells ±SD.
Proliferating CD4+ and CD8+ T-cells were present in all infected animals by 6–8 dpi. CD8+ T cell proliferative responses (Ki67+ cells) were evident at 6 dpi, maximal at 8–9 dpi and returned to background levels by 14 dpi (Fig 7B and 7D). Both central memory and effector memory CD4+ T cell proliferative responses were maximal at 7 dpi (Fig 7A and 7C) but took longer to return to baseline levels compared to the CD8+ T cells. Consistent with these findings, Granzyme B expression in naïve and central memory CD4+ and CD8+ T cells peaked between 7 and 10 days post infection (S5 Fig).
Fig 7. Rhesus adaptive immune cell proliferative responses following ZIKV infection.
Total peripheral blood mononuclear cells were analyzed by flow cytometry for the presence of T and B cell proliferative responses following infection. T cells were identified by staining with antibodies directed against the cellular markers CD3, CD4, CD8β, CD95, CD28, CD127 and for intracellular levels of Ki67 (proliferation marker) to assess changes the proliferation of A) CD4+ central memory T cells; B) CD8+ central memory T cells; C) CD4+ effector memory T cells; and D) CD8+ effector memory T cells. B cells were stained with antibodies directed against CD3, CD20, CD27, IgD and HLA-DR as well as Ki67 in order to compare the proliferative responses in E) naïve B cell; F) memory B cells; and G) marginal-zone like B cells. The percentage of actively proliferating cells (Ki67+) was calculated using FlowJo and the data was graphed in GraphPad Prism v6 software.
B cell proliferative burst responses were maximal at 14 dpi (Fig 7E–7G). Interestingly, when comparing the cohort 1 animals, the B-cell proliferative responses in RM 24961 (1x104 ffu) were observed slightly earlier than in RMs 25147 (1x105 ffu) and 25421 (1x106 ffu) and represented a greater percentage of cells within each subset. Proliferating T-cells also appeared for a longer time post infection in this animal (Fig 7A–7D).
ZIKV virion-reactive IgM and IgG antibodies in sera were quantified by ELISA. Levels of anti-ZIKV IgM became detectable between 7–10 dpi and were maintained through 28 dpi in 2 of 3 animals, while one animal (#25421) showed reduced titers after 10 dpi. (Fig 8A). IgG levels increased beginning between d8 to d14 pi and plateaued around 21 dpi (animals #25147 and #25421) or continued to increase in the low dose animal (#24961). Western blotting of ZIKV infected cell lysates revealed that antibody responses targeted at least two proteins that were 38 and 55 kDa, respectively, consistent with viral proteins NS1 and E (S6 Fig), which elicit antibody responses during ZIKV infection of humans and are also major antibody targets during other flavivirus infections [35,36]. The neutralizing capacity of the ZIKV-directed antibodies was quantitated, and robust neutralizing antibody responses were detected at 28 or 35 dpi (Fig 8C and 8D) in all animals, regardless of the infectious dose.
Fig 8. Detection of anti-ZIKV antibody responses in Rhesus plasma.
Rhesus macaques infected with ZIKV were analyzed for the presence of antibodies directed against ZIKV-PRABC59 by ELISA using whole virus as capture antigen with an HRP-conjugated anti-Rhesus IgM (A) or IgG (B) secondary antibody. C. Sera from indicated animals obtained pre-infection (d0) or at terminal bleed (d28 or 35 pi) were tested for neutralizing activity via plaque reduction neutralization titer (PRNT) assay. D. Fold dilution giving 50% reduction in infectious titer for each serum sample.
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