Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues

<div><p>Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1–7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.</p></div

Detection of anti-ZIKV antibody responses in Rhesus plasma.

<p>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 <b>(A)</b> or IgG <b>(B)</b> secondary antibody. <b>C.</b> 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. <b>D.</b> Fold dilution giving 50% reduction in infectious titer for each serum sample.</p

Viral loads in plasma and urine from ZIKV-infected rhesus macaques.

<p>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. 1/10^th 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 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006219#ppat.1006219.s002" target="_blank">S1 Fig</a>) as indicated by the dotted line. Asterisks indicate infectious virus co-cultured from plasma harvested on day shown.</p

Viral loads in the tissues following necropsy of ZIKV-infected rhesus macaques.

<p>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 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006219#ppat.1006219.s002" target="_blank">S1 Fig</a>) 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.</p

Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques

<div><p>Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.</p></div

CHIKV mAb treatment did not cause significant changes in CD4⁺ or CD8⁺ T cell proliferation.

<p>Rhesus macaques were inoculated with CHIKV and treated with control antibody SVIR002 or CHIKV mAb SVIR001. Blood was drawn daily 0–7 dpi, and PBMCs were examined for proliferative responses of different <b>(A-C)</b> CD4⁺ and <b>(D-F)</b> CD8⁺ T cell subsets. T cell subsets were defined in <b><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005637#pntd.0005637.s002" target="_blank">S2 Fig</a></b> as Naïve (NV), Central Memory (CM), and Effector Memory (EM). The Ki67⁺ proliferative status was plotted as a percentage of the total population. <b>(G)</b> IFNγ ELISpot analysis was performed on PBMCs from rhesus macaques at 7 dpi. PBMCs from animals treated with SVIR001 (5 mg/kg or 15 mg/kg) or SVIR002 (15 mg/kg) were stimulated with CHIKV peptide pools (10 μg/well), inactivated CHIKV (iCHIKV) (10 μg/well), or PMA/Ionomycin as a positive control. DMSO was used as a negative control to establish the baseline number of IFNγ-producing T cells for each animal. Spots were quantified on an AID ELISpot plate reader (n = 4/group).</p

Tissue viral load following CHIKV mAb therapy.

<p>Animals were euthanized at day 7 post-infection, and viral RNA was isolated from tissues and quantified by qRT-PCR. The viral load in <b>(A)</b> arm joints and muscles, <b>(B)</b> leg joints and muscles, and <b>(C)</b> lymphoid tissues, heart and kidney are reported. Statistical significance was determined on the log-transformed data using Dunnett’s multiple comparison test, and multiplicity-adjusted <i>P</i> values are reported (n = 4; ** <i>P</i> < 0.005, * <i>P</i> < 0.05).</p