ZIKV infection of 2 adult macaques.

<p>a) Virus inoculation and sampling timeline. Two West Nile virus-antibody negative non-pregnant female macaques received an intravenous inoculation of 5.0 log₁₀ PFU of a 2015 Brazilian ZIKV strain on day 0. Signs of clinical disease were recorded twice daily. Animals were anesthetized daily from 1 to 8 then on 10, 12 and 14 dpi for blood collection and euthanized for tissue collection 14 dpi. Plasma and whole blood ZIKV RNA and infectious virus levels and kinetics b) for animal 5021 and c) for animal 5242. ZIKV RNA levels in plasma (filled circles) and whole blood (filled squares) are reported in mean log₁₀ RNA copies/ml and were assayed in duplicate. Infectious virus levels in plasma (open circles) were measured as Vero cell plaque forming units/ml. Colored bars under the graph show the period plasma tested ZIKV RNA reactive by the Aptima<b>®</b> assay. NT indicates ‘not tested. Dotted line shows limits of detection for both assays 1.6 log₁₀ RNA copies or PFU per ml. d) Neutralizing and binding antibody kinetics and magnitude. ZIKV 80PRNT endpoint antibody titers are reported. The first plasma dilution tested was 1:20. The box shows the ZIKV IgG ELISA test results on plasma tested at a dilution of 1:50; plus and minus signs indicate positive or no reactivity, respectively. e) Saliva ZIKV RNA levels and kinetics. ZIKV RNA levels in saliva eluted from cotton swabs placed in the cheek of macaques, reported in mean log₁₀ RNA copies/ml or gram, were assayed in triplicate with standard deviations noted. The dotted line shows the limit of detection, 2.3 log₁₀ RNA copies/ml or gram.</p

ZIKV tissue distribution in macaques.

<p>ZIKV RNA was measured by qRT-PCR assay in triplicate with standard deviations noted for <b>a)</b> animal 5021 and <b>b)</b> animal 5242. The limit of detection varied depending on the weight of tissue sampled and volume of MEM needed to homogenize to liquefaction, with a mean of 2.3 (range 1.4–4) log₁₀ RNA copies per gram; non-reactive samples are reported at 2.0. log₁₀ RNA copies per gram. Arrows indicate qRT-PCR negative samples that tested positive by the qualitative Aptima<b>®</b> assay. nc indicates not collected.</p

Summary of radiological scoring.

All thorax radiographs were scored blinded by a veterinary radiologist, with scores of 0 to 3 assigned to each of the 7 lung lobes. For each time point, the total score of all lung lobes was tabulated. Thus, the maximum score per time point is 21. (DOCX)</p

Multivariable correlation analysis.

(A). Spearman r correlation matrix in heatmap format. For this analysis, lung pathology scores are the interstitial cellularity scores (from Fig 7). Lung ISH are the in situ hybridization data of Fig 6. Peak NT50 represents the peak neutralizing antibody titers up to day 5 (i.e., prior to possible de novo antibody responses). VITROS anti-spike total Ig represents the peak value for each animal (i.e., day 2; Fig 2). Nasal, oropharyngeal and BAL sgRNA values are based on AUC of the data in S7 Fig. Clinical scores (sedated and cage-side) are the tabulated scores of each animal over the 7-day observation period (Fig 3C and 3F). (B) Correlation between neutralizing antibody peak NT50 values and lung pathology scores (Spearman r = -0.87; p < 0.0005). The labels next to each symbol indicate the individual animal.</p

Lack of effect of convalescent plasma on sgRNA kinetics in nasal and oropharyngeal swabs and BAL of SARS-CoV-2 infected macaques.

A weighted average analysis was performed on the sgRNA data from nasal and oropharyngeal swabs and BAL (S7 Fig.) to calculate the relative decline of viral RNA (relative to cellular mRNA in the sample) from day 1 to day 7. For each animal, the AUC of relative sgRNA per cellular mRNA over time was tabulated using day 1 as baseline value, and then divided by 6 days to get the weighted average in the decline of sgRNA over the 6-day time period. Lines indicate mean values. On panel C, animal CCP-2 was excluded, as it had no detectable viral RNA in the BAL sample, which precluded this analysis. Statistical analysis revealed no effects between the control and CCP groups (panel A, p = 0.29; panel B: p = 0.30; panel C, p = 0.88; unpaired t-test). (TIFF)</p

Quantitation of SARS-CoV-2 RNA-positive cells in lung sections.

In situ hybridization (RNAScope) was used to detect viral RNA in lung sections. (A) For each animal, the number of positive cells was counted in approximately 20 fields of right caudal lung lobe. Lines indicate median values. There were no differences between both study groups (p = 0.89, Mann Whitney test). (B) Example of lung section of animal CCP-2, with the arrows indicating RNA-positive cells.</p

Time course of additional cytokines and chemokines in plasma of SARS-CoV-2 inoculated animals.

Cytokines and chemokines presented in this panel were ones that did not show consistent changes among animals. The legend is the same as that of S4 Fig. (TIFF)</p

Multivariable correlation analysis on CCP-treated animals.

Multivariate analysis was performed on the 8 CCP-treated animals only. (A). Spearman r correlation matrix in heatmap format. For this analysis, the markers used are the same ones as in Fig 8. (B) Correlation between neutralizing antibody peak NT50 values and clinical scores based on cage-side observations (Spearman r = -0.77; p = 0.04). (TIFF)</p

Clinical measurements collected at time of sedation.

Red and black arrows indicate time of virus inoculation and monoclonal antibody administration on days 0 and 1, respectively. (A) Body weight remained stable. (B) Rectal temperature; horizontal line indicates the cut-off of 103° F, above which ketoprofen treatment was administered. (C) Respiratory rate; the horizontal line indicates a cut-off value of 55 (per minute) as upper normal range. (D) Oxygen saturation measured by pulse oximetry; the horizontal line indicates 95% as the lower end cut-off of the normal range. Total clinical scores, including the markers not graphed above, are presented in Fig 3. (TIFF)</p

Animal demographics.

(DOCX)</p