Animals, ethics and clinical analysis

All infectious work with NHPs in this study was conducted with approval and oversight from the Rocky Mountain Laboratories (RML) Institutional Biosafety Committee and the RML Animal Care and Use Committee (ACUC). Studies were conducted in an AAALACi accredited facility following the guidelines and basic principles in the Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. The study design was constructed with consideration for the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, but with the knowledge that a limited number of NHPs were available. All methods are reported in accordance with these guidelines and all relevant NHP information is reported in Tables 1 and 2.

Mauritius origin cynomolgus macaques obtained from NIH colonies, were individually housed in adjoining primate cages that enabled social interactions. Marmosets obtained from NIH colonies were socially housed to mimic natural social structure. Commercially available nonhuman primate diets were provided twice daily and supplemented with treats, vegetables, or fruit daily. Water was available ad libitum. All animals were housed under controlled conditions of temperature, humidity, and light (12 h light:12 h dark cycles). Environmental enrichment consisted of a variety of manipulanda, commercial toys and human interactions. All procedures were conducted on anesthetized animals under the supervision of veterinary staff. Anesthetized was delivered for inoculation, blood draws and euthanasia at the indicated time points to cynomolgus macaques via intramuscular (i.m.) injection of ketamine (8-12 mg/kg) and to marmosets via i.m. injection of ketamine (20 m/kg)-xylazine (2 mg/kg) or isoflurane inhalation to effect per ACUC guidelines. Analgesics or other treatment methods could interfere with infection, the immune response to infection or the development of neurologic symptoms and were not used in this study^30,31,32.

For all animal studies, endpoint criteria were specified by RML ACUC-approved parameters to determine when animals were to be humanely euthanized. This unbiased approach removed the need for experimental blinding or randomization. Clinical scoring was performed twice daily and was based on the following criteria: general appearance, skin and coat appearance, discharge, respiration, neurological signs, food intake, feces and urine output and activity. A cumulative minimum clinical score was established to assist with early timepoint humane euthanasia decisions. In anticipation of viral infection of the brain, any sign of neurological disease was a clinical endpoint, although this was never reached by any study animal. Other nonviral pathologies were also considered. In the case of marmosets CM1 and CM2, hepatic issues at the 23 dpi timepoint were grounds for euthanasia. Additionally, lethargy and lack of grooming by marmoset CM3 at the same timepoint triggered euthanasia criteria. Although no cynomolgus macaques met endpoint criteria, WL-1, JL-1 and AL-2 were euthanized at 23 dpi such that histopathology could be performed on tissues. This decision was made, because WL-1 and JL-1 had demonstrated viral RNA in plasma at earlier timepoints (Table 1) and AL-2 was included as a comparison for the adult group. Euthanasia was performed under anesthesia (described above) by intravenous (i.v.) or intracardiac administration of Euthasol^® euthanasia solution (1mL/5kg, Virbac).

Virus stocks, NHP infections and whole blood collections

A 1978 human isolate of LACV was used for all experiments and has previously been described⁹. Infectious virus had not been passaged more than twice in vitro. LACV stocks were generated and titered by plaque assay as previously described³³. Cynomolgus macaques were infected subcutaneously (s.c.) along the dorsal thorax with 500µL of straight stock inoculum at a dose of 10⁷ plaque-forming units (PFU)/mL. Common marmosets were infected intranasally (i.n.) with 500µL of stock inoculum diluted 1:10 in sterile, pharmaceutical grade PBS at a dose of 10⁶ PFU/mL. 250µL of inoculum was applied to each naris. At indicated timepoints before and after LACV infection, whole blood was collected via i.v. puncture into EDTA-treated Vacutainer^® tubes (BD). Collected volumes did not exceed 10% of circulation blood volume in a two-week period for either species.

In vitro PBMC stimulation

Prior to LACV infection, EDTA whole blood from weanling, juvenile or adult cynomolgus macaques or adult common marmoset was collected. Most red blood cells (RBCs) were removed from each sample by diluting 1:2 into PBS containing 1% Dextran T-500, mixing and incubating each for 30 min at 37 °C. The partially clarified top layer containing PBMCs was collected, diluted 1:2 in PBS and spun for 5 min at 500 g. The pellet was resuspended in 3mL of ACK lysis buffer and incubated 5 min to remove any remaining RBCs. Lysis was halted by diluting the lysis buffer 1:3 with PBS and centrifuging for 5 min at 500 g. Pelleted cells were resuspended in warm RPMI-1640 supplemented with 10% fetal calf serum and 5% Penicillin-Streptomycin at a concentration of 2*10⁶ cells/mL. 500µL of PBMCs were added to each well of a 24 well plate. Treatment media for mock, high molecular weight (HMW) poly I: C (Invivogen) and virus were made up separately at a 2x concentration and 500µL was applied to each well. Mock media contained volume-matched Vero supernatant relative to the LACV media. HMW Poly I: C was diluted into 0.5 mg/mL Lyovec transfection reagent (Invivogen) to a concentration of 200 µg/mL and then diluted 100-fold into culture media such that the final concentration applied to PBMCs was 1 µg/mL. The final multiplicity of infection of LACV-contained media when applied to PBMCs was 0.01. PBMCs were incubated at 37 °C for 16 h after which cells were harvest into Eppendorf tubes and centrifuged for 5 min at 500 g. Cells were washed with PBS and repelleted at 500 g. Cells were then lysed with ZR RNA buffer (Zymo) for later quantitative real-time PCR analysis.

Flow cytometry

Following infection, PBMCs were isolated from EDTA whole blood of weanling, juvenile or adult NHPs as described above at the indicated time points. PBMCs were suspended in PBS with 0.05% BSA on ice at 10 × 10⁶ cells/mL following counting with a hemocytometer. 100µL of each sample was added to each required well of a 96 well plate to be immunolabeled. PBMCs were then incubated with TruStain FcX™ (Biolegend) Fc receptor blocking solution for 30 min and the immunolabeled with direct conjugate antibodies (Table 3). Controls included unlabeled cells, single antibody labeled cells and all antibodies minus-one labeled cells. PBMCs were then washed 3x with PBS, fixed with 2% paraformaldehyde for 30 min and washed again 3x with PBS and analyzed using a BD LSRII (BD Biosciences). Flow cytometric analysis was performed using FCS Express Research Edition version 5 (Denovo software). Gating strategies for specific cell types are shown in the respective figures.

RNA isolation from plasma and PBMCs and real-time PCR analysis

For viral RNA isolation from plasma at the indicated time points, EDTA whole blood was centrifuged at 2000 g for 10 min and the upper plasma fraction collected. RNA was extracted from 100µL of plasma. For viral RNA isolation from nasal swabs, the tip of the swab was incubated in 0.5mL of Viral RNA Buffer (Zymo) for 30 min with mixing. Subsequent RNA isolation from both plasma and nasal samples was performed using the Zymo ZR Viral RNA Isolation Kit (Zymo) per the manufacturers’ column-based protocol. Isolated RNA was eluted from the columns using 15µL of nuclease-free H₂0. For RNA from PBMCs, cells were isolated from EDTA whole blood as described above. PBCM RNA was isolated using the Quick-RNA™ Miniprep Kit (Zymo) per the manufacturers’ column-based protocol. Isolated RNA was eluted using 35mL of nuclease-free H₂0. Generation of cDNA from plasma and cell RNA samples and analysis of host transcripts was performed as previously described³⁴. For host genes, transcript expression was plotted relative to Gapdh expression. Analysis of viral RNA from plasma was done by comparing Ct values from plasma samples to standards established by spiking naïve species matched NHP plasma with null, 10¹, 10², and 10³ PFU. Samples were run in three triplicate groups and 6–9 wells with Ct values consistently near or above the 10¹ standard were considered positive, 2–5 wells were considered weak positive and 0–1 wells were negative (Table 1). Analysis of viral RNA from nasal swabs were similar, however, the analysis was only performed in triplicate with Ct values of all 3 wells consistently near or above the 10¹ standard being considered positive, 2 wells considered weak positive and 0–1 wells negative (Table 2). Primers for all transcripts are shown in Table 4.

Plasma cytokine analysis

Concentrations of 29 cytokines, chemokine and growth factors were measured in plasma from mock and LACV infected weanling, juvenile and adult cynomolgus macaque at 3, 7, 14 and 21 dpi using the Luminex magnetic bead-based assay (Monkey Cytokine Magnetic 29-Plex Panel (ThermoFisher)). Plasma was prepared as described above. The assay was run according to the manufacturer’s protocol using 50µL of sample. Protein concentrations were measuring using a Bio-Plex 200 system (BioRad).

Plaque assay and neutralizing antibody assays

Plasma from individual NHPs were collected and isolated as described above and stored at -80 °C. For plaque assays of NHP plasma, C17.2 cells (Sigma-Aldrich) were plated 1 day in advance into 24-well plates to confluency. Plasma samples were diluted serially at 1:10 decreasing concentrations for initial screening and 1:2 for final dilutions in DMEM (Gibco) supplemented with 2% FBS and penicillin/streptomycin and 200 µl of each dilution was applied the Vero cultures in duplicate. Plates were incubated 1 h to allow virus attachment and then each well was overlayed with 0.5 mL of 1.5% carboxymethyl cellulose in MEM (Gibco). Plates were incubated for 5 days and then fixed with 10% formaldehyde to a final concentration ≥ 4% formaldehyde per well for 1 h. Wells were rinsed with water, stained with 0.35% crystal violet and rinsed with water again. Plates were air dried and scanned for plaques.

For neutralization assay, plasma was diluted 1:10 and then subsequent 1:5 in DMEM, 2% FBS, 1% P/S. Samples were then mixed with 10² plaque-forming units (PFU) of LACV in DMEM/FBS/P/S. The entire mixture was incubated for 1 h at 37 °C to achieve neutralization. After neutralization, the mixture was added to confluent Vero cells in a 24-well plate and incubated again for 1 h at 37 °C. After incubation, 1.5% carboxymethylcellulose (CMC) in modified Eagle medium (MEM) was overlaid onto the Vero cells and incubated undisturbed at 37 °C. After 5 days the mixture was fixed by adding 10% formaldehyde to each well and incubated for 1 h at room temperature. After fixation, plates were rinsed with de-ionized water wash and stained with 0.35% crystal violet. Neutralizing titer was determined by the dilution that inhibited 50% viral PFUs compared to a 10² LACV-infected control well.

Histology and immunohistochemistry

Necropsies and tissue sampling was performed on cynomolgus macaques WL-1, JL-1 and AL-2 (Table 1) and all common marmosets (Table 2) at 23 dpi. Tissues collected included brain, spinal cord, dorsal root ganglia, draining lymph node, gut-associated lymph tissue, spleen, lung, muscle, liver and for common marmosets, nasal turbinates. Tissues were fixed in 10% neutral buffer formalin for a minimum of 7 days and processed for paraffin embedding, sectioning, H&E staining and immunohistochemistry as previously described³⁵. H&E sections were evaluated by a blinded, board-certified veterinary pathologist for evidence of virus-associated pathology or degeneration. Immunolabeling for LACV was performed with an inhouse-generated rabbit polyclonal antibody that has previously been described²⁰. Immunolabeled images were obtain with a Zeiss 710 (Zeiss) laser scanning microscope with a Plan-Apochromat 63x objective (NA of 1.40). Images of H&E-stained tissues were collected on a Nikon Eclipse 55i microscope (Nikon) with a Plan Fluor 40x objective (NA of 0.75) and a DS-Fi3 digital camera at 400x magnification.

Statistical analysis

All statistical analysis was performed with GraphPad Prism version 10.2.0 (GraphPad Software). Relevant tests are described in the associated figure legend. The authors acknowledge that all experiments have a small sample size due to the limitations of NHP research. It is possible that effects may be missed due to this small sample size, disallowing the rejection of the null hypothesis that may be significant with higher numbers of animals. The interpretation of the presented data takes this into account and thus focuses on both statistical differences and consistent trends that do not reach statistical significance.