Intranasal exposure of African green monkeys to SARS-CoV-2 results in acute phase pneumonia with shedding and lung injury still present in the early convalescence phase

The virus (SARS-CoV-2/INMI1-Isolate/2020/Italy) was isolated on January 30, 2020 from the sputum of the first clinical case in Italy, a tourist visiting from the Hubei province of China that developed respiratory illness while traveling [16]. The virus was initially passaged twice (P2) on Vero E6 cells; the supernatant and cell lysate were collected and clarified following a freeze/thaw cycle. This isolate is certified mycoplasma and Foot-and-Mouth Disease virus free. The complete sequence was submitted to GenBank (MT066156) and is available on the GISAID website (BetaCoV/Italy/INMI1-isl/2020: EPI_ISL_410545) upon registration. For in vivo challenge, the P2 virus was propagated on Vero E6 cells and the supernatant was collected and clarified by centrifugation making the virus used in this study a P3 stock.

SARS-CoV-2 seronegative AGMs (Chlorocebus aethiops) (6 females) (St Kitts origin, Worldwide Primates, Inc.) were randomized into two cohorts where one group (n = 3) was scheduled for euthanasia at 5 dpi and the other at 34 dpi. Animals were anesthetized with ketamine and inoculated with a target dose of 3.0 × 10⁶ PFU of SARS-CoV-2 (SARS-CoV-2/INMI1-Isolate/2020/Italy) using the LMA MAD, with the dose being equally divided between each nostril. All animals were longitudinally monitored for clinical signs of illness including temperature (measured by surgically implanted DST micro-T small implantable thermo loggers (Star-Oddi, Gardabaer, Iceland)), respiration quality, and clinical pathology. All measurements requiring physical manipulation of the animals were performed under sedation by ketamine. Mucosal swabs were obtained using sterile swabs inserted into the mucosal cavity, gently rotated to maximize contact with the mucosal surface, and deposited into 2.0 mL screw-top tubes containing sterile MEM media supplemented to 2% with FBS.

On specified procedure days (days 0, 2, 3, 4, 5, 7, 12, 15, 21, 28, 34), 100 μl of blood was added to 600 μl of AVL viral lysis buffer (Qiagen) for virus inactivation and RNA extraction. Following removal from the high containment laboratory, RNA was isolated from blood and swabs using the QIAamp viral RNA kit (Qiagen).

RNA was isolated from blood and mucosal swabs and assessed using the CDC SARS-CoV-2 N2 assay primers/probe for reverse transcriptase quantitative PCR (RT-qPCR) [17]. SARS-CoV-2 RNA was detected using One-step probe RT-qPCR kits (Qiagen) run on the CFX96 detection system (Bio-Rad), with the following cycle conditions: 50 °C for 10 min, 95 °C for 10 s, and 45 cycles of 95 °C for 10 s and 55 °C for 30 s. Threshold cycle (C_T) values representing SARS-CoV-2 genomes were analyzed with CFX Manager Software, and data are presented as GEq. To generate the GEq standard curve, RNA was extracted from supernatant derived from Vero E6 cells infected with SARS-CoV-2/INMI1-Isolate/2020/Italy was extracted and the number of genomes was calculated using Avogadro’s number and the molecular weight of the SARS-CoV-2 genome.

Infectious virus was quantitated by plaque assay on Vero E6 cells (ATCC CRL-1586) from all blood plasma and mucosal swabs, and bronchoalveolar lavage (BAL) samples. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero E6 cell monolayers in duplicate wells (200 μl). Cells were overlaid with EMEM medium plus 1.25% Avicel, incubated for 2 days, and plaques were counted after staining with 1% crystal violet in formalin. The limit of detection for this assay is 25 PFU/ml.

Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed from blood collected in tubes containing EDTA using a Vetscan HM5 hematologic analyzer (Abaxis). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), calcium, creatinine (CRE), C-reactive protein (CRP), gamma-glutamyltransferase (GGT), glucose, total protein, and uric acid by using a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis). Partial pressures of CO₂ and O₂ were obtained using an iSTAT Alinity hematological analyzer (Abbott).

Neutralization titers were calculated by determining the dilution of serum that reduced 50% of plaques (PRNT₅₀). A standard 100 PFU amount of SARS-CoV-2 was incubated with two-fold serial dilutions of serum samples for 1 hour. The virus-serum mixture was then used to inoculate Vero E6 cells for 60 min. Cells were overlaid with EMEM medium plus 1.25% Avicel, incubated for 2 days, and plaques were counted after staining with 1% crystal violet in formalin.

SARS-CoV-2-specific IgG antibodies to nucleoprotein were measured in sera by ELISA at the indicated time points. Nucleoprotein ELISA kits were kindly provided by Zalgen Labs, LLC. Sera were initially diluted 1:100 and then two-fold through 1:25,600 in 4 in (1 x PBS with 0.02% Tween-20). After a one-hour incubation, plates were washed six times with wash buffer (1 x PBS with 0.2% Tween-20) and incubated for an hour with a 1:5000 dilution of horseradish peroxidase conjugated anti-primate IgG antibody (Fitzgerald Industries International; Cat: 43R-IG020HRP). Tetramethylbenzidine was used to develop the reaction; the reaction was stopped with methane-sulfonic acid and plates were read at a wavelength of 450 nm. Absorbance values were normalized by blank-subtracting values from wells incubated with sera from a SARS-CoV-2-naïve animal at the corresponding serum dilution. End-point titers were defined as the reciprocal of the last adjusted serum dilution with a value ≥0.20.

Necropsy was performed on all subjects euthanized at 5 dpi and 34 dpi. Tissue samples of all major organs were collected for histopathologic and immunohistochemical (IHC) examination and were immersion-fixed in 10% neutral buffered formalin for > 7 days. Specimens were processed and embedded in paraffin and sectioned at 5 μm thickness. For IHC, specific anti-SARS immunoreactivity was detected using an anti-SARS nucleocapsid protein rabbit primary antibody at a 1:800 dilution for 60 min (Novusbio). The tissue sections were processed for IHC using the ThermoFisher Scientific Lab Vision Autostainer 360 (ThermoFisher Scientific). Secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories) at 1:200 for 30 min followed by Vector Streptavidin Alkaline Phosphatase at a dilution of 1:200 for 20 min (Vector Laboratories). Slides were developed with Bio-Red (Biopath) for 7 min and counterstained with hematoxylin for 1 minute. For IHC, specific anti-fibrin was detected using an anti-fibrin monoclonal mouse primary antibody at a 1:3200 dilution for 60 min (Sekisui Diagnostics). The tissue sections were processed for IHC using the ThermoFisher Scientific Lab Vision Autostainer 360 (ThermoFisher Scientific). Secondary antibody used was biotinylated goat anti-mouse IgG (Vector Laboratories) at 1:200 for 30 min followed by Vector Streptavidin Alkaline Phosphatase at a dilution of 1:200 for 20 min (Vector Laboratories). Slides were developed with Bio-Red (Biopath Laboratories) for 7 min and counterstained with hematoxylin for 1 minute. Tissues were stained following package instructions for collagen with the Trichrome One-Step Blue & Red Stain Kit (American MasterTech Scientific Laboratory Supplies).

We challenged six healthy, adult AGMs with a target dose of 3.0 × 10⁶ PFU of SARS-CoV-2 (SARS-CoV-2/INMI1-Isolate/2020/Italy) via intranasal inoculation with the LMA MAD (actual delivered dose of 2.8 × 10⁶ PFU). Three animals were euthanized at 5 days post-infection (dpi) which is thought to be the approximate time point of peak disease in AGMs [10], while the remaining three animals were euthanized at 34 dpi during early convalescence. Blood and mucosal swabs were sampled from all animals on days 0, 2, 3, 4, 5, and additionally on days 7, 9, 12, 15, 21, 28, and 34 for AGM-4, AGM-5, and AGM-6. BAL fluid collection was performed on days − 8, 3, and 5 for all animals, as well as 7 dpi for AGM-4, AGM-5 and AGM-6. Consistent with our previous report describing the development of the combined intranasal and intratracheal SARS-CoV-2 challenge model in AGMs [10], we did not observe overt signs of clinical illness in any AGMs in this study, other than decreased appetite or brief (single day) anorexia (Supp Table 1). Temperature was longitudinally monitored in 15 min increments for the entire study duration using surgically implanted temperature loggers; several animals (AGM-4, AGM-6) experienced brief (< 2 h) periods of mildly elevated temperatures at 3 dpi, and two animals (AGM-2, AGM-3) exhibited an abnormal temperature cycling pattern at 3 dpi (Supp Fig. 1).

As in our previous report, transient shifts in leukocyte populations, predominately manifested as lymphocytopenia (5/6 animals), thrombocytopenia (3/6 animals), and granulocytosis (defined by neutrophilia, eosinophilia, and/or basophilia) (6/6 animals) were observed, while markers for renal (BUN, CRE) and hepatic function (ALT, AST, ALP, GGT) remained unchanged for the most part, with the exception of mild (≤ 2-fold) increases in ALT (2/6 animals), and mild to moderate (1 to 16-fold) increases in CRP, a marker of acute systemic inflammation (5/6 animals) (Supp Table 1), although statistical significance was not reached for most parameters at most time points (Fig. 1). In addition, hypercapnia (defined here as ≥4 mmHg increase in dissolved CO₂) was observed in 3/6 animals (Supp Table 1), which as we observed previously [10], appeared to follow a biphasic pattern (Fig. 1a, data shown as fold-change from baseline]).

All animals exhibited normal prothrombin times (PT) as compared to their individual baseline values; however, mild to moderate prolongation of the activated partial thromboplastin time (aPTT) was also observed in all animals through the acute phase of disease, most prominently in AGM-1 and AGM-2, indicating possible disorder of the intrinsic coagulation pathway (Fig. 1h, i); this was mirrored by increased levels of circulating fibrinogen (Fig. 1j). We previously showed that the pathways connected to IL-6 production are activated during SARS-CoV-2 infection of AGMs [10], indicating possible mechanisms of coagulopathy in the current study.

All animals seroconverted, with weakly neutralizing titers (as quantified by PRNT₅₀) being detected as early as 5 dpi and gradually increasing in potency by 34 dpi, with terminal neutralizing antibody titers ranging from ~ 1:16–1:128 (Fig. 2a-e). We next quantified SARS-CoV-2 nucleoprotein specific IgG by ELISA (Fig. 2f). Seroconversion was not detected until day 15 in two animals (AGM-4 & AGM-5). Interestingly, not until 34 dpi was a modest level (1:800) of seroconversion detected in the third animal.

Viral RNA (vRNA) was purified from whole blood, oral, nasal and rectal mucosa, and BAL fluid from all collection days, as well as from lung tissue harvested at necropsy. As we previously reported [10], we were unable to detect SARS-CoV-2 vRNA in whole blood by RT-qPCR, nor were we able to recover infectious virus in the plasma fraction by plaque assay, confirming a lack of either cell-associated or freely-circulating virus in the peripheral blood. SARS-CoV-2 vRNA and infectious virus was detected in the nasal mucosa from all animals as early as 2 dpi, with vRNA persisting in a single animal up to 15 dpi (Fig. 3a, b). Likewise, vRNA was detected in oral swabs from all animals beginning 2–3 dpi before falling below the limit of detection by 7 dpi, while low quantities of infectious virus (1–2 log₁₀ PFU/mL) were only isolated from three animals (AGM-4, AGM-5, and AGM-6) (Fig. 3c, d). Remarkably, vRNA was transiently shed from the lower gastrointestinal tract up to 28 dpi (AGM-4 and AGM-6), although infectious virus could only be recovered from the rectal swab of a single animal (AGM-3) 4–5 dpi (Fig. 3e, f). vRNA was detected in BAL fluid from 4/6 animals 3 dpi and up to 7 dpi in all three animals held past 5 dpi, while infectious virus was recovered from 3/6 animals (Fig. 3g, h). Detectable quantities of vRNA were absent from lungs harvested during necropsy of AGMs euthanized 34 dpi, while 6–9 log₁₀ GEq/g were detected from all three animals euthanized at 5 dpi (Fig. 3i).

Necropsy was performed on all animals following euthanasia, and lungs were collected for gross examination and histopathological analysis. Consistent with our previous study utilizing a combined i.n. and i.t. inoculation route [10], all AGMs displayed varying degrees of pulmonary consolidation with hyperemia and hemorrhage, characterized by depressed and patchy dark red to light pink regions (Fig. 4, arrows). In all AGMs, the most severe lesions were located in the dorsal aspects of the lower lung lobes. A board-certified veterinary pathologist approximated lesion severity for each lung lobe (Supp Table 2). All AGMs at 5 dpi also had segmentally flaccid and gas distention of small intestines. There were no other significant gross lesions.

Histologically, all three AGMs euthanized at 5 dpi developed mild multifocal neutrophilic bronchointerstitial pneumonia (Fig. 5a-e, o). Histologic features include acute inflammation centered within the airways of terminal bronchioles with occasional flooding of adjacent alveolar spaces with neutrophils, macrophages, fibrin, edema, hemorrhage, mucous and rarely multinucleated giant cells (5A, B). In lesser-affected regions alveolar septate were expanded with mixed inflammatory cells and alveolar spaces contain increased numbers of alveolar macrophages with scattered red blood cells. Ulcerative tracheobronchitis was also present in all three AGMs and characterized by multifocal epithelial erosion associated with underlying hemorrhage, fibrin accumulation and infiltrating acute inflammation. Polymerized fibrin, highlighted by IHC, colocalized with acute inflammation within the bronchial lumen, alveolar spaces, alveolar walls and ulcerated regions of the trachea and bronchus (Fig. 5c). Fibrin was also present within medium and small caliber vessels but was not associated with an obvious adherent thrombus. Trichrome stain of representative lung sections identified modest collagen deposition within multifocal regions of alveolar septae (Fig. 5d). IHC for SARS-CoV-2 antigen was positive in all three AGMs associated with pulmonary lesions. Positive IHC labeling was noted diffusely within the cytoplasm of respiratory epithelium of the bronchus (Fig. 5o) and less in type I and type II pneumocytes (Fig. 5e).

Histologically, all three AGMs euthanized at 34 dpi developed moderate multifocal chronic interstitial pneumonia Fig. 5f-j). Histologic features include expansion of alveolar septae with macrophages, lymphocytes, and very rarely neutrophils (Fig. 5f, g). Wispy, pale eosinophilic, acellular material also multifocally expanded the alveolar walls and stained as immature collagen with trichrome staining (Fig. 5i). Polymerized fibrin was present within medium and small caliber vessels but was not associated with an obvious adherent thrombus (Fig. 5h). No immunolabeling for SARS-CoV-2 was noted with IHC in any of the examined tissue sections from this 34 dpi cohort (Fig. 5j).