key: cord-287372-ya5uvoki
authors: Böszörményi, Kinga P.; Stammes, Marieke A.; Fagrouch, Zahra C.; Kiemenyi-Kayere, Gwendoline; Niphuis, Henk; Mortier, Daniella; van Driel, Nikki; Nieuwenhuis, Ivonne; Zuiderwijk-Sick, Ella; Meijer, Lisette; Mooij, Petra; Remarque, Ed J.; Koopman, Gerrit; Hoste, Alexis C. R.; Sastre, Patricia; Haagmans, Bart L.; Bontrop, Ronald E.; Langermans, Jan A.M.; Bogers, Willy M.; Verschoor, Ernst J.; Verstrepen, Babs E.
title: Comparison of SARS-CoV-2 infection in two non-human primate species: rhesus and cynomolgus macaques
date: 2020-11-05
journal: bioRxiv
DOI: 10.1101/2020.11.05.369413
sha: 
doc_id: 287372
cord_uid: ya5uvoki

SARS-CoV-2 is a coronavirus that sparked the current COVID-19 pandemic. To stop the shattering effect of COVID-19, effective and safe vaccines, and antiviral therapies are urgently needed. To facilitate the preclinical evaluation of intervention approaches, relevant animal models need to be developed and validated. Rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) are widely used in biomedical research and serve as models for SARS-CoV-2 infection. However, differences in study design make it difficult to compare and understand potential species-related differences. Here, we directly compared the course of SARS-CoV-2 infection in the two genetically closely-related macaque species. After inoculation with a low passage SARS-CoV-2 isolate, clinical, virological, and immunological characteristics were monitored. Both species showed slightly elevated body temperatures in the first days after exposure while a decrease in physical activity was only observed in the rhesus macaques and not in cynomolgus macaques. The virus was quantified in tracheal, nasal, and anal swabs, and in blood samples by qRT-PCR, and showed high similarity between the two species. Immunoglobulins were detected by various enzyme-linked immunosorbent assays (ELISAs) and showed seroconversion in all animals by day 10 post-infection. The cytokine responses were highly comparable between species and computed tomography (CT) imaging revealed pulmonary lesions in all animals. Consequently, we concluded that both rhesus and cynomolgus macaques represent valid models for evaluation of COVID-19 vaccine and antiviral candidates in a preclinical setting. Author summary SARS-CoV-2 infection can have a wide range of symptoms. It can cause asymptomatic or mild disease, but can also have a severe, potentially deadly outcome. Vaccines and antivirals will therefore be crucial in fighting the current COVID-19 pandemic. For testing these prophylactic and therapeutic treatments, and investigating the progression of infection and disease development, animal models play an essential role. In this study, we compare the course of SARS-CoV-2 infection in rhesus and cynomolgus macaques. Both species showed moderate disease symptoms as shown by pulmonary lesions by CT imaging. Shedding of infectious virus from the respiratory system was also documented. This study provides a detailed description of the pathogenesis of a low-passage SARS-CoV-2 isolate in two macaque models and suggests that both species represent an equally good model in research for both COVID-19 prophylactic and therapeutic treatments.

were described in proven their value in research on the related coronaviruses that caused the SARS and MERS 112 epidemics [27, 28] , and thus are considered relevant NHP models for preclinical studies. Cynomolgus macaques have been deployed in studies describing aspects of CoV-2 pathogenesis [23, 29, 30] , and have been utilized to evaluate the efficacy of 115 hydroxychloroquine as an antiviral compound [31] . Rhesus macaques have also been applied 116 in COVID-19 pathogenesis studies [22, 24, 32, 33] , and to test the efficacy of remdesivir in the 117 treatment of SARS-CoV-2 infection [34] . Additionally, several prototype COVID- 19 vaccine 118 candidates have received their first efficacy evaluation in the rhesus macaque model [35] [36] [37] [38] [39] [40] [41] [42] . 119 Some research groups [23, 24] shed light on the heterogeneity in SARS-CoV-2 infection and 120 investigated disease progression in different NHP species. Most of these studies were 121 conducted by different research teams, and a controlled comparative approach is lacking thus 122 far. 123

In other NHP disease models, like those developed for AIDS, TB, and influenza research, the 124 choice of macaque (sub)species can influence the disease outcome considerably [43] [44] [45] [46] [47] . The 125 choice of a specific NHP species for research on a new and complex disease, like COVID-19, is 126 therefore not a trivial one and the key question which macaque species is best suited to 127 investigate specific aspects of COVID-19 research needs to be answered. To address this issue, 128

we compared SARS-CoV-2 replication in rhesus and cynomolgus macaque species and 129 monitored signs of COVID-19-like disease symptoms for three weeks after infection. The 130 macaques were infected in parallel with the same virus stock, received completely identical 131 treatment, and the course of infection was followed using the same analyses, including 132 monitoring of lung pathology using computed tomography (CT), and continuous telemetric 133 recording of body temperature and activity of the animals. 134 135

After administration of the virus in the upper trachea and nose, levels of viral RNA were 138 detectable in the tracheal and nasal swabs of all monkeys at day 1 pi. Viral RNA remained 139 evident in swab samples for several days. In the tracheal swab sample of rhesus macaque 140 R14002, viral RNA was first time below the detection time at 10 days pi. (Fig 1A, S1 Table) . The 141 individual variation of SARS-CoV-2 RNA levels detected in the macaques, regardless of species, 142 was considerable. Peak viral RNA levels in the trachea varied between 1.7 x 10 4 copies/ml 143 (R15096; day 1 pi) and 1.8 x 10 8 copies/ml (J16017; day 2 pi). The time frame in which viral 144 genetic material could be detected varied from only one day (R15096; day 1 pi) up to day 10 145 pi. (animal R14002, RNA in the trachea). 146

Peak viral loads detected in nasal swabs were generally lower than levels observed in the 147 throat samples and did not exceed 9.5 x 10 4 copies/ml (R15090; day 1 pi). The high virus loads 148 measured in the first two days post-infection may suggest that some remaining RNA from the 149 original inoculum was still present. However, in all macaques, viral RNA was also isolated from 150 nasal swabs at later time points, showing that SARS-CoV-2 was excreted via the nose, and thus 151

indicative for viral replication. The total viral RNA production over time is shown in Fig 1B. 152

The patterns of viral RNA detection in swabs also varied between individuals. The most 153 outstanding observation was made for cynomolgus macaque J16017 that was positive in the 154 nose at day 1 pi, then had no detectable viral RNA for a period of three days, but later the 155 animal became again positive in the nose swabs for three consecutive days. Other animals 156 (R15096, J16004, J16012, and Ji40805) also became PCR-positive again after a period of one 157 or more days characterized by undetectable levels. In the anal swabs, viral genetic material 158 was rarely detected. Only at a few time points, three macaques tested positive, with a 159 maximum viral RNA load of 3 x 10 3 copies/ml at day 1 pi., namely in cynomolgus macaque 160 J16017. One animal tested positive for viral RNA in blood at a single time point; R15080 at day 161 5 pi. (S1 Table) . Notably, no significant differences in viral RNA loads were calculated between 162 the macaque species (Fig 1B) . 163 164 Body temperature, activity, clinical symptoms and blood parameters after SARS-CoV-2 165 infection 166

Body temperature and activity of each animal was continuously monitored using a Physiotel 167

Digital telemetric device during the entire study. Upon infection, elevated body temperatures 168

were measured in both macaque species, which could be correlated to the episodes of viral 169 replication in the nose and trachea as was evidenced by qRT-PCR. In Fig 2, we show the body 170 temperature alterations from the baseline during the study. In both groups of animals, the 171 body temperature was significantly higher during the first two weeks after infection as 172 compared to later time points (Fig 2) . The temperature curves for the individual animals are 173 depicted in the supplementary data (S1 Fig). The group of cynomolgus macaques showed 174 elevated body temperature in the first 8 to 10 days following infection. This is in contrast with 175 the measurements of the rhesus macaques where no substantial rise in temperature was 176 measured, except for two animals (R14002 and R15090) that showed a sudden peak in body 177 temperature of 0.7°C at day 8 pi. We applied a clinical scoring list to enumerate clinical symptoms that may be caused by the 185 SARS-CoV-2 infection (S2 Table) . The cumulative clinical scores per week did not exceed 50 (of 186 a maximum 490 score per week; data not shown), confirming the absence of serious COVID-187 19-related symptoms. However, in the second week of infection, cynomolgus macaques 188 showed more, but still mild, clinical symptoms than rhesus macaques. This was less evident 189 during the first and third weeks, probably due to outlier clinical scores of individual animals 190 (Fig 3) . 191

Blood samples were analyzed for changes in cell subsets and in biochemical parameters. These 192 data were related to a set of normal (standard) values derived from uninfected, healthy 193 rhesus, and cynomolgus macaques from the same breeding colony. No significant deviations 194 from the normal values were seen in blood cell subsets of the infected monkeys. C-reactive 195 protein levels, which are increased in COVID-19 patients with pneumonia [48], were not found 196 higher in infected macaques. In humans, acute kidney injury has been related to 50] , and elevated levels of serum creatinine and blood urea were detected in 198 10-15% of a cohort of . Hence, we measured creatinine and urea levels 199 in macaque blood samples at days 0, 5, 10, 14, and 22 pi., but did not find evidence of kidney 200 malfunction in the infected, but otherwise seemingly healthy monkeys. Equally, depending on 201 the severity of the disease, blood coagulation disorders, like highly elevated D-dimer levels, 

Chest CTs of the macaques after infection revealed several manifestations of COVID-19 with 209 a variable time course and lung involvement ( Table 1 ). The most common lesion types that 210 were found in both rhesus and cynomolgus macaques were ground glass opacities (GGO), 211 consolidations, and crazy paving patterns (CCP) (Fig 4) . 212 Table 1 Lung lesions (max. CT score 2/35) were already seen in CTs early after infection on day 2 in 5 215 out of 8 monkeys, three rhesus, and two cynomolgus macaques. Thereafter, lung involvement 216 was seen in most animals and CT scores increased. Around days 8 and 10 pi., lung lesions were 217 manifest in all animals, and in several macaques the coverage had increased (Table 1) The cytokine profiles after SARS-CoV-2 were highly comparable between species, except for 306 IP-10 and MCP-1, suggesting differential involvement of monocyte activation between the 307 two species. The similarity in cytokine response after SARS-CoV-2 infection contrasts with 308 observations made after infection of macaques with another respiratory virus, pandemic 309 H1N1 influenza [47] . In that study, macaque species-specific cytokine responses (IL-6, MCP-1, 310 IL-15, IL-1Ra, MIP-1α, and IL-8) were induced upon infection with pH1N1, highlighting the virus 311 type-specific reaction of the chemokine system. 312

Unlike most published studies, we decided not to conduct a necropsy on animals early, 4-5 313 days, post-infection. At that time point after infection, evidence was found for acute viral 314 interstitial pneumonia [30, 32, 34, 54] . Instead, we performed CT imaging to visualize lung 315 pathology induced by SARS-CoV-2. In humans, the sensitivity of CT scanning for lung pathology 316 is high (positive predictive value of 92%), but the type of lesions found are not COVID- specific, and can also be observed in a number of other infectious and non-infectious diseases 318 [57, 58] . In this study, we used purpose-bred NHPs with a well-documented health status and 319 we could compare the scans with a CT obtained just before infection. Therefore, CT imaging 320 provides a valuable tool to specifically monitor the progression of COVID-19-related lung 321 pathology during the entire course of the study. Based on the criteria set to determine clinical 322 severity [59], the macaques in our panel featured moderate disease levels as all eight 323 individuals show levels of pneumonia. In another study using only cynomolgus macaques and 324 using CT imaging as well, lesions were found as early as 2 days post-infection in infected 325 animals [29] . Type-wise, the lung lesions described in that report were comparable to the ones 326 in this communication, but they tend to be located deeper in the lungs. An explanation for 327 this difference may be that the method of instillation of the virus is the underlying cause. studies [22, 24, 34, 38] . This demonstrates that tracheal swabs are a good alternative for BAL 338 sampling. In addition, the collection of tracheal swabs is a less invasive technique that causes 339 relatively minor discomfort to the animals. 340

In most SARS-CoV-2 studies in non-human primates, the animals are euthanized shortly after 341 infection in the first week, or after a period of 3 weeks. The animals from this study were not 342 euthanized to be able to perform re-infection studies or to monitor them for late clinical signs, 343 or co-morbidities related to We conclude that the course of SARS-CoV-2 infection of both macaque species is highly 345 similar, indicating that they are equally suitable models to test vaccines and antivirals in a 346 preclinical setting for safety and efficacy. The macaque model for SARS-CoV-2 infection in 347 humans manifests important virological aspects of this disease in humans. Given their 348 immunological and physiological resemblance to humans, NHPs likely will continue to play a 349 pivotal role in research for both COVID-19 prophylactic and therapeutic treatments. Four Indian-origin rhesus macaques and four cynomolgus macaques were used in this study 369 (S3 Table) . All macaques were mature, outbred animals, purpose-bred, and housed at the 370 BPRC. The animals were in good physical health with normal baseline biochemical and 371 hematological values. All were pair-housed with a socially compatible cage-mate in cages of 372 at least 4 m 3 with bedding to allow foraging and were kept on a 12-hour light/dark cycle. The 373 monkeys were offered a daily diet consisting of monkey food pellets (Ssniff, Soest, Germany) 374 supplemented with vegetables and fruit. Enrichment was provided daily in the form of pieces 375 of wood, mirrors, food puzzles, and a variety of other homemade or commercially available 376 enrichment products. Drinking water was available ad libitum via an automatic watering 377 system. Animal Care staff provided daily visual health checks before infection, and twice-daily 378 after infection. The animals were monitored for appetite, general behavior, and stool 379 consistency. All possible precautions were taken to ensure the welfare and to avoid any 380 discomfort to the animals. All experimental interventions (intratracheal and intranasal 381 infection, swabs, blood samplings, and CT scans) were performed under anesthesia. 382 383

The animals were infected with the SARS-CoV-2 strain BetaCoV/BavPat1/2020. This strain was 385 isolated from a patient who traveled from China to Germany, and an aliquot of a Vero E6 cell 386 culture was made available through the European Virus Archive-Global (EVAg). The viral stock 387 for the infection study was propagated on Vero E6 cells. For this study, a fifth passage virus 388 stock was prepared with a titer of 3.2x10 6 TCID50 per ml. The integrity of the virus stock was 389 confirmed by sequence analysis. 390 391

Three weeks before the experimental infection, a Physiotel Digital device (DSI Implantable 393 Telemetry, Data Sciences International, Harvard Bioscience, UK) was implanted in the 394 abdominal cavity of each animal. This device allowed the continuous real-time measurement 395 of the body temperature and the animals' activity remotely using telemetry throughout the 396 study. 397

At day 0, all animals were exposed to a dose of 1 x 10 6 TCID50 of SARS-CoV-2, diluted in 5 ml 398 phosphate buffered saline (PBS). The virus was inoculated via a combination of the 399 intratracheal route (4.5 ml) and intranasal route (0.25 ml per nostril). Virus infection was 400 monitored for 22 days, during which period the animals were checked twice-daily by the 401 animal caretakers and scored for clinical symptoms according to a previously published, 402 adapted scoring system [60] (S2 Table) . A numeric score of 35 or more per observation time 403 point was predetermined to serve as an endpoint and justification for euthanasia. Every time 404 an animal was sedated, the body weight was measured. Blood was collected using standard 405 aseptic methods from the femoral vein at regular time points post-infection (pi). In parallel, 406 tracheal, nasal, and anal swabs were collected using Copan FLOQSwabs (MLS, Menen, 407 Belgium). Swabs were placed in 1 ml DMEM, supplemented with 0.5% bovine serum albumin 408 (BSA), fungizone (2.5 μg/ml), penicillin (100 U/ml), and streptomycin (100 For the final reconstruction, the expiration phases were exclusively used and manually 445 selected. A semi-quantitative scoring system for chest CT evaluation was used to estimate 446 SARS-CoV-2-induced lung disease [29, 62] . Quantification of the CTs was performed 447 independently by two persons based on the sum of the lobar scores. The degree of 448 involvement in each zone was scored as: 0 for no involvement, 1 for <5%, 2 for 5-24%, 3 for 449 25-49%, 4 for 50-74% and 5 for >=75% involvement. An additional increase or decrease of 0.5 450 was used to indicate alterations in CT density of the lesions. By using this scoring system, a 

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A) Viral RNA quantification in tracheal and nasal swabs of 743 rhesus and cynomolgus macaques by qRT-PCR. The limit of quantification (154 RNA 744 copies/ml) is indicated by the dotted horizontal line. (B) Total virus loads in throat and nose 745 samples of macaques throughout the study. Horizontal bars represent geometric means

The different colors used for each animal as shown in the legend of 1A are used to denote 749 the same individual in all figures of this manuscript. The group of rhesus macaques is 750 indicated by yellow to red colors; cynomolgus macaques by green to blue

The body temperature was measured by 755 telemetry throughout the study. The daily average body temperature of rhesus and 756 cynomolgus macaques was calculated and the deviations from baseline body temperature 757 (in °C) are depicted

Cumulative clinical scores. The cumulative clinical scores were calculated per week 762 and per individual animal (day 1-7, 8-14 and 15-21). Horizontal bars represent medians

Types of lung lesions detected via CT scans in SARS-CoV-2-infected macaques

Ground glass opacities (GGO), (B) consolidations, and (C) crazy paving patterns (CCP)