Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen vaccines and treatments. We show that African green monkeys (AGMs) support robust SARS-CoV-2 replication and develop pronounced respiratory disease, which may more accurately reflect human COVID-19 cases than other nonhuman primate species. SARS-CoV-2 was detected in mucosal samples, including rectal swabs, as late as 15 days after exposure. Marked inflammation and coagulopathy in blood and tissues were prominent features. Transcriptome analysis demonstrated stimulation of interferon and interleukin-6 pathways in bronchoalveolar lavage samples and repression of natural killer cell-and T cell-associated transcripts in peripheral blood. Despite a slight waning in antibody titers after primary challenge, enhanced antibody and cellular responses contributed to rapid clearance after re-challenge with an identical strain. These data support the utility of AGM for studying COVID-19 pathogenesis and testing medical countermeasures.

12Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global 13 pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to 14 screen candidate vaccines and treatments. Nonhuman primates (NHP) are considered the gold standard model 15 for many infectious pathogens as they usually best reflect the human condition. Here, we show that African green 16 monkeys support a high level of SARS-CoV-2 replication and develop pronounced respiratory disease that may 17 be more substantial than reported for other NHP species including cynomolgus and rhesus macaques. In 18 addition, SARS-CoV-2 was detected in mucosal samples of all animals including feces of several animals as late 19 as 15 days after virus exposure. Importantly, we show that virus replication and respiratory disease can be 20 produced in African green monkeys using a much lower and more natural dose of SARS-CoV-2 than has been 21 employed in other NHP studies. 42varying degrees of non-lethal illness when the virus was delivered into the respiratory tract of these animals [7-43 13]. While each of these models has utility in the study of COVID-19, NHPs have the closest physiological 44 resemblance to humans allowing a better comparison of host responses to infection. This genetic similarity has 45 also contributed to the increased availability of reagents to perform in-depth analyses of the immune response. 46Recently, the first studies evaluating the pathogenic potential of SARS-CoV-2 in cynomolgus and rhesus 47 macaques were performed. Rhesus macaques developed pneumonia and clinical signs whereas disease in 48 cynomolgus macaques was fairly mild indicating the former appears to better reflect more severe cases of 49 . These results suggest certain NHP species may serve as better models than others for 50 4 coronavirus infections. For SARS, the disease caused by SARS-CoV-1, African green monkeys (AGMs) were 51 found to support the highest level of viral replication, followed by cynomolgus macaques and rhesus macaques 52 when all three species were challenged in parallel [14]. Only AGMs had notable replication in the lower 53 respiratory tract following SARS-CoV-1 inoculation; necropsy of these animals indicated focal interstitial 54 mononuclear inflammatory infiltrates and edema in the lung consistent with human SARS. As SARS-CoV-1 and 55 SARS-CoV-2 share the same putative host receptor angiotensin-converting enzyme 2 (ACE2) [15, 16], we 56 reasoned that AGMs might serve as a useful model for COVID-19. 57Here, we infected AGMs with a low passage isolate of SARS-CoV-2 (SARS-CoV-2/INMI1- 58Isolate/2020/Italy) and evaluated their potential as a model for COVID-19. SARS-CoV-2/INMI1-Isolate/2020/Italy 59 was isolated from the first clinical case in Italy [17] and is the first V clade virus (GISAID) to be experimentally 60 inoculated into NHPs. We demonstrate AGMs mimic several aspects of human disease including a high degree 61 of viral replication and severe pulmonary lesions. T...

Existing models of Ebola virus disease (EVD) suggest antigen-presenting cells are initial targets of Zaire ebolavirus (ZEBOV). In vitro studies have shown that ZEBOV infection of monocytes and macrophages results in the production of inflammatory mediators, which may cause lymphocyte apoptosis. However, these findings have not been corroborated by in vivo studies. In this study, we report the first longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. In addition, we report a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. In contrast, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs) and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis and strategies to suppress virus replication or modify innate responses to infection in these cells should be a priority for therapeutic intervention.

Zaire Ebolavirus (ZEBOV) continues to pose a significant threat to human health as highlighted by the recent epidemic that originated in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. Although the ZEBOV variant responsible for this epidemic (Makona) shares significant genetic similarity with previously identified variants (Kikwit and Mayinga), recent reports suggest slower disease progression in nonhuman primates. However, the pathogenesis caused by the new variant is not fully understood. We present the first comprehensive approach in understanding ZEBOV-Makona pathogenesis in cynomolgus macaques by measuring changes in immune cell frequencies, plasma levels of immune mediators, and differentially expressed genes (DEGs) within whole blood (WB) and peripheral blood mononuclear cells (PBMC). Our combined approach revealed a link between: 1) increased interferon-stimulated gene expression, IFNα levels, and activated plasmacytoid dendritic cells; 2) higher proinflammatory gene expression, cytokine and chemokine levels, and non-classical monocytes; 3) gene signature of leukocyte activation and increased granulocytes; and 4) decreased expression of lymphocyte related genes and lymphopenia. In addition, our data strongly indicate delayed disease progression as well as limited overlap (~30%) in host transcriptome changes following ZEBOV-Makona infection compared to ZEBOV-Kikwit. These observations provide novel insight into the molecular mechanisms of ZEBOV-Makona pathogenesis.

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We recently reported the development of the first African green monkey (AGM) model for COVID-19 based on a combined liquid intranasal (i.n.) and intratracheal (i.t.) exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we followed up on this work by assessing an i.n. particle only route of exposure using the LMA mucosal atomization device (MAD). Six AGMs were infected with SARS-CoV-2; three animals were euthanized near the peak stage of virus replication (day 5) and three animals were euthanized during the early convalescence period (day 34). All six AGMs supported robust SARS-CoV-2 replication and developed respiratory disease. Evidence of coagulation dysfunction as noted by a transient increases in aPTT and circulating levels of fibrinogen was observed in all AGMs. The level of SARS-CoV-2 replication and lung pathology was not quite as pronounced as previously reported with AGMs exposed by the combined i.n. and i.t. routes; however, SARS-CoV-2 RNA was detected in nasal swabs of some animals as late as day 15 and rectal swabs as late as day 28 after virus challenge. Of particular importance to this study, all three AGMs that were followed until the early convalescence stage of COVID-19 showed substantial lung pathology at necropsy as evidenced by multifocal chronic interstitial pneumonia and increased collagen deposition in alveolar walls despite the absence of detectable SARS-CoV-2 in any of the lungs of these animals. These findings are consistent with human COVID-19 further demonstrating that the AGM faithfully reproduces the human condition.

A recombinant vesicular stomatitis virus (rVSV) expressing the Marburg virus (MARV) Musoke variant glycoprotein fully protects macaques against 2 MARV variants and Ravn virus as a preventive vaccine and MARV variant Musoke as a postexposure treatment. To evaluate postexposure efficacy against the most pathogenic MARV variant, Angola, we engineered rVSVs expressing homologous Angola glycoprotein. Macaques were challenged with high or low doses of variant Angola and treated 20–30 minutes after exposure. A total of 25% and 60%–75% of treated macaques survived the high-dose and low-dose challenges, respectively. The more rapid disease progression of variant Angola versus variant Musoke may account for the incomplete protection observed.

Postexposure immunization can prevent disease and reduce transmission following pathogen exposure. The rapid immunostimulatory properties of recombinant vesicular stomatitis virus (rVSV)-based vaccines make them suitable postexposure treatments against the filoviruses Ebola virus and Marburg virus (MARV); however, the mechanisms that drive this protection are undefined. Previously, we reported 60-75% survival of rhesus macaques treated with rVSV vectors expressing MARV glycoprotein (GP) 20-30 minutes after a low dose exposure to the most pathogenic variant of MARV, Angola. Survival in this model was linked to production of GP-specific antibodies and lower viral load. To confirm these results and potentially identify novel correlates of postexposure protection, we performed a similar experiment, but analyzed plasma cytokine levels, frequencies of immune cell subsets, and the transcriptional response to infection in peripheral blood. In surviving macaques (80-89%), we observed induction of genes mapping to antiviral and interferon-related pathways early after treatment and a higher percentage of T helper 1 (Th1) and NK cells. In contrast, the response of non-surviving macaques was characterized by hypercytokinemia; a T helper 2 signature; recruitment of low HLA-DR expressing monocytes and regulatory T-cells; and transcription of immune checkpoint (e.g., PD-1, LAG3) genes. These results suggest dysregulated immunoregulation is associated with poor prognosis, whereas early innate signaling and Th1-skewed immunity are important for survival. Members of the genera Marburgvirus (MARV) and Ebolavirus (EBOV) are pathogens in the family Filoviridae that cause a similar life-threatening hemorrhagic disease in humans and non-human primates (NHPs) 1. More than 30,000 people have been infected with EBOV, whereas 469 cumulative cases and 376 recorded deaths are attributed to Marburg virus disease (MVD) 2-4. Although fewer cases are recorded for MARV, future outbreaks and spread of the virus into non-endemic regions are of great concern. MVD has an overall mortality rate of 81% and imported cases have occurred in Germany, the former Yugoslavia (presently Serbia), the Netherlands, and the United States 1-4. Moreover, the Egyptian fruit bat host reservoir has a wide geographic distribution 5. While MARV is thought to be limited to equatorial Africa, a research group that surveyed a large South African bat colony found that ~53% of these animals were seropositive for the virus, and recently MARV was isolated from bats in West Africa for the first time 6,7. Surveillance in the latter region also revealed serological evidence of filoviruses (MARV and EBOV) circulating in human subjects prior to the 2013-2016 EBOV outbreak 8,9. The likelihood of spillover events and spread into human populations emphasizes the need for adequate countermeasures against this deadly virus. One of the most promising vaccine candidates against MARV and EBOV uses a live, attenuated recombinant vesicular stomatitis virus (rVSV) platform to express filovirus ...