Respiratory disease in rhesus macaques inoculated with SARS-CoV-2.

An outbreak of coronavirus disease 2019 (COVID-19), which is caused by a novel coronavirus (named SARS-CoV-2) and has a case fatality rate of approximately 2%, started in Wuhan (China) in December 20191,2. Following an unprecedented global spread3, the World Health Organization declared COVID-19 a pandemic on 11 March 2020. Although data on COVID-19 in humans are emerging at a steady pace, some aspects of the pathogenesis of SARS-CoV-2 can be studied in detail only in animal models, in which repeated sampling and tissue collection is possible. Here we show that SARS-CoV-2 causes a respiratory disease in rhesus macaques that lasts between 8 and 16 days. Pulmonary infiltrates, which are a hallmark of COVID-19 in humans, were visible in lung radiographs. We detected high viral loads in swabs from the nose and throat of all of the macaques, as well as in bronchoalveolar lavages; in one macaque, we observed prolonged rectal shedding. Together, the rhesus macaque recapitulates the moderate disease that has been observed in the majority of human cases of COVID-19. The establishment of the rhesus macaque as a model of COVID-19 will increase our understanding of the pathogenesis of this disease, and aid in the development and testing of medical countermeasures.

Single-Dose Treatment With Vesicular Stomatitis Virus-Based Ebola Virus Vaccine Expressing Ebola Virus-Specific Artificial Micro-RNA Does Not Protect Mice From Lethal Disease.

Although significant progress has been made in the development of therapeutics against Ebola virus (EBOV), we sought to expand upon existing strategies and combine an RNA interference-based intervention with the approved vesicular stomatitis virus-based Ebola virus (VSV-EBOV) vaccine to conjointly treat and vaccinate patients during an outbreak. We constructed VSV-EBOV vectors expressing artificial micro-RNAs (amiRNAs) targeting sequences of EBOV proteins. In vitro experiments demonstrated a robust decrease in EBOV replication using a minigenome system and infectious virus. For in vivo evaluation, mouse-adapted EBOV-infected CD-1 mice were treated 24 hours after infection with a single dose of the VSV-EBOV amiRNA constructs. We observed no difference in disease progression or survival compared to the control-treated mice. In summary, while amiRNAs decrease viral replication in vitro, the effect is not sufficient to protect mice from lethal disease, and this therapeutic approach requires further optimization.

Ebola Virus IgG Seroprevalence in Southern Mali.

Mali had 2 reported introductions of Ebola virus (EBOV) during the 2013-2016 West Africa epidemic. Previously, no evidence for EBOV circulation was reported in Mali. We performed an EBOV serosurvey study in southern Mali. We found low seroprevalence in the population, indicating local exposure to EBOV or closely related ebola viruses.

Development and Characterization of Broadly Cross-reactive Monoclonal Antibodies Against All Known Ebolavirus Species.

As of 25 March 2015, the largest recorded outbreak of Ebola virus infection is ongoing, with almost 25 000 cases and >10 000 deaths. There are 5 genetically and antigenically distinct species within the genus Ebolavirus. Limited cross-reactivity and protection is observed between these 5 Ebolavirus species, which complicates vaccine development. However, on the basis of sequence homology between the 5 Ebolavirus species, we hypothesize that conserved epitopes are present on the viral glycoprotein (GP), which can be targeted by antibodies. In the current study, a panel of mouse monoclonal antibodies was isolated and characterized using an enzyme-linked immunosorbent assay (ELISA) to determine cross-reactivity, avidity, and competition for epitope binding; Western blot analysis was also performed. Four monoclonal antibodies were identified by ELISA as cross-reacting with the GPs of all 5 Ebolavirus species. The identification of cross-reactive antibodies that bind the GPs of all known Ebolavirus species will give us important insight into the presence of conserved epitopes on the viral GP. These data will be crucial for the development of novel therapeutics and diagnostic assays.

Soluble Glycoprotein Is Not Required for Ebola Virus Virulence in Guinea Pigs.

Ebola virus (EBOV) uses transcriptional editing to express several glycoproteins (GPs), including secreted soluble GP (sGP) and structural GP1,2, from a single gene. Recombinant viruses predominantly expressing GP1,2 are known to rapidly mutate and acquire an editing site predominantly expressing sGP in vivo, suggesting an important role of this protein during infection. Therefore, we generated a recombinant virus that is no longer able to express sGP and assessed its virulence in the EBOV guinea pig model. Surprisingly, although this virus remained genetically stable, it did not show any significant attenuation in vivo, showing that sGP is not required for virulence in this model.

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

UNLABELLED: Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. IMPORTANCE: Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

Combined molnupiravir-nirmatrelvir treatment improves the inhibitory effect on SARS-CoV-2 in macaques.

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have recently been approved as monotherapy for use in high-risk patients with COVID-19. As preclinical data are only available for rodent and ferret models, here we assessed the efficacy of MK-4482 and PF-07321332 alone and in combination against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Macaques were infected with the SARS-CoV-2 Delta variant and treated with vehicle, MK-4482, PF-07321332, or a combination of MK-4482 and PF-07321332. Clinical exams were performed at 1, 2, and 4 days postinfection to assess disease and virological parameters. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs, resulting in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated in the closest COVID-19 surrogate model of human infection.

Immunological correlates of protection afforded by PHV02 live, attenuated recombinant vesicular stomatitis virus vector vaccine against Nipah virus disease.

Introduction: Immune correlates of protection afforded by PHV02, a recombinant vesicular stomatitis (rVSV) vector vaccine against Nipah virus (NiV) disease, were investigated in the African green monkey (AGM) model. Neutralizing antibody to NiV has been proposed as the principal mediator of protection against future NiV infection. Methods: Two approaches were used to determine the correlation between neutralizing antibody levels and outcomes following a severe (1,000 median lethal doses) intranasal/intratracheal (IN/IT) challenge with NiV (Bangladesh): (1) reduction in vaccine dose given 28 days before challenge and (2) challenge during the early phase of the antibody response to the vaccine. Results: Reduction in vaccine dose to very low levels led to primary vaccine failure rather than a sub-protective level of antibody. All AGMs vaccinated with the nominal clinical dose (2 × 107 pfu) at 21, 14, or 7 days before challenge survived. AGMs vaccinated at 21 days before challenge had neutralizing antibodies (geometric mean titer, 71.3). AGMs vaccinated at 7 or 14 days before challenge had either undetectable or low neutralizing antibody titers pre-challenge but had a rapid rise in titers after challenge that abrogated the NiV infection. A simple logistic regression model of the combined studies was used, in which the sole explanatory variable was pre-challenge neutralizing antibody titers. For a pre-challenge titer of 1:5, the predicted survival probability is 100%. The majority of animals with pre-challenge neutralizing titer of ≥1:20 were protected against pulmonary infiltrates on thoracic radiograms, and a majority of those with titers ≥1:40 were protected against clinical signs of illness and against a ≥fourfold antibody increase following challenge (indicating sterile immunity). Controls receiving rVSV-Ebola vaccine rapidly succumbed to NiV challenge, eliminating the innate immunity stimulated by the rVSV vector as a contributor to survival in monkeys challenged as early as 7 days after vaccination. Discussion and conclusion: It was concluded that PHV02 vaccine elicited a rapid onset of protection and that any detectable level of neutralizing antibody was a functional immune correlate of survival.

Distinct VSV-based Nipah virus vaccines expressing either glycoprotein G or fusion protein F provide homologous and heterologous protection in a nonhuman primate model.

BACKGROUND: Nipah virus (NiV) causes recurrent outbreaks of lethal respiratory and neurological disease in Southeast Asia. The World Health Organization considers the development of an effective vaccine against NiV a priority. METHODS: We produced two NiV vaccine candidates using the licensed VSV-EBOV vaccine as a backbone and tested its efficacy against lethal homologous and heterologous NiV challenge with Nipah virus Bangladesh and Nipah virus Malaysia, respectively, in the African green monkey model. FINDINGS: The VSV-EBOV vaccine expressing NiV glycoprotein G (VSV-NiVG) induced high neutralising antibody titers and afforded complete protection from homologous and heterologous challenge. The VSV-EBOV vaccine expressing NiV fusion protein F (VSV-NiVF) induced a lower humoral response and afforded complete homologous protection, but only partial heterologous protection. Both vaccines reduced virus shedding from the upper respiratory tract, and virus replication in the lungs and central nervous system. None of the protected animals vaccinated with VSV-NiVG or VSV-NiVF showed histological lesions in the CNS, but one VSV-NiVF-vaccinated animal that was not protected developed severe meningoencephalitis. INTERPRETATION: The VSV-NiVG vaccine offers broad protection against NiV disease. FUNDING: This study was supported by the Intramural Research Program, NIAID, NIH.

Clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection.

Nipah virus (NiV) and Hendra virus (HeV) are emerging zoonotic viruses and the causative agents of severe respiratory disease and encephalitis in humans. Little is known about the mechanisms that govern the development of respiratory and neurological disease. Using a hamster model of lethal NiV and HeV infection, we describe the role of the route and dose of infection on the clinical outcome and determine virus tropism and host responses following infection. Infection of hamster with a high dose of NiV or HeV resulted in acute respiratory distress. NiV initially replicated in the upper respiratory tract epithelium, whereas HeV initiated infection primarily in the interstitium. In contrast, infection with a low dose of NiV or HeV resulted in the development of neurological signs and more systemic spread of the virus through involvement of the endothelium. The development of neurological signs coincided with disruption of the blood-brain barrier (BBB) and expression of tumor necrosis alpha (TNF-α) and interleukin 1 ß (IL-1ß). In addition, interferon-inducible protein 10 (IP-10) was identified as playing an important role in NiV and HeV pathogenesis. These studies reveal novel information on the development and progression of NiV and HeV clinical disease, provide a mechanism for the differences in transmission observed between NiV and HeV outbreaks, and identify specific cytokines and chemokines that serve as important targets for treatment.

Vesicular stomatitis virus-based Ebola vaccines with improved cross-protective efficacy.

For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d'Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)-based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig-adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.

Combined Molnupiravir and Nirmatrelvir Treatment Improves the Inhibitory Effect on SARS-CoV-2 in Rhesus Macaques.

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have each recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we originally assessed the efficacy of MK-4482 and PF-07321332 alone and then in combination Against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs. Combined treatment resulted in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated here in the closest COVID-19 surrogate model. One Sentence Summary: The combination of molnupiravir and nirmatrelvir inhibits SARS-CoV-2 replication and shedding more effectively than individual treatments in the rhesus macaque model.

Development of a nonhuman primate model for mammalian bornavirus infection.

Until recently, it was assumed that members of the family Bornaviridae could not induce severe disease in humans. Today, however, Borna disease virus 1 (BoDV-1), as well as the more recently emerged variegated squirrel bornavirus 1 (VSBV-1), are known as causative agents of lethal encephalitis in humans. In order to establish animal models reflecting the pathogenesis in humans and for countermeasure efficacy testing, we infected twelve rhesus macaques (Macaca mulatta) either with VSBV-1 or with BoDV-1. For each virus, three monkeys each were inoculated with 2 × 104 focus forming units by the intracerebral route or by multiple peripheral routes (intranasal, conjunctival, intramuscular, and subcutaneous; same dose in total). All BoDV-1 and VSBV-1 intracerebrally infected monkeys developed severe neurological signs around 5 to 6 or 8 to 12 weeks postinfection, respectively. Focal myoclonus and tremors were the most prominent observations in BoDV-1 and VSBV-1-infected animals. VSBV-1-infected animals also showed behavioral changes. Only one BoDV-1 peripherally infected animal developed similar disease manifestations. All animals with severe clinical disease showed high viral loads in brain tissues and displayed perivascular mononuclear cuffs with a predominance of lymphocytes and similar meningeal inflammatory infiltrates. In summary, rhesus macaques intracerebrally infected with mammalian bornaviruses develop a human-like disease and may serve as surrogate models for human bornavirus infection.

Three-Week Old Pigs Are Not Susceptible to Productive Infection with SARS-COV-2.

As the COVID-19 pandemic moves into its third year, there remains a need for additional animal models better recapitulating severe COVID to study SARS-CoV-2 pathogenesis and develop countermeasures, especially treatment options. Pigs are known intermediate hosts for many viruses with zoonotic potential and are susceptible to infection with alpha, beta and delta genera of coronaviruses. Herein, we infected young (3 weeks of age) pigs with SARS-CoV-2 using a combination of respiratory and parenteral inoculation routes. Pigs did not develop clinical disease, nor macroscopic or microscopic pathologic lesions upon SARS-CoV-2 infection. Despite occasional low levels of SARS-CoV-2 genomic RNA in the respiratory tract, subgenomic RNA and infectious virus were never found, and SARS-CoV-2-specific adaptive immune responses were not detectable over the 13-day study period. We concluded that pigs are not susceptible to productive SARS-CoV-2 infection and do not serve as a SARS-CoV-2 reservoir for zoonotic transmission.

Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague.

The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.

A novel model of lethal Hendra virus infection in African green monkeys and the effectiveness of ribavirin treatment.

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.

Favipiravir (T-705) Protects IFNAR-/- Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner.

Zika virus (ZIKV), a member of the Flaviviridae family, is an important human pathogen that has caused epidemics in Africa, Southeast Asia, and the Americas. No licensed treatments for ZIKV disease are currently available. Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) and ribavirin (1-(ß-D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide) are nucleoside analogs that have exhibited antiviral activity against a broad spectrum of RNA viruses, including some flaviviruses. In this study, we strengthened evidence for favipiravir and ribavirin inhibition of ZIKV replication in vitro. Testing in IFNAR-/- mice revealed that daily treatments of favipiravir were sufficient to provide protection against lethal ZIKV challenge in a dose-dependent manner but did not completely abrogate disease. Ribavirin, on the other hand, had no beneficial effect against ZIKV infection in this model and under the conditions examined. Combined treatment of ribavirin and favipiravir did not show improved outcomes over ribavirin alone. Surprisingly, outcome of favipiravir treatment was sex-dependent, with 87% of female but only 25% of male mice surviving lethal ZIKV infection. Since virus mutations were not associated with outcome, a sex-specific host response likely explains the observed sex difference.

Single Dose of a VSV-Based Vaccine Rapidly Protects Macaques From Marburg Virus Disease.

Marburg virus (MARV) is a member of the filovirus family that causes hemorrhagic disease with high case fatality rates. MARV is on the priority list of the World Health Organization for countermeasure development highlighting its potential impact on global public health. We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) and previously demonstrated uniform protection of nonhuman primates (NHPs) with a single dose. Here, we investigated the fast-acting potential of this vaccine by challenging NHPs with MARV 14, 7 or 3 days after a single dose vaccination with VSV-MARV. We found that 100% of the animals survived when vaccinated 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of whole blood samples indicated activation of B cells and antiviral defense after VSV-MARV vaccination. In the day -14 and -7 groups, limited transcriptional changes after challenge were observed with the exception of day 9 post-challenge in the day -7 group where we detected gene expression profiles indicative of a recall response. In the day -3 group, transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity. In summary, our data demonstrate that the VSV-MARV is a fast-acting vaccine suitable for the use in emergency situations like disease outbreaks in Africa.

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques.

The deployment of a vaccine that limits transmission and disease likely will be required to end the Coronavirus Disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally-administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S) in the upper and lower respiratory tract of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged one month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induced neutralizing antibodies and T cell responses and limited or prevented infection in the upper and lower respiratory tract after SARS-CoV-2 challenge. As this single intranasal dose vaccine confers protection against SARS-CoV-2 in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.

UK B.1.1.7 (Alpha) variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, we compared African green monkeys infected intranasally with either the UK B.1.1.7 (Alpha) variant or its contemporary D614G progenitor. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.