There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

41In December 2019 an outbreak of coronavirus disease emerged in 42 Wuhan, China. The causative agent was subsequently identified and named severe 43 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread 44 worldwide causing a pandemic. Currently there are no licensed vaccines or 45 therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in 46 development and repurposed drugs are being tested in the clinic. There is a vital need 47 for authentic COVID-19 animal models to further our understanding of pathogenesis 48 and viral spread in addition to pre-clinical evaluation of candidate interventions. 49 50Here we report a dose titration study of SARS-CoV-2 to determine the most suitable 51 infectious dose to use in the ferret model. We show that a high (5x10 6 pfu) and medium 52 (5x10 4 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral 53 RNA shedding in both groups of six challenged animals, whilst a low dose (5x10 2 pfu) 54 resulted in only one of six displaying signs of URT viral RNA replication. The URT 55 shedding lasted up to 21 days in the high dose animals with intermittent positive signal 56 from day 14. Sequential culls revealed distinct pathological signs of mild multifocal 57 bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and 58 medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 59 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were 60 observed although animals did present with a consistent post-viral fatigue lasting from 61 day 9-14 in the medium and high dose groups. After virus shedding ceased, re-62 challenged ferrets were shown to be fully protected from acute lung pathology. The 63Page 4 of 39 endpoints of URT viral RNA replication in addition to distinct lung pathology and post 64 viral fatigue were observed most consistently in the high dose group. This ferret model 65 of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of 66 patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 67 14-21 parallel observations reported in a minority of clinical cases. 68 69 70 71 Word count: 327 72 Introduction 73 74 Coronaviruses are positive sense, single stranded RNA viruses belonging to the family 75 Coronaviridae 1 . These viruses can infect a range of animals, including humans and 76 usually cause a mild respiratory infection, much like the common cold. Two highly 77 pathogenic coronaviruses have emerged in the human population in the last 20 years; 78 severe acute respiratory syndrome (SARS) CoV and middle eastern respiratory 79 syndrome (MERS) CoV. SARS-CoV infected approximately 8,000 people worldwide with 80 a case fatality rate (CFR) of 10%, while MERS-CoV has infected approximately 2,500 81 people with a CFR of 36% 2 .82 83 In December 2019 several pneumonia cases of unknown cause emerged in Wuhan, 84 Hubei, China. Deep sequencing analysis from lower respiratory tract samples from ...

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo. Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an “antiviral state.” Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

Background The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. Methods SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 10 6 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). Findings We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs ( p =<0.0001) by up to a 24 fold (96% reduction) and in nasal wash ( p =0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. Interpretation The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. Funding This work was funded by Ena Respiratory, Melbourne, Australia.