Characterisation and natural progression of SARS-CoV-2 infection in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3-7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies.

Serological evidence of a pararubulavirus and a betacoronavirus in the geographically isolated Christmas Island flying-fox (Pteropus natalis).

Due to their geographical isolation and small populations, insular bats may not be able to maintain acute immunizing viruses that rely on a large population for viral maintenance. Instead, endemic transmission may rely on viruses establishing persistent infections within hosts or inducing only short-lived neutralizing immunity. Therefore, studies on insular populations are valuable for developing broader understanding of viral maintenance in bats. The Christmas Island flying-fox (CIFF; Pteropus natalis) is endemic on Christmas Island, a remote Australian territory, and is an ideal model species to understand viral maintenance in small, geographically isolated bat populations. Serum or plasma (n = 190), oral swabs (n = 199), faeces (n = 31), urine (n = 32) and urine swabs (n = 25) were collected from 228 CIFFs. Samples were tested using multiplex serological and molecular assays, and attempts at virus isolation to determine the presence of paramyxoviruses, betacoronaviruses and Australian bat lyssavirus. Analysis of serological data provides evidence that the species is maintaining a pararubulavirus and a betacoronavirus. There was little serological evidence supporting the presence of active circulation of the other viruses assessed in the present study. No viral nucleic acid was detected and no viruses were isolated. Age-seropositivity results support the hypothesis that geographically isolated bat populations can maintain some paramyxoviruses and coronaviruses. Further studies are required to elucidate infection dynamics and characterize viruses in the CIFF. Lastly, apparent absence of some pathogens could have implications for the conservation of the CIFF if a novel disease were introduced into the population through human carriage or an invasive species. Adopting increased biosecurity protocols for ships porting on Christmas Island and for researchers and bat carers working with flying-foxes are recommended to decrease the risk of pathogen introduction and contribute to the health and conservation of the species.

ACE2 Expression in Organotypic Human Airway Epithelial Cultures and Airway Biopsies.

Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.

Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SARS-CoV-2 spike protein.

The 'D614G' mutation (Aspartate-to-Glycine change at position 614) of the SARS-CoV-2 spike protein has been speculated to adversely affect the efficacy of most vaccines and countermeasures that target this glycoprotein, necessitating frequent vaccine matching. Virus neutralisation assays were performed using sera from ferrets which received two doses of the INO-4800 COVID-19 vaccine, and Australian virus isolates (VIC01, SA01 and VIC31) which either possess or lack this mutation but are otherwise comparable. Through this approach, supported by biomolecular modelling of this mutation and the commonly-associated P314L mutation in the RNA-dependent RNA polymerase, we have shown that there is no experimental evidence to support this speculation. We additionally demonstrate that the putative elastase cleavage site introduced by the D614G mutation is unlikely to be accessible to proteases.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Supporting pandemic response using genomics and bioinformatics: A case study on the emergent SARS-CoV-2 outbreak.

Pre-clinical responses to fast-moving infectious disease outbreaks heavily depend on choosing the best isolates for animal models that inform diagnostics, vaccines and treatments. Current approaches are driven by practical considerations (e.g. first available virus isolate) rather than a detailed analysis of the characteristics of the virus strain chosen, which can lead to animal models that are not representative of the circulating or emerging clusters. Here, we suggest a combination of epidemiological, experimental and bioinformatic considerations when choosing virus strains for animal model generation. We discuss the currently chosen SARS-CoV-2 strains for international coronavirus disease (COVID-19) models in the context of their phylogeny as well as in a novel alignment-free bioinformatic approach. Unlike phylogenetic trees, which focus on individual shared mutations, this new approach assesses genome-wide co-developing functionalities and hence offers a more fluid view of the 'cloud of variances' that RNA viruses are prone to accumulate. This joint approach concludes that while the current animal models cover the existing viral strains adequately, there is substantial evolutionary activity that is likely not considered by the current models. Based on insights from the non-discrete alignment-free approach and experimental observations, we suggest isolates for future animal models.