SARS-CoV and SARS-CoV-2 display limited neuronal infection and lack the ability to transmit within synaptically connected axons in stem cell-derived human neurons.

Sarbecoviruses such as SARS and SARS-CoV-2 have been responsible for two major outbreaks in humans, the latter resulting in a global pandemic. While sarbecoviruses primarily cause an acute respiratory infection, they have been shown to infect the nervous system. However, mechanisms of sarbecovirus neuroinvasion and neuropathogenesis remain unclear. In this study, we examined the infectivity and trans-synaptic transmission potential of the sarbecoviruses SARS and SARS-CoV-2 in human stem cell-derived neural model systems. We demonstrated limited ability of sarbecoviruses to infect and replicate in human stem cell-derived neurons. Furthermore, we demonstrated an inability of sarbecoviruses to transmit between synaptically connected human stem cell-derived neurons. Finally, we determined an absence of SARS-CoV-2 infection in olfactory neurons in experimentally infected ferrets. Collectively, this study indicates that sarbecoviruses exhibit low potential to infect human stem cell-derived neurons, lack an ability to infect ferret olfactory neurons, and lack an inbuilt molecular mechanism to utilise retrograde axonal trafficking and trans-synaptic transmission to spread within the human nervous system.

Henipavirus-induced neuropathogenesis in mice.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses that can cause fatal encephalitis in humans. Many animal models have been used to study henipavirus pathogenesis. In the mouse, HeV infection has previously shown that intranasal challenge can lead to neurological infection, however mice similarly challenged with NiV show no evidence of virus infecting the brain. We generated recombinant HeV (rHeV) and NiV (rNiV) where selected proteins were switched to examine their role in neuroinvasion in the mouse. These viruses displayed similar growth kinetics when compared to wildtype in vitro. In the mouse, infection outcomes with recombinant virus did not differ to infection outcomes of wildtype viruses. Virus was detected in the brain of 5/30 rHeV-challenged mice, but not rNiV-challenged mice. To confirm the permissiveness of mouse neurons to these viruses, primary mouse neurons were successfully infected in vitro, suggesting that other pathobiological factors contribute to the differences in disease outcomes in mice.

A Time-Series Metabolomic Analysis of SARS-CoV-2 Infection in a Ferret Model.

The global threat of COVID-19 has led to an increased use of metabolomics to study SARS-CoV-2 infections in animals and humans. In spite of these efforts, however, understanding the metabolome of SARS-CoV-2 during an infection remains difficult and incomplete. In this study, metabolic responses to a SAS-CoV-2 challenge experiment were studied in nasal washes collected from an asymptomatic ferret model (n = 20) at different time points before and after infection using an LC-MS-based metabolomics approach. A multivariate analysis of the nasal wash metabolome data revealed several statistically significant features. Despite no effects of sex or interaction between sex and time on the time course of SARS-CoV-2 infection, 16 metabolites were significantly different at all time points post-infection. Among these altered metabolites, the relative abundance of taurine was elevated post-infection, which could be an indication of hepatotoxicity, while the accumulation of sialic acids could indicate SARS-CoV-2 invasion. Enrichment analysis identified several pathways influenced by SARS-CoV-2 infection. Of these, sugar, glycan, and amino acid metabolisms were the key altered pathways in the upper respiratory channel during infection. These findings provide some new insights into the progression of SARS-CoV-2 infection in ferrets at the metabolic level, which could be useful for the development of early clinical diagnosis tools and new or repurposed drug therapies.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Detection of filovirus-reactive antibodies in Australian bat species.

Bats have been implicated as the reservoir hosts of filoviruses in Africa, with serological evidence of filoviruses in various bat species identified in other countries. Here, serum samples from 190 bats, comprising 12 different species, collected in Australia were evaluated for filovirus antibodies. An in-house indirect microsphere assay to detect antibodies that cross-react with Ebola virus (Zaire ebolavirus; EBOV) nucleoprotein (NP) followed by an immunofluorescence assay (IFA) were used to confirm immunoreactivity to EBOV and Reston virus (Reston ebolavirus; RESTV). We found 27 of 102 Yinpterochiroptera and 19 of 88 Yangochiroptera samples were positive to EBOV NP in the microsphere assay. Further testing of these NP positive samples by IFA revealed nine bat sera that showed binding to ebolavirus-infected cells. This is the first report of filovirus-reactive antibodies detected in Australian bat species and suggests that novel filoviruses may be circulating in Australian bats.

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.

Evaluation of three rapid low-resource molecular tests for Nipah virus.

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/µL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000-100,000 TCID50/mL (100-200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

Challenges and Opportunities in the Use of High and Maximum Biocontainment Facilities in Developing and Licensing Risk Group 3 and Risk Group 4 Agent Veterinary Vaccines.

New solutions are necessary for the singular global health security threat formed by endemic, epidemic, and emerging/re-emerging zoonoses, coupled with epizootic and enzootic transboundary animal diseases (TADs). This One Health issue is related to the daily interactions between wildlife, domesticated and indigenous livestock, and humans primarily associated with global trade, transboundary co-movement of humans and diverse livestock/livestock products, and agriculture production intensification and penetration into previously uninhabited areas. The World Health Organization defines Risk Group 3 (RG-3) and RG-4 pathogens as mainly viruses but also bacteria that serve as the foundation for approximately 60% of emerging infectious diseases that are zoonoses. The World Organisation for Animal Health defines trade-notifiable TADs, and subsets of these are zoonotic. Livestock vaccination policies mainly focus on TADs that are promulgated by the United Nations Food and Agriculture Organization and government agriculture agencies. The development, licensure, and product manufacturing of next-generation molecular-based RG-3 and RG-4 veterinary vaccines largely ignored by the global animal health biopharmaceutical sector can have an important positive impact on food security and One Health. There have been sharp increases in the global demand for livestock meat and milk products, especially in low- and middle-income countries in Africa and Asia. This relatively recent market driver-coupled with scientific advances in human EID and zoonotic disease vaccine platform technologies and increases in the number of high (US biosafety level 3 agriculture) and maximum (US animal biosafety level 4) biocontainment facilities with supporting workforce capabilities-offers new investment opportunities to the animal health biopharmaceutical sector. Moreover, a growing number of One Health public-private partnerships have moved the net present value calculus in favor of the financial feasibility of RG-3 and RG-4 veterinary vaccine product development and licensure. This article highlights the challenges and opportunities in the use of high and maximum biocontainment facilities in developing and licensing RG-3 and RG-4 veterinary vaccines that are safe and effective against epizootic and enzootic TADs and zoonotic diseases.

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.

Evaluation of henipavirus chemical inactivation methods for the safe removal of samples from the high-containment PC4 laboratory.

Henipaviruses, Hendra (HeV) and Nipah (NiV), are highly pathogenic zoonotic agents that pose a serious health risk to human life, and as such are restricted to physical containment 4 (PC4) laboratories. For further analysis of virus-infected biological specimens, it is necessary to ensure absolute inactivation of any infectious virus present before removal from the PC4 laboratory. To evaluate the inactivation of HeV and NiV within infected samples, two chemical inactivation methods were assessed. Henipavirus-infected cell monolayers treated with 4 % paraformaldehyde (PFA) showed the complete inactivation of infectious virus, with an inactivation period of 15 min resulting in more than 8-log decrease in infectious titre. NiV-infected tissue samples treated with 10 % neutral-buffered formalin (NBF) showed a complete reduction of infectious virus in 7/8 ferret organs incubated for 24 h, with the remaining tissue demonstrating complete virus inactivation after 48 h. The chemical inactivation methods described herein evaluated two simple methods of henipavirus inactivation, resulting in the complete inactivation of infectious virus - an essential requirement for the safe removal and handling of biological samples from the PC4 laboratory.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection.

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management.

Erratum: Johnson et al. Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets. Viruses 2018, 10, 675.

The authors wish to make the following corrections to this paper [...].

Antagonism of STAT3 signalling by Ebola virus.

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.

Type I Hypersensitivity in Ferrets Following Exposure to SARS-CoV-2 Inoculum: Lessons Learned.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes.

Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with "fine-tuning" to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.

Metabolic Profiling from an Asymptomatic Ferret Model of SARS-CoV-2 Infection.

Coronavirus disease (COVID-19) is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n = 6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic Multiple Reaction Monitoring (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other metabolomic-based findings in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or repurposed drug therapies.

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.

Achimota Pararubulavirus 3: A New Bat-Derived Paramyxovirus of the Genus Pararubulavirus.

Bats are an important source of viral zoonoses, including paramyxoviruses. The paramyxoviral Pararubulavirus genus contains viruses mostly derived from bats that are common, diverse, distributed throughout the Old World, and known to be zoonotic. Here, we describe a new member of the genus Achimota pararubulavirus 3 (AchPV3) and its isolation from the urine of African straw-coloured fruit bats on primary bat kidneys cells. We sequenced and analysed the genome of AchPV3 relative to other Paramyxoviridae, revealing it to be similar to known pararubulaviruses. Phylogenetic analysis of AchPV3 revealed the failure of molecular detection in the urine sample from which AchPV3 was derived and an attachment protein most closely related with AchPV2-a pararubulavirus known to cause cross-species transmission. Together these findings add to the picture of pararubulaviruses, their sources, and variable zoonotic potential, which is key to our understanding of host restriction and spillover of bat-derived paramyxoviruses. AchPV3 represents a novel candidate zoonosis and an important tool for further study.

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.