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BackgroundIn 2014, Western Africa experienced an unanticipated explosion of Ebola virus infections. What distinguishes fatal from non-fatal outcomes remains largely unknown, yet is key to optimising personalised treatment strategies. We used transcriptome data for peripheral blood taken from infected and convalescent recovering patients to identify early stage host factors that are associated with acute illness and those that differentiate patient survival from fatality.ResultsThe data demonstrate that individuals who succumbed to the disease show stronger upregulation of interferon signalling and acute phase responses compared to survivors during the acute phase of infection. Particularly notable is the strong upregulation of albumin and fibrinogen genes, which suggest significant liver pathology. Cell subtype prediction using messenger RNA expression patterns indicated that NK-cell populations increase in patients who survive infection. By selecting genes whose expression properties discriminated between fatal cases and survivors, we identify a small panel of responding genes that act as strong predictors of patient outcome, independent of viral load.ConclusionsTranscriptomic analysis of the host response to pathogen infection using blood samples taken during an outbreak situation can provide multiple levels of information on both disease state and mechanisms of pathogenesis. Host biomarkers were identified that provide high predictive value under conditions where other predictors, such as viral load, are poor prognostic indicators. The data suggested that rapid analysis of the host response to infection in an outbreak situation can provide valuable information to guide an understanding of disease outcome and mechanisms of disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1137-3) contains supplementary material, which is available to authorized users.

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 ...

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH N /A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH N /A or recombinant LH N /A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH N /A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved. The clostridial neurotoxin (CNT)1 family includes tetanus toxin (TeNT), produced by Clostridium tetani, and the seven antigenically distinct botulinum neurotoxins produced from strains of Clostridium botulinum (BoNTs). These proteins are responsible for the conditions of tetanus and botulism, respectively, that develop as a direct result of inhibition of Ca 2ϩ -dependent neurotransmitter release, a mechanism of action common to all the CNTs. In the case of BoNTs, intoxication of the neuromuscular junction is thought to occur in at least three phases: an initial binding phase, an internalization phase, and finally a neurotransmitter blockade phase (1).All CNTs have a similar structure and consist of a heavy chain (ϳ100 kDa) covalently joined to a light chain (ϳ50 kDa) by a single disulfide bond. Proteolytic cleavage of the heavy chain of C. botulinum neurotoxin type A (BoNT/A) generates two fragments of ϳ50 kDa each. The C-terminal domain (H C ) is required for target cell binding, with the N-terminal domain (H N ) being proposed to be involved in intracellular membrane translocation (2). Under conditions in which the disulfide bond between the light and heavy chains is maintained, trypsin cleavage results in a 100-kDa species termed LH N /A (light chain plus N-terminal heavy chain domain) representing a catalytically active, non-cell binding, non-toxic derivative of BoNT/A. In addition to obtainin...