Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses.

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2.

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate.

BACKGROUND: Bluetongue virus (BTV) infection causes haemorrhagic disease in ruminants and induces cell death. The pathogenesis in animals and in cell culture has been linked to BTV-induced apoptosis. RESULTS: In this report, we investigated BTV-induced apoptosis in cell culture in depth and show that both extrinsic (caspase-8 activation) and intrinsic (caspase-9 activation) pathways play roles in BTV apoptosis. Further, by using chemical inhibitors and knock-out cell lines, we show that these pathways act independently of each other in BTV infected cells. In addition to activation of caspase-8, -9 and executioner caspase-3, we also identified that BTV infection causes the activation of caspase-7, which results in the cleavage of poly (ADP-ribose) polymerase (PARP). BTV-induced cell death appears to be due to apoptosis rather than necrosis, as the HMBG-1 was not translocated from the nucleus. We also examined if NF-κB response is related to BTV-induced apoptosis as in reovirus. Our data suggests that NF-κB response is not linked to the induction of apoptosis. It is controlled by the degradation of only IκBα but not IκBß, resulting in a rapid transient response during BTV infection. This was supported using an NF-κB dependent luciferase reporter gene assay, which demonstrated early response, that appeared to be suppressed by the late stage of BTV replication. Furthermore, virus titres were higher in the presence of NF-κB inhibitor (SN50), indicating that NF-κB has a role in initiating an antiviral environment. In addition, we show that BTV infection induces the translocation of interferon regulatory factors (IRF-3 and IRF-7) into the nucleus. The induction of IRF responses, when measured by IRF dependent luciferase reporter gene assay, revealed that the IRF responses, like NF-κB response, were also at early stage of infection and mirrored the timing of NF-κB induction. CONCLUSION: BTV triggers a wide range of caspase activities resulting in cell apoptosis. Although both NF-κB and IRF responses are induced by BTV infection, they are not sustained.

Analysis of Jembrana disease virus mRNA transcripts produced during acute infection demonstrates a complex transcription pattern.

Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute disease syndrome with a 20% case fatality rate after a short incubation period in Bos javanicus (Bali cattle) in Indonesia. Analysis of tat mRNA transcription patterns has identified up to six differently spliced transcripts indicating that, in common with other lentiviruses, JDV uses a complex splicing pattern. RT-PCR analysis of mRNA transcripts produced during the acute phase of infection with JDV(TAB/87) revealed at least 12 differently spliced transcripts involving 9 different splice sites. A single unspliced gag/pol transcript, singly spliced vif and tmx specific transcripts and alternatively spliced env, tat and rev transcripts were identified. A 67 nucleotide putative non-coding exon was identified that shared the same splice acceptor (SA) as vif and was incorporated into alternative transcripts of tat, rev and env.

Sequence analysis of mRNA transcripts encoding Jembrana disease virus Tat-1 in vivo.

Jembrana disease virus (JDV) is a lentivirus which replicates to very high titres in vivo and its Tat-1 protein has been shown to be a potent transactivator in vitro. Analysis of tat mRNA transcripts produced early in infection studies identified four predominant species which were generated by multiple splicing events. The use of a splice donor downstream of tat-1 was common indicating that a Tat-1 protein of 97 amino acids is expressed during the acute phase of Jembrana disease. The presence of an in-frame stop codon between tat-1 and tat-2 was identified in transcripts from three different strains of JDV confirming that expression of a single exon Tat-1 correlates with high viral titres in vivo. Sequence conservation in the regions of tat-1 that are critical for RNA binding and transcription activation in three different virus strains was high and the tat-2 sequences were completely conserved.

Sequence analysis of Jembrana disease virus strains reveals a genetically stable lentivirus.

Jembrana disease virus (JDV) is a lentivirus associated with an acute disease syndrome with a 20% case fatality rate in Bos javanicus (Bali cattle) in Indonesia, occurring after a short incubation period and with no recurrence of the disease after recovery. Partial regions of gag and pol and the entire env were examined for sequence variation in DNA samples from cases of Jembrana disease obtained from Bali, Sumatra and South Kalimantan in Indonesian Borneo. A high level of nucleotide conservation (97-100%) was observed in gag sequences from samples taken in Bali and Sumatra, indicating that the source of JDV in Sumatra was most likely to have originated from Bali. The pol sequences and, unexpectedly, the env sequences from Bali samples were also well conserved with low nucleotide (96-99%) and amino acid substitutions (95-99%). However, the sample from South Kalimantan (JDV(KAL/01)) contained more divergent sequences, particularly in env (88% identity). Phylogenetic analysis revealed that the JDV(KAL/01)env sequences clustered with the sequence from the Pulukan sample (Bali) from 2001. JDV appears to be remarkably stable genetically and has undergone minor genetic changes over a period of nearly 20 years in Bali despite becoming endemic in the cattle population of the island.

Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI.

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses.

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283-287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas.

Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme.

Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2'-O-methyltransferase (2'-O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2'-O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of in vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2'-O MTase activity in vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects on in vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2'-O MTase function for any member of the Reoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2'-O MTase activity and in turn, for virus fitness.

Analysis of Jembrana disease virus replication dynamics in vivo reveals strain variation and atypical responses to infection.

Jembrana disease virus (JDV) is an acute lentiviral infection of Bali cattle in Indonesia. Data generated during a series of cattle infection experiments was examined and significant differences were identified in the mean plasma viral load on the first and second days of the febrile response in cattle infected with JDV(TAB/87) compared to those infected with JDV(PUL/01). The peak and total viral loads >or=10(6) genome copies/ml during the acute stage of the disease were significantly higher in JDV(TAB/87) infected cattle. JDV(PUL/01) infected cattle developed peak rectal temperatures earlier than the JDV(TAB/87) cattle but there were no differences in the duration of the febrile responses observed for the 2 groups of animals. The plasma viremia was above 10(6) genome copies/ml for almost 3 days longer in JDV(TAB/87) compared to JDV(PUL/01) infected cattle. Atypical responses to infection occurred in approximately 15% of experimentally infected animals, characterized by reduced viral loads, lower or absent febrile responses and absence of p26-specific antibody responses. Most of these cattle developed normal Tm-specific antibody responses between 4-12 weeks post-infection.

Identification of a novel circovirus in Australian ravens (Corvus coronoides) with feather disease.

The complete genome of a novel Circovirus isolated from an Australian raven (Corvus coronoides) with feather lesions similar to those that occur in psittacine beak and feather disease is reported. Degenerate polymerase chain reaction primers were designed to amplify and sequence novel Circovirus DNA from affected feathers. Sequence analysis indicated that the tentatively named raven circovirus (RaCV) was 1898 nucleotides in size with two major open reading frames synonymous with other avian circoviruses, ORF C1 and ORF V1, likely to encode a putative capsid protein (Cap) and replicase-associated protein (Rep), respectively. In common with other circoviruses was the conservation of several nucleotide structures and amino acid motifs implicated in virus replication. Comparison with other members of the Circoviridae demonstrated that RaCV shares the greatest sequence homology with canary circovirus (CaCV) and pigeon circovirus (PiCV) and was more distantly related to the beak and feather disease virus, goose circovirus, duck circovirus and the two porcine circoviruses, PCV1 and PCV2. Phylogenetic analysis of the genome and the putative Cap and Rep proteins provided further evidence of the close relationship of RaCV with CaCV and PiCV.