One for all - Human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses

Despite the vast increase in research activity in the coronavirus field over the past two years, researchers are still heavily reliant on non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or not naturally susceptible cell lines overexpressing receptor ACE2 and other accessory factors, such as TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain and have limited suitability for high-throughput analysis. In vitro investigations of host-pathogen interactions of viruses is highly reliant on suitable cell and tissue culture models and results are only as good as the model they have been validated in. Here, we show the use of a highly characterized human kidney cell line, Caki-1, for infection with three human coronaviruses: Betacoronaviruses severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cells lines for the cultivation of the respective virus. Furthermore, we used a panel of antibodies generated against 21 SARS-CoV-2-encoded proteins to identify their location in the infected Caki-1 cells using immunocytochemistry. Most importantly, Caki-1 cells are also susceptible to two other respiratory viruses, Influenza A virus and RSV, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general.

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCEWe explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-{kappa}B signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

Face Coverings and Respiratory Tract Droplet Dispersion

Respiratory droplets are the primary transmission route for SARS-CoV-2. Evidence suggests that virus transmission can be reduced by face coverings, but robust evidence for how mask usage might affect safe distancing parameters is lacking. Accordingly, we investigate the effectiveness of surgical masks and single-layer cotton masks on mitigating dispersion of large respiratory droplets (i.e. non aerosol). We tested a manikin ejecting fluorescent droplets and human volunteers in speaking and coughing conditions. We quantified the number of droplets in flight using laser sheet illumination and UV-light for those that had landed at table height at up to 2m. For human volunteers, expiratory droplets were caught on a microscope slide 5cm from the mouth. Whether manikin or human, wearing a face covering decreased the number of projected droplets by >1000-fold. We estimated that a person standing 2m from someone coughing without a mask is exposed to over 1000 times more respiratory droplets than from someone standing 5 cm away wearing a basic single layer mask. Our results indicate that face coverings show consistent efficacy at blocking respiratory droplets. If aerosol transmission is later determined to be a significant driver of infection, then our findings may overestimate the effectiveness of face coverings.

Intra-genome variability in the dinucleotide composition of SARS-CoV-2

CpG dinucleotides are under-represented in the genomes of single stranded RNA viruses, and coronaviruses, including SARS-CoV-2, are no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development, and if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the newly emerged SARS-CoV-2 genome was characterised in the context of other coronaviruses. CpG suppression amongst coronaviruses does not significantly differ according to genera of virus, but does vary according to host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. While ORF10 is only present in the genomes of a subset of coronaviruses, E is essential for virus replication. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. Phylogeny indicates that this is an ancestrally-derived trait reflecting their origin in bats, rather than something selected for after zoonotic transfer. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.