Highlights d A SARS-CoV-2 variant with Spike G614 has replaced D614 as the dominant pandemic form d The consistent increase of G614 at regional levels may indicate a fitness advantage d G614 is associated with lower RT PCR Cts, suggestive of higher viral loads in patients d The G614 variant grows to higher titers as pseudotyped virions

Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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Bright-red colors in vertebrates are commonly involved in sexual, social, and interspecific signaling [1-8] and are largely produced by ketocarotenoid pigments. In land birds, ketocarotenoids such as astaxanthin are usually metabolically derived via ketolation of dietary yellow carotenoids [9, 10]. However, the molecular basis of this gene-environment mechanism has remained obscure. Here we use the yellowbeak mutation in the zebra finch (Taeniopygia guttata) to investigate the genetic basis of red coloration. Wild-type ketocarotenoids were absent in the beak and tarsus of yellowbeak birds. The yellowbeak mutation mapped to chromosome 8, close to a cluster of cytochrome P450 loci (CYP2J2-like) that are candidates for carotenoid ketolases. The wild-type zebra finch genome was found to have three intact genes in this cluster: CYP2J19A, CYP2J19B, and CYP2J40. In yellowbeak, there are multiple mutations: loss of a complete CYP2J19 gene, a modified remaining CYP2J19 gene (CYP2J19(yb)), and a non-synonymous SNP in CYP2J40. In wild-type birds, CYP2J19 loci are expressed in ketocarotenoid-containing tissues: CYP2J19A only in the retina and CYP2J19B in the beak and tarsus and to a variable extent in the retina. In contrast, expression of CYP2J19(yb) is barely detectable in the beak of yellowbeak birds. CYP2J40 has broad tissue expression and shows no differences between wild-type and yellowbeak. Our results indicate that CYP2J19 genes are strong candidates for the carotenoid ketolase and imply that ketolation occurs in the integument in zebra finches. Since cytochrome P450 enzymes include key detoxification enzymes, our results raise the intriguing possibility that red coloration may be an honest signal of detoxification ability.

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5 ′ UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 ′ end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/− cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.