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.

Identifying the genes underlying phenotypic variation in natural populations can provide novel insight into the evolutionary process. The candidate gene approach has been applied to studies of a number of traits in various species, in an attempt to elucidate their genetic basis. Here, we test the application of the candidate gene approach to identify the loci involved in variation in gastrointestinal parasite burden, a complex trait likely to be controlled by many loci, in a wild population of Soay sheep. A comprehensive literature review, Gene Ontology databases, and comparative genomics resources between cattle and sheep were used to generate a list of candidate genes. In a pilot study, these candidates, along with 50 random genes, were then sequenced in two pools of Soay sheep; one with low gastrointestinal nematode burden and the other high, using a NimbleGen sequence capture experiment. Further candidates were identified from single nucleotide polymorphisms (SNPs) that were highly differentiated between high- and low-resistance sheep breeds. A panel of 192 candidate and control SNPs were then typed in 960 individual Soay sheep to examine whether they individually explained variation in parasite burden, as measured as faecal egg count, as well as two immune measures (Teladorsagia circumcincta-specific antibodies and antinuclear antibodies). The cumulative effect of the candidate and control SNPs were estimated by fitting genetic relationship matrices (GRMs) as random effects in animal models of the three traits. No more significant SNPs were identified in the pilot sequencing experiment and association study than expected by chance. Furthermore, no significant difference was found between the proportions of candidate or control SNPs that were found to be significantly associated with parasite burden/immune measures. No significant effect of the candidate or control gene GRMs was found. There is thus little support for the candidate gene approach to the identification of loci explaining variation in parasitological and immunological traits in this population. However, a number of SNPs explained significant variation in multiple traits and significant correlations were found between the proportions of variance explained by individual SNPs across multiple traits. The significant SNPs identified in this study may still, therefore, merit further investigation.

Although single nucleotide polymorphisms (SNPs) are increasingly being recognized as powerful molecular markers, their application to non-model organisms can bring significant challenges. Among these are imperfect conversion rates of assays designed from in silico resources and the enhanced potential for genotyping error relative to pre-validated, highly optimized human SNPs. To explore these issues, we used Illumina's GoldenGate assay to genotype 480 Antarctic fur seal (Arctocephalus gazella) individuals at 144 putative SNPs derived from a 454 transcriptome assembly. One hundred and thirty-five polymorphic SNPs (93.8%) were automatically validated by the program GenomeStudio, and the initial genotyping error rate, estimated from nine replicate samples, was 0.004 per reaction. However, an almost tenfold further reduction in the error rate was achieved by excluding 31 loci (21.5%) that exhibited unclear clustering patterns, manually editing clusters to allow rescoring of ambiguous or incorrect genotypes, and excluding 18 samples (3.8%) with unreliable genotypes. After stringent quality filtering, we also found a counter-intuitive negative relationship between in silico minor allele frequency and the conversion rate, suggesting that some of our assays may have been designed from paralogous loci. Nevertheless, we obtained over 45 000 individual SNP genotypes with a final error rate of 0.0005, indicating that the GoldenGate assay is eminently capable of generating large, high-quality data sets for non-model organisms. This has positive implications for future studies of the evolutionary, behavioural and conservation genetics of natural populations.

The invasion of the giant Madagascar day gecko Phelsuma grandis has increased the threats to the four endemic Mauritian day geckos (Phelsuma spp.) that have survived on mainland Mauritius. We had two main aims: (i) to predict the spatial distribution and overlap of P. grandis and the endemic geckos at a landscape level; and (ii) to investigate the effects of P. grandis on the abundance and risks of extinction of the endemic geckos at a local scale. An ensemble forecasting approach was used to predict the spatial distribution and overlap of P. grandis and the endemic geckos. We used hierarchical binomial mixture models and repeated visual estimate surveys to calculate the abundance of the endemic geckos in sites with and without P. grandis. The predicted range of each species varied from 85 km2 to 376 km2. Sixty percent of the predicted range of P. grandis overlapped with the combined predicted ranges of the four endemic geckos; 15% of the combined predicted ranges of the four endemic geckos overlapped with P. grandis. Levin's niche breadth varied from 0.140 to 0.652 between P. grandis and the four endemic geckos. The abundance of endemic geckos was 89% lower in sites with P. grandis compared to sites without P. grandis, and the endemic geckos had been extirpated at four of ten sites we surveyed with P. grandis. Species Distribution Modelling, together with the breadth metrics, predicted that P. grandis can partly share the equivalent niche with endemic species and survive in a range of environmental conditions. We provide strong evidence that smaller endemic geckos are unlikely to survive in sympatry with P. grandis. This is a cause of concern in both Mauritius and other countries with endemic species of Phelsuma.