Fundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of subgenomic RNAs (sgRNAs) that are required for expression of most viral genes. We developed a bioinformatic pipeline to analyze metatranscriptomic data from 20 independent studies encompassing 588 individual samples and 10 coronavirus species. This comparative analysis defined a core sgRNA repertoire for SARS-CoV-2 and found novel sgRNAs that could encode functional short peptides. Relevant to coronavirus infectivity and transmission, we also observed that the ratio of Spike sgRNA to Nucleocapsid one is highest in SARS-CoV-2, among the β-coronaviruses examined. Furthermore, the adjustment of this ratio can be made by modifications to the viral RNA replication machinery, representing a form of viral gene regulation that may be involved in host adaption.

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Adaptation to various stresses during infection is important for Salmonella Typhimurium virulence, while the fitness determinants under infection-relevant stress conditions remain unknown. Here, we simulated conditions Salmonella encountered within the host or in the environment by 15 individual stresses as well as two model cell lines (epithelium and macrophage) to decipher the genes and pathways required for fitness. By high-resolution Tn-seq analysis, a total of 1242 genes were identified as essential for fitness under at least one stress condition. The comparative analysis of fitness determinants in 17 stress conditions indicated the essentiality of genes varied in different mimicking host niches. A total of 12 genes were identified as fitness determinants in all stress conditions, including recB , recC , and xseA (encode three exonuclease subunits necessary for DNA recombination repair) and a novel essential fitness gene yheM . YheM is a putative sulfurtransferase subunit that is responsible for tRNA modification, and our results showed that Salmonella lacking yheM accumulated more aggregates of endogenous protein than wild-type. Moreover, we established a scoring scheme for sRNA essentiality analysis and found STnc2080 of unknown function was essential for resistance to LL-37. In summary, we systematically dissected Salmonella gene essentiality profiling and demonstrated the general and specific adaptive requirements in infection-relevant niches. Our data not only provide valuable insights on how Salmonella responds to environmental stresses during infections but also highlight the potential clinical application of fitness determinants in vaccine development.

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12SARS-COV-2 and all other coronaviruses express its 3 prime genes by forming sub-genomic RNA. As 13 the genome of these virus exist in RNA form, only by profiling the relative abundance of these sgRNAs, can 14 the viral transcriptome be revealed. Utilizing publically available meta-transcriptomic data generated from 15 patient samples, we were able to infer the viral transcriptome in vivo, which is distinct from the in vitro one 16 derived from cell culture. Inter-sample diversity was also observed and a sample specific transcript was 17 identified. By doing the same analysis to MERS and SARS data, we were able to compare the three in terms 18 of transcription. Among the differences, SARS-COV-2 has significantly elevated expression of the Spike gene, 19 which may contribute to its high transmissibility. 20 21Highlights 22 1) The in vivo transcriptome of SARS-CoV-2 revealed by sgRNA profiling, for 25 patient samples around 23 the globe. 242) The Spike protein expression is an order of magnitude higher in SARS-CoV-2 than MERS-CoV or SARS-25CoV, possibly contributing to the virus' elevated transmissibility. 263) The in vivo SARS-CoV-2 transcriptomes, as inferred from human patient data was distinct from the in 27 vitro one derived from cell line culture, all the accessory genes were up-regulated in vivo, suggesting 28 intricate expression regulation mechanism for the small viral genome.