To identify genome-based features characteristic of the avian and human pathogen Chlamydia(C.) psittaci and related chlamydiae, we analyzed whole-genome sequences of 33 strains belonging to 12 species. Using a novel genome analysis tool termed Roary ILP Bacterial Annotation Pipeline (RIBAP), this panel of strains was shown to share a large core genome comprising 784 genes and representing approximately 80% of individual genomes. Analyzing the most variable genomic sites, we identified a set of features of C. psittaci that in its entirety is characteristic of this species: (i) a relatively short plasticity zone of less than 30,000 nt without a tryptophan operon (also in C. abortus, C. avium, C. gallinacea, C. pneumoniae), (ii) a characteristic set of of Inc proteins comprising IncA, B, C, V, X, Y (with homologs in C. abortus, C. caviae and C. felis as closest relatives), (iii) a 502-aa SinC protein, the largest among Chlamydia spp., and (iv) an elevated number of Pmp proteins of subtype G (14 in C. psittaci, 14 in Cand. C. ibidis). In combination with future functional studies, the common and distinctive criteria revealed in this study provide important clues for understanding the complexity of host-specific behavior of individual Chlamydia spp.

RNA-Seq enables the identification and quantification of RNA molecules, often with the aim of detecting differentially expressed genes (DEGs). Although RNA-Seq evolved into a standard technique, there is no universal gold standard for these data’s computational analysis. On top of that, previous studies proved the irreproducibility of RNA-Seq studies. Here, we present a portable, scalable, and parallelizable Nextflow RNA-Seq pipeline to detect DEGs, which assures a high level of reproducibility. The pipeline automatically takes care of common pitfalls, such as ribosomal RNA removal and low abundance gene filtering. Apart from various visualizations for the DEG results, we incorporated downstream pathway analysis for common species as Homo sapiens and Mus musculus. We evaluated the DEG detection functionality while using qRT-PCR data serving as a reference and observed a very high correlation of the logarithmized gene expression fold changes.

Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. Here, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We substituted the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev Response Element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125-2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also re-established Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in an eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly, we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation. IMPORTANCE Synonymous substitutions can influence the virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125-2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

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Although bats are increasingly becoming the focus of scientific studies due to their unique properties, these exceptional animals are still among the least studied mammals. Assembly quality and completeness of bat genomes vary a lot and especially non-coding RNA (ncRNA) annotations are incomplete or simply missing. Accordingly, standard bioinformatics pipelines for gene expression analysis often ignore ncRNAs such as microRNAs or long antisense RNAs. The main cause of this problem is the use of incomplete genome annotations. We present a complete screening for ncRNAs within 16 bat genomes. NcRNAs affect a remarkable variety of vital biological functions, including gene expression regulation, RNA processing, RNA interference and, as recently described, regulatory processes in viral infections. Within all investigated bat assemblies, we annotated 667 ncRNA families including 162 snoRNAs and 193 miRNAs as well as rRNAs, tRNAs, several snRNAs and lncRNAs, and other structural ncRNA elements. We validated our ncRNA candidates by six RNA-Seq data sets and show significant expression patterns that have never been described before in a bat species on such a large scale. Our annotations will be usable as a resource (rna.uni-jena.de/supplements/bats) for deeper studying of bat evolution, ncRNAs repertoire, gene expression and regulation, ecology and important host–virus interactions.