Metagenomics analysis reveals presence of the Merida-like virus in Georgia.

Arbovirus surveillance is fundamental for the discovery of novel viruses and prevention of febrile vector-borne illnesses. Vector-borne pathogens can rapidly expand and adapt in new geographic and environmental conditions. In this study, metagenomic surveillance was conducted to identify novel viruses in the Country of Georgia. A total of 521 mosquitoes were captured near a military training facility and pooled from species Culex pipiens (Linnaeus) (87%) and Aedes albopictus (Skuse) (13%). We decided to further analyze the Culex pipiens mosquitoes, due to the more extensive number of samples collected. Our approach was to utilize an unbiased total RNA-seq for pathogen discovery in order to explore the mosquito virome. The viral reads from this analysis were mostly aligned to Insect-specific viruses from two main families, the Iflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a negative- and single-stranded RNA virus. Our pathogen discovery analysis revealed viral reads aligning to the Merida-like virus Turkey (MERDLVT) strain among the Rhabdoviridae. To further validate this result, we conducted a BLAST sequence comparison analysis of our samples with the MERDLVT strain. Our positive samples aligned to the MERDLVT strain with 96-100% sequence identity and 99.7-100% sequence coverage. A bootstrapped maximum-likelihood phylogenetic tree was used to evaluate the evolutionary relationships among these positive pooled specimens with the (MERDLVT) strain. The Georgia samples clustered most closely with two strains from Turkey, the Merida-like virus KE-2017a isolate 139-1-21 and the Merida-like virus Turkey isolate P431. Collectively, these results show the presence of the MERDLVT strain in Georgia.

A region of SLBP outside the mRNA-processing domain is essential for deposition of histone mRNA into the Drosophila egg.

Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. Stemloop-binding protein (SLBP) binds the 3' end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte. The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. We report that in wild-type stage 10B oocytes, the histone locus bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, which is normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10-amino-acid deletion, fail to deposit sufficient histone mRNA in the oocyte, and do not transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion phenocopy the deletion. We conclude that a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription.This article has an associated First Person interview with the first author of the paper.

Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast.

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3 The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 3'-5' exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40 We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40 The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40 Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wild-type and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.