Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in humans and a wide range of animal species. Its rapid global spread has resulted in a major public health emergency, necessitating commensurately rapid research to improve control strategies. In particular, the ability to effectively retrace transmission chains in outbreaks remains a major challenge, partly due to our limited understanding of the virus’ underlying evolutionary dynamics within and between hosts. We used high-throughput sequencing whole-genome data coupled with bottleneck analysis to retrace the pathways of viral transmission in two nosocomial outbreaks that were previously characterised by epidemiological and phylogenetic methods. Additionally, we assessed the mutational landscape, selection pressures, and diversity at the within-host level for both outbreaks. Our findings show evidence of within-host selection and transmission of variants between samples. Both bottleneck and diversity analyses highlight within-host and consensus-level variants shared by putative source-recipient pairs in both outbreaks, suggesting that certain within-host variants in these outbreaks may have been transmitted upon infection rather than arising de novo independently within multiple hosts. Overall, our findings demonstrate the utility of combining within-host diversity and bottleneck estimations for elucidating transmission events in SARS-CoV-2 outbreaks, provide insight into the maintenance of viral genetic diversity, provide a list of candidate targets of positive selection for further investigation, and demonstrate that within-host variants can be transferred between patients. Together these results will help in developing strategies to understand the nature of transmission events and curtail the spread of SARS-CoV-2.

The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL-α-difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G1 (~15 hours post-invasion) by blocking G1/S transition. Cell cycle-arrested parasites enter a quiescent G0-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We subsequently used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca2+-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.

It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq. Chromium 10X provided a superior assembly, in terms of number of scaffolds, N50 and number of genes recovered. It required less DNA than other methods and was sequenced and assembled at a lower cost. Chromium sequencing could provide a solution to sequence and assemble genomes of obligate plant pathogens where the amount of available DNA is a limiting factor.

CD8+ T-cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T-cells expresses CXCR5, the chemokine receptor required for cell migration into B cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T-cells (fCD8s) frequencies, trafficking pattern and CXCR5 regulation. We show that, although HIV infection results in a marginal increase of fCD8s in LN, the majority of HIV-specific CD8+ T-cells are CXCR5 negative (non-fCD8s) (p<0.003). Mechanistic investigations using ATAC-seq showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprints analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In-vitro stimulation of non-fCD8s with recombinant TGF-β resulted in significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-β signaling as a viable strategy for increasing fCD8s frequencies in follicular areas of the LN where they are needed to eliminate HIV infected cells, with implications for HIV cure strategies.