Identification and functional characterisation of Aedes albopictus genes against Chikungunya virus

Chikungunya virus (CHIKV) of Alphavirus genus, has caused several outbreaks around the world in the last decade. Once a relatively unknown virus, it now causes seasonal infections in tropical and some temperate regions. This change in epidemiology is attributed to vector switch from Aedes aegypti to Aedes albopictus, an invasive pest leading to infections in temperate regions. Although recent research has identified mosquito factors influencing infections, our understanding of interaction between CHIKV and its new vector is limited. Using whole transcriptome sequencing of CHIKV infected mosquitoes, we studied differential expression of genes in the midgut and head and thorax, the two critical barrier sites of the mosquito at two time points. We identified several up and down regulated transcripts in the mosquito host genome in response to the viral infection. Two days post-infection, in the midgut tissue of the mosquitoes, 250 differentially expressed transcripts (25 when the next-generation sequencing (NGS) reads were aligned to the published reference genome and 225 when the reads were aligned to a de novo custom transcriptome we generated) were identified. From the head and thorax tissue of the mosquitoes, 8 days post-infection, 159 differentially expressed transcripts (96 when the NGS reads were aligned to the published reference genome and 63 when the reads were aligned to the de novo custom transcriptome) were identified. Twenty-seven of the targets (13 from 2dpi/midgut and 14 from 8dpi/head & thorax) identified to be differentially expressed were validated separately via qRT-PCR. Seven transcripts found to be differentially expressed in midguts of Ae. albopictus two days post-infection were also assessed for changes in expression in midguts of Ae. aegypti two days post-infection. Apart from differential expression in genes, we also identified down regulation of long non-coding RNAs that may also have functional relevance. The comparison between Ae. albopictus and Ae. aegypti also showed that the expression patterns of the same targets are different between the two species of mosquitoes after CHIKV infection. From the targets we validated, two were selected for further functional studies. Niemann-Pick 2 (NPC2) gene homologue was found to be significantly upregulated in the midguts of both Ae. albopictus and Ae. aegypti mosquitoes, two days post-infection with CHIKV. Known cytoplasmic lipid transporters, NPC family proteins had previously been implicated in pathogenesis of several viruses including dengue, Ebola and HIV. In fact, NPC2 protein was found to be essential for successful replication of CHIKV in human fibroblasts. To characterise the role of NPC2 during CHIKV in Ae. albopictus mosquito, the gene was over expressed in C6/36 mosquito cells 24 hours prior infection with the virus. The infectivity titres of extracellular mature virus and intracellular viral RNA levels were compared between wildtype cells and cells over expressing the protein. The expressed NPC2 protein and the virus were also labelled using antibodies and studied under confocal microscopy. While significant differences were not observed in the viral RNA levels or infectivity titres, confocal microscopy showed partial co-localisation of NPC2 protein and the virus. Inhibitor of Bruton¿s tyrosine kinase (BTKi) was identified to be significantly upregulated 8 days post-infection in the head and thorax of Ae. albopictus mosquitoes. To assess its functional significance, BTKi was knocked-down using double-stranded RNA in RML12, a mosquito cell line. While no significant difference in viral RNA levels or infectivity titers was detected, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. BTK is a pro-inflammatory cytoplasmic Tec kinase and is known to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis. The upregulation of BTKi a known anti-inflammatory protein post viral infection and increase in cellular apoptosis when the gene is knocked down may suggest a possible conserved mechanism at play between mosquitoes and mammals. We also studied changes in the viral genome during mosquito infection. We detected changes in viral diversity, as shown by number of mutations in the viral genome, with increase in number of mutations in the midgut compared with mammalian host (Vero cell culture), followed by reduction in the number of mutations in head and thorax at 8 dpi, indicating a possible genomic bottleneck. Taken together, these results will help in understanding Ae. albopictus interactions with CHIKV and can lead to development of novel disease control strategies

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing

Chromothripsis is a mutational phenomenon characterized by massive, clustered genomic rearrangements that occurs in cancer and other diseases. Recent studies in selected cancer types have suggested that chromothripsis may be more common than initially inferred from low-resolution copy-number data. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we analyze patterns of chromothripsis across 2,658 tumors from 38 cancer types using whole-genome sequencing data. We find that chromothripsis events are pervasive across cancers, with a frequency of more than 50% in several cancer types. Whereas canonical chromothripsis profiles display oscillations between two copy-number states, a considerable fraction of events involve multiple chromosomes and additional structural alterations. In addition to non-homologous end joining, we detect signatures of replication-associated processes and templated insertions. Chromothripsis contributes to oncogene amplification and to inactivation of genes such as mismatch-repair-related genes. These findings show that chromothripsis is a major process that drives genome evolution in human cancer

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Zika vector transmission risk in temperate Australia: a vector competence study

Background: Zika virus is an emerging pathogen of global importance. It has been responsible for recent outbreaks in the Americas and in the Pacific region. This study assessed five different mosquito species from the temperate climatic zone in Australia and included Aedes albopictus as a potentially invasive species. Methods Mosquitoes were orally challenged by membrane feeding with Zika virus strain of Cambodia 2010 origin, belonging to the Asian clade. Virus infection and dissemination were assessed by quantitative PCR on midgut and carcass after dissection. Transmission was assessed by determination of cytopathogenic effect of saliva (CPE) on Vero cells, followed by determination of 50% tissue culture infectious dose (TCID50) for CPE positive samples. Additionally, the presence of Wolbachia endosymbiont infection was assessed by qPCR and standard PCR. Results Culex mosquitoes were found unable to present Zika virus in saliva, as demonstrated by molecular as well as virological methods. Aedes aegypti, was used as a positive control for Zika infection and showed a high level of virus infection, dissemination and transmission. Local Aedes species, Ae. notoscriptus and, to a lesser degree, Ae. camptorhynchus were found to expel virus in their saliva and contained viral nucleic acid within the midgut. Molecular assessment identified low or no dissemination for these species, possibly due to low virus loads. Ae. albopictus from Torres Strait islands origin was shown as an efficient vector. Cx quinquefasciatus was shown to harbour Wolbachia endosymbionts at high prevalence, whilst no Wolbachia was found in Cx annulirostris. The Australian Ae. albopictus population was shown to harbour Wolbachia at high frequency. Conclusions The risk of local Aedes species triggering large Zika epidemics in the southern parts of Australia is low

RNASeq Analysis of Aedes albopictus Mosquito Midguts after Chikungunya Virus Infection

Chikungunya virus (CHIKV) is an emerging pathogen around the world and causes significant morbidity in patients. A single amino acid mutation in the envelope protein of CHIKV has led to a shift in vector preference towards Aedes albopictus. While mosquitoes are known to mount an antiviral immune response post-infection, molecular interactions during the course of infection at the tissue level remain largely uncharacterised. We performed whole transcriptome analysis on dissected midguts of Aedes albopictus infected with CHIKV to identify differentially expressed genes. For this, RNA was extracted at two days post-infection (2-dpi) from pooled midguts. We initially identified 25 differentially expressed genes (p-value &lt; 0.05) when mapped to a reference transcriptome. Further, multiple differentially expressed genes were identified from a custom de novo transcriptome, which was assembled using the reads that did not align with the reference genome. Thirteen of the identified transcripts, possibly involved in immunity, were validated by qRT-PCR. Homologues of seven of these genes were also found to be significantly upregulated in Aedes aegypti midguts 2 dpi, indicating a conserved mechanism at play. These results will help us to characterise the molecular interaction between Aedes albopictus and CHIKV and can be utilised to reduce the impact of this viral infection

Whole transcriptome analysis of Aedes albopictus mosquito head and thorax post-chikungunya virus infection

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton&rsquo;s tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV

Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients

INTRODUCTION: Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system. METHODS: Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel. RESULTS: Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified. CONCLUSIONS: Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients

PathOS: a decision support system for reporting high throughput sequencing of cancers in clinical diagnostic laboratories

Abstract Background The increasing affordability of DNA sequencing has allowed it to be widely deployed in pathology laboratories. However, this has exposed many issues with the analysis and reporting of variants for clinical diagnostic use. Implementing a high-throughput sequencing (NGS) clinical reporting system requires a diverse combination of capabilities, statistical methods to identify variants, global variant databases, a validated bioinformatics pipeline, an auditable laboratory workflow, reproducible clinical assays and quality control monitoring throughout. These capabilities must be packaged in software that integrates the disparate components into a useable system. Results To meet these needs, we developed a web-based application, PathOS, which takes variant data from a patient sample through to a clinical report. PathOS has been used operationally in the Peter MacCallum Cancer Centre for two years for the analysis, curation and reporting of genetic tests for cancer patients, as well as the curation of large-scale research studies. PathOS has also been deployed in cloud environments allowing multiple institutions to use separate, secure and customisable instances of the system. Increasingly, the bottleneck of variant curation is limiting the adoption of clinical sequencing for molecular diagnostics. PathOS is focused on providing clinical variant curators and pathology laboratories with a decision support system needed for personalised medicine. While the genesis of PathOS has been within cancer molecular diagnostics, the system is applicable to NGS clinical reporting generally. Conclusions The widespread availability of genomic sequencers has highlighted the limited availability of software to support clinical decision-making in molecular pathology. PathOS is a system that has been developed and refined in a hospital laboratory context to meet the needs of clinical diagnostics. The software is available as a set of Docker images and source code at https://github.com/PapenfussLab/PathOS