Enhanced Arbovirus Surveillance with High-Throughput Metatranscriptomic Processing of Field-Collected Mosquitoes.

Surveillance programs are essential for the prevention and control of mosquito-borne arboviruses that cause serious human and animal diseases. Viral metatranscriptomic sequencing can enhance surveillance by enabling untargeted, high-throughput arbovirus detection. We used metatranscriptomic sequencing to screen field-collected mosquitoes for arboviruses to better understand how metatranscriptomics can be utilised in routine surveillance. Following a significant flood event in 2016, more than 56,000 mosquitoes were collected over seven weeks from field traps set up in Victoria, Australia. The traps were split into samples of 1000 mosquitoes or less and sequenced on the Illumina HiSeq. Five arboviruses relevant to public health (Ross River virus, Sindbis virus, Trubanaman virus, Umatilla virus, and Wongorr virus) were detected a total of 33 times in the metatranscriptomic data, with 94% confirmed using reverse transcription quantitative PCR (RT-qPCR). Analysis of metatranscriptomic cytochrome oxidase I (COI) sequences enabled the detection of 12 mosquito and two biting midge species. Screening of the same traps by an established public health arbovirus surveillance program corroborated the metatranscriptomic arbovirus and mosquito species detections. Assembly of genome sequences from the metatranscriptomic data also led to the detection of 51 insect-specific viruses, both known and previously undescribed, and allowed phylogenetic comparison to past strains. We have demonstrated how metatranscriptomics can enhance surveillance by enabling untargeted arbovirus detection, providing genomic epidemiological data, and simultaneously identifying vector species from large, unsorted mosquito traps.

Developing a non-destructive metabarcoding protocol for detection of pest insects in bulk trap catches.

Metabarcoding has the potential to revolutionise insect surveillance by providing high-throughput and cost-effective species identification of all specimens within mixed trap catches. Nevertheless, incorporation of metabarcoding into insect diagnostic laboratories will first require the development and evaluation of protocols that adhere to the specialised regulatory requirements of invasive species surveillance. In this study, we develop a multi-locus non-destructive metabarcoding protocol that allows sensitive detection of agricultural pests, and subsequent confirmation using traditional diagnostic techniques. We validate this protocol for the detection of tomato potato psyllid (Bactericera cockerelli) and Russian wheat aphid (Diuraphis noxia) within mock communities and field survey traps. We find that metabarcoding can reliably detect target insects within mixed community samples, including specimens that morphological identification did not initially detect, but sensitivity appears inversely related to community size and is impacted by primer biases, target loci, and sample indexing strategy. While our multi-locus approach allowed independent validation of target detection, lack of reference sequences for 18S and 12S restricted its usefulness for estimating diversity in field samples. The non-destructive DNA extraction proved invaluable for resolving inconsistencies between morphological and metabarcoding identification results, and post-extraction specimens were suitable for both morphological re-examination and DNA re-extraction for confirmatory barcoding.

Coding-Complete Genome Sequence of Yada Yada Virus, a Novel Alphavirus Detected in Australian Mosquitoes.

Here, we report the detection of a novel alphavirus in Australian mosquitoes, provisionally named Yada Yada virus (YYV). Phylogenetic analysis indicated that YYV belongs to the mosquito-specific alphavirus complex. The assembled genome is 11,612 nucleotides in length and encodes two open reading frames.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini).

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool.

The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Practical application of this technique, however, requires a clear understanding of its analytical sensitivity and specificity. To assess this, five dilutions (1:1, 1:20, 1:400, 1:8,000 and 1:160,000) of Ross River virus (RRV) and Umatilla virus (UMAV) isolates were spiked into subsamples of a pool of 100 Culex australicus mosquitoes. The 1:1 dilution represented the viral load of one RRV-infected mosquito in a pool of 100 mosquitoes. The subsamples underwent nucleic acid extraction, mosquito-specific ribosomal RNA depletion, and Illumina HiSeq sequencing. The viral load of the subsamples was also measured using reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Metatranscriptomic sequencing detected both RRV and UMAV in the 1:1, 1:20 and 1:400 subsamples. A high specificity was achieved, with 100% of RRV and 99.6% of UMAV assembled contigs correctly identified. Metatranscriptomic sequencing was not as sensitive as RT-qPCR or RT-ddPCR; however, it recovered whole genome information and detected 19 other viruses, including four first detections for Australia. These findings will assist arbovirus surveillance programs in utilising metatranscriptomics in routine surveillance activities to enhance arbovirus detection.

Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance.

Trap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations. DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

Accurate identification of Australian mosquitoes using protein profiling.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

A novel group A rotavirus associated with acute illness and hepatic necrosis in pigeons (Columba livia), in Australia.

Cases of vomiting and diarrhoea were reported in racing pigeons in Western Australia in May, 2016. Morbidity and mortality rates were high. Similar clinical disease was seen in Victoria in December and by early 2017 had been reported in all states except the Northern Territory, in different classes of domestic pigeon-racing, fancy and meat bird-and in a flock of feral pigeons. Autopsy findings were frequently unremarkable; histological examination demonstrated significant hepatic necrosis as the major and consistent lesion, often with minimal inflammatory infiltration. Negative contrast tissue suspension and thin section transmission electron microscopy of liver demonstrated virus particles consistent with a member of the Reoviridae. Inoculation of trypsin-treated Vero, MDBK and MA-104 cell lines resulted in cytopathic changes at two days after infection. Next generation sequencing was undertaken using fresh liver samples and a previously undescribed group A rotavirus (genotype G18P[17]) of avian origin was identified and the virus was isolated in several cell lines. A q-RT-PCR assay was developed and used to screen a wider range of samples, including recovered birds. Episodes of disease have continued to occur and to reoccur in previously recovered lofts, with variable virulence reported. This is the first report of a rotavirus associated with hepatic necrosis in any avian species.

Metagenomic arbovirus detection using MinION nanopore sequencing.

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2.

Internal Transcribed Spacer 2 (ITS2) is a popular DNA barcoding marker; however, in some animal species it is hypervariable and therefore difficult to sequence with traditional methods. With next-generation sequencing (NGS) it is possible to sequence all gene variants despite the presence of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), homopolymeric regions, and microsatellites. Our aim was to compare the performance of Sanger sequencing and NGS amplicon sequencing in characterizing ITS2 in 26 mosquito species represented by 88 samples. The suitability of ITS2 as a DNA barcoding marker for mosquitoes, and its allelic diversity in individuals and species, was also assessed. Compared to Sanger sequencing, NGS was able to characterize the ITS2 region to a greater extent, with resolution within and between individuals and species that was previously not possible. A total of 382 unique sequences (alleles) were generated from the 88 mosquito specimens, demonstrating the diversity present that has been overlooked by traditional sequencing methods. Multiple indels and microsatellites were present in the ITS2 alleles, which were often specific to species or genera, causing variation in sequence length. As a barcoding marker, ITS2 was able to separate all of the species, apart from members of the Culex pipiens complex, providing the same resolution as the commonly used Cytochrome Oxidase I (COI). The ability to cost-effectively sequence hypervariable markers makes NGS an invaluable tool with many applications in the DNA barcoding field, and provides insights into the limitations of previous studies and techniques.

Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia.

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p-distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the 'Mosquitoes of Australia-Victoria' project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.

High-throughput sequencing of plasma microRNA in chronic fatigue syndrome/myalgic encephalomyelitis.

BACKGROUND: MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. RESULTS: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients. CONCLUSION: Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.