The Australian Biosecurity Genomic Database: a new resource for high-throughput sequencing analysis based on the National Notifiable Disease List of Terrestrial Animals.

The Australian Biosecurity Genomic Database (ABGD) is a curated collection of reference viral genome sequences based on the Australian National Notifiable Disease List of Terrestrial Animals. It was created to facilitate the screening of high-throughput sequencing (HTS) data for the potential presence of viruses associated with notifiable disease. The database includes a single verified sequence (the exemplar species sequence, where relevant) for each of the 60 virus species across 21 viral families that are associated with or cause these notifiable diseases, as recognized by the World Organisation for Animal Health.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (=46) or during collection efforts between January 2015 and June 2022 (=149). The sampled hosts were classified as crop (=155), weed (=24), ornamental (=7), native plant (=6), and insect (=3) species.

Hybrid RNA sequencing of broad bean wilt virus 2 from faba beans.

Globally, viral diseases impair the growth and vigor of cultivated crops such as grains, leading to a significant reduction in quality, marketability, and competitiveness. As an island nation, Australia has a distinct advantage in using its border to prevent the introduction of damaging viruses, which threaten the continental agricultural sector. However, breeding programs in Australia rely on imported seeds as new sources of genetic diversity.

Non-destructive insect metabarcoding for surveillance and biosecurity in citrus orchards: recording the good, the bad and the psyllids.

Background: The Australian citrus industry remains one of the few in the world to be unaffected by the African and the Asian citrus psyllids, Del Guercio and Kuwayama, respectively, and the diseases their vectored bacteria can cause. Surveillance, early detection, and strict quarantine measures are therefore fundamental to safeguard Australian citrus. However, long-term targeted surveillance for exotic citrus pests can be a time-consuming and expensive activity, often relying on manually screening large numbers of trap samples and morphological identification of specimens, which requires a high level of taxonomic knowledge.

LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites.

Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V.

LAMP assay for the detection of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psylloidea: Psyllidae).

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid (ACP), can vector the bacterium Candidatus Liberibacter asiaticus (CLas), agent of Huanglongbing (HLB): an incurable disease affecting citrus trees worldwide. In citrus growing regions where ACP and HLB are absent, such as Australia, the risk of an incursion and consequent economic damage to citrus industries make this psyllid one of the top-priority pests. Due to ACP's small dimensions and the generally poorly studied native psylloid fauna worldwide, morphological identification of this insect to distinguish it from harmless species is challenging, especially in the field, and with immature, partial or damaged specimens.

Fine-scale genomic tracking of Ross River virus using nanopore sequencing.

Background: Ross River virus (RRV) is Australia's most common and widespread mosquito-transmitted arbovirus and is of significant public health concern. With increasing anthropogenic impacts on wildlife and mosquito populations, it is important that we understand how RRV circulates in its endemic hotspots to determine where public health efforts should be directed. Current surveillance methods are effective in locating the virus but do not provide data on the circulation of the virus and its strains within the environment.

Genome Characterisation of the CGMMV Virus Population in Australia-Informing Plant Biosecurity Policy.

The detection of cucumber green mottle mosaic (CGMMV) in the Northern Territory (NT), Australia, in 2014 led to the introduction of strict quarantine measures for the importation of cucurbit seeds by the Australian federal government. Further detections in Queensland, Western Australia (WA), New South Wales and South Australia occurred in the period 2015-2020. To explore the diversity of the current Australian CGMMV population, 35 new coding sequence complete genomes for CGMMV isolates from Australian incursions and surveys were prepared for this study.

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.

Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing.

Rapid and reliable detection tools are essential for disease surveillance and outbreak management, and genomic data is essential to determining pathogen origin and monitoring of transmission pathways. Low virus copy number and poor RNA quality can present challenges for genomic sequencing of plant viruses, but this can be overcome by enrichment of target nucleic acid. A targeted whole genome sequencing (TWG-Seq) approach for the detection of cucumber green mottle mosaic virus (CGMMV) has been developed where overlapping amplicons generated using two multiplex RT-PCR assays are then sequenced using the Oxford Nanopore MinION.

Rapid, in-field deployable, avian influenza virus haemagglutinin characterisation tool using MinION technology.

Outbreaks of avian influenza virus (AIV) from wild waterfowl into the poultry industry is of upmost significance and is an ongoing and constant threat to the industry. Accurate surveillance of AIV in wild waterfowl is critical in understanding viral diversity in the natural reservoir. Current surveillance methods for AIV involve collection of samples and transportation to a laboratory for molecular diagnostics.

Disentangling bias for non-destructive insect metabarcoding.

A fast and reliable method for obtaining a species-level identification is a fundamental requirement for a wide range of activities, from plant protection and invasive species management to biodiversity assessments and ecological studies. For insects, novel molecular techniques such as DNA metabarcoding have emerged as a rapid alternative to traditional morphological identification, reducing the dependence on limited taxonomic experts. Until recently, molecular techniques have required a destructive DNA extraction, precluding the possibility of preserving voucher specimens for future studies, or species descriptions.

A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is a highly polyphagous invasive plant pest that has expanded its global geographic distribution, including recently into much of Australia. Rapid diagnostic tests are required for identification of FAW to assist subsequent management and control. We developed a new loop-mediated isothermal amplification (LAMP) assay based on the mitochondrial cytochrome c oxidase subunit I (COI) gene for accurate and timely diagnosis of FAW in the field.

A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium).

Background: Khapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA.

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses.

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; (PEBV), (CMV), (BYMV) and (PSbMV).

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors.

A LAMP assay for the detection of Bactrocera tryoni Queensland fruit fly (Diptera: Tephritidae).

LAMP assays are targeted molecular tests for the rapid detection of species in the laboratory and field. We developed a LAMP assay for an economically important fruit fly species, Queensland fruit fly, Bactrocera tryoni. This assay was assessed against a broad panel of target and non-target species and found to be specific, only amplifying the target species and closest relatives, in a portable real-time fluorometer (Genie III) in under 15 minutes with an anneal derivative temperature of 82.

Genome Analysis of Melon Necrotic Spot Virus Incursions and Seed Interceptions in Australia.

Melon necrotic spot virus (MNSV) was detected in field-grown (rockmelon) and (watermelon) plants in the Sunraysia district of New South Wales and Victoria, Australia, in 2012, 2013, and 2016, and in two watermelon seed lots tested at the Australian border in 2016. High-throughput sequencing was used to generate near full-length genomes of six isolates detected during the incursions and seed testing. Phylogenetic analysis of the genomes suggests that there have been at least two incursions of MNSV into Australia and none of the field isolates were the same as the isolates detected in seeds.

Screening for Resistance to PVY in Australian Potato Germplasm.

Potatoes are an important human food crop, but have a number of yield limiting factors, including disease susceptibility. (PVY) is found worldwide, and is one of the main virus problems for potato growers. PVY is transmitted by aphids and mechanically by machinery, tools and people, and symptoms are variable across cultivars and strains, including being symptomless in some cultivars.

Genome Characterization of Two Isolates from Australia.

The near-complete genome sequence of the original (CarVY) type isolate (CarVY-Vic) collected in 1999 in Victoria, Australia, and a near-complete genome sequence from an isolate collected in 2019 from the same region (CarVY-2-22) were determined following deep sequencing. The two CarVY genome sequences shared 98% nucleotide identity.

Updating the Quarantine Status of Infecting Viruses in Australia.

One hundred trees, including almond (), apricot (), nectarine (), peach (), plum (), purple leaf plum () and sweet cherry (), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: (ACLSV) (13/100), (ApMV) (1/100), (CGRMV) (4/100), (CNRMV) (2/100), (CVA) (14/100), (LChV2) (3/100), (PBNSPaV) (4/100), (PDV) (3/100), (PNRSV) (52/100), (HSVd) (9/100) and (PLMVd) (6/100).

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.

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.

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.

Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus.