Quantitative PCR assay for the detection of Aedes vigilax in mosquito trap collections containing large numbers of morphologically similar species and phylogenetic analysis of specimens collected in Victoria, Australia.

BACKGROUND: Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia's coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus, which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax. We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax, with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps. METHODS: Trapping was performed during the 2017-2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to. RESULTS: Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax, with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia. CONCLUSIONS: Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus, making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species.

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. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Characterization of Fitzroy River Virus and Serologic Evidence of Human and Animal Infection.

In northern Western Australia in 2011 and 2012, surveillance detected a novel arbovirus in mosquitoes. Genetic and phenotypic analyses confirmed that the new flavivirus, named Fitzroy River virus, is related to Sepik virus and Wesselsbron virus, in the yellow fever virus group. Most (81%) isolates came from Aedes normanensis mosquitoes, providing circumstantial evidence of the probable vector. In cell culture, Fitzroy River virus replicated in mosquito (C6/36), mammalian (Vero, PSEK, and BSR), and avian (DF-1) cells. It also infected intraperitoneally inoculated weanling mice and caused mild clinical disease in 3 intracranially inoculated mice. Specific neutralizing antibodies were detected in sentinel horses (12.6%), cattle (6.6%), and chickens (0.5%) in the Northern Territory of Australia and in a subset of humans (0.8%) from northern Western Australia.