Forty percent of the world's population is at risk of contracting dengue virus, which produces dengue fever with a potentially fatal hemorrhagic form. The wMelPop Wolbachia infection of Drosophila melanogaster reduces life span and interferes with viral transmission when introduced into the mosquito Aedes aegypti, the primary vector of dengue virus. Wolbachia has been proposed as an agent for preventing transmission of dengue virus. Population invasion by Wolbachia depends on levels of cytoplasmic incompatibility, fitness effects, and maternal transmission. Here we characterized these traits in an outbred genetic background of a potential target population of Ae. aegypti using two crossing schemes. Cytoplasmic incompatibility was strong in this background, and the maternal transmission rate of Wolbachia was high. The infection substantially reduced longevity of infected adult females, regardless of whether adults came from larvae cultured under high or low levels of nutrition or density. The infection reduced the viability of diapausing and nondiapausing eggs. Viability was particularly low when eggs were laid by older females and when diapausing eggs had been stored for a few weeks. The infection affected mosquito larval development time and adult body size under different larval nutrition levels and densities. The results were used to assess the potential for wMelPop-CLA to invade natural populations of Ae. aegypti and to develop recommendations for the maintenance of fitness in infected mosquitoes that need to compete against field insects.

Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies ofCulicoides(Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence ofWolbachia(1/33) andCardinium(8/33) infections. Here, we screened 20 species ofCulicoidesforWolbachiaandCardinium, utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels ofCardiniumDNA were detected in females of all but one of theCulicoidesspecies screened, and low levels ofWolbachiawere detected in females of 9 of the 20Culicoidesspecies. Sequence analysis based on partial 16S rRNA gene andgyrBsequences identified “CandidatusCardinium hertigii” from group C, which has previously been identified inCulicoidesfrom Japan, Israel, and the United Kingdom.Wolbachiastrains detected in this study showed 98 to 99% sequence identity toWolbachiapreviously detected fromCulicoidesbased on the 16S rRNA gene, whereas a strain with a novelwspsequence was identified inCulicoidesnarrabeenensis. Cardiniumisolates grouped to geographical regions independent of the hostCulicoidesspecies, suggesting possible geographical barriers toCardiniummovement. Screening also identifiedAsaiabacteria inCulicoides. These findings point to a diversity of low-level endosymbiont infections inCulicoides, providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

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. 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. 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. The risk of local Aedes species triggering large Zika epidemics in the southern parts of Australia is low. The potentially invasive Ae. albopictus showed high prevalence of virus in the saliva and constitutes a potential threat if this mosquito species becomes established in mainland Australia. Complete risk analysis of Zika transmission in the temperate zone would require an assessment of the impact of temperature on Zika virus replication within local and invasive mosquito species.

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.

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.