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Aedes aegypti is the principal mosquito vector of dengue, yellow fever, Zika and chikungunya viruses. The w Mel strain of the endosymbiotic bacteria Wolbachia pipientis was introduced into the vector as a novel biocontrol strategy to stop transmission of these viruses. Mosquitoes with Wolbachia have been released in the field in Northern Queensland, Australia since 2011, at various locations and over several years, with populations remaining stably infected. Wolbachia infection is known to alter gene expression in its mosquito host, but whether (and how) this changes over the long-term in the context of field releases remains unknown. We sampled mosquitoes from Wolbachia -infected populations with three different release histories along a time gradient and performed RNA-seq to investigate gene expression changes in the insect host. We observed a significant impact on gene expression in Wolbachia -infected mosquitoes versus uninfected controls. Fewer genes had significantly upregulated expression in mosquitoes from the older releases (512 and 486 from the 2011 and 2013/14 release years, respectively) versus the more recent releases (1154 from the 2017 release year). Nonetheless, a fundamental signature of Wolbachia infection on host gene expression was observed across all releases, comprising upregulation of immunity (e.g. leucine-rich repeats, CLIPs) and metabolism (e.g. lipid metabolism, iron transport) genes. There was limited downregulation of gene expression in mosquitoes from the older releases (84 and 71 genes from the 2011 and 2013/14 release years, respectively), but significantly more in the most recent release (509 from the 2017 release year). Our findings indicate that at > 8 years post-introgression into field populations, Wolbachia continues to profoundly impact expression of host genes, such as those involved in insect immune response and metabolism. If Wolbachia -mediated virus blocking is underpinned by these differential gene expression changes, our results suggest it may remain stable long-term.

A fatal case of Japanese encephalitis (JE) occurred in northern Australia in early 2021. Sequence studies showed that the virus belonged to genotype IV (GIV), a genotype previously believed to be restricted to the Indonesian archipelago. This was the first locally acquired case of Japanese encephalitis virus (JEV) GIV to occur outside Indonesia, and the second confirmed fatal human case caused by a GIV virus. A closely related GIV JEV strain subsequently caused a widespread outbreak in eastern Australia in 2022 that was first detected by fetal death and abnormalities in commercial piggeries. Forty-two human cases also occurred with seven fatalities. This has been the first major outbreak of JEV in mainland Australia, and geographically the largest virgin soil outbreak recorded for JEV. This outbreak provides an opportunity to discuss and document the factors involved in the virus’ spread and its ecology in a novel ecological milieu in which other flaviviruses, including members of the JE serological complex, also occur. The probable vertebrate hosts and mosquito vectors are discussed with respect to virus spread and its possible endemicity in Australia, and the need to develop a One Health approach to develop improved surveillance methods to rapidly detect future outbreak activity across a large geographical area containing a sparse human population. Understanding the spread of JEV in a novel ecological environment is relevant to the possible threat that JEV may pose in the future to other receptive geographic areas, such as the west coast of the United States, southern Europe or Africa.

In early 2022, the Japanese encephalitis virus (JEV) was identified as the cause of stillborn and mummified piglets in pig farms in southeastern Australia. Human cases and additional pig farms with infected piglets were subsequently identified across a widespread area encompassing four states. To inform surveillance and control programs, we synthesized existing information on Australian vectors of JEV, much of which was generated in response to incursions of JEV into the northern state of Queensland between 1995 and 2005. Members of the Culex sitiens subgroup, particularly Culex annulirostris, should be considered the primary vectors of JEV in Australia, as they yielded >87% of field detections of JEV, were highly efficient laboratory vectors of the virus, readily fed on pigs and birds (the key amplifying hosts of the virus) when they were available, and are widespread and often occur in large populations. Three introduced species, Culex quinquefasciatus, Culex gelidus and Culex tritaeniorhynchus may also serve as vectors, but more information on their geographical distribution, abundance and bionomics in the Australian context is required. Mosquitoes from other genera, such as Aedes and Verrallina, whilst considered relatively poor vectors, could play a regional or supplemental role in transmission, especially facilitating vertical transmission as a virus overwintering mechanism. Additional factors that could impact JEV transmission, including mosquito survival, dispersal and genetics, are also discussed. Possible directions for investigation are provided, especially in the context of the virus emerging in a region with different mosquito fauna and environmental drivers than northern Australia.

Background Insect-specific viruses do not replicate in vertebrate cells, but persist in mosquito populations and are highly prevalent in nature. These viruses may naturally regulate the transmission of pathogenic vertebrate-infecting arboviruses in co-infected mosquitoes. Following the isolation of the first Australian insect-specific flavivirus (ISF), Palm Creek virus (PCV), we investigated routes of infection and transmission of this virus in key Australian arbovirus vectors and its impact on replication and transmission of West Nile virus (WNV). Methods Culex annulirostris , Aedes aegypti and Aedes vigilax were exposed to PCV, and infection, replication and transmission rates in individual mosquitoes determined. To test whether the virus could be transmitted vertically, progeny reared from eggs oviposited by PCV-inoculated Cx. annulirostris were analysed for the presence of PCV . To assess whether prior infection of mosquitoes with PCV could also suppress the transmission of pathogenic flaviviruses, PCV positive or negative Cx. annulirostris were subsequently exposed to WNV. Results No PCV-infected Cx. annulirostris were detected 16 days after feeding on an infectious blood meal. However, when intrathoracically inoculated with PCV, Cx. annulirostris infection rates were 100 %. Similar rates of infection were observed in Ae. aegypti (100 %) and Ae. vigilax (95 %). Notably, PCV was not detected in any saliva expectorates collected from any of these species. PCV was not detected in 1038 progeny reared from 59 PCV-infected Cx. annulirostris . After feeding on a blood meal containing 10 7 infectious units of WNV, significantly fewer PCV-infected Cx. annulirostris were infected or transmitted WNV compared to PCV negative mosquitoes. Immunohistochemistry revealed that PCV localized in the midgut epithelial cells, which are the first site of infection with WNV. Conclusions Our results indicate that PCV cannot infect Cx. annulirostris via the oral route, nor be transmitted in saliva or vertically to progeny. We also provide further evidence that prior infection with insect-specific viruses can regulate the infection and transmission of pathogenic arboviruses.

Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission. The function on subgenomic flaviviral RNA (sfRNA) in the mosquito vector is not well understood. Here, Slonchak et al. show that sfRNA affects virus-induced apoptosis and dissemination of ZIKV in Aedes aegypti mosquitoes, suggesting a role of sfRNA in Zika virus replication and transmission.

Dengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites. We used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival. The saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.

Surveillance is critical for the prevention and control of mosquito-borne arboviruses. Detection of elevated or emergent virus activity serves as a warning system to implement appropriate actions to reduce outbreaks. Traditionally, surveillance of arboviruses has relied on the detection of specific antibodies in sentinel animals and/or detection of viruses in pools of mosquitoes collected using a variety of sampling methods. These methods, although immensely useful, have limitations, including the need for a cold chain for sample transport, cross-reactivity between related viruses in serological assays, the requirement for specialized equipment or infrastructure, and overall expense. Advances have recently been made on developing new strategies for arbovirus surveillance. These strategies include sugar-based surveillance, whereby mosquitoes are collected in purpose-built traps and allowed to expectorate on nucleic acid preservation cards which are submitted for virus detection. New diagnostic approaches, such as next-generation sequencing, have the potential to expand the genetic information obtained from samples and aid in virus discovery. Here, we review the advancement of arbovirus surveillance systems over the past decade. Some of the novel approaches presented here have already been validated and are currently being integrated into surveillance programs. Other strategies are still at the experimental stage, and their feasibility in the field is yet to be evaluated.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is a readily transmissible and potentially deadly pathogen which is currently re-defining human susceptibility to pandemic viruses in the modern world. The recent emergence of several genetically distinct descendants known as variants of concern (VOCs) is further challenging public health disease management, due to increased rates of virus transmission and potential constraints on vaccine effectiveness. We report the isolation of SARS-CoV-2 VOCs imported into Australia belonging to the B.1.351 lineage, first described in the Republic of South Africa (RSA), and the B.1.1.7 lineage originally reported in the United Kingdom, and directly compare the replication kinetics of these two VOCs in Vero E6 cells. In this analysis, we also investigated a B.1.1.7 VOC (QLD1516/2021) carrying a 7-nucleotide deletion in the open reading frame 7a (ORF7a) gene, likely truncating and rendering the ORF7a protein of this virus defective. We demonstrate that the replication of the B.1.351 VOC (QLD1520/2020) in Vero E6 cells can be detected earlier than the B.1.1.7 VOCs (QLD1516/2021 and QLD1517/2021), before peaking at 48 h post infection (p.i.), with significantly higher levels of virus progeny. Whilst replication of the ORF7a defective isolate QLD1516/2021 was delayed longer than the other viruses, slightly more viral progeny was produced by the mutant compared to the unmutated isolate QLD1517/2021 at 72 h p.i. Collectively, these findings contribute to our understanding of SARS-CoV-2 replication and evolutionary dynamics, which have important implications in the development of future vaccination, antiviral therapies, and epidemiological control strategies for COVID-19.

Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. After 14 days incubation at 28°C and high relative humidity, infection, dissemination and transmission rates were assessed. Infection in Culex annulirostris and Cx. sitiens could not be detected. 8% of Cx. quinquefasciatus were infected, but the virus did not disseminate in this species. Despite having infection rates > 50%, Aedes notoscriptus and Ae. vigilax did not transmit ZIKV. In contrast, Ae. aegypti had infection and transmission rates of 57% and 27%, respectively. In susceptibility trials, the virus dose required to infect 50% (ID50) of Ae. aegypti was106.4 tissue culture infectious dose50 (TCID50)/mL. Additionally, a threshold viral load within the mosquito of at least 105.1 TCID50 equivalents/mL had to be reached before virus transmission occurred. We confirmed Ae. aegypti to be the most likely mosquito vector of ZIKV in Australia, although the restricted distribution of this species will limit the receptive zone to northern Queensland where this species occurs. Importantly, the role in ZIKV transmission of Culex and other Aedes spp. tested will be negligible. Despite being the implicated vector, the relatively high ID50 and need for a high titer disseminated infection in Ae. aegypti suggest that high mosquito population densities will be required to facilitate epidemic ZIKV transmission among the currently immunologically naïve human population in Australia.

Mosquito-borne diseases are a major public health concern globally and early detection of pathogens is critical to implement vector management and control strategies. Existing methods for pathogen detection include screening sentinel animals for antibodies and analyzing mosquitoes for pathogen presence. While these methods are effective, they are also expensive, labor-intense, and logistically challenging. To address these limitations, a new method was developed whereby mosquito saliva is collected on honey-coated nucleic acid preservation cards which are analyzed by molecular assays for detection of pathogens. However, mosquitoes only expel small amounts of saliva when feeding on these cards, potentially leading to false negatives. Another bodily fluid that is expelled by mosquitoes in larger volumes than saliva is excreta, and recent laboratory experiments have demonstrated that a range of mosquito-borne pathogens can be detected in mosquito excreta. In the current study, we have modified light and passive mosquito traps to collect their excreta and assessed their efficacy in field evaluations. From these field-collections, we detected West Nile, Ross River, and Murray Valley encephalitis viruses. Our findings suggest that mosquito traps are easily modified to collect excreta and, that this system has the potential to enhance detection of pathogens.