Jay Nicholson

Aedes albopictus (Diptera: Culicidae) as a Potential Vector of Endemic and Exotic Arboviruses in Australia

ABSTRACT In 2005, established populations of Aedes albopictus (Skuse) were discovered in the Torres Strait, the region that separates Papua New Guinea from northern Australia. This increased the potential for this species to be introduced to mainland Australia. Because it is an arbovirus vector elsewhere, we undertook laboratory-based infection and transmission experiments to determine the potential for Ae. albopictus from the Torres Strait to become infected with and transmit the four major Australian endemic arboviruses—Murray Valley encephalitis virus, West Nile virus Kunjin strain (WNVKUN), Ross River virus (RRV), and Barmah Forest virus—as well as the exotic Japanese encephalitis virus. Ae. albopictus is susceptible to infection with all viruses, with infection rates ranging between 8% for WNVKUN and 71% for RRV. Transmission rates of ≈25% were observed for RRV and Barmah Forest virus, but these were <17% for Murray Valley encephalitis virus, WNVKUN, and Japanese encephalitis virus. Given its relative vector competence for alphaviruses, we also examined the replication kinetics and extrinsic incubation periods required for transmission of RRV and Chikungunya virus. Despite lower body titers, more mosquitoes reared and maintained at 28°C became infected with and transmitted the virus than those reared and maintained at 22°C. The minimum time between Ae. albopictus consuming an infected bloodmeal and transmitting Chikungunya virus was 2 d at 28°C and 4 d at 22°C, and for RRV, it was 4 d, irrespective of the temperature. Given its opportunistic feeding habits and aggressive biting behavior, the establishment of Ae. albopictus on the Australian mainland could have a considerable impact on alphavirus transmission.

High-resolution metatranscriptomics reveals the ecological dynamics of mosquito-associated RNA viruses in Western Australia

ABSTRACT Mosquitoes harbor a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a metatranscriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes of the Aedes genus exhibiting substantially less diversity and lower abundances than those of mosquitoes of the Culex genus, within which the viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that the host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95 to 98% nucleotide identity) to those from Indonesia and China. Finally, using abundance-level and phylogenetic relationships, we were able to distinguish potential mosquito viruses from those present in coinfecting bacteria, fungi, and protists. In sum, our metatranscriptomics approach provides important insights into the ecology of mosquito RNA viruses. IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize metatranscriptomic approaches to reveal both complete virus diversity and the relative abundance of these viruses. We used such a metatranscriptomic approach to determine key aspects of the ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are some of the most important components of the mosquito transcriptome, and we identified 19 new virus species from a diverse set of virus families. A key result was that host genetic background plays a more important role in shaping virus diversity than sampling location, with Culex species harboring more viruses at higher abundance than those from Aedes mosquitoes.

Ten years of the Tiger: Aedes albopictus presence in Australia since its discovery in the Torres Strait in 2005

The “Asian tiger mosquito”, Aedes albopictus, is highly invasive, an aggressive biter and a major arbovirus vector. It is not currently present on mainland Australia despite being intercepted on numerous occasions at international ports and infesting the Torres Strait of Australia since at least 2004. In the current paper, we describe the invasion and current status of Ae. albopictus in the Torres Strait, as well as research conducted to assess the threat of this species becoming established in arbovirus transmission cycles on the Australian mainland. Genetic analysis of the invading population demonstrated that the Indonesian region was the likely origin of the invasion and not Papua New Guinea (PNG) as initially suspected. There was also intermixing between Torres Strait, PNG and Indonesian populations, indicating that the species could be re-introduced into the Torres Strait compromising any successful eradication programme. Vector competence experiments with endemic and exotic viruses revealed that Ae. albopictus from the Torres Strait are efficient alphavirus vectors, but less efficient flavivirus vectors. Ae.albopictus obtains blood meals from a range of vertebrate hosts (including humans), indicating that it could play a role in both zoonotic and human-mosquito arbovirus transmission cycles in Australia. Predictive models coupled with climate tolerance experiments suggest that a Torres Strait strain of Ae. albopictus could colonise southern Australia by overwintering in the egg stage before proliferating in the warmer months. Cohabitation experiments demonstrated that the presence of Aedes notoscriptus larvae in containers would not prevent the establishment of Ae. albopictus. Evidence from these studies, coupled with global experience suggests that we need to be prepared for the imminent invasion of Australia by Ae. albopictus by thoroughly understanding its biology and being willing to embrace emerging control technologies.

Ability for aedes albopictus (Diptera: Culicidae) to survive at the climatic limits of its potential range in Eastern Australia

ABSTRACT Aedes albopictus (Skuse) is one of the most invasive mosquito species in the world and has infested islands in the Torres Strait, off the northern coast of Australia since at least 2004. This has led to fears that it may establish on the Australian mainland, including highly populated cities in southern temperate regions. To supplement theoretical projections addressing the range expansion of Ae. albopictus into Australia, laboratory-based trials were conducted to assess the performance of a Torres Strait Ae. albopictus population under a range of Australian conditions. First-instar larvae were placed in individual microcosms and maintained on a natural food resource, under average climatic conditions representing different regions of Australias east coast. Larvae could not survive winter conditions in southern Australia. As the population performance index was >1.0 for tropical winter and summer conditions, and temperate summer conditions, populations would likely increase during these times. To test whether Ae. albopictus could overwinter during adverse conditions as eggs, we exposed cohorts to four different temperature (7, 17, 27, and 33°C) and relative humidity (35, 55, and 80%) combinations for up to 3 mo. High temperatures and low humidity were most detrimental to egg survival. However, those eggs maintained under cooler climates remained viable after 3 mo, including 17% of eggs kept at 7°C. Overall, this study suggests that a Torres Strait Ae. albopictus strain could proliferate all year round under northern tropical conditions and could overwinter in the egg stage before proliferating in the summer in southern temperate regions.

Investigation of the First Case of Dengue Virus Infection Acquired in Western Australia in Seven Decades: Evidence of Importation of Infected Mosquitoes?

In October 2013, a locally-acquired case of dengue virus (DENV) infection was reported in Western Australia (WA) where local dengue transmission has not occurred for over 70 years. Laboratory testing confirmed recent DENV infection and the case demonstrated a clinically compatible illness. The infection was most likely acquired in the Pilbara region in the northwest of WA. Follow up investigations did not detect any other locally-acquired dengue cases or any known dengue vector species in the local region, despite intensive adult and larval mosquito surveillance, both immediately after the case was notified in October 2013 and after the start of the wet season in January 2014. The mechanism of infection with DENV in this case cannot be confirmed. However, it most likely followed a bite from a single infected mosquito vector that was transiently introduced into the Pilbara region but failed to establish a local breeding population. This case highlights the public health importance of maintaining surveillance efforts to ensure that any incursions of dengue vectors into WA are promptly identified and do not become established, particularly given the large numbers of viraemic dengue fever cases imported into WA by travellers returning from dengue-endemic regions.

Deployable Molecular Detection of Arboviruses in the Australian Outback.

The most common causes of human infection from the arboviruses that are endemic in Australia are the arthritogenic alphaviruses: Ross River virus (RRV) and Barmah Forest virus (BFV). The most serious infections are caused by the neurotropic flaviviruses, Murray Valley encephalitis virus (MVEV) and the Kunjin subtype of West Nile virus. The greatest individual risk of arbovirus infection occurs in tropical/subtropical northern Australia because of the warm, wet summer conditions from December to June, where conventional arbovirus surveillance is difficult due to a combination of low population density, large distances between population centers, poor roads, and seasonal flooding. Furthermore, virus detection requires samples to be sent to Perth up to 2,000 km away for definitive analysis, causing delays of days to weeks before test results are available and public health interventions can be started. We deployed a portable molecular biology laboratory for remote field detection of endemic arboviruses in northern Queensland, then in tropical Western Australia and detected BFV, MVEV, and RRV RNA by polymerase chain reaction (PCR) assays of extracts from mosquitoes trapped in Queensland. We then used a field-portable compact real-time thermocycler for the samples collected in the Kimberley region of Western Australia. Real-time field PCR assays enabled concurrent endemic arbovirus distribution mapping in outback Queensland and Western Australia. Our deployable laboratory method provides a concept of operations for future remote area arbovirus surveillance.

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.

Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher’s exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Mosquito (Diptera: Culicidae) communities of metropolitan Perth, Western Australia, and implications for managing pest and public health risks

Mosquito population data in the Perth Metropolitan area are limited. This research aimed to determine mosquito species abundance and composition within three local government areas of inner metropolitan Perth. A secondary aim was to determine whether mosquito species abundance and composition in the study area were related to proximity of known saltmarsh mosquito breeding sites along two major tidal estuary systems. Eleven mosquito species were collected, of which four were found in high abundance: Aedes vigilax, Culex quinquefasciatus, Culex annulirostris and Aedes notoscriptus. The majority of mosquitoes were collected within 1 km of the tidal estuary systems, suggesting that productive mosquito breeding habitat is likely to be located along the fringe of the estuaries. The implication for mosquito management is that saltmarsh mosquitoes are not the only type of mosquito species in the study area. Broadening surveillance and management methods to include freshwater and container breeding species will reduce nuisance concerns for the public and is also likely to reduce incidence of disease in the area.