Characterization of a Western Pacific Zika Virus Strain in Australian Aedes aegypti

Abstract

Zika virus (ZIKV) is a globally emerging arbovirus responsible for widespread epidemics in the western Pacific, the Americas, and Asia. The virus predominately circulates in urban transmission cycles between Aedes aegypti and humans. Australia is considered at risk to outbreaks of ZIKV due to the presence of A. aegypti populations in northern areas of the state of Queensland. Furthermore, close proximity to epidemic regions has led to almost 50% of imported cases reported since 2012 originating in the Pacific region. We conducted the first vector competence experiments with A. aegypti from three Australian populations for a western Pacific strain of ZIKV. When exposed to bloodmeals containing between 10⁵ and 10⁸ tissue culture infectious dose (TCID)₅₀/mL of virus, infection, dissemination, and transmission, rates were <10%. In comparison to using frozen virus stock, exposing mosquitoes to freshly cultured virus also did not increase infection or transmission rates. It was only when bloodmeal titers exceeded 10⁸ TCID₅₀/mL that infection rates approached 50% and transmission rates increased to >20%. However, this concentration of virus is considerably higher than levels previously reported in blood samples from viremic humans. The Australian A. aegypti tested appear to express a midgut barrier to ZIKV infection, as 50% of mosquitoes that became infected developed a disseminated infection, and 50% of those mosquitoes transmitted the virus. Overall, these results suggest that while Australian A. aegypti strains are able to transmit the western Pacific ZIKV strain, they are relatively inefficient vectors of the virus.

Introduction

Zika virus (ZIKV) has gained notoriety in the last 10 years for causing widespread epidemics in the western Pacific, South and Central America, and Asia (Aliota et al. 2017, Baud et al. 2017). Originally thought to be the etiological agent of a relatively benign, mild febrile illness, the recent dramatic emergence of ZIKV globally and its implication in more severe disease syndromes, including congenital malformations of the central nervous system (particularly microcephaly) and Guillain–Barré syndrome (Cao-Lormeau et al. 2016, Rasmussen et al. 2016), has redefined ZIKV as a major human pathogen. Urban ZIKV transmission predominately involves a cycle between humans and the domestic mosquito Aedes aegypti, although alternative modes of transmission, such as sexual, congenital, and neonatal, have been reported (Aliota et al. 2017, Baud et al. 2017).

Australia is at risk of a ZIKV outbreak because populations of A. aegypti are present in the state of Queensland and recent studies have shown that these populations are competent vectors of the prototype Ugandan and 2010 Cambodian ZIKV strains (Hall-Mendelin et al. 2016, Duchemin et al. 2017). The close proximity to epidemic foci in the western Pacific and increased travel and commercial trade from this region, and elsewhere in Southeast Asia and the Americas, potentially increases the risk of ZIKV outbreaks. Indeed, since 2012, over 130 overseas-acquired cases have been reported in Australia, with almost 50% of travelers infected in the western Pacific (Australian Government Department of Health 2018).

ZIKV isolation from clinical patient samples through conventional cell culture can be challenging with low success rates, even after detection of ZIKV RNA by real-time RT-PCR or inoculation of samples at the same day of their collection (Lanciotti et al. 2008, Bonaldo et al. 2016). Indeed, at our diagnostic facility, ZIKV could not be isolated in either mosquito C6/36 or mammalian Vero cell culture systems from 19 ZIKV RT-PCR-positive infected travelers between 2015 and 2016. It was only after a patient serum was inoculated into suckling mouse brains that an isolate was obtained from a traveler from Tonga in February 2016 (Pyke et al. 2016). During this time, Tonga was experiencing a ZIKV outbreak most likely driven by the vector A. aegypti (P. Whelan, Biting Insect Technical Extension Services, personal communication) that ultimately resulted in over 2,200 suspected cases (Ministry of Health, Tonga 2016). Phylogenetic analysis revealed that the Tongan ZIKV belonged to the Asian lineage and clustered with other recent strains circulating in the Pacific and the Americas.

The objective of the current study was to assess the ability for populations of A. aegypti from north Queensland to become infected with and transmit the 2016 Tongan ZIKV strain. Testing this mosquito–virus combination is important, as considerable variation in vector competence has been observed between different ZIKV and mosquito strains (Ciota et al. 2017, Pompon et al. 2017). While undertaking these experiments, we examined several factors that can affect vector competence assessments of mosquitoes for arboviruses, and included degradation of the virus during the blood feeding period and a comparison of fresh versus frozen virus stock used to prepare the infectious bloodmeals (Weger-Lucarelli et al. 2016, Ciota et al. 2017).

Materials and Methods

Mosquitoes

Eggs of A. aegypti were collected using ovitraps (Ritchie 2001) from three locations in north Queensland, Australia. Townsville (19° 15′ S 146° 49′ E) A. aegypti (TVL15) were collected in March 2015 and used in the F_3–5 generations. Collections of A. aegypti eggs were obtained from Innisfail (17° 31′ S, 146° 1′ E) in March 2016 (INN16) and April 2017 (INN17), and were used in the F_2–3 and F₀ generations, respectively. The 2017 collections were undertaken to obtain mosquitoes to replace the INN16 line, which was displaying an appreciable decline in colony health. Mosquito eggs (CNS17) from the Cairns suburb of Holloways Beach (16° 50 S 145° 44′ E), were obtained in March 2017 and were used in the F₁ generation. These collection locations in north Queensland have recently been the site of releases of Wolbachia-infected A. aegypti, which are being deployed as a new biocontrol strategy (Hoffmann et al. 2011). However, the mosquito egg collections from all locations in the current study were undertaken before the release of Wolbachia-infected A. aegypti. While Wolbachia releases have been conducted in Cairns and surrounding suburbs since 2011, Holloways Beach is an isolated community with no history of releases. Larvae were reared at 26°C and 12:12 L:D, and were fed Hikari^® Cichlid Staple pellets (Kyorin Co. Ltd., Himeji, Japan). Adults were maintained at 26°C and 12:12 L:D for 3–7 days postemergence. Mosquitoes were offered 15% honey water ad libitum before being starved for 24 h before virus exposure.

Virus

The ZIKV strain (TS17-2016) was isolated from an infected traveler who had arrived in Australia from Tonga in 2016 (Pyke et al. 2016). The virus strain was isolated in suckling mouse brains before being passaged five times in C6/36 (Aedes albopictus) cells. The final virus stocks were stored at −80°C and had a titer of 10^10.8 tissue culture infectious dose (TCID)₅₀/mL.

Exposure of mosquitoes to ZIKV

As ZIKV is exotic to Australia, all vector competence experiments were undertaken in the physical containment (PC3) insectary at the Forensic and Scientific Services, Department of Health, Queensland Government, Brisbane, Australia. Mosquitoes were exposed to infectious bloodmeals containing stock virus diluted in commercially available defibrinated sheep blood (Applied Biological Products Management- Australia, Aldinga Beach, Australia) housed in a Hemotek feeding apparatus (Discovery Workshops, Accrington, Lancashire, UK) fitted with pig intestine as the membrane. All experiments used frozen stock virus, except where mentioned in the text. Pre- and postfeeding samples of all blood/virus mixtures were diluted 1:10 in growth medium (GM) supplemented with 3% fetal bovine serum (FBS), and antibiotics and antimycotics, and stored at −80°C.

A number of different experiments were undertaken to characterize ZIKV TS17-2016 in A. aegypti. In the first experiment, INN16 and TVL15 A. aegypti were exposed for 2 h to stock virus diluted to produce predicted bloodmeal titers of ∼10⁷ TCID₅₀/mL. Following feeding, blood-engorged females were placed in 900-mL gauze-covered containers and maintained in an environmental growth cabinet at 28°C, 70–80% RH and 12:12 L:D cycle, and offered 15% honey water as a nutrient source.

We next tested whether degradation of the virus and reduction in titer occurs in the infectious bloodmeal, and whether this affects the quantity of virus imbibed by mosquitoes. During two replicate feeds, INN16 A. aegypti were offered an infectious bloodmeal over a 2-h period. At 30-min intervals, blood/virus mix and freshly blood-fed mosquitoes were collected. At the start of the feeding period, the blood/virus mixture was placed into the separate Hemotek reservoirs, which were then placed on the gauze-covered containers housing the mosquitoes. At the end of each time point, the container was removed and blood-engorged mosquitoes were placed in individual 2-mL U-bottomed tubes containing 1 mL GM plus 3% FBS, and a 5 mm stainless steel ball. Using a pipette tip, the pig intestine membrane was punctured, and an aliquot of the bloodmeal was taken and diluted 1:10 in GM plus 3% FBS. Engorged mosquitoes and blood/virus mixtures were stored at −80°C. As there were sufficient engorged mosquitoes that had fed between 31 and 60 min, a subsample of mosquitoes were placed in fresh gauze-covered containers and maintained for 7 days as described previously.

It has previously been demonstrated that using frozen virus for vector competence experiments can lead to reduced infection rates when compared with freshly propagated virus (Weger-Lucarelli et al. 2016, Ciota et al. 2017). Therefore, an experiment was conducted with INN16 and TVL15 A. aegypti to compare frozen virus stock with virus freshly harvested from infected C6/36 cell monolayers grown in 25-cm²flasks, which had been inoculated 96 h previously.

In the final experiment, INN17 and CNS17 A. aegypti were exposed to a bloodmeal containing ZIKV stock at a titer >10⁸ TCID₅₀/mL, which was considerably higher than the previous experiments.

Assessment of transmission potential

With the exception of the virus degradation experiment, mosquitoes were tested at 14–18 days postexposure for their ability to transmit the virus using the saliva expectorate collection method of Aitken (1977). Briefly, mosquitoes were anesthetized with CO₂, and the legs and wings removed before the proboscis was inserted into a capillary tube containing ∼25 μL of GM plus 20% FBS. After 30 min, the contents of the capillary tube were expelled into a 1.5-mL tube containing 0.5 mL GM plus 3% FBS. Bodies, legs, and wings were placed separately into 2-mL U-bottomed tubes containing 1 mL GM plus 3% FBS and a 5 mm stainless steel ball. Only bodies, legs, and wings were collected for the 7-day mosquitoes from the virus degradation experiment. All samples were stored at −80°C.

Virus assay

Blood/virus mixtures were titrated as 10-fold dilutions and inoculated onto C6/36 monolayers grown in 96-well microtiter plates. Bodies, legs, and wings were homogenized in a QIAGEN TissueLyser II (Qiagen, Hilden, Germany), centrifuged at 14,000 g for 5 min and filtered through a 0.2 μm Supor^® membrane filter (Pall Corporation, Ann Arbor, MI). Fifty microliters of filtered homogenate was inoculated in quadruplicate into the wells of a 96-well microtiter plate seeded with C6/36 cells. In addition, the freshly engorged whole mosquitoes from the virus degradation experiment and saliva expectorates from all experiments were titrated as described above. After 7 days incubation at 28°C, cells were fixed in 20% acetone and the plates were stored at −20°C. ZIKV infection was detected using a tissue culture enzyme immunoassay (TC-EIA; [Broom et al. 1998]) and the pan-flavivirus reactive monoclonal antibody 4G2 (provided by Roy Hall, University of Queensland, Australia).

Analysis

The titers of the blood/virus mixtures, blood/virus-engorged mosquitoes, and saliva expectorates were calculated using the 50% endpoint method of Reed and Muench (1938) and were expressed as TCID₅₀/mL. For the mosquitoes, infection, dissemination, and transmission rates between mosquito strains were compared using Fisher's exact tests with two-tailed p-values. Virus titers in saliva expectorates were compared between mosquito strains using a two-tailed t-test. Virus titers for the engorged mosquitoes collected at the different time points of the virus degradation experiment were compared using the Kruskal–Wallis test. All statistics were performed with GraphPad Prism Version 7.02 (GraphPad Software, La Jolla, California, www.graphpad.com).

Results

Relatively low infection rates were observed for the TVL15 and INN16 mosquitoes (Table 1) exposed to bloodmeals containing pre- and postfeeding titers of 10^5.9 TCID₅₀/mL and 10^4.2 TCID₅₀/mL, respectively. No TVL15 A. aegypti was infected on day 14, whereas only 1 out of 30 (3%) INN16 was infected at 14 and 18 days postexposure. Both of the infected mosquitoes transmitted the virus, with saliva titers of 10^2.0 TCID₅₀/mL and 10^2.8 TCID₅₀/mL for mosquitoes sampled at days 14 and 18 postexposure, respectively.

Table 1.

Infection, Dissemination, and Transmission Rates in Townsville (TVL15) and Innisfail (INN16) Aedes aegypti Exposed to Bloodmeals Containing Pre- and Postfeeding Titers of 10 ^5.9 TCID₅₀ /ml and 10^4.2 TCID₅₀/ml, Respectively, of Zika Virus TS17-2016

Strain	Days postexposure	% infection ^a	% dissemination ^b	% transmission ^c	% transmission/dissemination ^d
Townsville	14	0 (0/30)	0 (0/30)	0 (0/30)	0 (0/0)
Innisfail	14	3 (1/30)	3 (1/30)	3 (1/30)	100 (1/1)
Innisfail	18	3 (1/30)	3 (1/30)	3 (1/30)	100 (1/1)

Percentage of mosquitoes containing virus in their bodies (number positive/number tested).

Percentage of mosquitoes containing virus in their legs and wings (number positive/number tested).

Percentage of mosquito expectorates in which virus was detected (number of positive expectorates/number tested).

Percentage of mosquitoes with a disseminated infection in which virus was detected in the expectorate (number of positive expectorates/number disseminated).

TCID, tissue culture infectious dose.

We next assessed whether the virus degraded in the bloodmeal and whether this degradation led to mosquitoes imbibing a lower quantity of virus, a phenomenon which may have affected the results of the first experiment. We observed that the virus titer dropped by up to 10^2.2 TCID₅₀/mL during the 2 h that INN16 A. aegypti were exposed to the infectious bloodmeal (Table 2). Despite this virus titer, the quantity of virus that mosquitoes imbibed was not significantly different (p = 0.6295) between time points. The day 7 postexposure infection and dissemination rates were 10% and 5%, respectively, for mosquitoes from the 31- to 60-min exposure period. This cohort had imbibed the highest virus quantity of the time points tested.

Table 2.

Virus Titers of Infectious Bloodmeals and Engorged Mosquitoes Sampled at 4 Time Points During a 2-H Blood Feed Undertaken to Assess Whether Virus Degradation Affects Infection and Dissemination of Zika Virus TS17-2016 in Innisfail (INN16) Aedes aegypti

Time period of exposure	Titer of bloodmeal ^b	% infection ^c	Titer of engorged mosquitoes ^d	% dissemination ^e	% dissemination/Infection ^f
0–30 min	6.2 ± 0.2	92 (11/12)	2.7 ± 0.4	Not tested	Not tested
31–60 min	6.0 ± 0.1	92 (11/12)	3.0 ± 0.7	Not tested	Not tested
61–90 min	5.2 ± 0.9	100 (12/12)	2.6 ± 0.3	Not tested	Not tested
91–120 min	5.3 ± 1.0	100 (12/12)	2.7 ± 0.4	Not tested	Not tested
7 days^a	Not applicable	10 (4/40)	Not applicable	5 (2/40)	50 (2/4)

The starting titer of the bloodmeal was 10^7.5 TCID50/mL.

The 7-day mosquitoes were from the cohort that had been exposed to the infectious bloodmeal between 31 and 60 min.

Mean ± SD titer (log₁₀TCID₅₀/mL) of the infectious bloodmeal to which mosquitoes were exposed. Bloodmeals and engorged mosquitoes were sampled at the end of the time period.

Percentage of mosquitoes containing virus in their bodies (number positive/number tested).

Titer expressed as mean ± SD log₁₀TCID₅₀/mL.

Percentage of mosquitoes containing virus in their legs and wings (number positive/number tested).

Percentage of infected mosquitoes with a disseminated infection (number disseminated/number infected).

Next we examined whether the use of frozen virus stocks was responsible for the relatively low infection rates, by exposing TVL15 and INN16 mosquitoes to bloodmeals containing 10^7.7 or 10^7.2 TCID₅₀/mL of frozen or freshly harvested virus, respectively. Regardless of whether fresh or frozen virus was used, infection, dissemination, and transmission rates were still relatively low (Table 3).

Table 3.

Day 14 Infection, Dissemination, and Transmission Rates in Townsville (TVL15) and Innisfail (INN16) Aedes aegypti exposed to Bloodmeals Containing Frozen or Freshly Harvested Zika Virus TS17-2016 at Titers of 10^7.7 or 10^7.2 TCID₅₀/ml, Respectively

A. aegypti strain	Virus preparation	% infection ^a	% dissemination ^b	% transmission ^c	% transmission/dissemination ^d
Townsville	Frozen	10 (2/20)	10 (2/20)	10 (2/20)	50 (1/2)
	Fresh	0 (0/12)	0 (0/12)	0 (0/12)	0 (0/0)
Innisfail	Frozen	18 (3/17)	12 (2/17)	6 (1/17)	50 (1/2)
	Fresh	17 (3/18)	17 (3/18)	11 (2/18)	67 (2/3)

Percentage of mosquitoes containing virus in their bodies (number positive/number tested).

Percentage of mosquitoes containing virus in their legs and wings (number positive/number tested).

Percentage of mosquito expectorates in which virus was detected (number of positive expectorates/number tested).

Percentage of mosquitoes with a disseminated infection in which virus was detected in the expectorate (number of positive expectorates/number disseminated).

In the final experiment, INN17 and CNS17 A. aegypti were exposed to bloodmeals, which had pre- and postexposure titers of 10^8.7 TCID₅₀/mL and 10^8.2 TCID₅₀/mL, respectively. Back titration of mosquitoes collected immediately following feeding revealed that INN17 (n = 8) and CNS17 (n = 10) had imbibed 10^5.4 TCID₅₀/mL and 10^5.1 TCID₅₀/mL of ZIKV, respectively. Day 14 infection, dissemination, and transmission rates were not significantly different between the INN17 and CNS17 A. aegypti (Table 4). However, significantly more INN17 mosquitoes with a disseminated infection transmitted the virus compared with CNS17 (p = 0.0390). There was no significant difference (p = 0.4566) in the amount of ZIKV detected in the saliva of the two mosquito strains, with mean ± SD 10^2.4 ^± ^0.3 TCID₅₀/mL and 10^2.1 ^± ^0.3 TCID₅₀/mL expectorated by INN17 and CNS17 A. aegypti, respectively.

Table 4.

Infection, Dissemination, and Transmission Rates in Cairns (CNS17) and Innisfail (INN17) Aedes aegypti exposed to Bloodmeals Containing 10^8.5 ^± 0.4 TCID₅₀/ml of Zika Virus TS17-2016 and Tested at 14 days Postexposure

Strain	% infection ^a	% dissemination ^b	% transmission ^c	% transmission/dissemination ^d
Cairns	53 (16/30)	53 (16/30)	27 (8/30)	50 (8/16)
Innisfail	40 (12/30)	40 (12/30)	37 (11/30)	92 (11/12)

Percentage of mosquitoes containing virus in their bodies (number positive/number tested).

Percentage of mosquitoes containing virus in their legs and wings (number positive/number tested).

Percentage of mosquito expectorates in which virus was detected (number of positive expectorates/number tested).

Percentage of mosquitoes with a disseminated infection in which virus was detected in the expectorate (number of positive expectorates/number disseminated).

Discussion

The recent emergence of ZIKV across multiple continents has led to the evaluation of the vector competence of different populations of A. aegypti for different virus strains, with variable results (Diagne et al. 2015, Chouin-Carneiro et al. 2016, Richard et al. 2016, Weger-Lucarelli et al. 2016, Ciota et al. 2017, Pompon et al. 2017). Previous studies with Australian A. aegypti populations using the prototype MR 766 Uganda strain or a 2010 Cambodian strain have demonstrated considerably higher infection, dissemination, and transmission rates than were obtained in the current study (Hall-Mendelin et al. 2016, Duchemin et al. 2017). We used the same Townsville A. aegypti strain and similar experimental conditions used by Hall-Mendelin et al. (2016) who exposed mosquitoes to 10^6.7 TCID₅₀/mL of the MR 766 ZIKV strain and obtained infection and transmission rates of 57% and 27%, respectively. Even when exposed to ≥10 times more virus than these earlier experiments, infection and transmission rates for the Tongan ZIKV strain were still only 10% (Table 3).

The discrepancy in results between the Hall-Mendelin et al. (2016) and the current study, despite similar experimental conditions, could be due to several factors. The Hall-Mendelin et al. (2016) study used the highly passaged prototype MR 766 strain, which may be consequently better adapted to infecting mosquitoes, compared with the relatively low-passage Tongan strain. Evidence for a role of cell/host adaptation with this ZIKV strain is provided by the attempts to isolate it from patient serum in C6/36 cells, which were initially unsuccessful. It was only after passage in suckling mouse brain that an isolate could be obtained and subsequently passaged through C6/36 cells (Pyke et al. 2016). The increased sensitivity afforded by using real-time RT-PCR to detect mosquito infection as used by Hall-Mendelin et al. (2016) may also explain the discrepancy in infection and transmission rates between the two studies. However, preliminary assessment of the real-time RT-PCR assay and comparison with ZIKV detection by the TC-EIA did not lead to a higher rate of ZIKV detection (S. Hall-Mendelin, unpublished data), so the latter method was used for processing all mosquito samples.

Several factors that can influence the outcomes of vector competence experiments were investigated to help explain why our experiments yielded relatively low infection and transmission rates in the Australian A. aegypti tested. In contrast to recent studies, which demonstrated that ZIKV is relatively stable when incubated in growth media at 37°C (Goo et al. 2016, Kostyuchenko et al. 2016), the virus in our experiments degraded considerably in the bloodmeal over the 2-h feeding period from a starting titer of 10^7.5 TCID₅₀/mL to a final titer of 10^5.3 ^± ^1.0 TCID₅₀/mL. We have previously observed that it takes 10–15 min for the Hemotek feeders to heat to 37°C and for the mosquitoes to commence feeding. Thus, mosquitoes potentially imbibed closer to the 10^6.2 TCID₅₀/mL titer obtained at the end of the 0–30-min period than the 10^7.5 TCID₅₀/mL starting titer. Thus the difference in bloodmeal titers that mosquitoes actually ingested was ≤10^1.0 TCID₅₀/mL and may explain why we observed little difference in the titer of blood-engorged mosquitoes sampled at the different time points.

Interestingly, the use of fresh or frozen virus did not appear to influence the pattern of infection, as has been observed during other studies (Weger-Lucarelli et al. 2016, Ciota et al. 2017). The differences in experimental design and virus preparation may explain the disparity between the results of our experiments and the earlier work. In comparison with the other studies, which used only one virus stock prepared straight from culture without further concentration or purification to make subsequent fresh and frozen stocks, we prepared two separate stocks from the fourth virus passage. The frozen stock was prepared from 18 roller flasks and the virus was semipurified and concentrated to a high titer before freezing and the second was prepared straight from the culture flask. Thus, the highly concentrated virus stock may have had a sufficient titer to largely compensate for possible degradation effects afforded by frozen storage and thawing. Furthermore, ultracentrifugation may have further removed impurities and cellular debris/apoptosis factors, which can affect virus viability. Regardless, these factors highlight the complexity of undertaking vector competence experiments and interpreting results produced.

Overall, the results suggest that there was a high threshold of infection that must be reached to overcome the midgut infection barrier (Black et al. 2002) that is likely expressed by Australian A. aegypti strains. Once this midgut infection barrier was overcome, the virus readily disseminated in >50% of mosquitoes. Importantly >50% of mosquitoes with a disseminated infection transmitted the virus. Further evidence for a midgut barrier to infection in these mosquito strains is provided by Hall-Mendelin et al. (2017), who reported that 100% of INN16 A. aegypti were infected after being inoculated intrathoracically with 10^5.5 TCID₅₀/mL of the Tongan ZIKV strain. Intrathoracic inoculation is a method of virus exposure employed to circumvent the midgut infection and escape barriers.

Ultimately, the results of the current study suggest that the populations of A. aegypti from northern Australia are not efficient vectors of the Tongan strain of ZIKV, with bloodmeal titers of >10⁸ TCID₅₀/mL required to infect ≈50% of mosquitoes. Importantly, the relatively high titer of >10⁸ TCID₅₀/mL is considerably higher than the virus levels reported in blood sampled from viremic patients, which is ∼10⁶ TCID₅₀/mL (Musso et al. 2017). Despite being relatively inefficient vectors of the Tongan ZIKV strain, the role of Australian A. aegypti in virus transmission should not be discounted, as high populations, coupled with anthropophilic feeding behavior can negate the lack of intrinsic ability of this species to become infected with and transmit the virus. Furthermore, Australian A. aegypti may be able to serve as vectors of other contemporary ZIKV strains. For instance, Pompon et al. (2017) demonstrated that ZIKV from the Americas was more efficiently transmitted by Singapore A. aegypti than a 2013 French Polynesian strain. There is also a possibility that other Australian populations of A. aegypti may be more efficient vectors of the Tonga ZIKV strain, as has been reported for dengue viruses (Ye et al. 2014). Certainly, future experiments examining different Australian mosquito populations should utilize other ZIKV strains.

Footnotes

Acknowledgments

The authors wish to thank Helle Bielefeldt-Ohmann for early discussions regarding the study. They also thank Matt Bradford, Caleb Anning, Ben Purcel, and Di Morris of CSIRO for assisting in the collection of F₀ A. aegypti eggs from Innisfail, and Chris Paton (JCU), Sandy Taylor (CSIRO), and Jason Anderson (CSIRO) for providing the subsequent colony eggs used in this study. The authors also thank Bruce Harrower, Peter Burtonclay, and Tanya Constantino for cell culture maintenance. Funding for this study was provided by an internal Forensic and Scientific Services Research and Development Grant (Project number RSS17-005). S. Ritchie is supported by National Health and Medical Research Council Senior Research Fellowship 1044698.

Author Disclosure Statement

The authors are not aware of any commercial associations or biases that might be perceived as affecting the objectivity of this research article.

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