Australia Ae. aegypti mosquitoes are susceptible to a highly divergent and sylvatic dengue virus type 2 strain infection but are unlikely to transmit CURRENT STATUS:

Background: Humans are the primary hosts of the dengue virus; However, sylvatic cycles of transmission can occur among non-human primates and human to regions can be a source of emergence of new strains. We reported the isolation of a highly divergent and sylvatic DENV-2 strain (QML22) from a dengue fever patient returning Australia from Borneo. The objective of the present study was to evaluate the vector competence of Australian Ae. aegypti mosquitoes for this virus. Methods: Four-day old mosquitoes from two strains of Ae. aegypti from Queensland, Australia, were feed sheep blood meal containing 108 50% cell culture infectious dose per ml (CCID50/ml) of either QML22 or an Australian epidemic DENV serotype 2 strain (QML16) isolated from a dengue fever patient in 2015. Mosquitoes were maintained at 28°C, 75% relative humidity and sampled at 7, 10 and 14 days post-infection (DPI). Live virions in mosquito bodies (abdomen/thorax), legs and wings and saliva expectorates from individual mosquitoes were quantified using a Cell Culture Enzyme-linked Immunosorbant Assay (CCELISA) to determine infection, dissemination and transmission rates. Findings: The infection and dissemination rates of the sylvatic DENV2 strain, QML22, within mosquitoes were significantly lower than that for QML16. While the titres of virus in the bodies of mosquitoes infected with either of these viruses were similar, titres in legs and wings were significantly lower in mosquitoes infected with QML22 at most time points although they reached similar levels by 14 DPI. QML16 was detected in 16% (n = 25) and 28% (n = 25) of saliva expectorates at 10 and 14 DPI, respectively. In contrast, no virus was detected in the saliva expectorates of QML22 infected mosquitoes. Conclusions: Australia urban/peri-urban Ae.aegypti species are susceptible to infection by the sylvatic and highly divergent DENV-2 virus QML22. However, our results indicate that replication of QML22 is attenuated relative to the contemporary strain QML16 and/or a salivary gland infection or


Background
Dengue virus (DENV) has two ecologically and evolutionary discrete transmission cycles: sylvatic and urban endemic/epidemic [1]. The sylvatic cycle casts non-human primates as the host and several arboreal Aedes mosquito species as the transmission vectors [2,3]. Alternatively, the urban endemic/epidemic cycle sees humans as the host and the peri-domestic Ae. aegypti mosquito as the principal vector. These two kinds of DENV cycles are evolutionary distinct and all four serotypes of endemic/epidemic DENV are considered to have evolved independently from the sylvatic DENV progenitors over the past 1,000 years. Sylvatic DENV1-4 strains from Malaysia and DENV-2 from West Africa have been reported to be able to spill over to infect humans causing similar or relatively milder dengue symptoms compared with the classic endemic/epidemic DENV infections [4][5][6][7][8] Whether sylvatic DENV strains can overcome adaptive barriers to infect peri-domestic Ae. aegypti mosquitoes, then enter the urban human-mosquito-human transmission cycle to cause secondary human infection (spillover epidemics), has been a source of debate for over a decade [1,[9][10][11]. Previous studies testing the ability of sylvatic DENV strains to infect Ae. aegypti have produced a confusing picture in which the susceptibility of Ae. aegypti to infection with sylvatic DENV-2 has ranged from refractory to almost 100% susceptibility [12][13][14][15]. Noticeably, none of the viruses studied were recovered from patients (i.e. they were not known to be able to infect humans). Instead, viruses were isolated from non-human primates and/or mosquitoes. In addition, these studies used virus dissemination to mosquito legs, wings and heads as a proxy for virus transmission capability, based on the assumption that if the virus were able to disseminate from midgut to these tissues, that the virus would have infected the salivary glands and transmission could occur [15,16]. The detection of infectious virus from mosquito saliva provides a more accurate proxy for transmission [17].
In 2016, we reported the isolation of a sylvatic strain of DENV-2, QML22, from a patient returning to Australia from Borneo [5]. The complete genome of QML22 is clearly divergent from Asian and West African lineages of sylvatic DENV-2. It has been reported that DENV susceptibility for Ae. aegypti varied geographically in Australia [18][19][20]. Here we determined the vector competence of two lines of Ae. aegypti responsible for urban transmission in Australia for this sylvatic strain of DENV by oral infection of mosquitoes with virus and analysis of infection within mosquito bodies, legs and wings and saliva.

Methods
Cells, viruses and mosquitoes C6-36 (Ae. albopictus mosquito) cells were purchased from the American Type Culture Collection (ATCC) and cultured in 10% v/v heat inactivated foetal calf serum (FCS, Life Technology, USA)/RPMI 1640 medium (Sigma, USA). The DENV-2 strain QML16 was isolated from a dengue fever patient in Australia and QML22 was isolated from a dengue fever patient returning to Australia from Borneo [5].
The virus strains were passaged three times in C6-36 cells to a titer around of 10 8 CCID 50 /ml and the cell culture supernatant was stored at -80 °C for further use. Adults were provided with 10% sugar solution on cotton wool pledgets which was withheld two day prior to virus feeding. Prior to feeding, mosquitoes (4 day-old) were deprived of sucrose solution for 24 h.

Membrane feeding
Approximately one hundred 3-5 day old mosquitoes were placed into 750 ml containers with gauze covering the opening. DENV-2 strains QML16 and QML22 strains were mixed with defibrinated sheep blood to a titre of 10 8 CCID 50 /ml in C6/36 cells. The mosquitoes in containers were allowed 1 hr to feed on the blood/virus mixtures through bovine ceacum membrane using an artificial feeding apparatus maintained at 37 °C, as previously described [21]. After feeding, mosquitoes were anaesthetized using CO 2 , placed on a Petri dish on ice and fully engorged females were separated 5 from the unfed mosquitoes. The engorged mosquitoes were placed into the gauze covered containers, provided with cotton balls soaked with 10% sugar solution, and maintained within an Environmental Chamber (Panasonic) set at 28 °C, 75% relative humidity and 12:12 h day:night ligiht schedule with 30 min dawn:dusk periods.
In vitro transmission assays At 7, 10 and 14 dpi, female mosquitoes were anesthetized using CO 2 and ice; legs and wings were removed. In vitro transmission assays were performed as previously described [22,23]. USA) as the substrate. Wells with cell monolayers that stained blue were scored as positive for infection. The CCID 50 was determined from titration endpoints as described everywhere [27] and expressed as the CCID 50 /ml in C6/36 cells.
Infection rate was defined as the proportion of mosquitoes with DENV positive bodies/total number of engorged mosquitoes. Dissemination and transmission rates were defined as the proportions of infected mosquitoes with positive legs/wings and salivary secretions/ the total number of engorged mosquitoes. We compared the virus titers and proportions using Mann-Whitney U-test and Chi square test.

Mosquito immunohistochemistry
Histological analysis of DENV infection within mosquitoes using indirect immunofluorescence assay (IFA), microscopy was performed based on the methods of described in our previous publication [22].
Briefly, mosquitoes with legs and wings removed were fixed in 4% paraformaldehyde/0.5% Triton X

Results
Fewer mosquitoes of Townsville Ae. Aegypti colony became infected when fed QML22 (38.7%, n = 75) virus containing blood meals compared to that of QML16 containing blood meal fed (75%, n = 75) ( Fig. 1a and b. Chi square test, p < 0.0001), however body infection rates remained stable between 7 and 14 DPI time points for both virus strain.

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Despite the lower body infection rates among mosquitoes fed on the QML22 strain, body titres of infected mosquitoes were not significantly different to mosquitoes infected with the QML16 strain ( Fig. 1b) (Mann Whitney, p > 0.05).
DENV-2 QML22 virus was first detected from mosquito legs and wings at 10 DPI, in contrast to the QML16 strain which infected legs and wings by as early as 7 DPI. Furthermore, QML22 disseminated to legs and wings in fewer mosquitoes that QML16 and virus grew to lower titres than the QML16 strain at 7 and 10 DPI ( Fig. 1a and b, P < 0.01, chi squared test,). However QML22 reached similar titers to QML16 in legs and wings by 14 DPI (P > 0.05, chi square test, Fig. 1b).
No live virus was detected in saliva expectorates of mosquitoes fed on blood meals containing the QML22 strain at 7, 10 or 14 days post feeding. This was in contrast to mosquitoes fed on blood meals containing DENV2 QML16, which yielded 16% (4/25) and 28% (7/25) Fig. 1a&b). In keeping with the first experiment, the titres of virus in the infected bodies was similar between mosquitoes fed QML16 and QML22 strains (~ 10 7 CCID 50 /mosquito, Fig. 1b). This suggests that the Innisfail Ae. aegypti mosquitoes were less susceptible to infection with QML22 than those from Townsville. Low numbers of infected mosquitoes from Innisfail prevented statistical analysis.
Histological analysis of mosquitoes infected with dengue QML16 and QML22 supported the above results. Paraffin embedded sections were stained by indirect immunofluorescence employing anti-DENV envelope protein monoclonal antibody (4G2) and Alexa-fluor488-labelled anti-mouse 8 immunoglobulin (green) [22] (Fig. 2). Disseminated virus infection was observed in 88% (n = 25) mosquitoes ≥ 10 d post feeding on dengue QML16 and infection was observed in 50% of salivary glands (n = 6). In contrast, no virus dissemination had occurred beyond the midgut in any mosquitoes examined histologically at ≥ 10 d post feeding DENV2 QML22 (n = 13).

Discussion
The importation of a pathogenic, transmissible and highly divergent DENV-2 genotype into Australia with a human population largely susceptible to DENV outbreaks, plus the endemic of the primary Dengue transmission vector Aedes aegypti in north Queensland [28], could have significant public health implications. Here we demonstrated that strains of Ae. aegypti from two population centres in north Queensland, Australia; Townsville and Innisfail, are susceptible and able to transmit a contemporary epidemic DENV-2 strain but they are much less susceptible to a highly divergent and sylvatic strain, DENV-2 QML22, and are potentially unable to transmit it.
Variable vector competence of Aedes aegypti populations from around the world for sylvatic DENV has been reported. A sylvatic strain of DENV-2 isolated from a mosquito in Senegal in 1965 was shown to infect 50-91% of Ae. aegypti among eight different Senegalese Ae. aegypti populations.
Moreover, these results were achieved from blood meals containing substantially less virus than used in this study (approximately 10 6−7 PFU/ml virus) [14]. Our infection rates more closely resemble those achieved when Ae. aegypti collected from Galveston, United States and from Bolivia became infected when fed on blood meals containing 10 8 to 10 9.5 TCID 50 /ml of strains of sylvatic DENV-2 isolated from a mosquito in Burkina Faso, West Africa, and a sentinel monkey in Malaysia [29]. Variable vector susceptibility (0-26% infection rate) was also observed in another study testing the vector competence of six Senegalese Ae. aegypti populations after feeding a 10 6−7 PFU/ml virus of a sylvatic strain DENV-2 isolated from Senegal in 1999 [12]. Noticeably, all these studies used dissemination to distal body tissues to determine the virus transmission potential based on an assumption that these mosquitos were capable of transmitting DENV if the virus had disseminated from midgut into the hemocoel [15,16]. In our experiment, we observed the dissemination of the QML22 strain into legs 9 and wings but could not recover live virus from saliva at any time points. Our data also suggests that the vector competence of Australian Ae. aegypti mosquitoes for this sylvatic strain varies depending on the geographical origin of mosquito populations, even though they showed similar susceptibility for a contemporary epidemic DENV-2 QML16 strain. While it is possible that strains of Ae. aegypti from localities outside Australia may not have as stringent barriers to the dissemination and transmission of this highly divergent strain of DENV-2, it is unlikely that the barriers would be completely absent. Investigations to determine the mechanism underpinning the resistance of Ae.
aegypti to infection with this sylvatic strain of DENV-2 are likely to be complex, given the enormous differences between the nucleotide and amino acid sequences of it and other strains of DENV-2 for which Ae. aegypti is known to be able to be a competent vector [5].
In order to transmit the virus to an uninfected human, DENV must counteract the mosquito innate immune system to replicate and disseminate through the mosquito before infecting saliva [30].
Several physiological 'barriers' to this dissemination have been hypothesised, including midgut infection and escape barriers (MIB and MEB) and salivary gland infection and escape bariers (SGIB and SGEB). Earlier studies have indicated that the MIB is a major determinant of VC for Dengue virus [31,32]. The lower body infection rate of QML22 (Fig. 1a) suggests a MIB might be the first obstacle for the highly divergent QML22 where the virus/cell-receptor interaction and internalization into the midgut epithelial cells is potentially less compatible to that of the QML16 strain. When the MIB was overcome, the QML22 strain reached body similar virus titers to the QML16 strain (Fig. 1b). However, lower dissemination rates and virus titers in legs and wings indicate that replication way be attenuated in comparison to the QML16 strain. Although the QML22 strain reached the same titre in legs and wings as QML16 at 14DPI (Fig. 1b)

Conclusions
We demonstrated that both Ae. aegypti species from Townsville and Innisfail North Queensland are highly susceptible and able to transmit a contemporary epidemic DENV-2 strain but are much less susceptible to a highly divergent and sylvatic DENV-2 QML22 and potentially unable to transmit it.
Our findings support a conclusion that sylvatic DENV is unlikely to enter urban human -mosquito-

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