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. 2016 Jul 25;9(1):414.
doi: 10.1186/s13071-016-1683-2.

The insect-specific Palm Creek virus modulates West Nile virus infection in and transmission by Australian mosquitoes

Sonja Hall-Mendelin  1 , Breeanna J McLean  2 , Helle Bielefeldt-Ohmann  2   3 , Jody Hobson-Peters  2 , Roy A Hall  2 , Andrew F van den Hurk  4
Affiliations

The insect-specific Palm Creek virus modulates West Nile virus infection in and transmission by Australian mosquitoes

Sonja Hall-Mendelin et al. Parasit Vectors. .

Abstract

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.

Keywords: Aedes aegypti; Aedes vigilax; Culex annulirostris; Insect-specific flavivirus; Palm Creek virus; West Nile virus.

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Figures

Fig. 1
Fig. 1
Viral titers in the bodies of Culex annulirostris, Aedes aegypti and Aedes vigilax mosquitoes injected intrathoracically with PCV. Each point on the plot represents an individual infected mosquito and bars denote medians. P < 0.001 (***), Kruskill-Wallis test, with a Dunn’s post-hoc multiple comparisons test. The comparison between Ae. aegypti and Ae. vigilax was non-significant (P > 0.05)
Fig. 2
Fig. 2
Impact of PCV on WNV replication in bodies of Cx. annulirostris 10–12 days after being exposed to WNVKUN2009 via ingestion of an infectious blood meal (a) or intrathoracic inoculation (b), or WNVKUNMRM16 via intrathoracic inoculation (c). Each point on the plot represents an individual infected mosquito and bars denote medians. P < 0.01 (**), P < 0.05 (*) Mann-Whitney U test
Fig. 3
Fig. 3
Impact of PCV on WNV replication in saliva of Cx. annulirostris 10–12 days after being exposed to WNVKUN2009 via ingestion of an infectious blood meal (a) or intrathoracic inoculation (b), or WNVKUNMRM16 via intrathoracic inoculation (c). Each point on the plot represents an individual infected mosquito and bars denote medians
Fig. 4
Fig. 4
Immunohistochemical detection of PCV (a) and WNV (b) in midgut epithelial cells (red signal) of infected female Cx. annulirostris mosquitoes. c antibody isotype control. Hematoxylin was used as the counterstain
Fig. 5
Fig. 5
Comparative growth kinetics of PCV (circles) and WNV (squares) in C6/36 (RNAi-deficient) (a) and RML-12 (RNAi-competent) (b) Aedes albopictus cells. Cells were infected with either PCV or WNV at an MOI of 0.1 and infectious titers at each time-point (up to 5 or 7 days) determined by titration of culture supernatant on C6/36 cells and detection of infected wells by fixed cell ELISA. Error bars represent standard deviation and asterisks indicate significance (P < 0.0001) as determined by a two-way ANOVA
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