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. 2013;8(2):e56534.
doi: 10.1371/journal.pone.0056534. Epub 2013 Feb 27.

A new insect-specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells

Jody Hobson-Peters  1 , Alice Wei Yee Yam, Jennifer Wei Fei Lu, Yin Xiang Setoh, Fiona J May, Nina Kurucz, Susan Walsh, Natalie A Prow, Steven S Davis, Richard Weir, Lorna Melville, Neville Hunt, Richard I Webb, Bradley J Blitvich, Peter Whelan, Roy A Hall
Affiliations

A new insect-specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells

Jody Hobson-Peters et al. PLoS One. 2013.

Abstract

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10-43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic tree showing relationship between PCV and other insect-specific flaviviruses.
(A) Maximum likelihood tree constructed using NS5/3′UTR sequences of select insect-specific flaviviruses and four isolates of PCV. (B) Maximum likelihood tree constructed using the complete ORFs of a selection of insect-specific flaviviruses. For both trees, the numbers at the nodes represent bootstrap replicates as a percentage of 1000 replicates. Both trees have been mid-point rooted. CFAV and CxFV groups have been collapsed for clarity.
Figure 2
Figure 2. Transmission Electron Micrograph of PCV particles.
Culture supernatant from C6/36 cells infected with PCV was concentrated and round, enveloped virus particles with an electron-dense core were visualised using uranyl acetate staining. Scale bars are 100 nm and 50 nm for left and right panels respectively.
Figure 3
Figure 3. Reactivity of anti-PCV mAbs to PCV-infected C6/36 cells by IFA.
Mock-, PCV and WNVKUNV-infected C6/36 cell monolayers were fixed using acetone and probed with anti-PCV mAbs 3D6, 8G2 and 9G4 or with 4G4 which binds to WNVKUNV NS1 protein . The nucleus of each cell was stained by Hoechst. Images were taken using a ×40 objective lens.
Figure 4
Figure 4. Analysis of recombinant PCV proteins.
COS-7L cells were transiently transfected with pcDNA3.1-based plasmids encoding genes for the expression of PCV secreted prM/E, NS1 and partial NS5. Each expressed protein contained a C-terminal V5 tag. Mock cells were transfected with the empty pcDNA3.1 construct. A) Cells were fixed 21 hr post-transfection and probed with mAbs 3D6, 9G4 or anti-V5 using IFA. B) Lysates of COS-7L cells transfected with plasmids encoding PCV NS1, PCV secreted prM/E, WNV NS1 or pcDNA3.1 (M = mock) were treated (+) or untreated (−) with PNGaseF to remove N-linked glycans. The proteins were probed with anti-V5 (left panel) or anti-NS1 mAb 4G4 (right panel) in Western blot. C,D) Western blot analysis of supernatant and lysate of transiently transfected cells with PCV NS1 construct (C) or WNV NS1 construct (D). Protein detection was with anti-V5 and mAb 4G4 respectively. Lane 1 – NS1 lysate; Lane 2 – mock lysate; Lane 3 – NS1 supernatant; Lane 4 – mock supernatant. *Partial NS5 expressed. The construct is missing the sequence for the first 85 amino acids of the predicted full length PCV NS5 protein.
Figure 5
Figure 5. In vitro superinfection exclusion assay.
IFA and infectious titre assays showing reduced replication of the flaviviruses MVEV and WNVKUNV in C6/36 cells persistently infected with PCV when compared to mock infected (PCV-negative) cells. The alphavirus RRV replicates equally well in both. A–C Study 1; D–F Study 2. A) Co-staining was performed to detect the primary and secondary infecting viruses. The secondary viruses MVEV and RRV were detected with Alexa Fluor 488-labelled mAbs 4G4 and G8 respectively. The PCV primary infection was detected with the anti-PCV mAb 3D6. D) Dramatically less staining for the secondary infecting viruses WNVKUNV and MVEV was detected with mAb 4G4 in cells that were persistently infected with PCV compared with mock-infected cells. In comparison, RRV grew equally well in PCV- and mock-infected cells, as detected with mAb G8.
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Funding for this study was provided by the Australian Research Council (DP120103994). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.