Table 1.
Summary of PCV isolates from mosquito pools collected in Darwin.
Table 2.
Amino acid and nucleotide identities between PCV and other ISFs over the NS5/3′UTR region.
Table 3.
Amino acid and nucleotide identities between PCV and other ISFs over the entire coding region.
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.
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.
Table 4.
Replication of PCV56 in various cell lines.
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 [30]. The nucleus of each cell was stained by Hoechst. Images were taken using a ×40 objective lens.
Table 5.
Characterisation of anti-PCV mAbs.
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.
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.