Characterisation of anti-PCV mAbs.

*<p>Binding pattern of mAbs in fixed cell ELISA to PCV, Palm Creek virus; WNV, West Nile virus Kunjin subtype; MVEV, Murray Valley encephalitis virus; DENV, Dengue virus, YFV, yellow fever virus.</p><p>WB = Western blot.</p

Amino acid and nucleotide identities between PCV and other ISFs over the NS5/3′UTR region.

<p><b>Bold</b> text: amino acid identity.</p><p>Non bolded text : nucleotide identity.</p>*<p>A range is given for those viruses that have multiple isolates listed on Genbank.</p><p>PCV – Palm Creek virus; NAKV – Nakiwogo virus; CFAV – cell fusing agent virus; QBV – Quang Binh virus; CTFV – Culex theileri flavivirus; AeFV – Aedes flavivirus; CxFv - Culex flavivirus; KRV – Kamiti River virus.</p

Phylogenetic tree showing relationship between PCV and other insect-specific flaviviruses.

<p>(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.</p

Analysis of recombinant PCV proteins.

<p>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. <b>A</b>) Cells were fixed 21 hr post-transfection and probed with mAbs 3D6, 9G4 or anti-V5 using IFA. <b>B</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. <b>C,D</b>) Western blot analysis of supernatant and lysate of transiently transfected cells with PCV NS1 construct (<b>C</b>) or WNV NS1 construct (<b>D</b>). 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.</p

Susceptibility of Balb/c and C57BL/6 mice to ROCV infection.

<p>Mice were infected with different doses of ROCV (n = 10 mice per group). Survival rate and body weight loss for C57BL/6 (A and B) and for Balb/c (C and D). Comparison of mortality rate between C57BL/6 and Balb/c (Fig 1E). Survival rate and body weight loss were measured for up to 21 days post-infection. Statistically significant differences in B and D: ****p<0.0001 was determined by one-way ANOVA with Dunnett's multiple comparisons test, in E: *p<0.01 by Log-rank (Mantel-Cox) test.</p

Evaluation of cross-protective immunity against ROCV after prior infections with different flaviviruses.

<p>Immunization and challenge regime (A). Mice (n = 40 per group) were infected twice with the different flaviviruses known to circulate in Brazil, and were then challenged (n = 20 per group) with 2.76x10⁷ PFU of ROCV and survival rates (B and E), clinical scores (C and F) and body weight loss (D and G) were determined up to 21 days post-infection. Statistically significant differences in B and E: *p<0.01, **p<0.001 and ****p<0.0001 was determined by Log-rank (Mantel-Cox) test. In F: ****p<0.0001 was analyzed by a student t-test. In D-G *p<0.01, **p<0.001 and ****p<0.0001 was determined by One-way ANOVA with Dunnett's multiple comparisons test. All statistically significant differences were with group control (Mock).</p

Amino acid identity between the E proteins of Brazilian flaviviruses used in this study.

<p>Amino acid identity between the E proteins of Brazilian flaviviruses used in this study.</p

Ilheus and Saint Louis encephalitis viruses elicit cross-protection against a lethal Rocio virus challenge in mice

<div><p>Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.</p></div