<i>in vivo</i> validation of the biological properties of the IC virus in susceptible mice, histology.

<p>Histology performed on mouse brain 7 days p.i revealed inflammatory lesions in the meninges characterized by diffuse lymphocytic or lymphoplasmocytic infiltrates, characteristic of lymphocytic meningitis (right panel, arrows). Lymphocytic perivascular cuffs were also visible in the brain parenchyma, indicative of encephalitis (left panel, arrows). These lesions were observed in two of four animals or in one mouse for the infectious clone and the Israeli strain, respectively. Tissue sections were stained with hematoxylin, eosin and saffron.</p

<i>in vivo</i> validation of the biological properties of the IC virus in susceptible mice, viremia.

<p>Viral RNA copy number in blood of BALB/c outbred female mice (n = 5) injected i.p with 1, 10, 100, 1000 PFU of parental (P) or recombinant (IC) IS-98-ST1 virus, 3 days post-infection. Quantification was performed in duplicate.</p

<i>in vivo</i> validation of the biological properties of the IC virus in susceptible mice, viral load.

<p>Viral RNA copy number in brains of BALB/c outbred female mice (n = 5) injected i.p with 1, 10, 100, 1000 PFU of parental (P) or recombinant (IC) IS-98-ST1 virus. Quantification was performed in duplicate.</p

Schematic representation of the cloning strategy.

<p>The flavivirus genome is represented approximately to scale. Unique or rare restriction sites used for cloning and their location (numbering based on the sequence from Genbank no. AF481864) are shown at the top. Four cDNA fragments represented by thick lines were synthesized from IS-98-ST1 viral genomic RNA by RT-PCR to cover the complete coding WNV genome. The cDNA fragments were first cloned into the pCR2.1 plasmid, and then digested using unique restrictions sites and subcloned into the destination plasmid. The full length infectious clone was obtained by the extraction of fragment 2+3+4 from plasmid B after an enzymatic digestion with <i>XmaI</i> and <i>BamHI</i> followed by its insertion into plasmid A downstream of fragment 1+2. One silent mutation (shown in lower case) was engineered in the pCR2.1+ fragment 3 plasmid to create a <i>SnaBI</i> site.</p

Viral load in different organs of chicken embryo infected with IS-98-ST1 WNV.

<p>Groups of 6 ten-day-old pathogen-free chicken eggs were infected with 1 PFU of IS-98-ST1 parental virus via the intra-vascular route. Viral load in different organs was quantified by quantitative RT-PCR. PBS controls were negative for all organs.</p

<i>in vivo</i> validation of the biological properties of the IC virus in resistant mice.

<p>Groups of 5 adult female or male outbred MBT/Pas, congenic C.MBT-Oas1b or BALB/c mice were injected i.p. with 1000 PFU of parental (P) or recombinant (IC) IS-98-ST1 virus. The mice were monitored for 15 days p.i. (a) Survival curves and (b) viral RNA copy number in blood of mice 3 days p.i. * shows significant differences between groups. ns: not significant.</p

<i>in vivo</i> validation of the biological properties of the IC virus in chicken embryo.

<p>Groups of 6 ten-day-old pathogen-free chicken eggs were infected with 1 PFU of either parental (P) or recombinant (IC) IS-98-ST1 or Kunjin (Ku) virus by the intra-vascular route. (a) Survival curves and (b) viral RNA load in brains and hearts at day 4 p.i, quantified by quantitative RT-PCR, are shown. PBS controls were negative for all organs. Data are representative of two independent experiments, except RNA quantification at day 4 p.i for Kunjin, which was performed once. * shows significant differences between groups.</p

<i>in vitro</i> validation of the biological properties of the IC virus.

<p>(a) Plaque morphology of parental and IC-derived WNV on Vero cells. Vero cells in six-well plates were infected with 100 PFU of parental or recombinant virus. Plaques were visualized 3 days post-infection by staining with crystal violet. Data shown are representative of two independent experiments. (b) <i>SnaBI</i> digestion profile of the 1198 bp RT-PCR (5743F–6941R) fragment amplified from parental or IC-derived WNV stocks. (Quick Load 1 kb DNA Ladder, New England Biolabs). (c) Growth kinetics of parental (P) and infectious clone (IC) viruses in Vero cells at an MOI of 1. At the indicated time post-infection, culture supernatants were collected and viral titers were determined by plaque assay on Vero cells. Error bars represent the standard deviation of triplicates. (d) Immunofluorescence with anti-NS1 antibodies on Vero cells infected at an MOI of 1 with either the parental or IC-derived WNV 3 days post-infection. Data shown are representative of two independent experiments.</p

<i>in vivo</i> validation of the biological properties of the IC virus in susceptible mice, survival curves.

<p>Groups of 5 adult female BALB/c outbred mice were injected i.p with 1, 10, 100, 1000 PFU of parental (P) or recombinant (IC) IS-98-ST1 virus. Mice were monitored for 15 days post-infection. Data shown are representative of two independent experiments.</p

Localization of the E1 Changes on the 3D Structure Modelled from the Crystal Structure of SFV E1

<div><p>(A) Ribbon diagram of E1, with domain I coloured red, domain II yellow, and domain III blue. Green tubes mark the disulfide bonds. The fusion peptide, at the tip of the molecule (in domain II) is coloured orange and labelled. The N-terminus and the C-terminus observed in the crystal (which is 30 aa upstream of the trans-membrane region) are also labelled. The two unique changes observed in the Indian Ocean isolates are indicated by stars and labelled: positions 226 (white) and 284 (magenta).</p> <p>(B) Partial representation (one octant, slightly extended) of the icosahedral E1 scaffold at the surface of the virion, viewed down a 5-fold symmetry axis. One E1 protomer is highlighted in colours, as in (A); all the others are represented in grey. The location of the some of the icosahedral symmetry axes are drawn as solid black symbols: pentagon for 5-fold axis, triangle for 3-fold axes, ellipse for 2-fold axes (which in the T = 4 lattice of alphaviruses are coincident with quasi 6-fold axes). Open triangles indicate roughly the location of the E2 trimers that interact tightly with E1, covering domain II and the fusion peptide, and presenting the main antigenic sites. The open triangles mark also quasi 3-fold symmetry axes of the T = 4 surface icosahedral lattice. A magenta ball marks the location of Glu 284, at an inter-E1 protomer contact site. This contact is propagated 240 times at the surface lattice (note all pink balls drawn on the grey protomers). Note that the fusion peptide, in orange, is pointing up and away from contacts with other E1 protomers. This is more easily seen at the periphery of the virion, where one of them is labelled (FP). In the virion, this region of E1 is not accessible, covered underneath the E2 molecule [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030263#pmed-0030263-b020" target="_blank">20</a>]. </p></div