Schematic representation of recombinant lentiviral TRIP/JEV vectors.

<p>The organization of JEV genomic RNA is shown in top. The codon-optimized sequence encoding prM and E from JEV G3 strain RP-9 was cloned into a TRIP lentiviral vector under the control of human cytomegalovirus immediate early promoter (P_CMVie). The TRIP/JEV.prME vector includes the signal peptide sequence of prM (SP) followed by the entire prM and E gene regions of JEV strain RP-9 of G3. The TRIP/prME^ΔTM vector includes the same JEV sequence except that E was deleted from its two transmembrane domains TMD1 and TMD2.</p

Anti-JEV IgG responses of piglets immunized with TRIP/JEV.prME.

<p><i>In</i> (A), two groups of four piglets were immunized intramuscularly with 6 (low dose) or 7 log₁₀ TU (high dose) of TRIP/JEV.prME. As a control, two animals were inoculated with either low or high dose of TRIP/GFP. Animals were boosted 4 weeks after primary immunization with the same initial dose (vertical arrow). Serum samples were collected weekly and tested at a dilution of 1:400 for the presence of anti-JEV E IgGs by indirect ELISA. <i>In</i> (B), a group of three animals were experimentally infected with JEV strain Nakayama. The immune sera were tested at a dilution of 1:400 for the presence of anti-JEV E IgGs by indirect ELISA. <i>In</i> (C, D), box plots of the anti-JEV E IgG1/IgG2 from 1 to 10 weeks after immunization with the low (C) or high (D) dose of TRIP/JEV.prME are depicted. The vertical arrow indicates the boost. <i>In</i> (E), the levels of anti-JEV E IgG1/IgG2 in immune sera from piglets infected with JEV strain Nakayama.</p

Reactivity of JEV prM and E with anti-TRIP/JEV antibody.

<p>Lysates obtained from SK-N-SH cells infected with JEV strain RP-9 (JEV) or mock-infected (m.i.) were tested with pooled immune sera (antisera) from BALB/c mice twice inoculated with 5 log₁₀ TU of TRIP/JEV.prME or TRIP/JEV.prME vector by immunoblot assay. TRIP/JEV antisera were collected 3 weeks after the boosting inoculation. Mouse polyclonal serum directed against JEV strain RP-9 of G3 (JEV antiserum) served as a positive control. The bands corresponding to JEV E, NS1/NS1’ and prM proteins are indicated with arrows to the left of the blot.</p

Analysis of recombinant JEV antigens.

<p><i>In</i> (A), sequence alignment of EDIII from JEV of G1, G3, and G5. <i>In</i> (B, C), equal amount (100 ng) of highly purified recombinant proteins JEV.E^ΔTM-SNAP (B) and SNAP-JEV.EDIII of G1, G3 or G5 (C) were analyzed by immunoblotting with an anti- SNAP tag antibody. Purified SNAP protein served as a control. The bands corresponding to JEV.E^ΔTM-SNAP, SNAP-JEV.EDIII, and SNAP proteins are indicated with arrows to the right of the blot.</p

Protective capacity of TRIP/JEV antisera in a mouse model.

<p>Groups of 3-week-old C56BL/6 mice received i.p. inoculation with 0.1 ml of DPBS containing 0.01 ml of pooled immune sera collected from JEV-infected mice (JEV G3 antiserum) or TRIP/JEV-inoculated mice two months after boosting. Mice inoculated with DPBS (PBS) served as a group control. One day later, the mice were i.p. challenged with JEV strain RP-9 and observed daily for mortality. Survival was recorded for 21 days. The asterisk indicates that the differences between survival curves are statistically significant (*, <i>P</i> < 0.05, n.s.: <i>P</i> > 0.05).</p

Neutralizing activities of anti-TRIP/JE vector antisera.

<p>¹. The neutralization activities of pooled immune sera against three live JEV [G3 (strain RP-9), G1/3 chimera (strains CNS769_Laos_2009 and RP-9) and G5/3 chimera (strains XZ0934 and RP-9)] were evaluated using a standard FRNT assay in Vero cells. FRNT₅₀ values representative of <i>n</i> = 3 independent experiments are given.</p><p>². Immune sera were collected from groups of 3-week-old BALB/c mice (<i>n</i> = 6) at 21 days after intraperitoneal inoculation with 3 log₁₀ FFU of JEV G3 (strain RP-9).</p><p>³. Immune sera were collected from groups of 6-week-old BALB/c mice (<i>n</i> = 6) at 21 days after intraperitoneal inoculation with 5 log₁₀ TU of lentiviral TRIP/JE vectors.</p><p>Neutralizing activities of anti-TRIP/JE vector antisera.</p

Neutralizing antibody response in piglets immunized with TRIP/JEV.prME.

<p>Sera from piglets immunized with a low or high dose of TRIP/JEV.prME were tested for neutralization ability against JEV by PRNT50. <i>In</i> (A), the piglet sera collected prior immunization, 3 weeks after priming or 6 weeks after the boost were tested against the JEV G3 strain RP-9. <i>In</i> (B), the TRIP/JEV.prME antisera collected after the boost were tested for their cross-neutralizing capacity against JEV G1 and G3 strains, and JEV chimera G5/G3. <i>In</i> (C) the neutralizing activity of anti-JEV antibodies from animals experimentally infected with JEV G3 strain Nakayama was tested against JEV G1, G3, and the JEV chimera G5/G3 by PRNT50.</p

Transduction of HEK-293T cells with lentiviral TRIP/JEV vectors.

<p><i>In</i> (A), immunofluorescence analysis of transduced cells using the anti-E mAb 4G2 as primary antibody. <i>In</i> (B, C), immunoblot analysis of prM and E accumulation in lysates (B) or supernatants (C) of cells transduced with TRIP/JEV vectors. TRIP/GFP vector served as a control. The intracellular prM and E were detected with a mouse polyclonal serum directed against JEV strain RP-9 (JEV antiserum). Detection of calnexin (CNX) served as a loading control. JEV E and prM in supernatants of transduced cells were detected with mAb 4G2 or with the JEV antiserum, respectively. JEV VLPs were purified from supernatants of TRIP/JEV vector-transduced cells and analyzed by immunoblotting using anti-E mAb 4G2 and JEV antiserum. TRIP/GFP served as a negative control. The bands corresponding to prM, E or E^ΔTM and CNX are indicated with arrows to the right of the blots.</p

Construction strategy of chimeric live JEV and their recognition by TRIP/JEV antisera.

<p><i>In</i> (A), schematic diagram of chimeric JEV G1/3 and G5/3 which include the sequences encoding C, prM and E from JEV of G1 or G5 into the backbone of JEV of G3. The parental JEV was designed as JEV G3. <i>In</i> (B), recognition of chimeric JEV proteins by JEV antiserum or TRIP/JEV antisera. Lysates obtained from SK-N-SH cells infected with parental JEV G3, chimeric JEV G1/3 and G5/3, or mock-infected (m.i.) were analyzed by immunoblotting with the indicated antibodies. Anti-JEV NS5 (NS5) and anti-calnexin (CNX) antibodies served as controls.</p

Japanese encephalitis virus tropism in experimentally infected pigs.

Pigs are considered to be the main amplifying host for Japanese encephalitis virus (JEV), and their infection can correlate with human cases of disease. Despite their importance in the ecology of the virus as it relates to human cases of encephalitis, the pathogenesis of JEV in pigs remains obscure. In the present study, the localization and kinetics of virus replication were investigated in various tissues after experimental intravenous infection of pigs. The data demonstrate a rapid and broad spreading of the virus to the central nervous system (CNS) and various other organs. A particular tropism of JEV in pigs not only to the CNS but also for secondary lymphoid tissue, in particular the tonsils with the overall highest viral loads, was observed. In this organ, even 11 days post infection, the latest time point of the experiment, no apparent decrease in viral RNA loads and live virus was found despite the presence of a neutralizing antibody response. This was also well beyond the clinical and viremic phase. These results are of significance for the pathogenesis of JEV, and call for further experimental studies focusing on the cellular source and duration of virus replication in pigs