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A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Yun SI, Song BH, Kim JK, Yun GN, Lee EY, Li L, Kuhn RJ, Rossmann MG, Morrey JD, Lee YM - PLoS Pathog. (2014)

Bottom Line: Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain.In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication.Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.

ABSTRACT
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

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Discovery of a single locus that leads to the reversion of SA14-14-2MCV to lethal neurovirulence.(A-C) In vitro replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or each mutant cDNA, as indicated. RNA infectivity (in PFU/µg) at 4 dpt was determined by infectious center assay, combined with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was measured by plaque titration on BHK-21 cells (A); viral protein accumulation at 18 hpt was examined by immunoblotting of cell lysates with a panel of antibodies as indicated (B). For viral growth analysis, BHK-21 cells were infected at an MOI of 0.1 with the parent or each mutant virus obtained from the corresponding RNA-transfected cells. At the indicated time points, culture supernatants harvested for virus titration on BHK-21 cells (C). (D-E) In vivo neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were inoculated IC with doses of the virus stock serially diluted 10-fold, and the LD50 values were determined (D). For immunohistochemical staining, groups of the mice (n = 15 per group) were mock-infected or infected IC with 103 PFU of each virus. At 3, 5, and 7 dpi, five mice were processed for brain section staining with an α-NS1 antiserum. Shown are representative hippocampal slides (E).
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ppat-1004290-g004: Discovery of a single locus that leads to the reversion of SA14-14-2MCV to lethal neurovirulence.(A-C) In vitro replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or each mutant cDNA, as indicated. RNA infectivity (in PFU/µg) at 4 dpt was determined by infectious center assay, combined with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was measured by plaque titration on BHK-21 cells (A); viral protein accumulation at 18 hpt was examined by immunoblotting of cell lysates with a panel of antibodies as indicated (B). For viral growth analysis, BHK-21 cells were infected at an MOI of 0.1 with the parent or each mutant virus obtained from the corresponding RNA-transfected cells. At the indicated time points, culture supernatants harvested for virus titration on BHK-21 cells (C). (D-E) In vivo neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were inoculated IC with doses of the virus stock serially diluted 10-fold, and the LD50 values were determined (D). For immunohistochemical staining, groups of the mice (n = 15 per group) were mock-infected or infected IC with 103 PFU of each virus. At 3, 5, and 7 dpi, five mice were processed for brain section staining with an α-NS1 antiserum. Shown are representative hippocampal slides (E).

Mentions: To identify a key point mutation(s) in three variants of SA14-14-2MCV that leads to the acquisition of neurovirulence, we generated eight derivatives of SA14-14-2MCV, each containing one of the eight point mutations found in our three variants, by cloning them individually into the infectious SA14-14-2 cDNA and transfecting the synthetic RNAs derived from each mutant cDNA into BHK-21 cells. In all cases, the mutant RNA was as infectious as the parent RNA, with a specific infectivity of 6.4–8.3×105 PFU/µg; the sizes of the foci/plaques produced by each mutant RNA were indistinguishable from those generated by the parent RNA, paralleling their levels of virus production, with an average yield of 2.1–4.5×105 PFU/ml at 22 hpt (Fig. 4A). In agreement with these results, no difference was observed in the profile or expression level of the viral proteins, i.e., three structural (C, prM, and E) and one nonstructural (NS1), as determined by immunoblotting of RNA-transfected cells at 18 hpt (Fig. 4B). All the mutant viruses grew as efficiently as did the parental virus over the course of 96 h after infection at an MOI of 0.1 in BHK-21 cells (Fig. 4C). Thus, there was no apparent effect of any of the eight introduced genetic changes on virus replication.


A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Yun SI, Song BH, Kim JK, Yun GN, Lee EY, Li L, Kuhn RJ, Rossmann MG, Morrey JD, Lee YM - PLoS Pathog. (2014)

Discovery of a single locus that leads to the reversion of SA14-14-2MCV to lethal neurovirulence.(A-C) In vitro replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or each mutant cDNA, as indicated. RNA infectivity (in PFU/µg) at 4 dpt was determined by infectious center assay, combined with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was measured by plaque titration on BHK-21 cells (A); viral protein accumulation at 18 hpt was examined by immunoblotting of cell lysates with a panel of antibodies as indicated (B). For viral growth analysis, BHK-21 cells were infected at an MOI of 0.1 with the parent or each mutant virus obtained from the corresponding RNA-transfected cells. At the indicated time points, culture supernatants harvested for virus titration on BHK-21 cells (C). (D-E) In vivo neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were inoculated IC with doses of the virus stock serially diluted 10-fold, and the LD50 values were determined (D). For immunohistochemical staining, groups of the mice (n = 15 per group) were mock-infected or infected IC with 103 PFU of each virus. At 3, 5, and 7 dpi, five mice were processed for brain section staining with an α-NS1 antiserum. Shown are representative hippocampal slides (E).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4117607&req=5

ppat-1004290-g004: Discovery of a single locus that leads to the reversion of SA14-14-2MCV to lethal neurovirulence.(A-C) In vitro replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or each mutant cDNA, as indicated. RNA infectivity (in PFU/µg) at 4 dpt was determined by infectious center assay, combined with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was measured by plaque titration on BHK-21 cells (A); viral protein accumulation at 18 hpt was examined by immunoblotting of cell lysates with a panel of antibodies as indicated (B). For viral growth analysis, BHK-21 cells were infected at an MOI of 0.1 with the parent or each mutant virus obtained from the corresponding RNA-transfected cells. At the indicated time points, culture supernatants harvested for virus titration on BHK-21 cells (C). (D-E) In vivo neurovirulence. Groups of 3-week-old ICR mice (n = 10 per group) were inoculated IC with doses of the virus stock serially diluted 10-fold, and the LD50 values were determined (D). For immunohistochemical staining, groups of the mice (n = 15 per group) were mock-infected or infected IC with 103 PFU of each virus. At 3, 5, and 7 dpi, five mice were processed for brain section staining with an α-NS1 antiserum. Shown are representative hippocampal slides (E).
Mentions: To identify a key point mutation(s) in three variants of SA14-14-2MCV that leads to the acquisition of neurovirulence, we generated eight derivatives of SA14-14-2MCV, each containing one of the eight point mutations found in our three variants, by cloning them individually into the infectious SA14-14-2 cDNA and transfecting the synthetic RNAs derived from each mutant cDNA into BHK-21 cells. In all cases, the mutant RNA was as infectious as the parent RNA, with a specific infectivity of 6.4–8.3×105 PFU/µg; the sizes of the foci/plaques produced by each mutant RNA were indistinguishable from those generated by the parent RNA, paralleling their levels of virus production, with an average yield of 2.1–4.5×105 PFU/ml at 22 hpt (Fig. 4A). In agreement with these results, no difference was observed in the profile or expression level of the viral proteins, i.e., three structural (C, prM, and E) and one nonstructural (NS1), as determined by immunoblotting of RNA-transfected cells at 18 hpt (Fig. 4B). All the mutant viruses grew as efficiently as did the parental virus over the course of 96 h after infection at an MOI of 0.1 in BHK-21 cells (Fig. 4C). Thus, there was no apparent effect of any of the eight introduced genetic changes on virus replication.

Bottom Line: Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain.In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication.Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.

ABSTRACT
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

Show MeSH
Related in: MedlinePlus