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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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Characterization of biological properties of the molecularly cloned virus SA14-14-2MCVin vitro and in vivo.(A-D) BHK-21 cells were mock-infected or infected at an MOI of 1 with the molecularly cloned virus (SA14-14-2MCV) or the original parental virus (SA14-14-2). At the time points indicated, cells were lysed to analyze the accumulation levels of viral genomic RNA by Northern blotting (A) and viral proteins by immunoblotting (B), and culture supernatants were harvested to examine the production levels of progeny virions by plaque titration on BHK-21 cells (C). At 4 dpi, cell monolayers were first immunostained with a mouse α-JEV antiserum to visualize the infectious foci, and the same monolayers were then restained with crystal violet to observe the infectious plaques (D). The average plaque sizes (mean ± SD) were estimated by counting 10 representative plaques. (E) Groups of 3-week-old ICR mice (n = 20 per group) were infected IC, IM, or IP with serial 10-fold dilutions of each virus as indicated. The LD50 values (in PFU) were calculated by the Reed and Muench method [119]. CNU/LP2, a virulent JEV strain used as a reference.
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ppat-1004290-g002: Characterization of biological properties of the molecularly cloned virus SA14-14-2MCVin vitro and in vivo.(A-D) BHK-21 cells were mock-infected or infected at an MOI of 1 with the molecularly cloned virus (SA14-14-2MCV) or the original parental virus (SA14-14-2). At the time points indicated, cells were lysed to analyze the accumulation levels of viral genomic RNA by Northern blotting (A) and viral proteins by immunoblotting (B), and culture supernatants were harvested to examine the production levels of progeny virions by plaque titration on BHK-21 cells (C). At 4 dpi, cell monolayers were first immunostained with a mouse α-JEV antiserum to visualize the infectious foci, and the same monolayers were then restained with crystal violet to observe the infectious plaques (D). The average plaque sizes (mean ± SD) were estimated by counting 10 representative plaques. (E) Groups of 3-week-old ICR mice (n = 20 per group) were infected IC, IM, or IP with serial 10-fold dilutions of each virus as indicated. The LD50 values (in PFU) were calculated by the Reed and Muench method [119]. CNU/LP2, a virulent JEV strain used as a reference.

Mentions: In cell cultures [75], [76], we assessed the in vitro growth properties of the molecularly cloned virus (SA14-14-2MCV) rescued from the infectious cDNA, as compared to those of the uncloned parental virus (SA14-14-2) used for cDNA construction. In hamster kidney BHK-21 cells, which are used most frequently for JEV propagation in laboratories, SA14-14-2MCV replicated as efficiently as SA14-14-2, with no noticeable difference in the accumulation of viral genomic RNA (Fig. 2A) and proteins (Fig. 2B) over the first 24 h after infection at a multiplicity of infection (MOI) of 1 plaque-forming unit (PFU) per cell. These observations were consistent with their growth kinetics, which were essentially identical for 4 days following infection at three different MOIs: 0.1, 1, and 10 PFU/cell (Fig. 2C and data not shown). Similarly, there was no difference in focus/plaque morphology between SA14-14-2MCV and SA14-14-2 at 4 days post-infection (dpi) (Fig. 2D); as expected, their foci/plaques were ∼30% smaller than those produced by CNU/LP2, a virulent JEV strain used as a reference (Fig. S1). Also, their growth properties were equivalent in two other cell lines, human neuroblastoma SH-SY5Y and mosquito C6/36 cells, which are potentially relevant to JEV pathogenesis and transmission, respectively (Fig. S2). These data suggest that the uncloned parental and molecularly cloned viruses are indistinguishable in viral replication and spread in both mammalian and insect cells.


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)

Characterization of biological properties of the molecularly cloned virus SA14-14-2MCVin vitro and in vivo.(A-D) BHK-21 cells were mock-infected or infected at an MOI of 1 with the molecularly cloned virus (SA14-14-2MCV) or the original parental virus (SA14-14-2). At the time points indicated, cells were lysed to analyze the accumulation levels of viral genomic RNA by Northern blotting (A) and viral proteins by immunoblotting (B), and culture supernatants were harvested to examine the production levels of progeny virions by plaque titration on BHK-21 cells (C). At 4 dpi, cell monolayers were first immunostained with a mouse α-JEV antiserum to visualize the infectious foci, and the same monolayers were then restained with crystal violet to observe the infectious plaques (D). The average plaque sizes (mean ± SD) were estimated by counting 10 representative plaques. (E) Groups of 3-week-old ICR mice (n = 20 per group) were infected IC, IM, or IP with serial 10-fold dilutions of each virus as indicated. The LD50 values (in PFU) were calculated by the Reed and Muench method [119]. CNU/LP2, a virulent JEV strain used as a reference.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004290-g002: Characterization of biological properties of the molecularly cloned virus SA14-14-2MCVin vitro and in vivo.(A-D) BHK-21 cells were mock-infected or infected at an MOI of 1 with the molecularly cloned virus (SA14-14-2MCV) or the original parental virus (SA14-14-2). At the time points indicated, cells were lysed to analyze the accumulation levels of viral genomic RNA by Northern blotting (A) and viral proteins by immunoblotting (B), and culture supernatants were harvested to examine the production levels of progeny virions by plaque titration on BHK-21 cells (C). At 4 dpi, cell monolayers were first immunostained with a mouse α-JEV antiserum to visualize the infectious foci, and the same monolayers were then restained with crystal violet to observe the infectious plaques (D). The average plaque sizes (mean ± SD) were estimated by counting 10 representative plaques. (E) Groups of 3-week-old ICR mice (n = 20 per group) were infected IC, IM, or IP with serial 10-fold dilutions of each virus as indicated. The LD50 values (in PFU) were calculated by the Reed and Muench method [119]. CNU/LP2, a virulent JEV strain used as a reference.
Mentions: In cell cultures [75], [76], we assessed the in vitro growth properties of the molecularly cloned virus (SA14-14-2MCV) rescued from the infectious cDNA, as compared to those of the uncloned parental virus (SA14-14-2) used for cDNA construction. In hamster kidney BHK-21 cells, which are used most frequently for JEV propagation in laboratories, SA14-14-2MCV replicated as efficiently as SA14-14-2, with no noticeable difference in the accumulation of viral genomic RNA (Fig. 2A) and proteins (Fig. 2B) over the first 24 h after infection at a multiplicity of infection (MOI) of 1 plaque-forming unit (PFU) per cell. These observations were consistent with their growth kinetics, which were essentially identical for 4 days following infection at three different MOIs: 0.1, 1, and 10 PFU/cell (Fig. 2C and data not shown). Similarly, there was no difference in focus/plaque morphology between SA14-14-2MCV and SA14-14-2 at 4 days post-infection (dpi) (Fig. 2D); as expected, their foci/plaques were ∼30% smaller than those produced by CNU/LP2, a virulent JEV strain used as a reference (Fig. S1). Also, their growth properties were equivalent in two other cell lines, human neuroblastoma SH-SY5Y and mosquito C6/36 cells, which are potentially relevant to JEV pathogenesis and transmission, respectively (Fig. S2). These data suggest that the uncloned parental and molecularly cloned viruses are indistinguishable in viral replication and spread in both mammalian and insect cells.

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