scholarly journals Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice

2015 ◽  
Vol 89 (12) ◽  
pp. 6328-6337 ◽  
Author(s):  
Gregory D. Gromowski ◽  
Cai-Yen Firestone ◽  
Stephen S. Whitehead
Keyword(s):  
Tissue Culture ◽  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Vaccine Strain ◽  
Japanese Encephalitis ◽  
Second Generation ◽  
Virus Genome ◽  
Vaccine Virus ◽  
Wild Type ◽  
Virulence Attenuation

ABSTRACTThe safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5′ UTR (5′-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5′-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety.IMPORTANCEThe live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

scholarly journals Chimeric Japanese Encephalitis Virus SA14/SA14-14-2 Was Virulence Attenuated and Protected the Challenge of Wild-Type Strain SA14

2019 ◽  
Vol 2019 ◽  
pp. 1-7
Author(s):  
Rong Huang ◽  
Shengling Leng ◽  
Yalan Feng ◽  
Liping Tang ◽  
Lei Yuan ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Vaccine Strain ◽  
Japanese Encephalitis ◽  
Molecular Mechanisms ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Chimeric Virus ◽  
Dependent Manner ◽  
Wild Type ◽  
Dose Dependent

The attenuated Japanese encephalitis virus (JEV) live vaccine SA14-14-2 prepared from wild-type (WT) strain SA14 was licensed to prevent Japanese encephalitis (JE) in 1989 in China. Many studies showed that the premembrane (prM) and envelope (E) protein were the crucial determinant of virulence and immunogenicity of JEV. So we are interested in whether the substitution of prM/E of JEV WT SA14 with those of vaccine strain SA14-14-2 could decrease neurovirulence and prevent the challenge of JEV WT SA14. Molecular clone technique was used to replace the prM/E gene of JEV WT strain SA14 with those of vaccine strain SA14-14-2 to construct the infectious clone of chimeric virus (designated JEV SA14/SA14-14-2), the chimeric virus recovered from BHK21 cells upon electrotransfection of RNA into BHK21 cells. The results showed that the recovered chimeric virus was highly attenuated in mice, and a single immunization elicited strong protective immunity in a dose-dependent manner. This study increases our understanding of the molecular mechanisms of neurovirulence attenuation and immunogenicity of JEV.

scholarly journals Molecular mechanisms underlying attenuation of live attenuated Japanese encephalitis virus vaccine SA14-14-2

2021 ◽  
Author(s):  
Pooja Hoovina Venkatesh ◽  
Saurabh Kumar ◽  
Naveen Kumar ◽  
Krishna Chaitanya ◽  
Lance Turtle ◽  
...  
Keyword(s):  
Immune Responses ◽  
Er Stress ◽  
Japanese Encephalitis Virus ◽  
Vaccine Strain ◽  
Japanese Encephalitis ◽  
Virus Vaccine ◽  
Envelope Glycoprotein ◽  
Nonstructural Protein ◽  
Wild Type ◽  
Virus Particles

ABSTRACTThe live attenuated Japanese encephalitis virus vaccine SA14-14-2 demonstrated ≥ 95 % efficacy and is today the vaccine of choice against JEV globally. Relative to its parent strain SA14, SA14-14-2 carries 46 nucleotide and 24 amino acid alterations, with 8 of the latter located within the envelope glycoprotein. The vaccine strain also fails to synthesize the nonstructural protein NS1’ owing to a silent mutation that abrogates a-1-frameshifting event close to the 5’ end of the NS2A coding sequence. Previous studies employing reverse genetics and mouse models implicated both absence of NS1’ and mutated E, in attenuation of SA14-14-2. We demonstrate progressive reduction in ER stress sensor PERK levels and increased expression of CEBP-homologous protein (CHOP), accompanied by dephosphorylation of eIF2α, inhibition of autophagy maturation and necroptosis following infection of cultured cells with wild-type JEV strain P20778. Autonomous expression of NS1’ caused constitutive up-regulation of CHOP and loss of PERK. Conversely, infection with SA14-14-2 led to significantly increased IRE-1α activation, ER chaperone levels and autophagy. We report labile conformational epitopes accompanied by drastically reduced folding kinetics of intracellular SA14-14-2 envelope protein engendered by sluggish oxidation of cysteine sulfhydryl groups to form disulfide bonds within the endoplasmic reticulum along with altered envelope epitopes in extracellular SA14-14-2 viral particles. We also demonstrate near total conversion of prM to pr and M in SA14-14-2 virus particles. These alterations were accompanied by enhanced activation of mouse and human antigen presenting cells by SA14-14-2 along with superior CD8+ recall T cell responses to viral structural proteins in volunteers vaccinated with SA14-14-2.Author SummaryThe random process of cell culture passage adopted in generation of most live attenuated virus vaccines leads to fixation of multiple nucleotide changes in their genomes and renders it difficult if not impossible to pinpoint those mutations primarily responsible for their attenuated phenotype. Identifying the precise attenuating mutations and their modi operandi should aid in developing rationally attenuated vaccines for other viruses. We discovered that wild type (WT) JEV uses the nonstructural protein NS1’ to take over the host protein synthesis machinery to produce viral proteins. Loss of NS1’ in SA14-14-2 deprives the vaccine strain of this ability. Viruses uniformly target host death pathways to avoid generating potent antiviral immune responses. WT JEV prevents autophagy maturation. Conversely the SA14-14-2 vaccine activates autophagy due to unresolved ER stress caused by inability of its envelope glycoprotein to fold promptly post synthesis. Combined with enhanced proteolytic cleavage of the viral prM protein in SA14-14-2, this resulted in altered envelope epitopes on extracellular SA14-14-2 virus particles. These changes culminated in enhanced activation of innate and adaptive immune responses by SA14-14-2.

scholarly journals Phenotypic and Genotypic Comparison of a Live-Attenuated Genotype I Japanese Encephalitis Virus SD12-F120 Strain with Its Virulent Parental SD12 Strain

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 552 ◽  
Author(s):  
Muhammad Naveed Anwar ◽  
Xin Wang ◽  
Muddassar Hameed ◽  
Abdul Wahaab ◽  
Chenxi Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Clinical Symptoms ◽  
Encephalitis Virus ◽  
Amino Acid Substitutions ◽  
Genotype I ◽  
Genetic Changes ◽  
Genotypic Characteristics ◽  
Insight Into

The phenotypic and genotypic characteristics of a live-attenuated genotype I (GI) strain (SD12-F120) of Japanese encephalitis virus (JEV) were compared with its virulent parental SD12 strain to gain an insight into the genetic changes acquired during the attenuation process. SD12-F120 formed smaller plaque on BHK-21 cells and showed reduced replication in mouse brains compared with SD12. Mice inoculated with SD12-F120 via either intraperitoneal or intracerebral route showed no clinical symptoms, indicating a highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. SD12-F120 harbored 29 nucleotide variations compared with SD12, of which 20 were considered silent nucleotide mutations, while nine resulted in eight amino acid substitutions. Comparison of the amino acid variations of SD12-F120 vs. SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the variations at E138 and E176 positions of E protein were identified in four and three pairs, respectively, while the remaining amino acid variations were almost unique to their respective strain pairs. These observations suggest that the genetic changes acquired during the attenuation process were likely to be strain-specific and that the mechanisms associated with JEV attenuation/virulence are complicated.

scholarly journals Amino Acid at Position 166 of NS2A in Japanese Encephalitis Virus (JEV) Is Associated with In Vitro Growth Characteristics of JEV

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 709
Author(s):  
Shigeru Tajima ◽  
Satoshi Taniguchi ◽  
Eri Nakayama ◽  
Takahiro Maeki ◽  
Takuya Inagaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Neuroblastoma Cells ◽  
Encephalitis Virus ◽  
Growth Potential ◽  
Sequencing Analysis ◽  
Murine Neuroblastoma ◽  
Growth Ability

We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2AMuar) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A166) could rescue the low replication ability of NS1-3Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A166 (NS2AY166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2AMuar-based mutant virus, NS2AMuar-H166Y, showed a higher growth ability than NS2AMuar in Neuro-2a cells. Thus, these results indicate that the NS2A166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.

scholarly journals Comparison of genomic and amino acid sequences of eight Japanese encephalitis virus isolates from bats

2013 ◽  
Vol 158 (12) ◽  
pp. 2543-2552 ◽  
Author(s):  
Shan Liu ◽  
Xing Li ◽  
Zhiyong Chen ◽  
Yixiong Chen ◽  
Qionghua Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Amino Acid Sequences ◽  
Encephalitis Virus ◽  
Virus Isolates

scholarly journals Towards Obtaining a Nanoscale Structure of Terminal Regions of japanese Encephalitis Virus Genome

2019 ◽  
Vol 116 (3) ◽  
pp. 354a
Author(s):  
Tyler Mrozowich ◽  
Vanessa Meier-Stephen ◽  
Justin Vigar ◽  
Astha ◽  
Janusz M. Bujnicki ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Genome ◽  
Encephalitis Virus ◽  
Nanoscale Structure ◽  
Japanese Encephalitis Virus Genome

scholarly journals A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus PropagationIn Vitrobut NotIn Vivo

2015 ◽  
Vol 89 (11) ◽  
pp. 6126-6130 ◽  
Author(s):  
Yuki Takamatsu ◽  
Kouichi Morita ◽  
Daisuke Hayasaka
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Single Amino Acid ◽  
Virus Propagation ◽  
Viral Loads ◽  
Unique Amino Acid

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagationin vitromay not reflect the level of virus neuroinvasivenessin vivo.

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