scholarly journals 3′UTR SL-IV and DB1 Regions Contribute to Japanese Encephalitis Virus Replication and Pathogenicity

2021 ◽  
Vol 8 ◽  
Author(s):  
Jinchao Xing ◽  
Youyue Zhang ◽  
Ziying Lin ◽  
Lele Liu ◽  
Qiang Xu ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Rna Secondary Structure ◽  
Rational Design ◽  
Case Fatality ◽  
Encephalitis Virus ◽  
Reverse Genetic System ◽  
Emerging Pathogens

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 is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3′ untranslated region (3′UTR). The 3′UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3′UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3′UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3′UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3′UTR of rSA14-3′UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, in vivo mouse experiments illustrated that the rSA14-3′UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3′UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.

scholarly journals Potential Role of Birds in Japanese Encephalitis Virus Zoonotic Transmission and Genotype Shift

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 357
Author(s):  
Muddassar Hameed ◽  
Abdul Wahaab ◽  
Mohsin Nawaz ◽  
Sawar Khan ◽  
Jawad Nazir ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Zoonotic Transmission ◽  
In Vivo Studies ◽  
Genotype I ◽  
Genotype Iii

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

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.

scholarly journals Fusion PCR generated Japanese encephalitis virus/dengue 4 virus chimera exhibits lack of neuroinvasiveness, attenuated neurovirulence, and a dual-flavi immune response in mice

2004 ◽  
Vol 85 (9) ◽  
pp. 2503-2513 ◽  
Author(s):  
Edward Gitau Matumbi Mathenge ◽  
Maria del Carmen Parquet ◽  
Yasutomo Funakoshi ◽  
Seiji Houhara ◽  
Pooi Fong Wong ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Line ◽  
Japanese Encephalitis ◽  
Secondary Infection ◽  
Neuronal Cell ◽  
Protein S ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Rapid Preparation

The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3·17×104 f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100×LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210 000 and 37 000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.

A tripeptide (NSK) inhibits Japanese encephalitis virus infection in vitro and in vivo

2013 ◽  
Vol 159 (5) ◽  
pp. 1045-1055 ◽  
Author(s):  
Chen Li ◽  
Ling-ling Ge ◽  
Ya-ling Yu ◽  
Li Huang ◽  
Yue Wang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Japanese Encephalitis Virus Infection

scholarly journals A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

2015 ◽  
Vol 90 (5) ◽  
pp. 2676-2689 ◽  
Author(s):  
Mélissanne de Wispelaere ◽  
Cécile Khou ◽  
Marie-Pascale Frenkiel ◽  
Philippe Desprès ◽  
Nathalie Pardigon
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Encephalitis Virus ◽  
Mutant Virus ◽  
M Protein ◽  
Single Amino Acid ◽  
Progeny Virus

ABSTRACTJapanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuationin vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virusin vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts.IMPORTANCEJEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

scholarly journals Humanized Monoclonal Antibodies Derived from Chimpanzee Fabs Protect against Japanese Encephalitis Virus In Vitro and In Vivo

2008 ◽  
Vol 82 (14) ◽  
pp. 7009-7021 ◽  
Author(s):  
Ana P. Goncalvez ◽  
Cheng-Hsin Chien ◽  
Kamolchanok Tubthong ◽  
Inna Gorshkova ◽  
Carrie Roll ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Neutralizing Antibodies ◽  
Therapeutic Potential ◽  
Encephalitis Virus ◽  
Domain Iii ◽  
Humanized Monoclonal Antibodies

ABSTRACT Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys179 (domain I), that for Fab B2 was Ile126 (domain II), and that for Fab E3 was Gly302 (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED50) of 0.84 μg, followed by MAb A3 (ED50 of 5.8 μg) and then MAb E3 (ED50 of 24.7 μg) for a 4-week-old mouse. Administration of 200 μg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.

scholarly journals Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

2014 ◽  
Vol 95 (4) ◽  
pp. 806-815 ◽  
Author(s):  
Xiao-Dan Li ◽  
Xiao-Feng Li ◽  
Han-Qing Ye ◽  
Cheng-Lin Deng ◽  
Qing Ye ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cdna Clone ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Full Length ◽  
Large Plaque ◽  
Adaptive Mutations ◽  
A Genome

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

scholarly journals Incidence of Japanese encephalitis virus among patients presenting with acute encephalitis syndrome in a tertiary care hospital, West Bengal, India

2021 ◽  
Vol 8 (12) ◽  
pp. 5947
Author(s):  
Arup Roy ◽  
Poulami Saha ◽  
Asraful Islam ◽  
Rajdeep Saha ◽  
Abhishek Sengupta ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
West Bengal ◽  
Tertiary Care ◽  
Case Fatality ◽  
Encephalitis Virus ◽  
Care Hospital ◽  
Serum Samples ◽  
Acute Encephalitis Syndrome ◽  
Acute Encephalitis

Background: Japanese encephalitis virus (JEV) is a flavivirus related to dengue, yellow fever and West Nile viruses, and is spread by mosquitoes. JEV is the main cause of viral encephalitis in many countries of Asia with an estimated 68000 clinical cases every year. Although symptomatic Japanese encephalitis (JE) is rare, the case-fatality rate among those with encephalitis can be as high as 30%. Permanent neurologic or psychiatric sequelae can occur in 30-50% of those with encephalitis. This study was conducted to find the incidence of JEV IgM in patients presenting with acute encephalitis syndrome (AES) in Raiganj govt. medical college and hospital, Uttar Dinajpur, West Bengal, India.Methods: Blood and CSF samples were collected from patients presenting with AES. IgM antibody capture ELISA was performed on the CSF and serum samples by JE virus MAC ELISA kit.Results: The overall prevalence of JEV IgM was 13.8%. Among the positive cases male comprised of 71.4% and female 28.5%. The most common age group affected was above 16 years of age. JEV prevalence was present throughout the year with high number of cases between the monsoon and post monsoon seasons.Conclusions: This study demonstrates the endemicity of JEV in Uttar Dinajpur district of West Bengal, India. As most of the JE cases are asymptomatic strengthening the existing surveillance system is required to find out the actual scenario of JEV in West Bengal. Control of vectors, early diagnosis and treatment, vaccinations are the key to decrease the morbidity and mortality caused by JEV. 

The NS3 and NS4A genes as the targets of RNA interference inhibit replication of Japanese encephalitis virus in vitro and in vivo

Gene ◽  
2016 ◽  
Vol 594 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Lei Yuan ◽  
Rui Wu ◽  
Hanyang Liu ◽  
Xintian Wen ◽  
Xiaobo Huang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus
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