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1.
J Vector Borne Dis ; 56(2): 105-110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31397385

RESUMO

Background & objectives: Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. Methods: Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. Results: The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. Interpretation & conclusion: The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.


Assuntos
Anticorpos Antivirais/sangue , Encefalite Japonesa/imunologia , Testes de Fixação do Látex/normas , Proteínas não Estruturais Virais/genética , Zoonoses/diagnóstico , Animais , Vírus da Encefalite Japonesa (Espécie)/imunologia , Índia , Testes de Fixação do Látex/veterinária , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Suínos , Proteínas não Estruturais Virais/imunologia , Zoonoses/imunologia , Zoonoses/virologia
2.
Biologicals ; 60: 36-41, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31178277

RESUMO

JE vaccination is the most effective and economical method of preventing JE. A live attenuated JE vaccine has been widely used in many countries since 1989, playing an important role in controlling JE outbreaks. However, whether the large-scale use of the live attenuated JE vaccine will lead to the dissemination of the vaccine virus in the environment and whether reversion of the neuroattenuation of the virus will occur during the transmission process remain major concerns for some researchers. To evaluate the transmission of a live attenuated JEV vaccine in mosquitoes and hosts, JE SA14-14-2 attenuated vaccine virus was intrathoracically (i.t.) inoculated into Culex tritaeniorhynchus, a native vector. Subsequently, virus harvested from inoculated mosquitoes was inoculated into pigs, a mammalian reservoir. The virus was isolated from the pigs and passaged once again in Culex tritaeniorhynchus. The genome sequences and virulence of the passaged viruses were then investigated. While a few nucleotide substitutions occurred during passaging, there was no change in the encoded amino acids. After intracerebral (i.c.) inoculation of mice with the vaccine, no pathological effects were observed. In addition, virus virulence remained low after inoculation of suckling mouse brains. These results indicate that vaccination of individuals with the live vaccine will not result in transmission of the live SA14-14-2 vaccine virus through mosquito biting and virus amplified in pigs.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa , Vacinas contra Encefalite Japonesa/imunologia , Animais , Linhagem Celular , Cricetinae , Culex/imunologia , Culex/virologia , Encefalite Japonesa/imunologia , Encefalite Japonesa/prevenção & controle , Feminino , Vacinas contra Encefalite Japonesa/efeitos adversos , Masculino , Camundongos , Suínos , Vacinas Atenuadas
3.
Virol Sin ; 34(5): 538-548, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31215000

RESUMO

Japanese encephalitis (JE) is a viral encephalitis disease caused by Japanese encephalitis virus (JEV) infection. Uncontrolled inflammatory responses in the central nervous system (CNS) are a hallmark of severe JE. Although the CCR2-CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8+ T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection (i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8+ T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8+ T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells (CD44+CD62L-). JEV infection dramatically enhanced the expression of programmed death 1 (PD-1) on infiltrating CD8+ T cells in the brain, as compared to that on peripheral CD8+ T cells in the spleen. This effect was more pronounced on infiltrating CCR2+CD8+ T cells than on CCR2-CD8+ T cells. In conclusion, we identified a new subset of CD8+ T cells (PD1+CCR2+CD8+ T cells) present in the CNS of mice during acute JEV infection. These CD8+ T cells might play a role in JE pathogenesis.


Assuntos
Encéfalo/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular , Encefalite Japonesa/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptores CCR2/imunologia , Doença Aguda , Animais , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Vírus da Encefalite Japonesa (Espécie) , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética , Receptores CCR2/genética , Subpopulações de Linfócitos T/imunologia , Regulação para Cima
4.
Arch Virol ; 164(6): 1535-1542, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30900070

RESUMO

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by Culex mosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 µg/mL) and mAb working concentration (1 µg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/diagnóstico , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/sangue , Anticorpos Antivirais/metabolismo , Clonagem Molecular , Encefalite Japonesa/imunologia , Encefalite Japonesa/veterinária , Feminino , Soroconversão , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/imunologia , Vacinação , Células Vero , Proteínas não Estruturais Virais/genética
5.
Nat Commun ; 10(1): 706, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30742008

RESUMO

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.


Assuntos
Quimases/metabolismo , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/imunologia , Encefalite Japonesa/metabolismo , Mastócitos/metabolismo , Mastócitos/virologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/virologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Quimases/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite Japonesa/mortalidade , Humanos , Imunidade Inata , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Morbidade , Permeabilidade , Análise de Sobrevida , Proteínas de Junções Íntimas
6.
Jpn J Infect Dis ; 72(2): 115-117, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30381682

RESUMO

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus da Encefalite Japonesa (Subgrupo)/imunologia , Encefalite Japonesa/imunologia , Genótipo , Soro/imunologia , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático , Criança , Pré-Escolar , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/imunologia , Doenças Transmissíveis Emergentes/virologia , Vírus da Encefalite Japonesa (Subgrupo)/classificação , Vírus da Encefalite Japonesa (Subgrupo)/genética , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/virologia , Feminino , Humanos , Masculino , Testes de Neutralização , Vietnã/epidemiologia , Adulto Jovem
7.
J Infect Dis ; 219(3): 375-381, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30165664

RESUMO

Background: Japanese encephalitis virus (JEV) is a zoonotic, mosquito-borne flavivirus, distributed across Asia. Infections are mostly mild or asymptomatic, but symptoms include neurological disorders, sequelae, and fatalities. Data to inform control strategies are limited due to incomplete case reporting. Methods: We used JEV serological data from a multicountry Asian dengue vaccine study in children aged 2-14 years to describe JEV endemicity, measuring antibodies by plaque reduction neutralization test (PRNT50). Results: A total 1479 unvaccinated subjects were included. A minimal estimate of pediatric JEV seroprevalence in dengue-naive individuals was 8.1% in Indonesia, 5.8% in Malaysia, 10.8% in the Philippines, and 30.7% in Vietnam, translating to annual infection risks varying from 0.8% (in Malaysia) to 5.2% (in Vietnam). JEV seroprevalence and annual infection estimates were much higher in children with history of dengue infection, indicating cross-neutralization within the JEV PRNT50 assay. Conclusions: These data confirm JEV transmission across predominantly urban areas and support a greater emphasis on JEV case finding, diagnosis, and prevention.


Assuntos
Dengue/epidemiologia , Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/imunologia , Adolescente , Ásia/epidemiologia , Criança , Pré-Escolar , Vacinas contra Dengue , Vírus da Dengue/imunologia , Humanos , Indonésia/epidemiologia , Malásia/epidemiologia , Testes de Neutralização , Filipinas/epidemiologia , Prevalência , Estudos Soroepidemiológicos , Vietnã/epidemiologia
8.
Viral Immunol ; 32(1): 68-74, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30585774

RESUMO

Japanese encephalitis (JE) is a vector-borne viral disease with clinical manifestations ranging from asymptomatic to severe neurological symptoms and even leading to death. The exact pathophysiology for diverse clinical spectrum of the disease is complex and has not yet been defined. Studies have postulated that during JE infection, inflammatory cytokines and chemokines are produced after the initial recognition of viral antigens through the engagement of toll-like receptors (TLR) pathways. However, there is paucity of knowledge on the expression levels of chemokines and TLRs among mild and severely affected JE patients. Hence, to better understand disease pathogenesis, we examined the mRNA expression of chemokines, CCL2 and CCL5, and their respective receptors CCR2 and CCR5 along with TLRs viz. TLR3, TLR7, TLR8, and TLR9 in context of mild and severely Japanese encephalitis virus (JEV)-infected (n = 19) and healthy (n = 19) individuals. Our study showed significant downregulation of CCL2, CCL5, CCR2, CCR5, and TLR3 by log 0.87, 1.02, 0.82, 0.68, and 0.37-fold respectively, among mild cases compared with controls. Significant difference of gene expression among mild and severe JE cases for CCL2 (p < 0.001), CCL5 (p < 0.01), and TLR7 (p < 0.05) was observed. In conclusion, our results proposes that chemokines viz. CCL2 and CCL5 along with TLR7 may be associated with degree of pathogenesis of JE and could be putative therapeutic targets for preventing severe inflammation during viral encephalitis.


Assuntos
Quimiocinas/genética , Encefalite Japonesa/imunologia , Expressão Gênica , Receptores Toll-Like/genética , Adolescente , Adulto , Idoso , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocinas/imunologia , Criança , Regulação para Baixo , Encefalite Japonesa/patologia , Feminino , Humanos , Índia , Inflamação , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Adulto Jovem
9.
Virol Sin ; 33(6): 515-523, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30542978

RESUMO

Japanese encephalitis virus (JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a 52-amino acid C-terminal extension of NS1, is generated with a -1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected (rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus (pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type I interferon (IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-ß and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/imunologia , Interferon beta/antagonistas & inibidores , Proteínas não Estruturais Virais/imunologia , Células A549 , Animais , Linhagem Celular , Cricetinae , Feminino , Células HeLa , Humanos , Evasão da Resposta Imune , Interferon beta/imunologia , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Células Vero , Virulência , Replicação Viral
10.
J Virol ; 92(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30282716

RESUMO

The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCE JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/veterinária , Mucosa Nasal/virologia , Doenças dos Suínos/virologia , Animais , Células Cultivadas , Quimiocinas/metabolismo , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Células Epiteliais/citologia , Mosquitos Vetores/virologia , Mucosa Nasal/citologia , Mucosa Nasal/imunologia , Suínos , Doenças dos Suínos/imunologia , Internalização do Vírus , Replicação Viral , Eliminação de Partículas Virais
11.
Ann Neurol ; 84(3): 386-400, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30246904

RESUMO

OBJECTIVE: The incidence of childhood onset myasthenia gravis (CMG) in China is higher than that in other countries; however, the reasons for this are unclear. METHODS: We investigated the clinical and immunological profiles of CMG, and assessed the potential precipitating factors. For the mouse studies, the possible implication of vaccination in the pathogenesis was explored. RESULTS: In our retrospective study, 51.22% of the 4,219 cases of myasthenia gravis (MG) were of the childhood onset type. The cohort study uncovered that the pathophysiology of CMG was mediated by immune deviation, rather than through gene mutations or virus infections. The administration of the live-attenuated Japanese encephalitis vaccine (LA-JEV), but not the inactivated vaccine or other vaccines, in mice induced serum acetylcholine receptor (AChR) antibody production, reduced the AChR density at the endplates, and decreased both muscle strength and response to repetitive nerve stimulation. We found a peptide (containing 7 amino acids) of LA-JEV similar to the AChR-α subunit, and immunization with a synthesized protein containing this peptide reproduced the MG-like phenotype in mice. INTERPRETATION: Our results describe the immunological profile of CMG. Immunization with LA-JEV induced an autoimmune reaction against the AChR through molecular mimicry. These findings might explain the higher occurrence rate of CMG in China, where children are routinely vaccinated with LA-JEV, compared with that in countries, where this vaccination is not as common. Efforts should be made to optimize immunization strategies and reduce the risk for developing autoimmune disorders among children. Ann Neurol 2018;84:386-400.


Assuntos
Encefalite Japonesa/etiologia , Miastenia Gravis/virologia , Vacinação/efeitos adversos , Vacinas Atenuadas/imunologia , Animais , Anticorpos Antivirais/sangue , Criança , Estudos de Coortes , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Humanos , Camundongos , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-30042931

RESUMO

The major structural envelope (E) protein of Japanese encephalitis virus (JEV) facilitates cellular binding/entry and is the primary target of neutralizing antibodies. JEV E protein has one N-linked glycosylation site at N154 (G2 site), but the related dengue virus E protein has two glycosylation sites at N67 (G1 site) and N153 (G2 site). We generated three recombinant JEVs with different glycosylation patterns on the E protein. As compared with wild-type (WT) JEV with G2 glycosylation, viral growth in culture cells as well as neurovirulence and neuroinvasiveness in challenged mice were reduced when infected with the G1 mutant (E-D67N/N154A) with glycosylation shifted to G1 site, and the G0 mutant (E-N154A) with non-glycosylation. The G1G2 mutant (E-D67N), with E-glycosylation on both G1 and G2 sites, showed potent in vitro viral replication and in vivo neurovirulence, but reduced neuroinvasiveness. Furthermore, the JEV mutants with G1 glycosylation showed enhanced DC-SIGN binding, which may then lead to reduced brain invasion and explain the reason why WT JEV is devoid of this G1 site of glycosylation. Overall, the patterns of N-linked glycosylation on JEV E proteins may affect viral interaction with cellular lectins and contribute to viral replication and pathogenesis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Encefalite Japonesa/patologia , Glicosilação , Camundongos , Ligação Proteica , Virulência , Replicação Viral
13.
PLoS Pathog ; 14(7): e1007166, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30016363

RESUMO

CCCH-type zinc-finger antiviral protein (ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP (isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus (JEV), dengue virus (DENV) and Zika virus (ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3'-5' RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3'-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Proteínas de Ligação a RNA/imunologia , Humanos
14.
Jpn J Infect Dis ; 71(5): 360-364, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-29962489

RESUMO

Japanese encephalitis (JE) is an acute viral disease caused by the Japanese encephalitis virus (JEV). JEV strains are classified into 5 genotypes (I-V). JEV genotype V strains have never been detected in Japan to date, but they were recently detected in South Korea. In the present analysis, we tried to determine if a JEV genotype V strain caused any JE case in Japan in 2016. Serum and cerebrospinal fluid samples were collected from 10 JE patients reported in Japan in 2016. JEV RNA was not detected in any of the samples. Although JEV is a single-serotype virus, it can be expected that the neutralizing antibody titers against JEV genotype V strains are higher than those against genotype I and III strains in the serum of patients with JE in Japan whose causative JEV was the genotype V strain. The neutralizing antibody titers against the JEV genotype V strain were not higher than those against the genotype I or III strain in any serum samples. Therefore, the evidence that the JEV genotype V strain caused any JE case in Japan in 2016 was absent.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus da Encefalite Japonesa (Espécie)/classificação , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Genótipo , Adulto , Idoso , Idoso de 80 Anos ou mais , Vírus da Encefalite Japonesa (Espécie)/genética , Feminino , Humanos , Japão , Masculino , Testes de Neutralização , RNA Viral/líquido cefalorraquidiano
15.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29899084

RESUMO

Flaviviruses account for most arthropod-borne cases of human encephalitis in the world. However, the exact mechanisms of injury to the central nervous system (CNS) during flavivirus infections remain poorly understood. Microglia are the resident immune cells of the CNS and are important for multiple functions, including control of viral pathogenesis. Utilizing a pharmacologic method of microglia depletion (PLX5622 [Plexxikon Inc.], an inhibitor of colony-stimulating factor 1 receptor), we sought to determine the role of microglia in flaviviral pathogenesis. Depletion of microglia resulted in increased mortality and viral titer in the brain following infection with either West Nile virus (WNV) or Japanese encephalitis virus (JEV). Interestingly, microglial depletion did not prevent virus-induced increases in the expression of relevant cytokines and chemokines at the mRNA level. In fact, the expression of several proinflammatory genes was increased in virus-infected, microglia-depleted mice compared to virus-infected, untreated controls. In contrast, and as expected, expression of the macrophage marker triggering receptor expressed on myeloid cells 2 (TREM2) was decreased in virus-infected, PLX5622-treated mice compared to virus-infected controls.IMPORTANCE As CNS invasion by flaviviruses is a rare but life-threatening event, it is critical to understand how brain-resident immune cells elicit protection or injury during disease progression. Microglia have been shown to be important in viral clearance but may also contribute to CNS injury as part of the neuroinflammatory process. By utilizing a microglial depletion model, we can begin to parse out the exact roles of microglia during flaviviral pathogenesis with hopes of understanding specific mechanisms as potential targets for therapeutics.


Assuntos
Encéfalo/patologia , Encéfalo/virologia , Encefalite Japonesa/patologia , Microglia/imunologia , Carga Viral , Febre do Nilo Ocidental/patologia , Animais , Encéfalo/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Camundongos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Análise de Sobrevida , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/isolamento & purificação
16.
Cell Death Dis ; 9(7): 719, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915260

RESUMO

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) poses a serious threat to the world's public health yet without a cure. Certain JEV-infected neural cells express a subset of previously identified intrinsic antiviral interferon stimulated genes (ISGs), indicating brain cells retain autonomous antiviral immunity. However, whether this happens in composited brain remains unclear. Human pluripotent stem cell (hPSC)-derived organoids can model disorders caused by human endemic pathogens such as Zika virus, which may potentially address this question and facilitate the discovery of a cure for JE. We thus generated telencephalon organoid and infected them with JEV. We found JEV infection caused significant decline of cell proliferation and increase of cell death in brain organoid, resulting in smaller organoid spheres. JEV tended to infect astrocytes and neural progenitors, especially the population representing outer radial glial cells (oRGCs) of developing human brain. In addition, we revealed variable antiviral immunity in brain organoids of different stages of culture. In organoids of longer culture (older than 8 weeks), but not of early ones (less than 4 weeks), JEV infection caused typical activation of interferon signaling pathway. Preferential infection of oRGCs and differential antiviral response at various stages might explain the much more severe outcomes of JEV infection in the younger, which also provide clues to develop effective therapeutics of such diseases.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Organoides/imunologia , Telencéfalo/crescimento & desenvolvimento , Telencéfalo/imunologia , Imunidade Adaptativa/fisiologia , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/virologia , Células Cultivadas , Cricetinae , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Humanos , Neurogênese/fisiologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/virologia , Telencéfalo/citologia , Telencéfalo/virologia
17.
Pediatr Infect Dis J ; 37(9): e233-e240, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29912844

RESUMO

BACKGROUND: An inactivated Vero cell culture derived Japanese encephalitis virus vaccine (IXIARO) requires a booster dose 1 year after primary schedule for long-term antibody persistence in adults. The aim of this study is to evaluate immunogenicity and safety of a booster dose in children 2 months to <18 years of age. METHODS: This is a randomized, controlled open-label study in the Philippines. Three hundred children vaccinated with IXIARO in a previous trial were randomized 1:1 to receive either no booster or a booster 12 months after initiation of the primary series. Neutralizing antibody titers were assessed before and after the booster and up to 3 years after primary series. Safety endpoints included the rate of subjects with solicited adverse events (AEs), unsolicited AEs and serious AEs within 1 month after the booster. RESULTS: Geometric mean titer declined by 1 year after the primary series, but titers remained above the established protective threshold in 85%-100% of children depending on age group. The booster led to a pronounced increase in geometric mean titer and 100% seroprotection rate in all age groups. The booster was well tolerated, with AE rates lower compared with the primary series. Most AEs were mild. CONCLUSIONS: A booster dose of IXIARO administered 12 months after the primary immunization was well tolerated and highly immunogenic.


Assuntos
Anticorpos Antivirais/sangue , Encefalite Japonesa/prevenção & controle , Imunogenicidade da Vacina , Vacinas contra Encefalite Japonesa/uso terapêutico , Adolescente , Anticorpos Neutralizantes/sangue , Criança , Pré-Escolar , Encefalite Japonesa/imunologia , Humanos , Imunização Secundária , Lactente , Vacinas contra Encefalite Japonesa/administração & dosagem , Masculino , Filipinas , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/uso terapêutico
18.
Sci Rep ; 8(1): 7481, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748549

RESUMO

Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Proteção Cruzada , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/terapia , Vacinas contra Encefalite Japonesa/uso terapêutico , Vacinação/métodos , Animais , Anticorpos Neutralizantes/genética , Células CHO , Células COS , Cricetinae , Cricetulus , Proteção Cruzada/genética , Proteção Cruzada/imunologia , Modelos Animais de Doenças , Vírus da Encefalite Japonesa (Espécie)/classificação , Encefalite Japonesa/genética , Encefalite Japonesa/imunologia , Feminino , Genótipo , Vacinas contra Encefalite Japonesa/genética , Vacinas contra Encefalite Japonesa/imunologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Viral/genética , Suínos , Vacinação/veterinária , Células Vero , Vírion/genética , Vírion/imunologia
19.
Antiviral Res ; 154: 58-65, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29665373

RESUMO

West Nile virus (WNV) is a positive-sense single-stranded RNA flavivirus belonging to the Japanese encephalitis virus (JEV) serocomplex of the Flaviviridae family and causes mosquito-borne infections. Although most human infection cases are asymptomatic, approximately one in 150 infected individuals develops meningoencephalitis, with a mortality rate of 4-14%. While the development of human neutralizing antibody therapeutics against WNV is strongly anticipated, WNV is difficult to study in conventional laboratories due to its high safety level requirement. In this study, we established fully human WNV-neutralizing monoclonal antibodies from the peripheral blood mononuclear cells of inactivated-JEV-vaccinated individuals, and these antibodies exhibited WNV neutralization both in vitro and in vivo. Our results demonstrate a new antibody cross-reactivity strategy to develop immunological therapeutic reagents for WNV and other JEV serotype viruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Vacinas contra Encefalite Japonesa/administração & dosagem , Leucócitos Mononucleares/imunologia , Vírus do Nilo Ocidental/imunologia , Adulto , Animais , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa/imunologia , Encefalite Japonesa/terapia , Feminino , Humanos , Técnicas Imunológicas , Vacinas contra Encefalite Japonesa/imunologia , Camundongos Endogâmicos C57BL , Testes de Neutralização , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/terapia
20.
Nat Rev Neurol ; 14(5): 298-313, 2018 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-29697099

RESUMO

Japanese encephalitis is a mosquito-borne disease that occurs in Asia and is caused by Japanese encephalitis virus (JEV), a member of the genus Flavivirus. Although many flaviviruses can cause encephalitis, JEV causes particularly severe neurological manifestations. The virus causes loss of more disability-adjusted life years than any other arthropod-borne virus owing to the frequent neurological sequelae of the condition. Despite substantial advances in our understanding of Japanese encephalitis from in vitro studies and animal models, studies of pathogenesis and treatment in humans are lagging behind. Few mechanistic studies have been conducted in humans, and only four clinical trials of therapies for Japanese encephalitis have taken place in the past 10 years despite an estimated incidence of 69,000 cases per year. Previous trials for Japanese encephalitis might have been too small to detect important benefits of potential treatments. Many potential treatment targets exist for Japanese encephalitis, and pathogenesis and virological studies have uncovered mechanisms by which these drugs could work. In this Review, we summarize the epidemiology, clinical features, prevention and treatment of Japanese encephalitis and focus on potential new therapeutic strategies, based on repurposing existing compounds that are already suitable for human use and could be trialled without delay. We use our newly improved understanding of Japanese encephalitis pathogenesis to posit potential treatments and outline some of the many challenges that remain in tackling the disease in humans.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa/terapia , Animais , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/imunologia , Encefalite Japonesa/fisiopatologia , Encefalite Japonesa/virologia , Humanos
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