Nakayama Strain Research Articles

Vector competence of Swedish Culex pipiens mosquitoes for Japanese encephalitis virus

BackgroundJapanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe.MethodsThis study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding.ResultsThe study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV.Conclusions Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes.Graphical

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-β was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

AB5-Type Toxin as a Pentameric Scaffold in Recombinant Vaccines against the Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is an enveloped icosahedral capsid virus with a prime neutralizing epitope present in E protein domain III (EDIII). E dimers are rearranged into a five-fold symmetry of icosahedrons. Cholera toxin B (CTB) and heat-labile enterotoxin B (LTB) of AB5-type toxin was used as the structural scaffold for emulating the pentameric axis of EDIII. We produced homo-pentameric EDIII through the genetic fusion of LTB or CTB in E. coli without recourse to additional refolding steps. Harnessing an RNA-mediated chaperone further enhanced the soluble expression and pentameric assembly of the chimeric antigen. The pentameric assembly was validated by size exclusion chromatography (SEC), non-reduced gel analysis, and a GM1 binding assay. CTB/LTB-EDIII chimeric antigen triggered high neutralizing antibodies against the JEV Nakayama strain after immunization in mice. Altogether, our proof-of-principle study creating a JEV-protective antigen via fusion with an AB5-type toxin as both a pentameric scaffold and a built-in adjuvant posits the bacterially produced recombinant chimeric antigen as a cost-effective alternative to conventional inactivated vaccines against JEV.

Belgian Anopheles plumbeus Mosquitoes Are Competent for Japanese Encephalitis Virus and Readily Feed on Pigs, Suggesting a High Vectorial Capacity.

Anopheles plumbeus, a day-active mosquito known to feed aggressively on humans, was reported as a nuisance species near an abandoned pigsty in Belgium. Since Japanese encephalitis virus (JEV) is an emerging zoonotic flavivirus which uses pigs as amplification hosts, we investigated (1) whether An. plumbeus would feed on pigs and (2) its vector competence for JEV, to investigate whether this species could be a potential vector. Three- to seven-day-old F0-generation adult mosquitoes, emerged from field-collected larvae, were fed on a JEV genotype 3 Nakayama strain spiked blood meal. Blood-fed mosquitoes were subsequently incubated for 14 days at two temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient. Our results show that An. plumbeus is a competent vector for JEV at the 25 °C condition and this with an infection rate of 34.1%, a dissemination rate of 67.7% and a transmission rate of 14.3%. The vector competence showed to be influenced by temperature, with a significantly lower dissemination rate (16.7%) and no transmission when implementing the temperature gradient. Moreover, we demonstrated that An. plumbeus readily feeds on pigs when the opportunity occurs. Therefore, our results suggest that Belgian An. plumbeus mosquitoes may play an important role in the transmission of JEV upon an introduction into our region if temperatures increase with climate change.

Relevant Day/Night Temperatures Simulating Belgian Summer Conditions Reduce Japanese Encephalitis Virus Dissemination and Transmission in Belgian Field-Collected Culex pipiens Mosquitoes.

Japanese encephalitis virus (JEV), a zoonotic mosquito-borne Flavivirus, can be considered an emerging infectious disease. Therefore, vector competence studies with indigenous mosquitoes from regions where JEV is not yet endemic are of great importance. In our study, we compared the vector competence of Culex pipiens mosquitoes emerged from Belgian field-caught larvae under two different temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient representing typical summer temperatures in Belgium. Three- to seven-day-old F0-generation mosquitoes were fed on a JEV genotype 3 Nakayama strain spiked blood-meal and incubated for 14 days at the two aforementioned temperature conditions. Similar infection rates of 36.8% and 35.2% were found in both conditions. The observed dissemination rate in the gradient condition was, however, significantly lower compared to the constant temperature condition (8% versus 53.6%, respectively). JEV was detected by RT-qPCR in the saliva of 13.3% of dissemination positive mosquitoes in the 25 °C condition, and this transmission was confirmed by virus isolation in 1 out of 2 RT-qPCR positive samples. No JEV transmission to saliva was detected in the gradient condition. These results suggest that JEV transmission by Culex pipiens mosquitoes upon an accidental introduction in our region is unlikely under current climatic conditions. This could change in the future when temperatures increase due to climate change.

Antiviral effect of 18-mer-peptide (1b-4/21-C12) on Japanese encephalitis virus and Akabane virus

Japanese encephalitis virus (JEV) and Akabane virus (AKAV) are mosquito-borne viruses that cause encephalitis and reproductive disorders in horses and cattle, respectively. There is no treatment for JEV or AKAV infections in animals. Therefore, we evaluated the antiviral activity of 18-mer amphipathic peptides in the 1b-4/21-C series on JEV and AKAV using Vero cells in vitro and evaluated their effects on JEV in mice. Of 6 peptides, 1b-4/21-C12 had the lowest IC50 of 0.313 against JEV and its use as an antiviral against JEV and AKAV was examined. The IC50 of 1b-4/21-C12 against JEV and AKAV was 0.78 and 1.14 μM, respectively. Mice treated with 5 or 2 mg/kg of 1b-4/21-C12 had 32% and 16% survival rates, respectively, and the surviving mice treated with 1b-4/21-C12 began to gain weight beginning 8 days post challenge with the virulent Nakayama strain. Moreover, 20 μM 1b-4/21-C peptide had no cytotoxic effects on Vero cells. Our in vitro and vivo results indicate that 1b-4/21-C12 has antiviral activity against enveloped JEV and AKAV and might be useful as a therapeutic substance.

Investigation into an annonymous, unclaimed Japanese encephalitis virus strain and, the biosafety and biosecurity lessons learnt.

Background: Japanese encephalitis (JE) is caused by the mosquito–borne Japanese encephalitis virus (JEV). The transmission cycle of the virus naturally takes place between wild birds and Culex tritaeniorhynchus mosquitoes, with pigs often serving as important natural host due to the high and prolonged viraemia. In year 2009, while searching for specimens in a freezer located in an unsecured room, we stumbled upon a vial labelled as “JEV” with no record of its origin. This vial was treated as a risk group 3 organism and immediately sent to the biocontainment level 3 facility, according to the standard operating procedures, for further investigation. Methods and materials: As a result of the vial label and to test the viability of the organism, the content was inoculated into Vero cell cultures and viewed daily for signs of cytopathic effect (CPE). A novel real time PCR specific for the NS3 region of JEV was designed to confirm the identity of the organism. Additionally, biosafety and biosecurity protocols were assessed and reviewed to address the storage of infectious materials in the laboratory. Results: Cells began to exhibit CPE at day 4 post-inoculation. Melting curve analysis revealed that the real time PCR was specific and sequencing of the purified PCR product confirmed the organism as JEV (strain MY8662). Phylogenetic analysis showed that MY8662 and the Nakayama strain were genetically very similar, evident by the high nucleotide (99.68%) and deduced amino acid (99.88%) sequence similarities.The root cause of the unsecured vial was obscure inventory that failed to record specimens that could have been stored for decades. To this end, an access controlled room with ventilation was constructed to house all freezers storing infectious materials, coupled with an inventory system with barcodes on every storage vial. Additionally, inventory accountability was instituted for the tracking and transportation of infectious materials within and outside of the institution. Conclusion: This unanticipated discovery of an unknown vial has precipitated the laboratory management to take significant measures to address the potential impact of the loss or intentional misuse of these agents. It is therefore, crucial to establish a sustainable culture of safety that involves every stakeholder in the laboratory.

Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo

In a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/β–γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response

BackgroundJapanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce.MethodsNine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS.ResultsBesides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed.ConclusionsThese findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300–500 μg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.

Reemergence of Japanese Encephalitis in South Korea, 2010-2015.

To the Editor: Japanese encephalitis (JE) is caused by a virus transmitted by Culex tritaeniorhynchus mosquitoes. JE was the major public health concern in South Korea until the late 1960s, with several thousand cases reported annually. The national vaccination program with the inactivated mouse brain–derived Nakayama strain was initiated in 1983 and targeted children <15 years of age. During 1983–2000, annual booster vaccinations were given to children <15 years of age, but in 2000, the booster schedule was changed to 2 doses (1 dose each) for children 6 and 12 years of age. The live attenuated JE vaccine SA 14-14-2 was introduced in 2002 and included in the national immunization program in 2014. After introduction of the mandatory immunization program, JE was nearly eliminated; during most of the past 3 decades, <0.02 cases per 100,000 population have been reported annually (1). However, since 2010, JE has reemerged in South Korea. We describe epidemiologic data for JE, focusing on the recent increase in number of cases in South Korea. We accessed demographic information from the disease web statistics system provided by the Korea Centers for Disease Control and Prevention (2). Our study was exempted from review by the Institutional Review Board at Seoul National University Hospital (E-1602-053-739).

Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene

Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V.

A Single Dose of Vero Cell-Derived Japanese Encephalitis (JE) Vaccine (Ixiaro) Effectively Boosts Immunity in Travelers Primed With Mouse Brain-Derived JE Vaccines

A significant part of the world population lives in areas with endemic Japanese encephalitis (JE). For travelers from nonendemic countries, Vero cell-derived vaccine (JE-VC; Ixiaro) has replaced traditional mouse brain-derived vaccines (JE-MB) associated with safety concerns. The 2 vaccines are derived from different viral strains: JE-VC from the SA14-14-2 strain and JE-MB from the Nakayama strain. No data exist regarding whether JE-VC can be used to boost immunity after a primary series of JE-MB; therefore, a primary series of JE-VC has been recommended to all travelers regardless of previous vaccination history. One hundred twenty travelers were divided into 4 groups: Volunteers with no prior JE vaccination received primary immunization with (group 1) JE-MB or (group 2) JE-VC, and those primed with JE-MB received a single booster dose of (group 3) JE-MB or (group 4) JE-VC. Immune responses were tested before and 4-8 weeks after vaccination using plaque reduction neutralization test (PRNT) against both vaccine strains. In vaccine-naive travelers, the vaccination response rate for test strains Nakayama and SA14-14-2 was 100% and 87% after primary vaccination with JE-MB and 87% and 94% after JE-VC, respectively. Antibody levels depended on the target virus, with higher titers against homologous than heterologous PRNT(50) target strain (P < .001). In travelers primed with JE-MB, vaccination response rates were 91% and 91%, and 98% and 95% after a booster dose of JE-MB or JE-VC, respectively. Subgroup analysis revealed that a higher proportion of primed (98%/95%) than nonprimed (39%/42%) volunteers responded to a single dose of JE-VC (P < .001). A single dose of JE-VC effectively boosted immunity in JE-MB-primed travelers. Current recommendations should be reevaluated. NCT01386827.

Phylogenetic analysis of poly and non structural protein in Japanese Encephalitis virus with other related viral families.

Japanese encephalitis (JE) causes inflammation of brain. The mortality rate due to JE is 30% while 10 -15 % of patients make full recovery. The disease spreads through infected mosquito bites breeding in rice fields and feeds on pigs, birds, and ducks. As proteins show important structure to function relationship the study was designed to carry out the identification of poly and non-structural proteins in the infective virus group using different strains of Japanese encephalitis virus i.e. JAOARS982, Nakayama Strain SA (V), Strain SA-14. With reference to non structural proteins we obtained protein sequences of the following Japanese encephalitis virus groups: Japanese encephalitis virus, Weatnile virus, Kunjin virus. Further comparative and phylogenetic analysis was performed to explore evolutionary relationship among these groups. Results of phylogeny of alignment score was found to be 375184 using multiple alignment, Jal view, ClustalW (1.83) and ClustalW2. However, the analysis among the non-structural proteins of Japanese Encephalitis Virus, Westnile Virus, and Kunjin Virus revealed the phylogeny alignment score to be 875 through multiple sequence alignment and Tree view respectively. Phylogenetic analysis revealed that these four strains are interrelated as well as showing high similarity with the other viruses of this group due to conserved regions among their sequences.

JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: Biological properties in the mouse model of neuroinvasive disease

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5′-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5′CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5′CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5′CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5′CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5′ UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5′ UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.

Acute Flaccid Paralysis as an Unusual Presenting Symptom of Japanese Encephalitis: A Case Report and Review of the Literature

Japanese encephalitis (JE) is an endemic disease in Taiwan. Acute JE virus infection characterized by acute flaccid paralysis in an adult has never been reported in Taiwan. We report a young adult man who received four doses of JEV (Nakayama strain) vaccination in childhood, but still developed acute JE virus infection, characterized with acute flaccid paralysis.He presented with fever, headache, progressive muscle weakness, and respiratory paralysis requiring mechanical ventilator. Deep tendon reflexes were decreased except for the Achilles reflex. After supportive care, he was weaned from the mechanical ventilator and at discharge 1 month later, his muscle power level and deep tendon reflexes recovered partially. The diagnosis of JE was based on the presence of anti-JE virus IgM in the CSF and seroconversion of IgM and IgG by the ELISA method. Electrophysiological findings were described. From the experience of this case, we caution that a history of vaccination for JE with the Nakayama strain may not provide a complete protection against natural infection in the community; and in Taiwan or any area where JE remains an endemic disease, Japanese virus encephalitis infection should be considered as a differential diagnosis in any adult presenting with acute flaccid paralysis.

Comparative Safety and Efficacy of Subcutaneous and Intradermal Administration of Inactivated Japanese Encephalitis Vaccine during Predeployment Preparations in the Australian Defence Force

Japanese encephalitis is a viral disease emerging in areas of influence for the Australian Defence Force immediately north of the continent, including the Torres Strait border of Australia and Papua, New Guinea. Only the mouse brain-derived, inactivated, Nakayama strain vaccine is commercially available to the Australian Defence Force. This vaccine has a high cost and significant adverse event profile, requiring restricted duties after administration. To address these issues, intradermal vaccination (either single intradermal administration or two intradermal injections at two separate sites) was assessed, compared with the conventional subcutaneous vaccination method, in a randomized controlled trial among soldiers preparing for deployment. Dual intradermal vaccination (0.1 mL at two sites) was found to have a slightly more favorable adverse event profile while maintaining comparable serological efficacy and reduced cost. An expansion of the concept and a test of logistics were conducted by vaccinating a battalion formation during predeployment medical preparations. The method of vaccination was well accepted and retained comparable immunogenicity.

Chimeric Japanese encephalitis virus/dengue 2 virus infectious clone: biological properties, immunogenicity and protection against dengue encephalitis in mice

A molecular clone of Japanese encephalitis virus (JE virus) was derived from the JE virus Nakayama strain and used to produce infectious JE virus in cell culture. The engineered JE virus resembled the parental JE virus in cell-culture properties and was related closely to other JE virus strains based on nucleotide sequence analysis. The JE virus clone was used as a genetic background for construction of a chimeric virus containing the structural proteins prM and E of Dengue virus, serotype 2. The chimeric JE/dengue 2 virus generated authentic dengue 2 structural proteins as assessed by immunoassays for the dengue E protein. It exhibited a small plaque size and less efficient growth in various cell lines than the parental JE virus. JE/dengue 2 virus was non-neuroinvasive for young adult mice, but displayed partial neurovirulence at doses up to 4 log p.f.u. given intracerebrally. Immunization of 3-week-old mice with JE/dengue 2 virus yielded neutralizing-antibody titres against dengue 2 virus and conferred protection against dengue encephalitis caused by neuroadapted dengue 2 virus. A rise in post-challenge neutralizing-antibody titres against dengue 2 virus in surviving mice suggests that immunization is associated with establishment of a memory antibody response in this model. This study demonstrates the capacity of JE virus to serve as a vector for expression of heterologous flavivirus structural proteins. Similar to previous studies with other chimeric flaviviruses, this approach may be useful as a genetic system for engineering experimental vaccines against Dengue virus and other medically important flaviviruses.

Mosquito Cells Infected with Japanese Encephalitis Virus Release Slowly‐Sedimenting Hemagglutinin Particles in Association with Intracellular Formation of Smooth Membrane Structures

Arthropod-borne flaviviruses can grow in both arthropod and mammalian cells. Virion morphogenesis, though well studied in mammalian cells, is still unclear in arthropod cells. Here, we compared a mosquito cell line C6/36 and a mammalian cell line Vero in extracellular virus particles and intracellular ultrastructures triggered by infection with Japanese encephalitis virus (JEV). Sedimentation analyses of virion and slowly-sedimenting hemagglutinin (SHA) particles released by infection with the Nakayama strain revealed that C6/36 cells produced higher envelope (E) antigen levels in the SHA than the virion fraction in contrast to Vero cells that showed the opposite pattern. Specific infectivities per ng of E were similar in both cells, whereas specific hemagglutinating activities in the SHA fraction were lower in C6/36 than Vero cells. The precursor membrane protein was less efficiently cleaved to the membrane protein in SHA particles released from C6/36 than Vero cells. Ultrastructural studies showed more remarkable production of smooth membrane structures (SMSs) in C6/36 than in Vero cells. The differences in sedimentation patterns of extracellular virus particles between Nakayama-infected C6/36 and Vero cells were consistently observed in 5 other strains (Beijing P1, Beijing P3, JaTH-160, KE-093 and JaGAr-O1), except for KE-093-infected C6/36 cells which exhibited the Vero-type sedimentation profile under conditions of open cultivation. By electron microscopy, the production of SMSs from KE-093-infected C6/36 cells under open conditions was markedly less than that under closed conditions where the cells exhibited the C6/36-type sedimentation profile. Thus, intracellular SMS formations were associated with extracellular SHA production in JEV-infected mosquito cells.

The efficacy of mouse-brain inactivated Nakayama strain Japanese encephalitis vaccine—Results from 30 years experience in Taiwan

An intensive mandatory vaccination program has been underway, combating Japanese encephalitis (JE) since 1968 in Taiwan. Long-term collection of immunization records has been developed from 1967 to 2000 in this study to retrospectively assess the efficacy of the mouse-brain inactivated Nakayama JE vaccine. The vaccine efficacy (VE) of completing at least two doses of the JE vaccine was 96.98%. Among 1 to 14-year-old children, the efficacy of completing 1, 2, and 3 doses of immunization was 85.59%, 91.07% and 98.51%, respectively. Furthermore, the long-term efficacy for a single dose vaccinated at least 25 years was 86.79%, and for 2 and 3 doses it was 88.10% and 95.54%, respectively. In contrast to previous studies that recommended at least two doses of JE vaccination to acquire necessary protection, the empirical results in this study indicated that even immunization with one single dose provides sufficient protection to the population. However, a single dose of JE vaccine might still be beneficial for some JE epidemic or endemic developing countries with limited resources for infectious disease control.