Mutation analysis of the cross-reactive epitopes of Japanese encephalitis virus envelope glycoprotein

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs

Partially Neutralizing Potency against Emerging Genotype I Virus among Children Received Formalin- Inactivated Japanese Encephalitis Virus Vaccine

Background: Genotype I (GI) Japanese encephalitis virus (JEV) that replaced GIII virus has become the dominant circulating virus in Asia. Currently, all registered live and inactivated JEV vaccines are derived from genotype III viruses. In Taiwan, the compulsory JEV vaccination policy recommends that children receives four doses of formalin-inactivated Nakayama (GIII) JEV vaccine. Methodology/Principal Findings: To evaluate the influence of genotype replacement on the post-vaccination viral neutralizing ability by GIII and GI viruses, the small panel of vaccinated-children serum specimens was assembled, and the reciprocal 50% plaque-reduction neutralizing antibody titers (PRNT50) were measured against Nakayama vaccine strain, CJN GIII human brain isolate and TC2009-1 GI mosquito isolate. The seropositivity rate (PRNT50$1:10) and geometric mean titers (GMT) against the TC2009-1 virus were the lowest among the three viruses. The protective threshold against the CJN and TC2009-1 viruses could only be achieved when the GMT against Nakayama virus was$1:20 or $1:80, respectively. Using undiluted vaccinees’ sera, the enhancement of JEV infection in K562 cells was observed in some low or non-neutralizing serum specimens. Conclusions/Significance: Our preliminary study has shown that neutralizing antibodies, elicited by the mouse brainderived and formalin-inactivated JEV Nakayama vaccine among a limited number of vaccinees, have reduced neutralizing capacity against circulating GI virus, but more detailed studies are needed to address the potential impact on the future vaccine policy

SHORT REPORT: DETECTION OF JAPANESE ENCEPHALITIS VIRUS IN MOUSE PERIPHERAL BLOOD MONONUCLEAR CELLS USING AN IN SITU REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION

Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase- polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% par formaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin- labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2-3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides

Bovine lactoferrin inhibits Japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein

AbstractLactoferrin is a natural anti-microbial protein which affects Japanese encephalitis virus (JEV) activity. Binding of lactoferrin to cell surface expressed heparan sulfate (HS), one possible receptor for JEV, has been postulated to be the possible mechanism of anti-JEV antiviral activity. In this study, we evaluate the effects of bovine lactoferrin (bLF) against JEV infection in vitro, using both wild-type (WT) and laboratory-adapted strains. bLF inhibited the infectivity of all the JEV strains tested. In particular the infectivity of the HS-adapted JEV strains was strongly reduced, whereas the non HS-adapted JEV strains were inhibited to lesser extent. Using both HS-adapted CJN-S1 and non HS-adapted CJN-L1 viruses, the results showed that bLF inhibited the early events essential to initiate JEV infection, which includes blocking virus attachment to cellular membranes and reducing viral penetration. This anti-JEV activity was the highest using HS-adapted CJN-S1 strain on HS-expressed CHO-K1 cells. Also, binding of bLF to heparin-sepharose blocked JEV binding; and soluble HS attenuated the anti-JEV activity of bLF. The results support the premise that the interaction of bLF with cell surface expressed glycosaminoglycans, in particular the highly sulfated HS, plays an essential role in the antiviral activity of bLF. However, bLF was functional in inhibiting CJN-S1 entry into HS-deficient CHO-pgsA745 cells, and bLF-treated CHO-K1 and -pgsA745 cells also prevented non HS-adapted CJN-L1 virus entry, indicating that a non-HS pathway may be involved in bLF inhibition of JEV entry. The low-density lipoprotein receptor (LDLR), possibly involved in the entry of several RNA viruses, also binds to bLF. We found that both rLDLR and anti-LDLR antibodies reduced the effectiveness of bLF inhibition of JEV infection. This finding provided evidence to suggest that cell surface-expressed LDLR may play a role in JEV infection, especially for non HS-adapted strains

Reduced neutralizing antibody titer against genotype I virus in swineimmunized with a live-attenuated genotype III Japanese encephalitisvirus vaccine

A shift in prevalence from Japanese encephalitis virus (JEV) genotype III (GIII) to GI virushas been observed in several Asian countries. Genotype I virus was first detected in Taiwanin 2008, and became the dominant JEV island-wide within a year. We conducted aserosurvey using swine serum specimens from multiple counties in Taiwan following thetransmission season in 2009 and results showed 67–100% of JEV seropositive swineshowed evidence of GI virus infection. The envelope (E) protein is a structural protein thatelicits protective neutralizing antibodies (Nt Ab). The GIII at222 (a live-attenuated swinevaccine) virus E protein differs at eight amino acid residues (E-123, E-129, E-138, E-176, E-209, E-222, E-327 and E-366) from that of the GI TC2009-1 strain (isolated in Taiwan in2009). Twenty piglets were vaccinated with two doses of at222 vaccine, and serumspecimens were collected to evaluate the strain-specific Nt Ab titer against GIII at222, GIIICJN, and GI TC2009-1 viruses. Seropositivity rates (Nt Ab titer 1:10) and geometric meantiters (GMT) were similar against at222 and CJN viruses. However, sera from swinevaccinated with at222 were least potently neutralizing against GI TC2009-1 virus. Theestimated protective threshold against GI virus was observed only when the PRNT50against at222 virus was 1:320. Thus, our current study indicates that the live-attenuatedat222 swine vaccine can be partially protective against GI virus, and suggests that theefficacy of GIII swine vaccines currently used may require a comprehensive reevaluation inthe field

Effectiveness of Live-Attenuated Genotype III Japanese Encephalitis Viral Vaccine against Circulating Genotype I Viruses in Swine

Expansion of genotype I (GI) Japanese encephalitis viruses (JEV) has resulted in the replacement of the dominant genotype III (GIII) viruses, raising serious public health concerns for using GIII virus-derived vaccines to effectively control JEV epidemics. Therefore, this study used swine as the model to estimate the effectiveness of GIII live-attenuated vaccine against GI virus infection by comparing the incidence of stillbirth/abortion in gilts from vaccinated and non-vaccinated pig farms during the GI-circulation period. In total, 389 and 213 litters of gilts were recorded from four vaccinated and two non-vaccinated pig farms, respectively. All viruses detected in the aborted fetuses and mosquitoes belonged to the GI genotype during the study period. We thus estimated that the vaccine effectiveness of GIII live-attenuated vaccine against GI viruses in naive gilts based on the overall incidence of stillbirth/abortion and incidence of JEV-confirmed stillbirth/abortion was 65.5% (50.8–75.7%) and 74.7% (34.5–90.2%), respectively. In contrast to previous estimates, the GIII live-attenuated vaccine had an efficacy of 95.6% (68.3–99.4%) to prevent the incidence of stillbirth/abortion during the GIII-circulating period. These results indicate that the vaccine effectiveness of GIII live-attenuated JEV vaccine to prevent stillbirth/abortion caused by GI viruses is lower than that against GIII viruses

First detection of the Africa/Caribbean/Latin American subtype of Culex flavivirus in Asian country, Taiwan

Culex flavivirus (CxFV), a member of the genus flavivirus, is a novel insectinsect-specific flavivirusesthat can be divided into two subtypes, the cytopathic Asia/U.S. and the noncytopathicAfrica/Caribbean/Latin American subtypes. The CxFV circulates in several Asian countries,and here we conducted the first study investigating CxFV in Taiwan. A total of 14,016mosquitoes were collected between 2010 and 2012 and 3.4% (6/179) of the pools wereCxFV-positive. The phylogenetic analyses indicate that the Taiwan isolates are closelyrelated to the Africa/Caribbean/Latin American subtype, but form an independent cluster. Inthe cytology experiments, the CxFV Taiwan isolate infected only mosquito cells and causedcell–cell fusion that might be associated with a unique glycine residue at position 117 withinthe envelope protein, which is shared with the cytopathic effect-causing Asia/US subtype.This study marks the first time the Africa/Caribbean/Latin American subtype of CxFV hasbeen identified in an Asian country and grouped into a novel cluster

Bovine Lactoferrin Inhibits Dengue Virus Infectivity by Interacting with Heparan Sulfate, Low-Density Lipoprotein Receptor, and DC-SIGN

Bovine lactoferrin (bLF) presents in milk and has been shown to inhibit several viral infections. Effective drugs are unavailable for the treatment of dengue virus (DENV) infection. In this study, we evaluated the antiviral effect of bLF against DENV infection in vivo and in vitro. Bovine LF significantly inhibited the infection of the four serotypes of DENV in Vero cells. In the time-of-drug addition test, DENV-2 infection was remarkably inhibited when bLF was added during or prior to the occurrence of virus attachment. We also revealed that bovine LF blocks binding between DENV-2 and the cellular membrane by interacting with heparan sulfate (HS), dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), and low-density lipoprotein receptors (LDLR). In addition, bLF inhibits DENV-2 infection and decreases morbidity in a suckling mouse challenge model. This study supports the finding that bLF may inhibit DENV infection by binding to the potential DENV receptors

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

<div><p>Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H₂O₂, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H₂O₂, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.</p></div