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.

Japanese encephalitis (JE) virus infects a number of host cells, either mosquitoes or vertebrates, in nature. The viral envelope (E) protein is known to interact with molecule(s) on the cell membrane during the early stage of virus infection. In this study, two sets of virus variants including T1P1-L4/T1P1-S1 and CJN-L1/CJN-S1 derived from two strains (T1P1 and CJN) of the JE virus were used to evaluate the effects of genomic variations on virus entry. Each set of virus variant (T1P1-L4/T1P1-S1 or CJN-L1/CJN-S1) possessed a single amino acid variation in the E protein. The variation of Glu/Lys at E-306 was found between T1P1-L4 and T1P1-S1 whereas the same variation at E-138 was seen between CJN-L1 and CJN-S1. The results showed that heparan sulfate (HS) differentially expressed on the surface of different types of host cells was essential for JE virus infection as shown in an evident difference in attachment efficiency between CHO-K1 cells and its mutant with defects in GAG biosynthesis. Furthermore, differential interaction of heparin with the envelope protein of JE virus variants implies the significance of virus mutations (especially Lys for E-138 and/or E306 in this case) that are rather likely involved in determining efficiencies of viral attachment, penetration, and eventual infection.

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.

Genetically different subpopulations were identified and purified from Japanese Encephalitis virus (JEV). Those with small plaques (SPs; <2 mm in diameter), derived from strains of T1P1, CJN, and CC27, were more competent than those with large plaques (LPs; >5 mm in diameter) when passaged in Neuro-2a cells. Differences in amino acids between SPs and LPs from each strain were shown in the viral envelope (E) protein. The amino acid at E-306 was Glu in LP but was substituted by Lys in SP in the T1P1 strain. A similar substitution occurred at E-138 in the CJN strain. However, the amino acid was Asp in LP but was substituted by Asn in SP at E-389 in the CC27 strain. All SPs were shown to have a higher affinity to the cellular membrane when compared to LPs, and this resulted in more-efficient infection of Neuro-2a cells, suggesting that the differential fitness of JEV variants to Neuro-2a cells appeared in the early phase of infection. In addition, glycosaminoglycans (GAGs) on the surface of many mammalian cells have been demonstrated to be critical for infection by JEV, especially SP variants. The present results suggest that T1P1-SP1 viruses infected Neuro-2a cells more efficiently in spite of the sparse distribution of cell surface GAGs. We conclude that highly sulfated forms of GAGs expressed by Neuro-2a cells play an important role in selecting JEV variants with specific mutations in the E glycoprotein.