Expression of Japanese encephalitis virus antigens in Escherichia coli

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P.C. McAda, P.W. Mason, C.S. Schmaljohn, J.M. Dalrymple, T.L. Mason, and M.J. Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector. Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome.     

Monoclonal antibody raised against envelope glycoprotein peptide neutralizes Japanese encephalitis virus

Summary.  Epitopes on envelope glycoprotein of Indian strain of Japanese encephalitis virus were delineated by prediction methods. Monoclonal antibodies (MAb) raised against a putative B cell epitope peptide, reacted with the virion in ELISA and immunofluorescence assays. One MAb was also able to neutralize the virus. The reactivity of this MAb against a Sri Lankan strain was checked, since this strain had a substitution within the B cell epitope at position Egp 153 (G → W). The MAb was able to bind to, but was not able to neutralize the Sri Lankan isolate. The data indicated that the predicted B cell epitope is a neutralizing epitope and may be included in a peptide-based vaccine against the virus. Received July 20, 2000 Accepted September 28, 2000     

Monoclonal antibody to Japanese encephalitis virus cross-reacting with histones present in the cell nuclei.

An immunoglobulin G (IgG2b) class of monoclonal antibody (MoAb, NHA-1) raised against Japanese encephalitis virus (JEV) E glycoprotein, reacted with the viral antigen expressed in cytoplasm of the infected cells and also with the cell nuclei, by an indirect fluorescent antibody technique (FA). The NHA-1 reactivity to nuclei was found to be due to its recognizing a JEV cross-reactive epitope present on the nuclear histones. Adsorption with calf thymus histones (type II-AS) showed a drop in NHA-1 reactivity to both JEV and histones by an enzyme-linked immunosorbent assay (ELISA) and indirect FA; the drop was higher against the histones. The MoAb recognized specifically the viral antigens expressed on the infected porcine kidney cell surface by a modified indirect FA. ELISA carried out with glutaraldehyde-fixed antigens showed an almost 2-fold increase in the reactivity over unfixed JEV antigen but none for the histones. Thus, the results indicate that histones share a sequential homology with E glycoprotein of JEV, which might lead to an autoimmune disorder induced due to the molecular mimicry between these two antigens.     

Japanese encephalitis and Japanese encephalitis virus in mainland China

Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000–50,000 people suffer from JE every year, with a mortality rate of 10,000–15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV. Copyright © 2012 John Wiley & Sons, Ltd.     

Japanese encephalitis virus glycoproteins

Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK₂ cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.     

Monoclonal antibodies to the structural glycoprotein of tick-borne encephalitis virus.

Hybridomas secreting antibodies to the structural glycoprotein of tick-borne encephalitis (TBE) virus were prepared by fusion of X63-Ag8/653 mouse myeloma cells with spleen cells from mice immunized with purified glycoprotein complexes of TBE virus. These antibodies were tested against 10 different TBE virus strains isolated in different European countries over a period of 26 years from different hosts. Quantitative evaluation of enzyme immunoassay results did not reveal any differences in reactivity among these strains, pointing further to the homogeneity of European TBE virus isolates, which has previously been inferred from results obtained by peptide mapping and competitive radioimmunoassay. Hybridomas defining three different antibody-combining sites (epitopes) on the glycoprotein of TBE virus were selected on the basis of cross-reactivity with another flavivirus. West Nile virus, as well as the ability to inhibit hemagglutination. Two epitopes were type specific, and the third was indistinguishably also present on West Nile virus. Hemagglutination was inhibited by monoclonal antibodies reacting with one of the type-specific epitopes as well as the cross-reactive determinant, which is apparently responsible for the broad cross-reactivity among different flaviviruses observed in hemagglutination inhibition tests with polyvalent immune sera.     

The proteins of Japanese encephalitis virus

Polyacrylamide gel electrophoresis of Japanese encephalitis virus (JEV) grown in both LLC-MK₂ and chick embryo cell culture revealed three principal polypeptides with molecular weights of 8,700, 13,500, and 53,000 (V-1, V-2, and V-3, respectively). Infected chick cells that were treated with actinomycin D and cycloheximide contained seven polypeptides not present in uninfected cells. In addition to V-2 and V-3, polypeptides with molecular weights of 10,500, 19,000, 45,000, 71,000, and 93,000 (NV-1 through NV-5) were found; V-1 was not regularly detected. A similar pattern of polypeptides was obtained by radioimmune precipitation of soluble antigens from cytoplasmic extracts of infected, actinomycin-D treated, chick cells. When virions were treated with NP-40, a dense, RNA-rich structure was detected which contained V-2. An extracellular, slowly sedimenting, RNA-poor, hemagglutinating particle with a density comparable to the virion was present in virus preparations from cell culture and contained V-1, V-3, and NV-2.     

A reverse passive haemagglutination test for detection of Japanese encephalitis virus antigens in cerebrospinal fluid

A rapid sensitive and specific reverse passive haemagglutination test (RPHA) was developed for the detection of Japanese encephalitis virus (JEV) antigens in human cerebrospinal fluid (CSF). Sheep red cells were sensitized with five monoclonal antibodies (109, 112, 203, 204 and 301) reactive with envelope glycoprotein of JEV. Viral antigens were detected in CSF from 35 of 58 (60%) clinical cases of JE when the five MAb coated cells were used in combination. An IgM capture ELISA detected JEV specific antibodies in CSF among 52 of these 58 cases (90%). While 29 specimens contained both antigen and IgM antibodies, 23 had only IgM antibodies and 6 had only antigen. RPHA proved valuable for detection of viral antigens in CSF samples obtained within 10 days after onset of clinical symptoms. Amongst the five MAbs used, the individual antigen detection rates were 44, 12, 43, 12 and 36%, for MAbs 109, 112, 203, 204 and 301, respectively.     

Studies on the neutralization of Japanese encephalitis virus

Summary The neutralization reaction of Japanese encephalitis virus with early serum was compared with that with late serum. The analysis of antiserum by Sephadex G200 gel filtration indicated that the neutralizing activity in early serum was present only in the IgM fraction, while that in late serum was present only in the IgG fraction. The antibody dose response curves in early serum were characterized by the early and high appearance of a persistent fraction. This fraction was found to consist of an infectious virus antibody complex (sensitized virus) which was neutralized by anti-IgM serum. The amount of virus neutralized by anti-IgM serum varied with the concentration of antiviral antibody employed for the sensitization. In contrast, it was a characteristic of the neutralization by late serum that the residual infectivity was inversely related to the concentration of antibody in the serum, resulting in a low level of a non-neutralized virus fraction. Therefore, the maximal reduction of residual infectivity by anti-IgG serum was attained under an optimal ratio of antibody to virus.Virus sensitized with early serum had a blocking effect against a high concentration of late serum antibody, but was neutralized by anti-IgM serum. Virus sensitized with an insufficient amount of late serum antibody was neutralized not only by high concentrations of late serum antibody, but also was supersensitized by early serum antibody.Since the sensitized virus which had been adsorbed on host cells was still neutralizable by anti--globulin, aggregation seemed to be excluded as the main factor in the mechanism of neutralization by anti--globulin serum.     

Japanese encephalitis virus infection in fetal mice at different stages of pregnancy. II. Resistance to Japanese encephalitis virus infection

The relationship between the stage of pregnancy of mice at the time of Japanese encephalitis (JE) virus inoculation and the resistance of JE virus infection of their offsprings was investigated. It was found that there was a stronger resistance to JE virus infection in offsprings born of mothers inoculated with JE virus at nine to sixteen days before parturition than in offsprings of mothers inoculated at one to eight days or at seventeen to twenty days before parturition. Resistance of the offsprings to JE virus infection lasted up to the age of 180 days after birth.     

Monoclonal antibody study of antigens E and NS1 of Western Siberian tick borne encephalitis virus strains]

Antigenic structure of tick-borne encephalitis virus proteins was studied by ELISA with monoclonal antibodies (MAb) to E and NS1 glycoproteins of strain Sofyin. Envelope proteins appeared to be conservative which corresponded to previously determined nucleotide sequences of E gene fragments and deduced primary structures of the corresponding E protein. Five of six studied MAb to NS1 nonstructural glycoprotein of strain Sofyin reacted with this protein of all studied strains. The only exception was MAb 17C3 which discriminates West Siberian strains from Far Eastern strain Sofyin.     

A rapid method for detection of flavivirus antigens: staphylococcal co-agglutination test using monoclonal antibodies to Japanese encephalitis virus

Staphylococcus aureus rich in protein A when coated with monoclonal antibodies (MoAb) to Japanese encephalitis virus (JEV) gave a highly specific reaction with flavivirus antigens. The bacteria coated with JEV species-specific MoAb gave a strong co-agglutination with fifty-six JEV isolates from various parts of China, but no co-agglutination with Murray Valley encephalitis (MVE) and Kunjin (Kun) virus antigens. The flavivirus- and subgroup-specific MoAbs were reactive with MVE and Kun, as well as with the majority of the JEV strains. Blocking test with homologous MoAbs abolished co-agglutination further confirming its specificity. Numerous virus particles were observed on the surface of MoAb-coated staphylococci under the electron microscope after co-agglutination. The test appeared rapid, specific, simple to perform, and useful for rapid detection and identification of flaviviruses.     

Studies on neutralization of Japanese encephalitis virus

The effect of anti-cellular rabbit serum (ACRS) on the neutralization of sensitized Japanese encephalitis virus (JEV) by anti-rabbit IgG serum was examined to elucidate the interaction between virus-antibody complex and the surface of the host cells during the process of neutralization. ACRS had no effect on the adsorption of either sensitized or non-sensitized virus, but was able to restore the lost infectivity of sensitized virus which occurred during the process of neutralization by anti-rabbit IgG serum. This restoration of infectivity was found to take place not only by the addition of ACRS to the reaction mixtures (virus-antibody, anti-rabbit IgG complex) but also by pretreatment of the host cells with ACRS. Although the restoration of lost infectivity varied in magnitude with the concentration of ACRS used, it never exceeded the infectivity titer of the sensitized virus befor incubatio with anti-rabbit IgG serum. This result suggests that ACRS has no ability to reverse the neutralization by anti-viral serum. Since the ACRS reacted only with anti-rabbit IgG serum treated sensitized virus, resulting in an increase of the number of infectious centers, the restoration of lost infectivity was explained as being due to the enhancement of adsorption of sensitized virus to the host cells by bridge formation of anti-rabbit IgG antibody between them.     

Partial nucleotide sequence of the Japanese encephalitis virus genome

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.