Expression of envelope glycoprotein (E) of Japanese encephalitis virus by recombinant vaccinia virus

Vaccinia virus recombinants inserted with cDNA clones of Japanese encephalitis (JE) virus envelope glycoprotein (E) gene were constructed. The E gene product was detected in the recombinant virus-infected BHK21 cells by immunofluorescence (IF) and Western blotting. The intensity of IF observed was higher by the recombinant of the TK promoter--P7.5 promoter--inserted cDNA construct than by the P7.5 promoter--TK promoter--inserted cDNA construct. The E gene product was hardly detected by the recombinant carrying the TK promoter only upstream to the inserted cDNA, although the glycoprotein E mRNA had been transcribed.     

Expression of Japanese encephalitis virus envelope protein in transgenic tobacco plants

The virus envelope (E) protein of Japanese encephalitis virus induces virus-neutralizing antibodies and is therefore a potential vaccine antigen. In a mammalian system, co-expression of another viral structural protein prM is necessary for proper expression and folding of E protein. Transgenic tobacco plants were produced carrying JEV cDNA encoding prM and E proteins under the control of the CaMV 35S promoter. E protein, however, was not detectable in these plants. In vitro translation studies showed that the presence of the prM sequence inhibited transgene expression in the plant system. Accordingly, JEV E protein could be expressed in transgenic tobacco plants only without the prM protein.     

DNA-binding property of recombinant capsid protein of Japanese encephalitis virus

Japanese encephalitis virus (JEV) is a member of the Flaviviridae family, and it is capable of inducing febrile syndrome, encephalitis and death. In this study, the cDNA of JEV-YL capsid (core) protein were cloned and expressed in E. coli. Three expressed recombinant clones (pET32/CORE, pET32/CD and pET32/C2D) including different parts of capsid protein, were constructed from JEV cDNA clone, pJE-S. These recombinant proteins (34, 31, and 26 kDa, respectively) containing an amino terminal tag of six histidines were isolated by the nickel chelate affinity chromatography and the purified products were identified by Western blotting with anti-serum of JEV-infected swine. To examine DNA binding property of the capsid protein, the purified recombinant proteins were assayed by electrophoretic mobility shift assay. The results showed the capsid proteins had the abilities to bind DNA, except the prominent product of pET32/C2D, which had deletion in the middle hydrophobic region of capsid protein and revealed the coding region of the amino acid, F46 to P61, is mediating the DNA binding ability. This study suggests a possible regulatory role for capsid protein in the pathway of JEV infection.     

Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses

Two recombinant baculoviruses were constructed by inserting via the transfer vector pAcYM1 the genes coding for the structural proteins of dengue (DEN)-2 virus downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of DEN-specific envelope (E) glycoprotein. Recombinant DEN-2 E glycoproteins were processed and transported to the surface of Spodoptera frugiperda Sf9 cells infected with both viruses. We show that about one-third of the E glycoprotein minus its whole C-terminal hydrophobic anchor domain was secreted into an endoglycosidase H-resistant form. The type-specific neutralizing epitopes were conserved in the recombinant proteins as shown with a panel of monoclonal antibodies.     

Louping ill virus envelope protein expressed by recombinant baculovirus and vaccinia virus fails to stimulate protective immunity.

We have constructed recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein alone or all of the structural proteins (core, membrane and envelope) of louping ill virus. Glycosylated viral envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of the envelope protein were observed between the envelope protein and structural protein constructs as well as between the insect and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either vector failed to induce, in mice or rabbits, either neutralizing or protective antibodies against louping ill virus.     

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus

Summary Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P_7·5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.     

Fragment of Japanese encephalitis virus envelope protein produced in Escherichia coli protects mice from virus challenge.

A fragment from the N-terminal part (E(A)) and a fragment from the C-terminal part (E(B)) of the envelope (E) protein of Japanese encephalitis virus (JEV) was synthesized in Escherichia coli. These two fragments were overlapping with each other by nine amino acids, however, they were not cross-reacting with each other at the antisera level. Both E(A)and E(B)are antigenic by themselves when injected into mice, but when tested against sera from mice, rabbit, swine and human that had been immunized or naturally infected with JEV, E(B)acted as a better antigen than E(A)by ELISA assays. E(B)also proved to be a better immunogen in protection against lethal JEV infection than E(A). The protection appears to be correlated with the neutralizing titres of the anti-JEV sera. The response elicited by E(B)is a Th1 response and the antibody produced contained higher neutralizing titre than E(A)fragment. The major difference between E(A)and E(B)fragments is the solubility during expression in E. coli, while E(B)fragment is soluble, E(A)was isolated from the insoluble inclusion bodies. Therefore the antigenicity and immunogenicity expressed by the E(B)fragment may probably be due to its proper folding to assume a correctly assembled form during expression in E. coli, a quality that is important for a protein to qualify as a good vaccine candidate.     

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus.

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.     

马乙型脑炎病毒E蛋白的原核表达及间接ELISA的初步应用

目的原核表达马乙型脑炎病毒(Japanese encephalitis virus,JEV)E蛋白,建立JEV间接ELISA诊断方法。方法根据GenBank(AF315119.1)公布的JEVSA14-14-2株E蛋白基因序列设计一对引物,提取病毒RNA经反转录和RT-PCR扩增获得E蛋白编码基因片段,将该片段插入表达载体pET30a(+),转化BL21(DE3)后经IPTG诱导蛋白表达,Western blot检测活性,以表达的E蛋白为包被抗原建立间接ELISA诊断方法。结果获得约1500bp目的片段,与GenBank上的序列同源性100%,其表达产物相对分子质量约为60000,Western blot结果显示该蛋白可与JEV阳性血清结合,具有免疫反应性。建立了间接ELISA检测方法,对不同省份的340份马血清进行检测,其中154份为阳性。结论原核表达JEV的E蛋白具有良好的免疫活性,建立的间接ELISA诊断方法特异性和灵敏性较好,可用于JEV的现地检测。     

Immunogenicity of peptides cleaved by cyanogen bromide from Japanese encephalitis virus envelope glycoprotein E

Envelope glycoprotein (E) prepared from purified Japanese encephalitis (JE) virus was cleaved with cyanogen bromide (CNBr) followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Mice were immunized with 36 kD, 27 kD, and 8 kD bands from CNBr-cleaved and 54 kD band from control specimens. Neutralization test was positive in one out of two anti-54 kD, in none of the anti-36 kD, and all of anti-27 kD and anti-8 kD sera at 1:10 dilution. Geometrical mean ELISA titre was the highest for anti-54 kD followed by anti-36 kD, anti-27 kD, and anti-8 kD sera. Reactivity of these sera to CNBr-cleaved fragments in Western blotting indicated that the 8 kD fragment was a part of the 27 kD but was not included in the 36 kD fragment, while the 27 kD and 36 kD fragments shared an overlapping part. These fragments were located on the E protein by N-terminal amino acid sequencing of each fragment purified by reversed-phase high-performance liquid chromatography and by comparison with the nucleotide sequence of the E protein gene. The 36 kD fragment was located between the third and the ninth methionine and covered most of the N-terminal side of the E protein. In contrast, the 27 kD fragment was located between the fourth and the tenth methionine and included the 8 kD fragment which was situated between the ninth and the tenth methionine near to the C-terminus of the E protein. Denaturation-resistant neutralizing epitope(s) appeared to be present on the 8 kD fragment, but not on the 36 kD fragment.     

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     

Locus of a virus neutralization epitope on the Japanese encephalitis virus envelope protein determined by use of long PCR-based region-specific random mutagenesis

We prepared recombinant Japanese encephalitis (JE) virus populations possessing random mutations at the envelope (E) protein region by a long PCR-based method. Neutralization-resistant mutants were selected from these populations by application of JE-specific virus neutralizing monoclonal antibody (mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, Ile-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, Ile-Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not complete recovery of reactivity to mAb 503. Our results indicate that the amino acid substitutions at amino acid positions 52, 126, 136, and 275 altered the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E(0)-e, D(0)-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome.     

Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein

Summary The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn²⁺ was found to be 20 times more effective than Mg²⁺ in coordinating the catalytic reaction of RdRp, while Ca²⁺ inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.     

Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: Inclusion of the largest protein VP1 in the core and virus-like particles

Bluetongue virus (BTV) VP1 protein, a component of the viral RNA-directed RNA polymerase, but not the VP4 or VP6 proteins, was specifically incorporated into baculovirus expressed BTV core-like particles (composed of VP3 and VP7) and BTV virus-like particles (composed of VP2, VP3, VP5, and VP7). The VP1 protein has been shown to be associated with subcore particles composed of VP3. The data suggest that the VP1 protein of BTV has both enzymatic and structural roles in the virus life cycle.     

Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus.

The nonstructural protein NS1 of Japanese encephalitis virus (JEV) was expressed at a high level under the control of the polyhedrin promoter in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus. Recombinant NS1 was designed to contain its natural signal sequence at its N-terminus and no C-terminal hydrophobic domain that could act as a membrane anchor. This recombinant protein exhibited similar size to native NS1 expressed in Aedes albopictus (C6/36) insect cells infected with wild-type JEV. The signal sequence of NS1 allowed translocation of the protein into the endoplasmic reticulum where it underwent glycosylation. A small fraction of synthesized NS1 was able, in the absence of any other viral protein, to associate as a homodimer, showing similar characteristics to the native dimer. Interestingly, this recombinant dimeric form seemed to be exported and released in the extracellular medium of infected cell culture. During its transport, one of the two N-linked oligosaccharides of the polymannose type was processed to an endoglycosidase H-resistant form, suggesting that the protein had passed through the Golgi compartment before reaching the cell surface. Moreover, Triton X-114 partitioning analysis showed that monomeric NS1 behaved essentially as a hydrophilic protein, whereas both intracellular and extracellular dimeric NS1 were either free of or associated to membraneous components.     

Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III

Immunologic Research - The Japanese encephalitis virus (JEV) is one of the vector borne causes of encephalitis found in southeastern Asia. This positive single-stranded RNA virus is a member of the...     

Characterization of haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus expressed by a recombinant baculovirus

A recombinant baculovirus containing a cDNA which encodes haemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) was constructed. Spodoptera frugiperda cells infected with this recombinant virus produced a large amount of HN glycoprotein similar to the authentic HN in size. The recombinant HN glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. The antigenic properties of the recombinant HN glycoprotein seemed to be slightly different from the authentic one, as judging by the reactivity with a panel of monoclonal antibodies specific to the antigenic sites responsible for neutralization of viral infectivity. Chickens inoculated with the cells infected with the recombinant virus developed haemagglutination-inhibition and virus neutralization antibodies, and were completely protected from the NDV challenge.     

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.