Antigenic analysis of Japanese encephalitis virus by using monoclonal antibodies

Hybridoma cells were produced by fusing P3X63Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with Japanese encephalitis (JE) virus, Nakayama-RFVL strain. The resulting 26 clones produced hemagglutination inhibition antibodies against the homologous strain. The hemagglutination inhibition reactivity of each clone was tested against six flaviviruses: JE, Murray Valley encephalitis (MVE), Egypt 101 strain of West Nile (WN), St. Louis encephalitis (SLE), Russian spring summer encephalitis, and dengue type 1. The 26 monoclonal antibodies fell into four groups: 14 JE species-specific antibodies, 6 antibodies reactive to JE and MVE viruses, 3 antibodies to three or four viruses in the JE-MVE-WN-SLE subgroup, and 3 antibodies to all six flaviviruses. Furthermore, antigenic comparison of 27 strains of JE virus was carried out by using five JE species-specific monoclonal antibodies. Of these, 24 strains were isolated in various parts of Japan, and 3 strains came from Southeast Asia. In reactivity, the 27 strains were classified into at least four antigenic groups. The results showed that the Nakayama-Yakken strain is a mutant strain which lacks the Nakayama strain-specific antigen and that the recently isolated strains are immunologically different from Nakayama and JaGAr 01 strains. One clone (NARMA 13) produced a JE species-specific antibody which showed almost the same titer against 26 JE virus strains, whereas one clone (NARMA 5) produced a Nakayama strain-specific antibody which reacted only to the Nakayama-RFVL and Nakayama-Yoken strains.     

Study of combined foci of mosquito-transmitted arbovirus infections

A total of 5227 serum specimens from humans, horses and swine collected in the seasons of 1968--1976 in 15 administrative areas of the Primorskiy Kray were examined with antigens of a number of mosquito-borne arboviruses: Japanese encephalitis (JE), West Nile (WN), Getah, and Sindbis. Both independent and combined circulation of these viruses in the region was established. Sindbis virus was found to be circulating separately most frequently, West Nile virus the least frequently. According to the results of the serological analysis, the conditions for combined circulation are most closely related for JE and Getah, and JE and WN viruses. The interpretation of the results of examinations of the sera reacting simultaneously with JE and WN virus is most difficult because of close antigenic relationship of these viruses. A set of methods for serological differentiation of bivalent sera to JE and WN viruses and the criteria for the assessment of the results are recommended.     

Seroepidemiological study of infection with West Nile virus in Karachi, Pakistan, in 1983 and 1985

The prevalence of West Nile (WN) virus infection in Karachi, Pakistan, was unknown until 1982. It had been noticed that there were more than a few patients with encephalitides in Karachi, and it was supposed that Japanese encephalitis (JE) cases would be found among them. Therefore, a seroepidemiological study was conducted to define the prevalence of WN virus infection and the possible occurrence of JE virus infection in the Karachi area. Prevalences of haemagglutination inhibition (HI) and neutralization (NT) antibodies against WN virus were studied among 81 serum samples (in July, 33 samples; in September, 48) during 1983, and among 156 paired serum samples that were collected twice, in July and October of 1985. Nearly the same antibody-positive rates were obtained in July of both years (1983: HI 55%; 1985: HI 53%; NT 50%); the rates increased slightly during September/October (1983: HI 65%; 1985: HI 59%, NT 54%). Among 156 paired samples in 1985, 20 (13%) and 12 (8%) showed positive- or negative-antibody conversion between July and October. Two serum samples from each of 156 residents obtained in July had a significantly higher NT antibody titre against JE virus than against WN virus (in case 1, JE 1:80, WN less than 1:10; in case 2, JE 1:40, WN less than 1:10). This is the first report to show the prevalence of WN virus infection in Karachi, Pakistan.     

Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.     

Terminal sequences of the replicative form of RNA of the Japanese encephalitis virus

The 5' and 3' terminal sequences of the replicative form (RF) of RNA of a flavivirus, the Japanese encephalitis (JE) virus, strain Ja0ArS982, have been determined by in vitro labelling and mobility shift analysis. The plus strand sequence was 5'AGAAGUUUAUCUGUGUGAA...UCUOH3', while the minus strand sequence was 5'AGAUCCUGUGUUCUUCCUCA...UCUOH3'. These sequences were similar to those of West Nile (WN) virus being identical in 12 nucleotides at the 5'terminal of the minus strand, and in the 5'terminal dinucleotides, 5'AG3'. Somewhat more internal hexanucleotides 5'CUGUGU3' are conserved among 3 flaviviruses, the JE, WN, and yellow fever viruses.     

Use of Immunoglobulin M Cross-Reactions in Differential Diagnosis of Human Flaviviral Encephalitis Infections in the United States

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.     

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.     

Persistence of tic-borne encephalitis virus in monkeys. III. Phenotypes of the persisting virus

The properties of tick-borne encephalitis (TBE) virus persisting for 90-383 days after intracerebral and subcutaneous inoculation of Macaca rhesus monkeys were studied, namely (1) the type of infection produced directly in the tissues of the experimental monkeys; (2) the activating effect of co-cultivation and explantation procedures; and (3) the phenotype of the isolates by a set of markers. The virus was detected and analysed in 52 instances. Directly in monkey tissues the virus induced a productive infection rarely (5.8%) but more frequently (71.2%) an abortive infection detectable by immunofluorescence (presence of virus-specific antigen). In 23% of instances a nonproductive infection was observed in monkey tissues. Like abortive infection it could be activated by the co-cultivation of cells and explantation procedures. The latter exerted a more marked activating effect than co-cultivation. The strains isolated from monkey tissues in productive infection or activated by explanation or co-cultivation were heterogeneous in their properties. The following virus phenotypes were found: virus highly virulent for mice, cytocidal and antigenically complete; a cytocidal virus of low virulence, possessing haemagglutinin; and a cytocidal virus apathogenic for mice, devoid of haemagglutinin but synthesizing complement-fixing antigen and an antigen detectable by immunofluorescence.     

Two-way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques.

Cross-protection between Japanese encephalitis (JE) and West Nile (WN) viruses was tested in bonnet macaques (Macaca radiata) immunized either with JE virus (JEV) or WN virus (WNV). JEV immunized monkeys were challenged by intranasal (i.n.) route with WNV and vice versa. Four control unimmunized monkeys were similarly infected either with WNV or JEV. Two of three control monkeys infected with WNV, developed paralysis followed by death. Virus was recovered from the central nervous system (CNS) of the both dead control monkeys and the histopathological examination of CNS revealed changes suggestive of viral encephalitis. The control monkey infected with JEV developed encephalitis and the virus was recovered from the blood and CNS. All the 3 JEV-immunized monkeys withstood WNV challenge, whereas only 2 of the 5 WNV immunized monkeys withstood the challenge with JEV. Out of 3 WNV-immunized monkeys surviving challenge with JEV, 2 revealed symptoms suggestive of mild encephalitis followed by complete recovery. The third monkey died on the 60th day post-infection (p.i.) without any symptoms and virus was recovered only from the olfactory lobe. These studies indicate that the immunization with JEV protects the bonnet macaques against WNV, whereas the WNV immunization only reduces the severity of the disease due to JEV.     

Chronic infection of HeLa cells with Japanese encephalitis virus. General characteristics of the system

Chronic infection of HeLa cells was induced by an attenuated variant of Japanese encephalitis (JE) virus (HeLa-K3 cell line). The chronic infection was characterized by alternating phases of degeneration and recovery of the cell monolayer. JE virus was regularly released into the medium of chronically infected cell cultures and virus-specific antigen was regularly demonstrated in the cytoplasm of 15--25% of cells. JE virus persisting in HeLa-K3 cells was sensitive to pancreatic ribonuclease and resistant to treatment with 4 M urea. HeLa-K3 cells did not undergo cytological or karyological transformation; they were susceptible to superinfection with heterologous viruses but resistant to reinfection with homologous virus.     

Persistence of Japanese encephalitis virus clones in continuous HeLa cells

Some virological and biological properties of the H-N and H-P systems (cells of HeLa line clone persistently infected with the Nakayama and Peking I strains of Japanese encephalitis virus, JE, respectively) were studied. A feature distinguishing these systems from all previously described models of persistent infection (PI) of vertebrate cells with JE virus consisted in the lack of visible signs of virus-specific degeneration of the cultures. Attempts to free PI cells from persisting viruses by changing cultivation conditions or with virus-specific antibodies failed. The "zone" phenomenon frequently observed in PI as well as resistance of the cultures to superinfection with isologous viruses only appeared to indicate the presence of interfering particles in the H-N and H-P systems.     

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.     

Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States,Europe, and the Middle East

The complete nucleotide sequences of eight West Nile (WN) virus strains (Egypt 1951, Romania 1996-MQ, Italy 1998-equine, New York 1999-equine, MD 2000-crow265, NJ 2000MQ5488, NY 2000-grouse3282, and NY 2000-crow3356) were determined. Phylogenetic trees were constructed from the aligned nucleotide sequences of these eight viruses along with all other previously published complete WN virus genome sequences. The phylogenetic trees revealed the presence of two genetic lineages of WN viruses. Lineage 1 WN viruses have been isolated from the northeastern United States, Europe, Israel, Africa, India, Russia, and Australia. Lineage 2 WN viruses have been isolated only in sub-Saharan Africa and Madagascar. Lineage 1 viruses can be further subdivided into three monophyletic clades.     

Differences in fusogenicity and mouse neurovirulence of Japanese encephalitis viruses

Summary The fusogenic capacity in AP-61 cell monolayers of 10 strains of Japanese encephalitis (JE) virus from different geographic locations was compared. One strain, isolated from Beijing (JE-Bei), did not fuse AP-61 cells after replication (fusion from within; FFWI), whereas all other strains fused these cells by 72 h post-infection. JE-Bei also readily established a non-cytolytic persistent infection in AP-61 cells. Differences in the envelope proteins of fusogenic and non-fusogenic virus were detected by haemagglutination-inhibition tests and by antigenic analysis using monoclonal antibodies. Yields of infectious virus in either AP-61 or Vero cell cultures were similar if JE-Bei was compared with the fusogenic strain (JE-Sar) but yields of haemagglutinin were 50–100 fold higher with the non-fusogenic virus, implying excessive generation of non-infectious particles. When added directly to AP-61 cell monolayers at pH 6, only JE-Bei produced significant fusion from without (FFWO) presumably reflecting the larger quantity of antigen.Cell monolayers persistently infected with JE-Bei or monolayers treated with UV-inactivated JE-Bei, were resistant to superinfection with JE, West Nile and dengue 2 viruses but were susceptible to infection with the alphavirus Sindbis. When administered intracerebrally (I/C) to newborn and weanling mice, the viruses were equally neurovirulent. However, fusogenic JE-Sar was significantly more neurovirulent than JE-Bei for weanling mice after intraperitoneal (I/P) or subcutaneous (S/C) inoculation. Mice given non-fusogenic JE-Bei, resisted the peritoneal challenge with fusogenic JE-Sar, and West Nile but not Semliki Forest virus when given 6 h after the first virus.The potential significance of cell fusion by JE virus and interference through over production of non-infectious virus, is discussed in the context of JE virus virulence.     

中国分离乙脑病毒与灭活疫苗株(P3株)E基因差异分析

目的 分析我国近年来从蚊虫及患者标本中分离的乙脑病毒与灭活疫苗株（P3株）之间在E基因区段核苷酸及氨基酸差异。方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列，通过Clustal X（1．8）、DNASTAR、GENEDOC（3．2）等生物学软件进行分析。结果 P3株与福建分离株之间核苷酸同源性在98．3％-98．5％之间、氨基酸同源性在98．2％-98．6％之间；P3株与上海分离株之间核苷酸同源性在88．0％-88．5％之间、氨基酸同源性在98．0％-98．4％之间。E基因区段500个氨基酸中P3株与所有新分离乙脑病毒株之间共存在19个位点的差异，其中在E-76、E-306、E-408处所有新分离毒株与P3株存在共同的差异；在E-160、E-487处福建分离株与P3株存在共同差异；上海分离株与P3株在E-129、E-222、E-227、E-366存在共同差异。结论 上海蚊虫中分离的Ⅰ型乙脑病毒和福建省脑炎患者中分离的Ⅲ型乙脑病毒与P3株在E基因的部分氨基酸位点存在差异，但均不处在影响病毒生物学特性的关键位点。     

Serological monitoring of arbovirus infections in the estuary of the Kuban River (the 2006-2007 data)

Solid-phase enzyme immunoassay, neutralization test, and the hemagglutination-inhibition test were used to study the sera from human beings (152 samples), agricultural animals (n = 77), hares (n = 3), and wild birds (n = 69), collected in 2006-2007 in the Kuban River estuary (Temryuk District, Krasnodar Territory). There were specific antibodies against viruses of West Nile (WH), tick-borne encephalitis (TBE) (Flaviviridae, Flavivirus), Sindbis (Togaviridae, Alphavirus), the antigenic complex of California, Batai (Bunyaviridae, Orthobunyavirus), Dhori (Orthomyxoviridae, Thogotovirus). The findings suggest the presence of arboviruses from 6 transmitting mosquitoes and ticks in the study area and human infection by the viruses of the antigenic complex of California (20-47%), Batai (3-15%), West Nile (3-12%), Dhori (2%). The index agricultural animals (horses, cattle) were observed to have specific antibodies to the viruses of WN (8-15%), TBE (0-2%), Sindbis (2-9%), the antigenic complex of California (27-54%). Out of the representatives of the wild fauna, virus-neutralizing antibodies to Sindbis virus were found in European hares (Lepus europaeus), California complex virus in gulls (Larus argentatus) and terns (Sterna hirundo), WN and Sindbis viruses in herons (Ardea purpurea), and WN and California complex viruses in bald-coots (Fulica atra).     

Replication of non-respiratory viruses in tracheal organ cultures.

Employing Hoorn''s technique, tracheal explant cultures were set from adult hamsters, rabbits and human foetuses. To determine the susceptibility of these cultures they were infected with nine different mainly non-respiratory viruses. Assay of virus was carried out in susceptible cell lines. The results of these studies indicated that herpes simplex type I (HSV-1) and vaccinia viruses multiplied in these cultures and caused ciliostasis. Herpes simplex type 2 (HSV-2) although multiplying in all, produced ciliostasis only in human foetal tracheal cultures. However, Chandipura (CHP), West Nile (WN), sandfly fever (SF-N) and polio-1 viruses multiplied without ciliostasis. These cultures did not support multiplication of Japanese encephalitis (JE) and Chikungunya (CHIK) viruses. The studies indicated that mammalian tracheal cultures support replication of the non-respiratory viruses. The continued and undiminished multiplication of viruses over long periods without ciliostasis suggests a role for the trachea in the transmission of viral infections by aerosol.     

Influenza virus infection in newborn rats: a possible marker of attenuation for man.

The growth of parent influenza viruses A/England/939/69 and A/PR/8/34, and clones 6, 7, and 64C, derived by recombination, was studied in newborn rats. Using an inoculum of 10(4.0) EID50, influenza virus A/England/939/69 produced the highest titres of virus in rat turbinates at 48 hours after inoculation; clones 6 and 7 and A/PR/8/34 grew to lower titres; and clone 64C grew to the lowest titre. These differences were less apparent when 10(2.0) EID50 of virus was used as an inoculum, and rats were not infected by smaller inoculum of any of the virus strains. Infection with 10(4.0) EID50 of all viruses produced lung infection; at 48 hours after infection, the highest titres were recovered from rats infected with A/PR/8/34 and A/England/939/69 virus. Prior infection with A/England/939/69 or A/PR/8/34 increased the incidence of bacteraemia and meningitis following intranasal inoculation of Haemophilus influenzae type b; infection with clone 64C did not enhance bacterial meningitis, while infection with clone 6 gave an intermediate result. Volunteer studies with these viruses have shown that influenza virus A/England/939/69 was virulent, clones 6 and 7 were attenuated, clone 64C was over-attenuated, and A/PR/8/34 virus was noninfective for man. The relative titres of virus recovered from turbinates taken 48 hours after infection with 10(4.0) EID50 of virus and the ability of virus infection to enhance bacterial infection correlated with the property of virus attenuation for man for four of the five strains tested; however, no correlation was seen for A/PR/8/34 virus, which is a result also found in other laboratory tests designed to measure virulence for man.