Neutralizing antibody responses to Japanese encephalitis vaccine in children

Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.     

Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.     

Human rotavirus K8 strain represents a new VP4 serotype.

The complete VP4 gene of the human rotavirus (HRV) K8 strain (G1 serotype) was cloned and inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. A K8VP4 recombinant baculovirus was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with transfer vector DNA containing the K8VP4 gene and wild-type baculovirus DNA. Infection of Sf9 cells with this VP4 recombinant baculovirus resulted in the production of a protein that is similar in size and antigenic activity to the authentic VP4 of the K8 strain. Guinea pigs immunized with the expressed VP4 developed antibodies that neutralized the infectivity of the K8 strain. This antiserum neutralized HRV strains belonging to VP4 serotypes 1A, 1B, and 2 with efficiency eightfold or lower than that of the homologous virus, indicating that the human rotavirus K8 strain represents a distinct VP4 serotype (P3). In addition, low levels of cross-immunoprecipitation of the K8VP4 and its VP5 and VP8 subunits with hyperimmune antisera to HRV strains representing different VP4 serotype specificities also suggested that the K8 strain possesses a unique VP4 with few epitopes in common with other P-serotype strains.     

利用单克隆抗体对乙型脑炎病毒不同毒株抗原分析的研究

实验发现,应用动物免疫血清(多克隆抗体,PcAb)进行抗原分析时,我国分离到的乙型脑炎病毒SA14、P3、A2和高株的抗原性无明显差别,与从日本分离到的中山株有些差别,但仍被PcAb所中和;而用单克隆抗体(McAb)进行分析,情况就明显不同:SA14和P3株抗原性相似,高株和中山株不能被51-8McAb所中和,A2株则介于两者之间。A2和高顺生株的寡核苷酸指纹图谱分析表明,高株比A2株多两个斑点,但大多数斑点是一样的,证实了上述结果。实验还发现不同毒株的保护效果不同,A2株的免疫效果明显优于高株。     

Serotypes of Vibrio parahaemolyticus from clinical and environmental sources in Togo (West Africa).

Serological analysis of O and K antigens was performed on 343 strains of Vibro parahaemolyticus isolated from clinical and environmental sources in Togo. Only two strains were not typable by the available O antisera. K untypable strains were found in 4.8% of isolates from gastroenteritis patients, in 11% from healthy carriers, and in 47% and 46% of isolates, respectively, from water and fish samples. Thirteen serotypes identified in Togo are not considered in the Japanese antigenic scheme. The suitability of the Japanese typing scheme for geographic areas outside of Japan is discussed and its extension is suggested.     

Antitoxic immunity against the so-called lung toxin produced by Escherichia coli.

Cross neutralization test with antisera to crude haemolysins produced by some Escherichia coli strains indicated that there were no antigenic differences among the haemolysins tested. These crude preparates showed definite cytotoxicity which could also be cross neutralized by "antihaemolysin" sera. Neutralization experiments were performed in mouse lung test with homologous and heterologous anti-haemolysin sera, and with O and OK sera to the wild type strain and its toxic R mutant. The results showed that the immunity in the mouse lung model is antitoxic and antibacterial.     

Immunochemical analysis of substance P using its synthetic fragments and analogs

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3.

Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. II. Prevalence of Japanese Encephalitis Antibody in Residents in Okinawa,Miyako and Ishigaki Islands

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.     

Identification of Flagellar (H) antigenci subfactors in Bacillus thuringiensis H serotypes 10, 18, and 24 isolated in Japan

A total of 95 Bacillus thuringiensis strains isolated from Japan and belonging to H serotypes 10, 18 and 24, were examined for their H antigenic subfactors. Of 84 H serotype 10 isolates, 83 were identified as the H serotype 10a: 10b (serovar darmstadiensis ) and only one isolate was assigned to the II serotype 10a: 10c (serovar londrina ). Among five isolates belonging to the H serotype 18, three were allocated to the H serotype 18a: 18b (serovar kumamotoensis ), while two isolates did not react to antisera against the two known H antigenic subfactors, 18b and 18c. All of the six H serotype 24 isolates were assigned to the H serotype 24a: 24b (serovar neoleonensis ).     

Serological relationships among genotypic variants of betanodavirus

Betanodaviruses, the causative agents of viral nervous necrosis or viral encephalopathy and retinopathy, are divided into 4 genotypes based on the coat protein gene (RNA2). In the present study, serological relationships among betanodavirus genotypic variants were examined by virus neutralization tests using rabbit antisera raised against purified virions of strains representative of each genotype. All 20 isolates examined shared epitopes for neutralizing, but they fell into 3 major serotypes (A, B, C). This sero-grouping is in part consistent with their genotypes, i.e. Serotype A for striped jack nervous necrosis virus (SJNNV) genotype, Serotype B for tiger puffer nervous necrosis virus (TPNNV) genotype, and Serotype C for both redspotted grouper nervous necrosis virus (RGNNV) and barfin flounder nervous necrosis virus (BFNNV) genotypes. The serological relatedness between RGNNV and BFNNV genotypes may result from their relatively higher similarity in RNA2 sequences. In neutralization tests using antisera of kelp grouper Epinephelus moara, which were raised against recombinant coat proteins representing each genotype, anti-SJNNV and anti-TPNNV sera neutralized only the homologous strain, and anti-RGNNV and anti-BFNNV sera reacted with both RGNNV and BFNNV strains. The present serological findings will be important in investigating the infectivity and host-specificity of betanodaviruses and in developing vaccines for the disease.     

Formation of mixed hemagglutinin trimers during double infection with influenza virus strains of the same subtype

Formation of hemagglutinin spikes in the course of the mixed infection of cell culture by two influenza virus strains belonging to the same antigenic subtype or to different subtypes was studied by means of immunoprecipitation of [14C]-labelled hemagglutinins from cell lysates. The immunoprecipitates were further analysed by polyacrylamide gel electrophoresis. Lysates of separately infected cells mixed before lysis were used as control samples. The analysis of immunoprecipitates revealed the formation of chimeric hemagglutinin spikes in the cells infected by the strains possessing hemagglutinins of the same subtype but not in the cells infected by the strains of different subtypes (H1 and H3). The results are discussed in connection with the homology of amino-acid sequences of influenza virus hemagglutinins.     

Occurrence ofAlexandrium cohorticula in Japanese coastal water

Two clones ofAlexandrium cohorticula were isolated at Aburatsubo, Sagami Bay, Japan. Cultured cells of both contained high amounts of paralytic shellfish toxins. The toxicity of these isolates was comparable with that of highly toxic Thai clones. No significant difference in toxin components or their proportions was observed between Japanese and Thai strains. The optimum growth temperature of both strains was around 25 °C. Japanese strains survived at 15 °C, whereas Thai strains did not; the latter grew faster than the former at 30 °C.     

Recovery and altered neutralization specificities of chimeric viruses containing capsid protein domain exchanges from antigenically distinct strains of feline calicivirus

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization.     

Evidence for natural reassortants of human rotaviruses belonging to different genogroups.

Of 335 rotavirus isolates associated with diarrheal disease in Bangladesh that were culture adapted and subsequently characterized for electropherotype, subgroup, and serotype, 9 had properties that suggested they may be natural reassortants between human rotaviruses belonging to different "genogroups." Two of these were examined in greater detail by RNA-RNA hybridization with prototype strains representative of each of the three proposed human rotavirus genogroups. One subgroup II isolate, 248, with a "long" electrophoretic pattern was neutralized by hyperimmune antisera to both serotype 2 and 4 strains. Consistent with these results, seven RNA segments of this isolate formed hybrids with human strains belonging to the Wa genogroup and four segments hybridized with strains belonging to the DS-1 genogroup. The second isolate examined, 456, belonged to subgroup II and had a long electrophoretic pattern but was found to be a serotype 2 strain. This isolate also appeared to be an intergenogroup reassortant because three of its segments formed hybrids with strains belonging to the Wa genogroup and eight hybridized with viruses of the DS-1 genogroup. On the basis of the relative migration rates of these RNA-RNA hybrids during gel electrophoresis, a suggested origin for each gene segment was proposed which was consistent with the results expected from electrophoretic, subgroup, and serotypic analyses.     

Global identification of three major genotypes of varicella-zoster virus: longitudinal clustering and strategies for genotyping

By analysis of a single, variable, and short DNA sequence of 447 bp located within open reading frame 22 (ORF22), we discriminated three major varicella-zoster virus (VZV) genotypes. VZV isolates from all six inhabited continents that showed nearly complete homology to ORF22 of the European reference strain Dumas were assigned to the European (E) genotype. All Japanese isolates, defined as the Japanese (J) genotype, were identical in the respective genomic region and proved the most divergent from the E strains, carrying four distinct variations. The remaining isolates carried a combination of E- and J-specific variations in the target sequence and thus were collectively termed the mosaic (M) genotype. Three hundred twenty-six isolates collected in 27 countries were genotyped. A distinctive longitudinal distribution of VZV genotypes supports this approach. Among 111 isolates collected from European patients, 96.4% were genotype E. Consistent with this observation, approximately 80% of the VZV strains from the United States were also genotype E. Similarly, genotype E viruses were dominant in the Asian part of Russia and in eastern Australia. M genotype viruses were strongly dominant in tropical regions of Africa, Indochina, and Central America, and they were common in western Australia. However, genotype M viruses were also identified as a minority in several countries worldwide. Two major intertypic variations of genotype M strains were identified, suggesting that the M genotype can be further differentiated into subgenotypes. These data highlight the direction for future VZV genotyping efforts. This approach provides the first simple genotyping method for VZV strains in clinical samples.     

Comparison of AP-PCR methods,ribotyping (PCR-ribotyping) and pulsed-field electrophoresis (PFGE) for strains of Clostridium difficile producing toxin B and not producing toxin A

In this study were used AP-PCR, PCR-ribotyping and pulsed-field elecrophoresis (PFGE) for comparative study of toxin A-negative/toxin B-posi-tive Clostridium difficile strains with deletion in toxin A gen. We investigated nine unrelated clinical strains, isolated from different units and different time from patients suffering to antibiotic associated diarrhea (AAD). We found that toxin A-negative/toxin B-positive C. difficile strains isolated in Poland belonging to a single genotype A, are being similar to the Japanese strains.     

Monitoring Antigenic Variations of Enterovirus 71: Implications for Virus Surveillance and Vaccine Development

Enterovirus 71 (EV71) causes life-threatening epidemics in Asia and can be phylogenetically classified into three major genogroups (A∼C) including 11 genotypes (A, B1∼B5, and C1∼C5). Recently, EV71 epidemics occurred cyclically in Taiwan with different genotypes. In recent years, human studies using post-infection sera obtained from children have detected antigenic variations among different EV71 strains. Therefore, surveillance of enterovirus 71 should include phylogenetic and antigenic analysis. Due to limitation of sera available from children with EV71 primary infection, suitable animal models should be developed to generate a panel of antisera for monitoring EV71 antigenic variations. Twelve reference strains representing the 11 EV71 genotypes were grown in rhabdomyosarcoma cells. Infectious EV71 particles were purified and collected to immunize rabbits. The rabbit antisera were then employed to measure neutralizing antibody titers against the 12 reference strains and 5 recent strains. Rabbits immunized with genogroup B and C viruses consistently have a lower neutralizing antibody titers against genogroup A (≧8-fold difference) and antigenic variations between genogroup B and C viruses can be detected but did not have a clear pattern, which are consistent with previous human studies. Comparison between human and rabbit neutralizing antibody profiles, the results showed that ≧8-fold difference in rabbit cross-reactive antibody ratios could be used to screen EV71 isolates for identifying potential antigenic variants. In conclusion, a rabbit model was developed to monitor antigenic variations of EV71, which are critical to select vaccine strains and predict epidemics.     

Genetic typing of Mycobacterium tuberculosis strains by spoligotyping and genome fingerprinting techniques

A total of 122 M. tuberculosis clinical drug-resistant strains isolated in Central Russia were studied by spoligotyping and genome fingerprinting techniques. According to spoligotyping results 77% of M. tuberculosis strains were distributed to 13 oligotypes, while 23% of these strains were found to form unique patterns. Most of them belonged to the families Beijing and Haarlem (43.4% and 13.9% respectively). The patterns of the strains of oligotype 12 (7F-7F-7E-0E-78-3E) were identical to those of the strains isolated in Brazil, France and the Netherlands. The strains of the spoligotype 22 (7F-1E-7F-7F-07-3E) had the patterns identical to those of the strains of group S13, also isolated in Brazil. According to genome fingerprinting 31.4% of the strains were found to belong to clusters with the similarity coefficient equal to 1. The strains belonging to genotypes W and A1 were found to prevail in the analyzed group.