RESUMEN
The diagnosis of human T -cell leukemia virus type 1 (HTLV-1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV-1 and/or HTLV-2 are used to confirm infection with HTLV-1 and/or HTLV-2 and are also used for the follow-up of HTLV-1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV-1 and HTLV-2, a multiplex quantitative polymerase chain reaction (qPCR) by large-scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV-1 provirus per 105 cells. Moreover, HTLV-1 provirus could be detected in 97.2% (205 of 211) of HTLV-1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV-2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV-1 and HTLV-2 provirus with extremely high sensitivity.
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Infecciones por HTLV-I/diagnóstico , Infecciones por HTLV-II/diagnóstico , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 2 Humano/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Provirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones por HTLV-I/virología , Infecciones por HTLV-II/virología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Japón , Provirus/genética , Sensibilidad y EspecificidadRESUMEN
In the original publication of article [1], '20 × 101 copies', which is in the sentence 'As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 101 copies in 2 µL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)' changes to '2.0 × 101 copies' in results section. The publisher apologizes to the readers and authors for the inconvenience.
RESUMEN
BACKGROUND: JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS: A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS: The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS: The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.
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Líquido Cefalorraquídeo/virología , Virus JC/aislamiento & purificación , Leucoencefalopatía Multifocal Progresiva/diagnóstico , Leucoencefalopatía Multifocal Progresiva/virología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus). METHODS: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay. RESULTS: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes. CONCLUSION: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.
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Anticuerpos Neutralizantes/inmunología , Callithrix/inmunología , Coinfección/inmunología , Virus del Dengue/genética , Virus del Dengue/inmunología , Dengue/inmunología , Genotipo , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Callithrix/virología , Coinfección/sangre , Reacciones Cruzadas/inmunología , Dengue/sangre , Dengue/prevención & control , Vacunas contra el Dengue/inmunología , Virus del Dengue/clasificación , Modelos Animales de Enfermedad , Pruebas de Neutralización , SerogrupoRESUMEN
Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Dengue/inmunología , Vacunas contra el Dengue/genética , Virus del Dengue/clasificación , Virus del Dengue/genética , Genotipo , Humanos , Pruebas de Neutralización , Filogenia , Serogrupo , Proteínas del Envoltorio Viral/clasificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.
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Callithrix , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Modelos Animales de Enfermedad , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Sangre/virología , Vacunas contra el Dengue/administración & dosificación , Virus del Dengue/aislamiento & purificación , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Enfermedades de los Monos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Viremia/prevención & controlRESUMEN
Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.
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Agua de Mar/microbiología , Vibrio cholerae/aislamiento & purificación , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio vulnificus/aislamiento & purificación , Algoritmos , Carga Bacteriana/métodos , Microbiología Ambiental , Humanos , Japón , Modelos Teóricos , Reacción en Cadena de la Polimerasa , Salinidad , Agua de Mar/química , Temperatura , Vibrio cholerae/clasificación , Vibrio cholerae/genética , Vibrio parahaemolyticus/clasificación , Vibrio parahaemolyticus/genética , Vibrio vulnificus/clasificación , Vibrio vulnificus/genéticaRESUMEN
Potency controls of inactivated rabies vaccines for human use are confirmed by the National Institutes of Health challenge test in which lethal infection with severe neurological symptoms should be observed in approximately half of the mice inoculated with the rabies virus. Weight loss, decreased body temperature, and the presence of rabies-associated neurological signs have been proposed as humane endpoints. The potential for reduction of animal suffering by introducing humane endpoints in the potency test for inactivated rabies vaccine for human use was investigated. The clinical signs were scored and body weight was monitored. The average times to death following inoculation were 10.49 and 10.99 days post-inoculation (dpi) by the potency and challenge control tests, respectively, whereas the average times to showing Score-2 signs (paralysis, trembling, and coma) were 6.26 and 6.55 dpi, respectively. Body weight loss of more than 15% appeared at 5.82 and 6.42 dpi. The data provided here support the introduction of obvious neuronal signs combined with a body weight loss of ≥15% as a humane endpoint to reduce the time of animal suffering by approximately 4 days.
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Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Rabia/inmunología , Vacunación/métodos , Potencia de la Vacuna , Animales , Peso Corporal/inmunología , Embrión de Pollo , Femenino , Humanos , Ratones , Rabia/mortalidad , Rabia/virología , Análisis de Supervivencia , Tasa de Supervivencia , Factores de Tiempo , Vacunas de Productos Inactivados/inmunología , Pérdida de Peso/inmunologíaRESUMEN
BACKGROUND: Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS: Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS: A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION: This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection.
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Virus del Dengue/genética , Dengue/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Serogrupo , Reacción a la Transfusión , Seguridad de la Sangre , Dengue/prevención & control , Dengue/transmisión , Humanos , Reacción en Cadena de la Polimerasa Multiplex , ARN Viral/análisis , ARN Viral/sangre , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus, and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination. METHODS: Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells. RESULTS: A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %). CONCLUSION: In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.
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Acrecentamiento Dependiente de Anticuerpo , Virus del Dengue/inmunología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Vacunas contra la Encefalitis Japonesa/inmunología , Adulto , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Chlorocebus aethiops , Cricetinae , Reacciones Cruzadas , Dengue/inmunología , Dengue/virología , Virus del Dengue/patogenicidad , Encefalitis Japonesa/virología , Femenino , Humanos , Vacunas contra la Encefalitis Japonesa/efectos adversos , Masculino , Persona de Mediana Edad , Vacunación , Vacunas de Productos Inactivados/inmunología , Células VeroRESUMEN
The characteristics of genotype V Japanese encephalitis virus (GV JEV) remain poorly understood as only two strains have been isolated to date. In this study, we examined the effects of the GV JEV Muar strain on in vitro growth and pathogenicity in mice; we also evaluated the efficacy of inactivated JEV vaccines against the Muar strain. Although growth of the Muar strain in mouse neuroblastoma N18 cells was clearly worse than that of the GIII Beijing-1 and GI Mie/41/2002 strains, neuroinvasiveness of the Muar strain was similar to that of the Beijing-1 strain and significantly higher than that of the Mie/41/2002 strain. The results of a plaque reduction neutralization test suggested that the neutralization ability of the JEV vaccines against the Muar strain was reduced compared with the GI and GIII strains. However, the protection potency of the JEV vaccine against the Muar strain was similar to that for the Beijing-1 strain in mice. Our data indicate that GV JEV has unique growth, virulence and antigenicity features.
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Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Japonesa (Especie)/crecimiento & desarrollo , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/virología , Animales , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/inmunología , Femenino , Genotipo , Humanos , Vacunas contra la Encefalitis Japonesa/administración & dosificación , Vacunas contra la Encefalitis Japonesa/inmunología , Masculino , Ratones , Pruebas de Neutralización , VirulenciaRESUMEN
Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. The critical factors determining mortality and severity of JE virus (JEV) infection remain unclear. We identified brain-infiltrating T cells associated with a fatal outcome of JEV infection in mice. Dying mice were defined as those that lost more than 25 % of their body weight by day 13 and died by day 21, while surviving mice were defined as those that lost less than 10 % by day 13, based on the result of the survival time course study. Two groups of five mice that demonstrated brain virus titers of >1 × 10(6) pfu/g were randomly selected from the dying and surviving groups and used in the analyses. Cytokine patterns in brains were first examined, revealing a higher ratio of Th1-related cytokine genes in dying mice. The expression levels of CD3, CD8, CD25, and CD69 increased in JEV-infected mice relative to mock-infected mice. However, expression levels of these cell-surface markers did not differ between the two groups. T-cell receptor (TCR) usage and complementary determining region 3 (CDR3) sequences were analyzed in the brain-infiltrating T cells. T cells expressing VA8-1, VA10-1, and VB2-1 increased in both groups. However, the dominant T-cell clones as defined by CDR3 amino acid sequence differed between the two groups. The results indicate that the outcome of JEV infection, death or survival, was determined by qualitative differences in infiltrating T-cell clones with unique CDR3 amino acid sequences.
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Encéfalo/inmunología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/mortalidad , Subgrupos de Linfocitos T/inmunología , Animales , Peso Corporal , Encéfalo/virología , Regiones Determinantes de Complementariedad/genética , Modelos Animales de Enfermedad , Ratones , Receptores de Antígenos de Linfocitos T/genética , Análisis de SupervivenciaRESUMEN
BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.
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Infecciones por Bunyaviridae/diagnóstico , Phlebovirus/aislamiento & purificación , Animales , Infecciones por Bunyaviridae/virología , Chlorocebus aethiops , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Phlebovirus/genética , Filogenia , Estudios Retrospectivos , Células VeroRESUMEN
We report a patient with congenital Chagas disease in Japan. This report reemphasizes the role of neglected and emerging tropical diseases in the era of globalization. It also indicates the need for increased vigilance for detecting Chagas disease in non-disease-endemic countries.
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Enfermedad de Chagas/transmisión , Transmisión Vertical de Enfermedad Infecciosa , Trypanosoma cruzi , Adolescente , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/tratamiento farmacológico , Humanos , Japón , Masculino , Enfermedades Desatendidas , Nitroimidazoles/administración & dosificación , Nitroimidazoles/uso terapéutico , Resultado del Tratamiento , Tripanocidas/administración & dosificación , Tripanocidas/uso terapéutico , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genéticaRESUMEN
There are four dengue virus (DENV) serotypes. Primary infection with one does not confer protective immunity against the others. We have reported previously that the marmoset (Callithrix jacchus) is a useful primary DENV infection model. It has been reported that secondary DENV infection with a heterotypic serotype induces viraemia kinetics and antibody responses that differ from those in primary infection. Thus, it is important to determine the utility of the marmoset as a model for secondary DENV infection. Marmosets were infected with heterologous DENV by secondary inoculation, and viraemia kinetics and antibody responses were analysed. The marmosets consistently developed high levels of viraemia after the secondary inoculation with heterologous DENV serotypes. IgM responses were lower compared with primary inoculation responses, whilst IgG responses were rapid and high. Neutralizing activities, which possessed serotype cross-reactive activities, were detected as early as 4 days after inoculation. In addition, infectious viraemia titres were higher when assayed with Fcγ receptor-expressing baby hamster kidney (BHK) cells than when assayed with conventional BHK cells, suggesting the presence of infectious virus-antibody immune complexes. After secondary infection with heterotypic DENV, the marmosets demonstrated viraemia kinetics, IgM and IgG responses, and high levels of serotype cross-reactive neutralizing antibody responses, all of which were consistent with secondary DENV infection in humans. The results indicate the marmoset as a useful animal for studying secondary, as well as primary, DENV infection.
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Anticuerpos Antivirales/inmunología , Callithrix , Coinfección/inmunología , Virus del Dengue/fisiología , Dengue/inmunología , Viremia/inmunología , Animales , Callithrix/inmunología , Callithrix/virología , Línea Celular , Coinfección/virología , Cricetinae , Reacciones Cruzadas , Dengue/virología , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Humanos , Viremia/virologíaRESUMEN
Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.
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Brotes de Enfermedades , Virus del Moquillo Canino/aislamiento & purificación , Moquillo/epidemiología , Moquillo/virología , Enfermedades de los Primates/epidemiología , Enfermedades de los Primates/virología , Animales , China/epidemiología , Chlorocebus aethiops , Análisis por Conglomerados , Moquillo/mortalidad , Moquillo/patología , Virus del Moquillo Canino/clasificación , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/patogenicidad , Heces/virología , Macaca fascicularis , Macaca mulatta , Datos de Secuencia Molecular , Filogenia , Enfermedades de los Primates/mortalidad , Enfermedades de los Primates/patología , ARN Viral/genética , Saliva/virología , Análisis de Secuencia de ADN , Análisis de Supervivencia , Células Vero , Esparcimiento de VirusRESUMEN
A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.
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Adaptación Fisiológica/genética , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Brotes de Enfermedades , Virus del Moquillo Canino/metabolismo , Macaca/virología , Enfermedades de los Monos/virología , Receptores de Superficie Celular/metabolismo , Sustitución de Aminoácidos , Animales , Chlorocebus aethiops , Moquillo/epidemiología , Moquillo/virología , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/patogenicidad , Perros , Células Epiteliales/metabolismo , Células Epiteliales/virología , Hemaglutininas Virales/genética , Humanos , Enfermedades de los Monos/epidemiología , Enfermedades de los Monos/mortalidad , Receptores Virales/metabolismo , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Células VeroRESUMEN
Dengue virus (DENV) causes a life-threatening illness, with a wide range of symptoms from mild febrile illness, dengue fever (DF), to life-threatening illness, dengue hemorrhagic fever (DHF). Antibody-dependent enhancement (ADE) is considered to be a risk factor for DHF. In the present study, we determined the parameters for ADE assays using FcγR-expressing BHK cells. Monoclonal antibodies and human serum samples were used in the assays. We examined antibody concentration and virus concentration and analyzed whether antibody concentration or DENV-antibody ratio determines ADE activity. Virus growth was quantified by a conventional plaque titration method using FcγR-expressing BHK cells. The assay allowed the detection of DENV growth with inoculation doses ranging from 10(2) PFU/ml to 10(6) PFU/ml using monoclonal antibodies and undiluted or diluted serum samples. The results indicate that antibody concentration rather than DENV-antibody ratio determines the demonstration of ADE activity. Thus, antibody concentration rather than multiplicity of infection was defined as the main determinant in ADE assays using FcγR-expressing BHK cells.
Asunto(s)
Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Virus del Dengue/inmunología , Receptores de IgG/inmunología , Pruebas Serológicas/métodos , Dengue Grave/inmunología , Animales , Anticuerpos Antivirales/análisis , Línea Celular , Cricetinae , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Humanos , Receptores de IgG/genética , Dengue Grave/diagnóstico , Dengue Grave/virologíaRESUMEN
We detected two viruses, Japanese encephalitis virus (JEV)/Kochi/01/2005 and Getah virus (GETV)/Kochi/01/2005 in the same culture supernatant obtained by inoculation of Vero cells with a swine serum sample and subsequent passaging of the supernatant in Vero cells. Phylogenetic analysis using the nucleotide sequences of the complete genome and the E2 region of GETV indicated that GETV/Kochi/01/2005 is most similar to a Mongolian strain. In contrast, a partial sequence of the nsP1 protein coding region of GETV/Kochi/01/2005 showed that it was similar to Japanese strains isolated in the 1980s. Alignment of the nucleotide sequence of the E region of JEV showed that JEV/Kochi/01/2005 has the highest similarity to a Japanese strain. We also examined the changes in the amount of JEV/Kochi/01/2005 and GETV/Kochi/01/2005 present after passaging in Vero cells. The RNA copy number and infectious titer of JEV/Kochi/01/2005 decreased, whereas those of GETV/Kochi/01/2005 increased, following repeated passages in Vero cells. Our results provide evidence for coinfection with JEV and GETV in the Kochi/01/2005 pig. This is the first report of incidental confection with JEV and GETV in a domestic animal.
Asunto(s)
Infecciones por Alphavirus/veterinaria , Alphavirus/fisiología , Coinfección/veterinaria , Virus de la Encefalitis Japonesa (Especie)/fisiología , Encefalitis Japonesa/veterinaria , ARN Viral/sangre , Enfermedades de los Porcinos/virología , Alphavirus/clasificación , Alphavirus/genética , Alphavirus/aislamiento & purificación , Infecciones por Alphavirus/virología , Animales , Chlorocebus aethiops , Coinfección/virología , Virus de la Encefalitis Japonesa (Especie)/clasificación , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Encefalitis Japonesa/virología , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Porcinos , Células VeroRESUMEN
Rabies is a viral disease transmitted through bites from rabid animals and can be prevented by vaccines. Clinically used rabies vaccines are prepared from inactivated rabies viruses grown in cell cultures or embryonated eggs. In Japan and across the world, tests that confirm complete inactivation, such as the in vivo suckling mouse assay, in which suckling mice are intracerebrally inoculated with vaccine products, are required for quality control. In this study, we developed a novel cell-based immunofluorescence assay that does not require mice for testing rabies vaccine inactivation for human use. The sensitivity of this cell-based in vitro assay was 5.7 times that of the in vivo suckling mouse assay, with a detection limit of one focus forming units per ml of test sample. This newly developed in vitro assay may replace the established in vivo suckling mouse assay for confirming viral vaccine inactivation.