RESUMO
Cultured mononuclear peripheral blood leukocytes (PBL) from nonimmune human beings and monkeys are nonpermissive to dengue 2 virus (D2V) infection at multiplicities of infection of 0.001-0.1, but become permissive when non-neutralizing dengue antibody is added to medium. D2V infection occurred in PBL prepared from anti-coagulated but not from defibrinated plasma. Infection enhancement was produced by multiple lots of heterotypic anti-dengue raised in several mammalian species. Homotypic anti-dengue neutralized D2V at high concentrations but enhanced at low concentrations; enhancement end point in one serum was 1:320,000. The infection-enhancing factor was a noncytophilic antibody of the IgG class. D2V infection occurred in the absence of heat-labile complement components but did not occur when complexes were prepared with anti- dengue F(ab)(2). Treatment of PBL with several proteases increased permissiveness to D2V infection by immune complexes but not by virus alone. Two rhesus monkey serums collected 14 days after D2V infection contained an IgG antibody with high-titered enhancing activity but with no hemagglutination-inhibition or neutralizing activity. Virus-antibody complexes are irreversibly attached to PBL within 15 min and completely internalized in 60 min. There was considerable variation in cellular infection in different experiments, however, maximum virus yields usually exceeded 1,000 plaque-forming units per 1 x 10(6) PBL occurring between 2 and 4 days in culture. In vitro antibody-dependent infection of PBL provides a possible model for study of pathogenetic mechanisms in infants with dengue shock syndrome who passively acquire maternal anti-dengue IgG.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Dengue/imunologia , Fagócitos/imunologia , Animais , Sítios de Ligação de Anticorpos , Haplorrinos , Testes de Inibição da Hemaglutinação , Heparina/farmacologia , Humanos , Imunidade Celular , Imunoglobulina G , Imunoglobulina M , Leucócitos/imunologia , Leucócitos/microbiologia , Macaca mulatta , Testes de Neutralização , Peptídeo Hidrolases , Fagócitos/microbiologia , Replicação ViralRESUMO
Studies were made on the identity of human and monkey mononuclear leukocytes permissive to antibody-enhanced dengue 2 virus (D2V) infection. In cultures of peripheral blood leukocytes (PBL) inoculated immediately after separation, it was concluded that only mononuclear phagocytes support dengue infection. This is based upon observations that D2V-permissive cells were resistant to 1,200 rads, were both plastic adherent and nonadherent, were removed when passed through nylon wool columns in 10 percent fetal bovine serum or 100 percent autologous serum, and were destroyed by incubation with 100 mug/ml particulate silica. On direct immunofluorescence staining, perinuclear dengue antigen was visualized at 24 h, becoming maximal at 60 h. Antigen-containing cells had ample cytoplasm, ruffled cytoplasmic membrane, and 73 percent were actively phagocytic. As further evidence of the infection of mononuclear phagocytes, antibody-enhanced D2V replication was observed in bone marrow cultures from five of five rhesus monkeys, but not in cell cultures of spleen, thymus, or lymph nodes prepared from the same animals. It is hypothesized that dengue virus complexed with non-neutralizing antibody is internalized by immune phagocytosis in a mononuclear phagocyte with a defective virus-destroying mechanism. Dengue permissiveness may depend upon cellular immaturity since bone marrow leukocytes could be infected even when held for 4 days before infection while PBL held for this time decreased in permissiveness. In vitro antibody-dependent infection of mononuclear phagocytes should prove useful as a model for study of immunopathologic mechanisms in human dengue.
Assuntos
Vírus da Dengue/imunologia , Leucócitos/imunologia , Fagócitos/imunologia , Animais , Antígenos , Medula Óssea/microbiologia , Células da Medula Óssea , Adesão Celular , Imunofluorescência , Raios gama , Haplorrinos , História do Século XVIII , Humanos , Imunização , Leucócitos/microbiologia , Leucócitos/efeitos da radiação , Linfonodos/citologia , Macaca mulatta , Nylons , Fagócitos/microbiologia , Dióxido de Silício/farmacologia , Baço/citologia , Timo/citologia , Replicação ViralRESUMO
PURPOSE OF REVIEW: Japanese encephalitis is the most common vaccine-preventable viral encephalitis in Asia. In view of the production cessation of the inactivated mouse brain-derived Japanese encephalitis vaccine, it is timely to provide an update on new Japanese encephalitis vaccines and revised vaccine recommendations. RECENT FINDINGS: A new inactivated, adjuvanted, Vero cell-culture-based Japanese encephalitis vaccine, IC51, was licensed in Europe and the United States in 2009. Administered in a two-dose regimen at 0 and 28 days, it was shown to be well tolerated and produce high seroconversion rates. In addition, Chimerivax Japanese encephalitis, a novel live-attenuated one-dose chimeric vaccine comprising the structural genes of SA 14-14-2 virus and nonstructural genes of yellow fever 17D virus, is in the process of getting licensed in Australia and in south east Asia. SUMMARY: Previous recommendations for Japanese encephalitis vaccination of travelers were predicated on minimizing exposure to a mouse-brain-derived vaccine with a poorly understood and worrisome safety profile, whereas the risk of acquiring Japanese encephalitis itself during travel was assessed to be relatively low. With the availability of a new cell-culture-derived vaccine IC51 with an excellent safety profile, it is appropriate to reconsider benefit-risk considerations for the vaccination of travelers. These considerations are reflected in the March 2010 revised recommendations by the United States Advisory Committee on Immunization Practices.
Assuntos
Encefalite Japonesa/prevenção & controle , Vacinas contra Encefalite Japonesa/imunologia , Vacinação/métodos , Ásia , Austrália , Europa (Continente) , Imunização Secundária/métodos , Vacinas contra Encefalite Japonesa/efeitos adversos , Viagem , Estados Unidos , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologiaRESUMO
Dengue viruses occur as four antigenically related but distinct serotypes transmitted to humans by Aedes aegypti mosquitoes. These viruses generally cause a benign syndrome, dengue fever, in the American and African tropics, and a severe syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), in Southeast Asian children. This severe syndrome, which recently has also been identified in children infected with the virus in Puerto Rico, is characterized by increased vascular permeability and abnormal hemostasis. It occurs in infants less than 1 year of age born to dengue-immune mothers and in children 1 year and older who are immune to one serotype of dengue virus and are experiencing infection with a second serotype. Dengue viruses replicate in cells of mononuclear phagocyte lineage, and subneutralizing concentrations of dengue antibody enhance dengue virus infection in these cells. This antibody-dependent enhancement of infection regulates dengue disease in human beings, although disease severity may also be controlled genetically, possibly by permitting and restricting the growth of virus in monocytes. Monoclonal antibodies show heterogeneous distribution of antigenic epitopes on dengue viruses. These epitopes serve to regulate disease: when antibodies to shared antigens partially neutralize heterotypic virus, infection and disease are dampened; enhancing antibodies alone result in heightened disease response. Further knowledge of the structure of dengue genomes should permit rapid advances in understanding the pathogenetic mechanisms of dengue.
Assuntos
Vírus da Dengue , Dengue/etiologia , Adolescente , Anticorpos Antivirais/fisiologia , Antígenos Virais/imunologia , Sudeste Asiático , Criança , Pré-Escolar , Cuba , Dengue/epidemiologia , Dengue/etnologia , Dengue/imunologia , Dengue/microbiologia , Dengue/prevenção & controle , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Vírus da Dengue/fisiologia , Feminino , Humanos , Lactente , Masculino , Porto Rico , SorotipagemRESUMO
Human peripheral blood mononuclear cells, cultured in the presence of 100 microgram/ml protein-coated silica particles, were studied to determine changes in number and function of monocytes, immunoglobulin bearing (B), sheep red blood cell rosetting (T) lymphocytes and the effector cells of antibody dependent cell-mediated cytotoxicity (ADCC). After 24-48 h, phagocytic cells were effectively eliminated from culture but there was no significant reduction in number or function of T or B lymphocytes or in ADCC to cell line targets. ADCC to erythrocyte targets was inhibited but not completely blocked. It is concluded that silica is a specific toxin for human peripheral blood mononuclear phagocytes and may be useful in in vitro immunological studies as a means of eliminating or determining the role of these cells without resort to separation methods which result in losses of cells other than monocytes.
Assuntos
Fagócitos/imunologia , Dióxido de Silício/toxicidade , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/imunologia , Sobrevivência Celular , Galinhas , Relação Dose-Resposta Imunológica , Eritrócitos/imunologia , Humanos , Formação de Roseta , Linfócitos T/imunologiaRESUMO
Concern that ADE of HIV infection could occur in vivo, as a result of HIV immunization, has arisen for several reasons. Immune-mediated disease enhancement occurs in several human and animal viral diseases, including lentiviral diseases. Tropism for host M/M cells is a common characteristic in these diseases. Sera from naturally infected, and possibly HIV-immunized, individuals have been shown to contain infection enhancing antibodies in vitro. Finally, there is considerable genetic, and potentially antigenic, diversity among HIV-1 isolates. This workshop was convened to evaluate these concerns regarding ADE of HIV infection in human HIV vaccine trials and to propose studies that would address this potential risk. Although there is currently no evidence that immune-mediated enhancement of disease occurs in HIV, there is clearly a need for carefully designed experiments to further evaluate this issue. As there are several notable diseases for which in vitro ADE does not correlate with ADE in vivo, in vitro data are insufficient to deter development of current HIV-1 vaccine candidates. In vivo correlates of protection/enhancement are necessary to evaluate the ADE risk accurately. The development of an HIV animal model that would allow testing of vaccine candidates is of primary importance.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Animais , Ensaios Clínicos como Assunto/métodos , Modelos Animais de Doenças , Variação Genética , HIV/genética , HIV/fisiologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/etiologia , Infecções por HIV/prevenção & controle , Humanos , Técnicas In Vitro , Fatores de Risco , Viroses/etiologia , Replicação Viral/imunologiaRESUMO
The International Clinical Epidemiology Network (INCLEN) was established in 1982 to strengthen the research capacity of medical schools in the developing world through the development of Clinical Epidemiology Units (CEUs). The role of these units is to promote a rational approach to clinical and health care decision making, drawing on the methods of clinical epidemiology, biostatistics, health economics and health social science. This paper summarizes the evolution of the INCLEN model and the experience to date. Progress with Phase 1, the designation of sites for CEU development and the provision of advanced research training by developed country training centres has been substantial. The network now consists of 27 units: 26 in developing country medical schools in Asia, Latin America, India and Africa and 1 in France. More than 60% of the target of 270 fellows have completed training and returned to take up faculty positions in their unit. The remainder will be trained and on site by 1995. The non-return rate of fellows (2%) is very low. Research productivity is significant given only 60 fellows have been working in their CEUs for more than 3 years following the completion of training. An appropriate balance between hospital and community-based research is evident and changes in clinical and health care policy have been made based on the research conducted. The educational responsibilities of all units include courses and workshops in critical appraisal and clinical epidemiology for medical trainees and colleagues. Graduate training programs have emerged in 3 units so far. Major challenges lie ahead as we move into Phase 2 of the project--self sustainability and the transfer of training responsibility to the CEUs. The problems encountered during Phase 1 will need to be addressed. These include time protection for research, the limited availability of research funds, the low priority given to research careers and the poor linkage between health researchers and government policy makers. Our experience echos the recommendations of the recent report of the Commission on Health Research for Development, namely that donors and national governments should give increased priority to the role of health research in less developed countries. We conclude that with continuing support and special attention to the problems encountered, the INCLEN approach can contribute to ensuring that the medical establishment is part of the solution rather than the problem faced by health systems in less developed countries.
Assuntos
Epidemiologia/tendências , Países em Desenvolvimento , Educação Médica Continuada , Educação de Pós-Graduação em Medicina , Epidemiologia/educação , Pesquisa sobre Serviços de Saúde , Humanos , Cooperação InternacionalRESUMO
The antiviral effects of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and 6-mercapto-9-tetrahydro-2-furylpurine (6-MPTF) against dengue viruses were examined in vitro. Ribavirin significantly reduced the growth of dengue virus types 1-4 in LLC-MK2 cells at concentrations well below cytotoxic levels (cell viability was determined by trypan blue dye exclusion) Addition of guanosine to ribavirin-treated dengue virus-infected cell cultures completely reversed the antiviral effect of the drug. In contrast, ribavirin had no effect on dengue virus replication in human peripheral blood leukocytes (PBL). 6-MPIF, a specific inhibitor of hypoxanthine-guanine phosphoribosyltransferase, did not significantly reduce the growth of dengue viruses in either LIC-MK2 cells or human PBL. However, synergistic effects of 6-MPTF and ribavirin were observed, as combined treatment of the drugs markedly suppressed the replication of dengue viruses in human PBL. The successful demonstration that dengue virus replication in mononuclear leukocytes is markedly suppressed by the combined treatment of ribavirin and 6-MPTF signals a need to evaluate the efficacy of this treatment against dengue virus infections in vivo.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Mercaptopurina/análogos & derivados , Ribavirina/farmacologia , Ribonucleosídeos/farmacologia , Animais , Células Cultivadas , Vírus da Dengue/crescimento & desenvolvimento , Sinergismo Farmacológico , Fibroblastos , Humanos , Leucócitos/efeitos dos fármacos , Macaca mulatta , Mercaptopurina/farmacologia , Nucleosídeos/farmacologiaRESUMO
Four participants in the American Army Philippine dengue (d) studies of Siler (1924-25) and five subjects from the studies of Simmons (1929-30) were bled 42 to 48 years after experimental infection. Sera were studied by hemagglutination-inhibition, complement-fixation and plaque reduction neutralization (PRNT) tests for antibodies to the known viral causes of the dengue syndrome in Asia and to St. Louis encephalitis virus. PRNT tests were done with and without fresh normal human serum (accessory factor). Serum from one of four Siler study volunteers had unequivocal evidence of only a previous d4 infection; the remainder had broadly reactive group B antibodies, including d4, suggesting past infection with two or more different dengue viruses. Sera from four of the Simmons study group had high-titered PRNT antibodies to d1 and the remaining serum had d1 antibody when accessory factor was added. In three sera there was monotypic PRNT antibody to d1. Acessory factor increased PRNT titers, particularly to d1 virus in the Simmons group. Only sera with antibody patterns suggesting past infections with two or more dengue viruses had high levels of SLE PRNT antibody. It is concluded that d4 was transmitted in the 1924 to 1925 and d1 in the 1929 to 1930 experiments. Notable differences in clinical features of dengue infections in the two studies suggest the possibility of the existence of an unique spectrum of responses to infection with different dengue virus type.
Assuntos
Dengue/etiologia , Adulto , Idoso , Anticorpos Antivirais/análise , Antígenos Virais , Testes de Fixação de Complemento , Dengue/história , Dengue/imunologia , Dengue/transmissão , Vírus da Dengue/imunologia , Testes de Inibição da Hemaglutinação , História do Século XX , Humanos , Masculino , Testes de Neutralização , Filipinas , Estudos Retrospectivos , Estados UnidosRESUMO
In August-September 1928 appoximately 650,000 residents of Athens and Piraeus contracted dengue fever, and 1,061 died. We were interested in the etiology of this severe epidemic in which many cases resembled dengue hemorrhagic fever or the dengue shock syndrome, and have attempted a retrospective seroepidemiological study. Serum specimens were obtained from 111 residents of Athens or Piraeus who were born in 1927 or 1928, and were studied by plaque reduction neutralization test for antibodies to dengue 1-4 viruses. Of 75 persons born in 1928, 20 (27%) had monospecific dengue 1, 10 (13%) had monospecific dengue 2, and 1 (1%) had dengue 1 and 2 neutralizing antibodies. When prevalence of neutralizing antibody was analyzed by month of birth in 42 individuals, evidence of both dengue 1 and 2 infections was found in persons born in January-July, but only dengue 2 antibody was detected in those who were born after July. This study dates dengue 1 and dengue 2 transmission to 1928, allowing for the possibility that sequential infections with these viruses could have played a pathogenetic role in the outbreak.
Assuntos
Dengue/história , Dengue/transmissão , Anticorpos Antivirais/análise , Grécia , Testes de Hemaglutinação , História do Século XX , Humanos , Testes de Neutralização , Estudos RetrospectivosRESUMO
Sequential blood samples were obtained from eight Thai children before, during and 3-5 months after hospitalization for dengue shock syndrome. All patients experienced a secondary-type antibody response as evidenced by hemagglutination-inhibition antibody responses in acute and convalescent sera. Dengue 2 viruses were recovered from two patients. In their pre-illness blood sample, all children had monotypic neutralizing antibodies; five to dengue 1, two to dengue 3 and one to dengue 4. The highest neutralizing antibody titers in acute phase and late convalescent sera were to the initial infecting virus type. This report documents for sequential dengue infections the existence of an original antigenic sin antibody response. It may be possible to apply this phenomenon to identify initial dengue serotype infection in individuals experiencing secondary dengue infections, thus helping to clarify the antecedents to dengue shock syndrome.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Dengue/imunologia , Dengue/imunologia , Adolescente , Criança , Pré-Escolar , Vírus da Dengue/classificação , Testes de Inibição da Hemaglutinação , Humanos , Memória Imunológica , Lactente , Testes de NeutralizaçãoRESUMO
Dengue viruses were cultivated in Balb 3T12-3 cells, a continuous line of cells derived from mouse embryo cell cultures. Optimal serum concentrations were determined for maximal kinetics of virus replication. All four types of dengue viruses replicate in these cells, and peak virus titers were obtained by 72 hours post-infection. The successful growth of dengue viruses in a continuous line of mouse cells should allow for more extensive studies examining the cellular immune response to dengue virus infection in murine models.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Animais , Camundongos , Camundongos Endogâmicos BALB C , Cultura de Vírus/métodosRESUMO
Enhanced dengue 2 virus (D2V) infection in suspension cultures of human peripheral blood mononuclear phagocytes (PBL) produced by subneutralizing concentrations of dengue antisera has been described previously. In this study, the enhancement phenomenon was found to be a general property of representative flavivirus antisera. All except one of 24 antisera, which had been raised by 1-3 injections of flaviviruses in rabbits, enhanced the growth of dengue 2 virus in human PBL. Flavivirus antisera showing the greatest level of cross-reactivity against a battery of 42 flavivirus antigens in the hemagglutination-inhibition test were most potent in enhancing dengue replication in PBL cultures. Cross-neutralizing reactivity did not relate to enhanced D2V infection. However nearly one-half of studied flavivirus antisera neutralized D2V at dilutions of 1:10 or 1:20. Heterotypic D2V neutralizing antibody could serve as a "brake" on infection enhancement in vivo. Observations should be made in the field to look for possible enhancement of dengue infection in heterotypic flavivirus immunes.
Assuntos
Infecções por Arbovirus/imunologia , Dengue/imunologia , Epitopos , Soros Imunes/imunologia , Monócitos/imunologia , Adulto , Anticorpos Antivirais/análise , Arbovírus/imunologia , HumanosRESUMO
Human sera collected from Nigerians were examined for plaque reduction neutralizing and infection-enhancing antibodies against dengue 2, yellow fever, and West Nile viruses. Neutralization tests showed that 17 of 19 sera contained flavivirus neutralizing antibody; 11 were positive to all 3 viruses, 5 to dengue and yellow fever, and 1 to dengue virus only. Two sera had no detectable neutralizing antibody to any of the flaviviruses. Enhancement assays showed that 17 flavivirus neutralizing antibody-positive sera contained infection-enhancing antibodies to dengue 2, and 16 had antibody to yellow fever. Although 11 sera were positive for West Nile neutralizing antibody, 17 enhanced this virus. Heterologous infection-enhancing antibody titers were lower than the homologous ones. Broadly reacting sera and those with high neutralizing antibody titers produced the highest infection-enhancing antibody titers.
Assuntos
Anticorpos Antivirais/análise , Flavivirus/imunologia , Infecções por Togaviridae/imunologia , Vírus da Dengue/imunologia , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Testes de Neutralização , Nigéria , Infecções por Togaviridae/epidemiologia , Vírus do Nilo Ocidental/imunologia , Vírus da Febre Amarela/imunologiaRESUMO
The effects of rimantadine on dengue virus replication were examined in a variety of tissue culture systems. The growth of dengue virus type 2 in human peripheral blood leukocytes (PBL) was completely suppressed when rimantadine was included in the culture medium at a concentration of 25 microgram/ml. Similarly, rimantadine caused a significant inhibition of dengue virus replicaton in cultures of rhesus monkey PBL. Addition of drug into virus-infected LLC-MK2 cell cultures caused a decrease in the production of all four types of dengue virus. Maximal inhibition of dengue virus replication by rimantadine was observed when the drug was added immediately following the viral adsorption period. Rimantadine did not induce may cytopathic effects on either LLC-MK2 cells or PBL at concentrations less than 75 microgram/ml. These findings demonstrate that rimantadine is an effective inhibitor of dengue virus replication in vitro, and indicate a need for further examination of the efficacy of rimantadine against severe dengue virus disease.
Assuntos
Adamantano/análogos & derivados , Vírus da Dengue/efeitos dos fármacos , Rimantadina/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Dengue/imunologia , Humanos , Leucócitos/microbiologia , Macaca mulatta , Fatores de TempoRESUMO
Sera from 52 young adults resident in a rural area in North Thailand were studied for plaque-reducing neutralizing antibodies against dengue (DEN) viruses types 1-4 and Japanese encephalitis (JE), and for DEN-2 infection-enhancing antibodies using a newly described microtest in the human monocyte cell line, U-937. Infection-enhancing antibody titers in U-937 cells using a simplified micromethod were similar to results obtained by published methods using human peripheral blood leukocytes and a macrotest using U-937 cells. In the sample, there were 23 with antibodies to one or more DEN viruses with or without accompanying JE antibodies; 16 sera demonstrated antibodies only to JE and 13 had no detectable antibodies to any flavivirus. All but two DEN antibody-containing sera enhanced DEN-2 infections in U-937 cells, often to titers of 1:10,000 or greater. By contrast, only one of 16 JE-immune sera enhanced DEN-2 infection in monocytes, and that at a dilution of 1:100. None of the flavivirus-negative sera had DEN-2 enhancing activity. The failure of human anti-JE contrasts with the ability of rabbit anti-JE to enhance DEN-2 infections, but correlates with the absence of recorded instances of dengue shock syndrome in human beings sequentially infected with JE and then a DEN virus. This report seemingly reconciles in vitro and in vivo phenomena, and may provide an opportunity to study mechanisms involved.
Assuntos
Anticorpos Antivirais/fisiologia , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/imunologia , Monócitos/microbiologia , Adulto , Linhagem Celular , Vírus da Dengue/imunologia , Humanos , Testes de Neutralização , TailândiaRESUMO
We tested three dengue type 2 (DEN-2) isolates from children with clinically apparent but mild secondary dengue infections, and 10 isolates from children with moderately severe dengue hemorrhagic fever, and noted significant growth differences in peripheral blood leukocytes, but not in C6/36 cells. We also observed cytopathic effects in C6/36 cells that correlated with disease severity. These preliminary observations suggest the possibility that viral factors, whether surface antigens, attachment sites for entry into leukocytes, or intrinsic replication properties in human mononuclear phagocytes, might contribute to enhanced DEN infection and to the severity of the disease.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Dengue/microbiologia , Leucócitos/microbiologia , Criança , Feminino , Humanos , Masculino , Replicação ViralRESUMO
Tissue culture-adapted dengue 2 virus (DEN 2), strain 16681, exhibits antibody-dependent enhancement of infection (ADE) in P388D1 cells, a mouse macrophage-like cell line. ADE is dependent upon maintaining DEN 2 multiplicity of infection at between 0.1 and 0.001, and can be simply measured in multi-well plastic plates. The assay uses either trypsinized or non-trypsinized P388D1 cells at 5 x 10(5) cells per ml, an appropriate dilution of DEN 2 virus, and a source of antibody, and is most conveniently performed without further washing of stationary cultures, which are incubated in 5% CO2. Trypsinization of P388D1 cells prior to the addition of virus-serum mixtures reduced infection in control cultures thus increasing ADE. When cells were washed after incubation of virus-serum mixtures for 1 hour, a paradoxical increase of infection in cultures exposed to virus plus normal serum was noted, which reduced the sensitivity of the ADE assay. Using human cord blood sera, ADE titers measured in human monocytes and P388D1 cells were closely similar. This convenient and economical assay will facilitate large scale biological and epidemiological studies of dengue virus enhancing antibodies.
Assuntos
Anticorpos Antivirais/análise , Bioensaio , Linhagem Celular , Macrófagos/microbiologia , Animais , Anticorpos Antivirais/fisiologia , Complexo Antígeno-Anticorpo , Vírus da Dengue/crescimento & desenvolvimento , Humanos , Camundongos , Monócitos/microbiologia , Tripsina/farmacologiaRESUMO
Attempts were made to attenuate prototype dengue (DEN) 4 (H-241) virus. The original viremic human serum was passed once in a susceptible monkey and twice in Aedes albopictus mosquitoes and then serially passed in primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Weekly transfers of undiluted virus were carried to the 50th passage in both primary cell cultures. Biological markers were studied at passages 7, 15, 30 and 50. Parental DEN-4 phenotype characteristics included large plaque formation in LLC-MK2 cells, plaque formation in GMK cells, cytopathic effect in LLC-MK2 cells, growth in human monocyte cultures, growth at 39 degrees C, consistent production of viremia in monkeys and short-incubation neurovirulence in mice. At the seventh passage in both PDK and GMK cell cultures, DEN-4 viruses exhibited reduced plaque-size in LLC-MK2, and failed to plaque in GMK, to produce cytopathic effect in LLC-MK2, or to grow in human monocytes. Serial passage in PDK, as opposed to GMK, resulted in a graduated loss of monkey virulence. Rhesus monkeys inoculated with the PDK 50 strain failed to develop detectable viremia and only 1 of 4 developed an antibody response. Also, replication of PDK 50 was completely shut-off at 39 degrees C. The graduated change in biological properties noted, particularly those in PDK cells, provide a range of potential vaccine candidates for evaluation in human beings.
Assuntos
Vírus da Dengue/patogenicidade , Aedes/microbiologia , Animais , Anticorpos Antivirais/biossíntese , Encéfalo/microbiologia , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , Dengue/imunologia , Dengue/microbiologia , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/imunologia , Cães , Humanos , Rim/microbiologia , Linfócitos/microbiologia , Macaca mulatta , Camundongos , Temperatura , Ensaio de Placa Viral , Viremia , Virulência , Cultura de VírusRESUMO
Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.