Human skin Langerhans cells are targets of dengue virus infection.

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.

Absence of dengue 2 infection enhancement in human sera containing Japanese encephalitis antibodies.

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.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at different dog kidney passage levels.

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.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine candidate in fetal rhesus lung cells.

A strain of primary dog kidney (PDK)-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 35-TD3) was propagated in large volumes in fetal rhesus lung (FRhL) cells to produce a candidate vaccine for evaluation in man. Production seed (FRhL p2) and candidate vaccine (FRhL p3) were subjected to rigorous safety tests to exclude contaminating microbial agents. There was no significant monkey neurovirulence of parental or PDK-passaged DEN-4 virus or of control fluid cultures. FRhL-passaged viruses retained the phenotypic characteristics: small (occasional medium) plaque; temperature sensitivity at 38.5 degrees C; and absence of plaque formation in African green monkey kidney cells, cytopathic effect in LLC-MK2 cells, and viral growth in human monocytes. FRhL p2 virus displayed low virulence for monkeys; only one of four animals was viremic and three of four developed low-titered antibody. FRhL p3 virus produced viremia in three monkeys and moderate to high hemagglutination-inhibition and neutralizing antibody titers in all animals. Virus at both passages in FRhL exhibited reduced neurovirulence in suckling mice as compared to parental DEN-4. Because of its safety and desirable monkey virulence attributes PDK 35-TD3 FRhL p3 is recommended for human phase I trial.

Haiti: absence of dengue hemorrhagic fever despite hyperendemic dengue virus transmission.

In 1994-1996, 185 strains of dengue (DEN) virus types 1, 2, and 4 were recovered from febrile United States and other United Nations military personnel in Haiti. We wondered whether risk factors for dengue hemorrhagic fever (DHF) existed and, if so, were DHF cases occurring among Haitian children. Dengue transmission rates were studied in 210 school children (6-13 years old) resident in Carrefour Borough, Port-au-Prince, Haiti. When sera were tested for plaque-reduction neutralizing antibodies to DEN 1-4 viruses, nearly 85% had antibodies to two or more DEN serotypes. The annual transmission rate was estimated at 30%, a rate observed in countries endemic for DHE Haitian DEN 2 isolates were genotype I, which are repeatedly associated with DHF cases in Southeast Asia and American regions. Despite positive virologic pre-conditions, DHF cases were not recorded by experienced Port-au-Prince pediatricians. These observations, which are reminiscent of those in Africa, provide further evidence of a dengue resistance gene in black populations.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. III. Reversion to virulence by passage of cloned virus in fetal rhesus lung cells.

Two strains of primary dog kidney-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 24-TD3 and 35-TD3) were propagated in fetal rhesus lung (FRhL) cells to produce candidate vaccine virus seeds. Both serial passage and prolonged replication of PDK 24-TD3 in FRhL resulted in appearance of medium and large plaques in LLC-MK2 assays. When picked, these plaques proved to contain temperature-resistant, monkey-virulent revertants. Serial passage and prolonged replication of PDK 24-TD3 in LLC-MK2 cells did not result in reversion; but, prolonged replication in PDK cells did. Passage of PDK 35-TD3 in FRhL cells resulted in appearance of medium size plaques which, when picked, yielded temperature sensitive (ts) (38.5 degrees C) viruses of low monkey-virulence. Because of its stability in monkeys and FRhL cells, reduced monkey virulence and ts property. PDK 35-TD3 is a promising candidate for trial in man.

Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization.

A newly modified semimicro plaque reduction neutralization test (PRNT) in BHK cells was compared with a standard PRNT in bottles with LLC-MK2 monolayers and with an LLC-MK2 PRNT adapted to semimicro methods. The BHK semimicro PRNT compared favorably in terms of sensitivity in detecting dengue antibody (96%), specificity at a screening dilution (95%), and ability to detect seroconversion to dengue viruses of three serotypes (93%). Disagreements between the BHK test and the LLC-MK2 tests were attributed to greater sensitivity of the BHK test in detecting dengue type 2 (DEN-2) antibody in acute-phase sera and to apparent low-level DEN-1/DEN-3 cross-reactions in some sera in all three tests. The BHK PRNT was easier, faster, and more economical than either of the LLC-MK2 tests. Many of the benefits of the BHK PRNT derive from the fact that cells are infected while still in suspension, at the time of cell splitting, hence the term "BHK suspension test."

Immunogenicity of live attenuated SA14-14-2 Japanese encephalitis vaccine--a comparison of 1- and 3-month immunization schedules.

Live attenuated SA14-14-2 Japanese encephalitis (JE) vaccine has been safe and effective in >100 million immunized children, but its current administration schedule of two doses given a year apart does not lend itself to inclusion in established Expanded Program of Immunization (EPI) schedules of childhood immunization. Immune responses to immunization at shorter intervals were compared in middle-school-aged children immunized with two doses separated by 1 month (n = 116) or 2.5 months (n = 115). Two vaccine lots were compared. Seroconversion to the vaccine was observed in 100% of vaccinees immunized in the 1-month schedule and in 94% (lot 2) and 100% (lot 1) of vaccinees immunized in the 2.5-month schedule. Geometric mean titers were almost 2-fold higher with the longer schedule. The routine administration of JE SA14-14-2 vaccine to infants in an EPI schedule should be possible using either interval.

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Human dendritic cells as targets of dengue virus infection.

Dengue virus infections are an emerging global threat. Severe dengue infection is manifested as dengue hemorrhagic fever and dengue shock syndrome, both of which can be fatal complications. Factors predisposing to complicated disease and pathogenesis of severe infections are discussed. Using immunohistochemistry, immunofluorescence, flow cytometry, and ELISA techniques, we studied the cellular targets of dengue virus infection, at both the clinical (in vivo) and the laboratory (in vitro) level. Resident skin dendritic cells are targets of dengue virus infection as demonstrated in a skin biopsy from a dengue vaccine recipient. We show that factors influencing infection of monocytes/macrophages and dendritic cells are different. Immature dendritic cells were found to be the cells most permissive for dengue infection and maybe early targets for infection. Immature dendritic cells exposed to dengue virus produce TNF-alpha protein. Some of these immature dendritic cells undergo TNF-alpha mediated maturation as a consequence of exposure to the dengue virus.