A comparison of the age distributions in the dengue hemorrhagic fever epidemics in Santiago de Cuba (1997) and Thailand (1998).

The age profiles of the infected populations of two dengue hemorrhagic fever (DHF) epidemics, the 1997 epidemic, in Santiago de Cuba and the 1998 epidemic in Thailand, are compared. Using an age-structured model of disease transmission, the dependence of the forces of infection on age was determined for each epidemic. The difference in the behavior of the two epidemics and the role of primary and secondary infection in the development of DHF are discussed.

Dengue virus specific T cell responses to live attenuated monovalent dengue-2 and tetravalent dengue vaccines.

The proliferative T cell responses to dengue vaccines were studied using the parental strains of dengue vaccines as antigens in 26 dengue immune individuals who resided in Bangkok which is the endemic area of dengue infection. The magnitude of the T cell responses in subjects with flavivirus cross-reactive neutralizing antibody was much higher and the cross-reactivity was broader than in those with dengue serotype-specific neutralizing antibodies, Japanese encephalitis (JE) specific antibodies or dengue cross-reactive antibodies. The T cell response in those with neutralizing antibody against a single serotype or in those who had dengue cross-reactive neutralizing antibody was relatively low, independent of the level or degree of cross-reactivity of the antibody. Evaluation of the proliferative T cell responses in 8 recipients of the monovalent dengue-2 (16681-PDK53) or the tetravalent dengue vaccines demonstrated that both vaccines induced high levels of neutralizing antibody as well as high levels of T cell responses to all serotypes of dengue virus. These results indicate that the evaluated dengue vaccines efficiently induced humoral and cell mediated immunity comparable to natural infection with dengue virus.

Serological study of hantavirus in the rodent population of Nakhon Pathom and Nakhon Ratchasima Provinces Thailand.

A serological survey has been carried out to detect evidence of hantavirus infection in rodents from two provinces of Thailand. This study aimed to examine virus antibody in 354 rodents trapped among 6 different villages of Nakhon Pathom Province (February-March, 1998) and in 326 rodents trapped among 14 villages of Nakhon Ratchasima Province (August-October, 1998). Seroprevalence among rodents from Nakhon Pathom Province (2.3%), was mostly find in Rattus norvegicus (3.8%) and Bandicota indica (2.6%). In Nakhon Ratchasima Province seroprevalence (4.0%) was mostly in Bandicota indica (19.1%) and Rattus exulans (3.5%).

Preliminary study on potential circulation of arenaviruses in the rodent population of Nakhon Pathom Province, Thailand and their medical importance in an evoluting environment.

Preliminary serological investigations were prefered to detect evidence of arenavirus infection in rodents. The study examined virus antibody in 367 rodents trapped in 6 different geographical areas of Nakhon Pathom Province, Thailand from February-March, 1998. The overall seroprevalence among rodents was 13.3%, mostly in Bandicota savilei (35.7%) and Rattus norvegicus (31.5%). Between ecology, behavior and sex of the rodents, seroprevalence was not significantly different (p>0.05), however the seroprevalence found among different geographical areas of Nakhon Pathom Province were significantly different (p<0.0001).

Dengue viruses induce cell proliferation and morphological changes of endothelial cells.

Replication of dengue viruses (type 1, 2, 3 and 4) in vitro in endothelial cells from human umbilical cord vein was demonstrated by virus titers and immunofluorescent antibody studies. Both showed highest peak at Day 6 after inoculation and declined to origin at Day 14. Some of the cultured endothelial cells detached from the culture well. Most of these floating cells were rarely viable as shown by failure in trypan blue exclusion whereas the adhering cells are mostly viable. More frequent and higher intensity of immunofluorescent positive cells were found in the detached cells as compared to adhering cells. The virus titers in the supernatant and in the adhering cell population were comparable, although floating cells were maximally 26.2% of the total cultured endothelial cells. Many floating cells and occasional adhering cells had numerous blebs on their surface. Endothelial cell proliferation was markedly increased after virus inoculation as compared with the control. Increased number of mitotic cells was also observed in the dengue virus-endothelial cell culture. Comparing among the four types, dengue type 4 induced highest peaks of cell proliferation and cell mitosis at Day 10 after inoculation. Dengue type 2 had the highest virus titers both in adhering cells and in supernatant at Day 6 as compared with other types.

In vivo and in vitro studies on the morphological change in the monkey epidermal Langerhans cells following exposure to dengue 2 (16681) virus.

A direct comparison of skin Langerhans cell (LC) morphologic change following in vivo and in vitro exposure to dengue-2 (DEN-2) virus (16681) was performed in the monkey to investigate any differences in functional activity profiles. Time-lapse study of skin biopsy at the intradermal (id) virus injection sites, and thin skin sheets removed from the monkey with exposure to virus in culture medium, revealed a highly active migration of epidermal LCs in both sets of experimental specimens. The migration led to a relatively higher number of dendritic cells (DC) which appeared in active migrational profiles, in the superficial dermis. Moreover, obvious cytoplasmic structural changes, corresponding to their immunologic function, were observed in these superficial dermal DCs 2 hours after exposure. Despite their similar changes, early and late endosomes with degraded virus-like particles could be seen in the skin sheets owing to lagging in cellular physiological process in vitro, but none in the skin biopsies. Existence of these endosomes, which was extremely difficult to visualize in vivo, highlighted the mode of antigen processing by the endocytic pathway. The present study showed that the epidermal LC was a potent antigen-presenting cell for eliciting the success of id immunization and carried out the immunological activity in vivo or in vitro in the like manner, in respect to the physiological conditions.

Dengue-3 (16562) PGMK 33 vaccine: neurovirulence, viremia and immune responses in Macaca fascicularis.

Investigation of monkey neurovirulence of dengue-3 viruses (DEN-3, 16562) was undertaken to provide an evaluation of the relative safety of virus strain attenuated for potential use of live virus vaccine. Ten flavivirus-negative, cynomolgus monkeys (Macacafascicularis) were used in the test. The animals were inoculated intrathalamically, intraspinally and intramuscularly with DEN-3 PGMK 33 attenuated live virus vaccine (6 monkeys): parent virus (2) and control cell culture fluid (2). Blood samples were collected on days 0, 1, 2, 4, 6, 8, 10, 12 and 21 for virus isolation and days 0 and 21 or 22 for serologic testing. One monkey with DEN-3 (16562) PGMK 33 candidate vaccine had detectable viremia on day 10. By day 21, all recipients of PGMK 33 and both monkeys with DEN-3 parent virus developed serum neutralizing antibodies to DEN-3 titers ranged from 56-320. The monkeys showed no evidence of illness and none died of dengue infection. Histopathological examination of tissue collected on day 21 or 22 revealed only minimal neurovirulence lesions as scored by the routine grading system. No differences were observed between the DEN-3 parent and vaccine viruses and it is concluded that neither virus is neurovirulent for cynomolgus monkeys.

Dengue virus serotypic identification using suckling mouse and western blot technique.

The suckling mouse which is used in the classical method to detect and propagate dengue viruses was evaluated in conjunction with the western blot and immunoenzymatic methods to detect the infecting strains of dengue viruses. After intracerebral inoculation of patients' sera into the suckling mice for 7 days, the mice were examined for the presence of dengue proteins, even though the mice did not have the neurological symptoms which usually serve as an indicator for the presence of dengue infection in the mouse brain. With a blind study of a set of 12 specimens, the suckling mice could detect the virus with the same frequency as the mosquito system but in shorter time of incubation period. The whole process to identify the type of infection takes 9 days. Another important finding is the demonstration of the virion antigen in the liver. The quantity and quality of viral proteins in liver are comparable to those in the brain suggesting that the virus may replicate in the liver as well as in the brain.

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 viral antigen. II. Observations for the antibody enhancement activity in human monocytes.

Peroxidase-antiperoxidase (PAP) staining was applied to measure the antibody enhancement activity in human monocytes. Increasing in number of infected cells can be seen with increasing of staining intensity of the cells by ordinary light microscope. Shifting of the optimum enhancement activity was found in previously tritiated antiserum indicated that for titration of antibody enhancement activity several dilutions of antiserum should be included in each experiment. Validity of the PAP method was made by the comparison of the results with Infectious Center Assay (ICA). With this technique, titration for antibody enhancement for dengue virus infection can be done with non-expensive equipment and can be kept for comparison for months.

Neurovirulence effects of dengue-2 viruses on the rhesus (Macaca mulatta) brain and spinal cord.

Vaccines prepared from attenuated virus can cause symptomatic viral infection of the central nervous system. In the present study, dengue-2 parental and its live attenuated viruses were tested by intrathalamic and intraspinal injections in rhesus monkeys. The dengue-2 viruses were found to be only very weakly neurovirulent when injected directly into the brain or spinal cord of rhesus monkeys.

Blood value changes in flavivirus-inoculated Macaca fascicularis monkeys.

Blood values were analysed in eighteen cynomolgus monkeys on pre-and post-neurovirulence testing of dengue-2 and yellow fever vaccine viruses, dengue-2 parental and Japanese encephalitis viruses. Certain changes between blood chemistry, hematology and serology were observed and briefly discussed.

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 antigen. I. In an endogenous peroxidase containing cell systems.

The unlabelled immunoperoxidase, peroxidase-anti-peroxidase (PAP), technique was used to detect dengue type-2 viral antigen in several cell systems including the endogenous peroxidase containing cells. These cells are the mosquito cell line (C6/36), continuous cell line of rhesus monkey kidney (LLC-MK2), human monocyte culture both cell suspension and monolayer, and human peripheral blood leukocytes. All of these specimens gave the same results that dengue-2 viral antigen presented in cytoplasm only and the patterns of marker presentation in positive cells varied depending on the duration after infection. The sensitivity of this method is extremely high since it can detect dengue-2 antigen after its attachment on mosquito cells (15 min) as seen in experiments with mosquito cell line, C6/36. False positive was not observed in all cell systems tested.