Phylogeographic analysis of rabies viruses in the Philippines.

Rabies still remains a public health threat in the Philippines. A significant number of human rabies cases, about 200-300 cases annually, have been reported, and the country needs an effective strategy for rabies control. To develop an effective control strategy, it is important to understand the transmission patterns of the rabies viruses. We conducted phylogenetic analyses by considering the temporal and spatial evolution of rabies viruses to reveal the transmission dynamics in the Philippines. After evaluating the molecular clock and phylogeographic analysis, we estimated that the Philippine strains were introduced from China around the beginning of 20th century. Upon this introduction, the rabies viruses evolved within the Philippines to form three major clades, and there was no indication of introduction of other rabies viruses from any other country. However, within the Philippines, island-to-island migrations were observed. Since then, the rabies viruses have diffused and only evolved within each island group. The evolutionary pattern of these viruses was strongly shaped by geographical boundaries. The association index statistics demonstrated a strong spatial structure within the island group, indicating that the seas were a significant geographical barrier for viral dispersal. Strong spatial structure was also observed even at a regional level, and most of the viral migrations (79.7% of the total median number) in Luzon were observed between neighboring regions. Rabies viruses were genetically clustered at a regional level, and this strong spatial structure suggests a geographical clustering of transmission chains and the potential effectiveness of rabies control that targets geographical clustering. Dog vaccination campaigns have been conducted independently by local governments in the Philippines, but it could be more effective to implement a coordinated vaccination campaign among neighboring areas to eliminate geographically-clustered rabies transmission chains.

Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines.

BACKGROUND: Ebolaviruses induce lethal viral hemorrhagic fevers (VHFs) in humans and non-human primates, with the exceptions of Reston virus (RESTV), which is not pathogenic for humans. In human VHF cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the IgG responses to nucleoprotein (NP) and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein (GP1,2) are the major indicator of protective immunity. On the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against NP and GP1,2 in RESTV-infected cynomolgus macaques is still unclear. In this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the RESTV epizootic in 1996 in the Philippines to expand our knowledge on the immune responses in naturally RESTV-infected non-human primates. RESULTS: The antibody responses were analyzed using IgG-ELISA, an indirect immunofluorescent antibody assay (IFA), and a pseudotyped VSV-based neutralizing (NT) assay. Antigen-capture (Ag)-ELISA was also performed to detect viral antigens in the serum specimens. We found that the anti-GP1,2 responses, but not the anti-NP responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the RESTV-infected cynomolgus macaques. Additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as IFNγ, IL8, IL-12, and MIP1α, in the convalescent phase sera. CONCLUSIONS: These results imply that both the antibody response to GP1,2 and the proinflammatory innate responses play significant roles in the recovery from RESTV infection in cynomolgus macaques.

Epizootology and experimental infection of Yokose virus in bats.

To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.

Antigen capture enzyme-linked immunosorbent assay for specific detection of Reston Ebola virus nucleoprotein.

Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.

Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells.

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.

Serologic evidence of Lyssavirus infections among bats, the Philippines.

Active surveillance for lyssaviruses was conducted among populations of bats in the Philippines. The presence of past or current Lyssavirus infection was determined by use of direct fluorescent antibody assays on bat brains and virus neutralization assays on bat sera. Although no bats were found to have active infection with a Lyssavirus, 22 had evidence of neutralizing antibody against the Australian bat lyssavirus (ABLV). Seropositivity was statistically associated with one species of bat, Miniopterus schreibersi. Results from the virus neutralization assays are consistent with the presence in the Philippines of a naturally occurring Lyssavirus related to ABLV.