The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8 ؉ T-cell responses to six Ebola virus proteins were examined. CD8 ؉ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8 ؉ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8؉ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.

Abstract. A candidate live-attenuated virus vaccine for protection against Venezuelan equine encephalitis (VEE) (designated V3526) was tested in mice to measure the magnitude, duration, and kinetics of virus replication in the blood and the central nervous system and its phenotypic stability after multiple passages in mice and cell culture. All results were compared to parallel experiments with parental virus and the existing VEE virus vaccine, TC-83. Maximum virus titers in the brains of V3526-inoculated mice were between 10-and 100-fold less than those observed in brains of mice inoculated intracranially (ic) with either the parental virus or TC-83. Neither V3526 nor TC-83 was lethal in BALB/c mice inoculated ic. However, mice inoculated with TC-83 developed acute symptoms lasting at least 14 days. In contrast, ic inoculation of TC-83 was uniformly lethal for C3H/HeN mice. V3526 was avirulent in both BALB/c and C3H/HeN mice after ic inoculation. The virulence characteristics of V3526 remained unchanged after five serial ic passages in mouse brains or after five cell culture passages. Finally, pathologic changes induced after ic inoculation of V3526 were consistently less severe and of shorter duration than those observed in TC-83-inoculated mice. Based on these results, V3526 is stable and appears to be significantly less neurovirulent in mice than TC-83.

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