Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

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.

Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.

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 (i.c.) with either the parental virus or TC-83. Neither V3526 nor TC-83 was lethal in BALB/c mice inoculated i.c.. However, mice inoculated with TC-83 developed acute symptoms lasting at least 14 days. In contrast, i.c. inoculation of TC-83 was uniformly lethal for C3H/HeN mice. V3526 was avirulent in both BALB/c and C3H/HeN mice after i.c. inoculation. The virulence characteristics of V3526 remained unchanged after five serial i.c. passages in mouse brains or after five cell culture passages. Finally, pathologic changes induced after i.c. 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.

Limited potential for mosquito transmission of genetically engineered, live-attenuated Venezuelan equine encephalitis virus vaccine candidates.

In an attempt to improve the current live-attenuated vaccine (TC-83) for Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts.

Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico.

Two outbreaks of encephalitis consistent with an etiology of Venezuelan equine encephalitis (VEE) virus occurred in equines on the Pacific coast of southern Mexico in 1993 (Chiapas State) and in 1996 (Oaxaca State). In Chiapas, there were 125 cases, of which 63 were fatal and in Oaxaca, there were 32 cases and 12 fatalities. Virus was isolated from two horses from each outbreak, including three brain isolates and one from blood. Virus isolates (93-42124, ISET-Chi93, Oax131, and Oax142) were shown by indirect immunofluorescence, hemagglutination inhibition, monoclonal antibody ELISA, and nucleotide sequencing to be VEE virus, subtype IE, a type previously thought to be equine-avirulent. Genetic characterization and phylogenetic analysis indicated that the outbreak viruses were identical or nearly identical to one another and that they were closely related to equine-avirulent IE strains from Guatemala and the Gulf coast of Mexico. In a plaque-reduction neutralization test, sera collected from healthy horses in Chiapas and Oaxaca reacted significantly better with isolate 93-42124 than with Guatemala IE isolate 68U201, suggesting that subtle genetic changes may have resulted in alteration of neutralization domains. It is not clear whether these differences may also influence equine virulence. However, renewed VEE virus subtype IE activity in Mexico, and its apparent conversion to equine virulence, underscores the need for increased surveillance, additional laboratory and epidemiologic studies in VEE-endemic regions, and possibly new vaccines.

A putative receptor for Venezuelan equine encephalitis virus from mosquito cells.

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.

Laboratory biology of Hyalomma truncatum (Acari: Ixodidae).

The life cycle of Hyalomma truncatum Koch (Acari: Ixodidae) required an average of 108 d at 26 +/- 1 degree C. 92-96% RH, and a 12:12 (L:D) photoperiod to complete. Mean weights of unfed larvae, nymphs, and females were 0.02, 0.19, and 11.1 mg, respectively. Weight of larvae, nymphs, and females increased 20-, 91-, and 48-fold, respectively, as a result of feeding on guinea pigs. All stages exhibited host-seeking behavior less than 1 d after emergence. The mean (+/- SE) feeding period of larvae, nymphs, and adults was 3.8 (+/- 0.1), 7.7 (+/- 0.3), and 8.3 (+/- 0.3) d, respectively. Larvae and nymphs molted an average of 11.0 (+/- 0.3), and 30.7 (+/- 0.2) d after engorgement, respectively. The female/male sex ratio, as determined from emerged adults, was 1.4:1. Oviposition started an average of 11.9 (+/- 0.8) d after engorgement, and a mean of 6.701 eggs per female was deposited. A total of only 48% of the eggs enclosed after a mean incubation of 35 (+/- 1.1) d. Females converted 56% of their engorged weight into eggs and produced an average of 12,614 (+/- 2.0) eggs/g of engorged body weight. On the first day of oviposition, an egg less than 24 h old weighed an average of 44.8 (+/- 1.5) micrograms. Egg weight was significantly (P less than 0.01) lower during peak egg production (days 2-8 after onset of oviposition) than during reduced egg production (days 14-20 after onset of oviposition).

Biology of Hyalomma impeltatum (Acari: Ixodidae) under laboratory conditions.

Completion of the life cycle of Hyalomma impeltatum Schulze & Schlottke required an average of 108 d at 26 +/- 1 degree C, 92-96% RH, and 12:12 (L:D) photoperiod. Weights of unfed larvae, nymphs, and females were 0.02, 0.16, and 15.4 mg, respectively, and increased 23-, 164-, and 55-fold, respectively, as a result of feeding on guinea pigs. Larvae and adults exhibited host-seeking behavior less than 1 d after hatching and molt, respectively; nymphs exhibited host-seeking behavior 2.9 d after molt. The mean (+/- SE) feeding period as larvae was 5.9 (+/- 2.23) d, nymphs 6.7 (+/- 1.10) d, and females 8.0 (+/- 0.19) d. Larvae molted 12.4 (+/- 0.26) d and nymphs molted 28.9 (+/- 0.22) d after engorgement. A sex ratio of 1.26:1 female/male was determined from emerged adults. Females began oviposition 8.9 (+/- 0.22) d after engorgement and produced 10,680 (+/- 300) eggs per female. Egg hatch was 84% (+/- 2.68) after an incubation period of 32.8 (+/- 0.19) d. Females converted 55% of engorged weight into eggs and produced 12,475 (+/- 188) eggs/g of engorged body weight. A freshly laid egg on the first day of oviposition weighted 47.7 (+/- 0.65) micrograms. An inverse relationship between egg weight and rate of egg production was observed.

Growth and metabolism of L cells in a chemically defined medium in a controlled environment culture system. I. Effects of O2 tension on L-cell cultures.

Six water-jacketed 500-ml Bellco spinner flasks were equipped to monitor and control environmental variables to study their effects on the growth and metabolism of mammalian cells. Studies with automated control of pO(2) levels of l-cell cultures, grown at pH 6.9 +/- 0.1, showed that dissolved O(2) tensions of ca. 9% were optimal for cell growth. At pO(2) values of 5 and 20%, maximum cell yields as well as growth rates were reduced by approximately 20%. Peak yields of L-cell cultures exceeded 5 x 10(6) cells/ml when grown for 4 days without medium renewal from inocula of ca. 10(6) cells/ml in a defined medium sparged with 5% CO(2) and maintained at 9% dissolved O(2) tension. The redox potentials of L-cell cultures reflected the pO(2) levels in the medium and ranged from -45 to +160 mv (versus calomel reference) for O(2) values ranging from 2 to 20% dissolved oxygen tension. Increased utilization of glucose per cell occurred in the presence of increased pO(2), whereas minimal accumulation of ammonia occurred with a pO(2) value maintained at 9%.