Vector competence of Culex (Melanoconion) taeniopus for allopatric and epizootic Venezuelan equine encephalomyelitis viruses.

The vector competence of Culex (Melanoconion) taeniopus was examined in the laboratory for "enzootic" allopatric and "epizootic" strains of Venezuelan equine encephalomyelitis viruses of hemagglutination inhibition subtypes I, II, III, and IV. Following bloodmeals from viremic hamsters, and extrinsic incubation of 20-22 days, mosquitoes were allowed to refeed for transmission attempts. Infection rates never exceeded 50% with oral doses of less than 10(4) chick embryo cell culture plaque forming units (CECPFU), and approached 100% only after ingestion of greater than or equal to 10(5.5) PFU. Transmission was achieved for some "epizootic" subtype IABC and "enzootic" subtype ID strains after bloodmeals containing greater than or equal to 10(3.4) CECPFU; subtypes II, III, and IV were never transmitted despite oral doses up to 10(5.0) CECPFU. These data contrast sharply with those reported previously for sympatric "enzootic" subtype IE Middle American Venezuelan encephalitis viruses.

Entomological studies at an enzootic Venezuelan equine encephalitis virus focus in Guatemala, 1977-1980.

The ecology of several potential mosquito vectors of Venezuelan equine encephalitis (VEE) alphavirus was studied in an enzootic focus of that virus on the Pacific coast of Guatemala over a four-year period. Four species-Culex taeniopus, Mansonia titillans, Culex nigripalpus and Aedes taeniorhynchus-were most prevalent during the wet season when transmission normally occurs. However, only Cx. taeniopus yielded VEE virus. The bloodfeeding patterns of these species revealed that Ae. taeniorhynchus and Ma. titillans fed almost exclusively on bovine and equine hosts. Conversely, Cx. nigripalpus was highly ornithophilic but occasionally fed on mammals. Cx. taeniopus exhibited a wide host range, utilizing both large and small mammals as well as birds and, rarely, reptiles. The versatility in feeding pattern displayed by this mosquito coupled with its ability to become infected with relatively low levels of enzootic VEE virus suggests that vertebrates other than rodents may serve as amplifying hosts in this habitat. Nepuyo virus was also isolated from Cx. taeniopus, suggesting that this mosquito might be an endemic vector of this rodent-associated bunyavirus. A single isolate of St. Louis encephalitis virus was made from Cx. nigripalpus.

Laboratory vector competence of Culex (Melanoconion) cedecei for sympatric and allopatric Venezuelan equine encephalomyelitis viruses.

Laboratory vector competence of Culex (Melanoconion) cedecei was examined for Venezuelan equine encephalomyelitis (VEE) viruses. Colonized adult female mosquitoes originating from a southern Florida population were given bloodmeals from viremic hamsters circulating various titers of 3 hemagglutination inhibition (HI) subtypes of VEE viruses. Following extrinsic incubation of about 3 weeks, mosquitoes were allowed to refeed on uninfected hamsters for transmission trials. Cx. cedecei was highly efficient in becoming infected with and transmitting its sympatric, HI subtype II "Everglades" virus. With bloodmeal titers of 10(0.9) chick embryo cell culture (CEC) plaque forming units (PFU), the infection rate was 9% and transmission occurred following extrinsic incubation. Infection rates were greater than or equal to 80% with oral doses of greater than or equal to 10(1.8), and all infected mosquitoes were capable of transmission following incubation. Cx. cedecei was also highly sensitive to infection with allopatric HI subtype IE Middle American VEE virus isolates. Infection rates were greater than or equal to 50% with bloodmeal titers undetectable by CEC assay. Rates were 100% with oral doses of greater than or equal to 10(0.8) CECPFU. Transmission rates were 100% in all experiments. Similar results were obtained with HI subtype IAB "epizootic" VEE virus isolates from the 1969 Middle American outbreak. Infection rates were 100% with oral doses of greater than or equal to 10(1.2), and transmission rates were 100% after extrinsic incubation. Comparisons with laboratory vector competence of the Middle American enzootic VEE virus vector, Culex (Melanoconion) taeniopus, are discussed.

Markers of Venezuelan encephalitis virus which distinguish enzootic strains of subtype I-D from those of I-E.

Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.

Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

Barriers to dissemination of Venezuelan encephalitis viruses in the Middle American enzootic vector mosquito, Culex (Melanoconion) taeniopus.

Barriers to dissemination of Middle American epizootic hemagglutination inhibition subtype I-AB, and enzootic, subtype IE, Venezuelan encephalitis (VE) viruses were examined in a colony of the enzootic vector mosquito, Culex (Melanoconion) taeniopus. This species is highly susceptible to oral infection with enzootic, but not epizootic, virus strains. Adult female mosquitoes were intrathoracically inoculated with epizootic virus suspensions to ascertain whether a mesenteron infection barrier exists to these subtype I-AB strains. All inoculated mosquitoes became infected, including those receiving only 10 chick embryo cell culture plaque-forming units (CEC pfu). This confirmed that a mesenteron infection barrier exists to epizootic, but not enzootic Middle American VE strains. Mosquitoes were also given high titer hamster bloodmeals of epizootic viruses and dissected at 2-day intervals to determine the location of virus in the few infected individuals. With mean bloodmeal titers of up to 10(5.3) CEC pfu, only 20% or less of the mosquitoes became infected, and virus replication was confined to the mesenteron. This indicated that a mesenteron escape barrier to epizootic VE viruses exists in this mosquito. Mosquitoes were also given large and small oral doses of enzootic virus strains to compare viral replication patterns. With high titer bloodmeals, virus disseminated from the mesenteron within 4 days of infection, and titers in mosquitoes peaked 7-9 days after infection. All mosquitoes that ingested large doses became infected. Mosquitoes receiving small oral doses of enzootic viruses showed a different pattern of virus replication.(ABSTRACT TRUNCATED AT 250 WORDS)

An unusual strain of Venezuelan encephalitis virus existing sympatrically with subtype I-D strains in a Peruvian rain forest.

In 1971, an unusual strain of Venezuelan encephalitis (VE) virus (71D1252) was recovered from the same small area of a rain forest in the western Amazon basin of South America near Iquitos, Loreto, Peru, from which strains of subtype I-D were recovered. The marker characteristics of this strain resembled most closely those of VE subtype III (Mucambo) and were distinctly different from coexisting I-D strains. Thus the concurrent presence of two different VE virus subtypes in one place was a striking exception to the usual geographic allopatry of VE virus subtypes. Strain 71D1252 also contained temperature sensitive (ts) (37 degrees C versus 39 degrees C) virions in the original mosquito suspension and first suckling mouse passage brain tissue suspensions. It thus represents one of the few so-far-reported ts strains of viruses found in nature, and the only natural ts strain of VE virus.

Marker characteristics of Venezuelan encephalitis virus strains isolated before and after epidemics and equine epizootics in Middle America.

Ninety-four strains of Venezuelan encephalitis (VE) virus isolated from sentinel hamsters exposed in the Middle American countries of Mexico, Guatemala, Belize, and Honduras were examined for the presence of virions with marker characteristics of strains that cause large epidemics and equine epizootics. Thirty-four strains came from before and 60 strains came from after the Middle American epidemics and equine epizootics of 1966 and 1969-1972. Twenty-three virion clones that resembled epizootic strains by hydroxylapatite chromatography and Vero monkey kidney cell plaque size determinations were characterized further. However, the predominant virions in these clones were like enzootic strains from Middle America north of the Panama Canal region, and not like Middle American epizootic VE strains, since they were in hemagglutination-inhibition antigenic subtype IE, usually had optimal pH of hemagglutination at 6.2, and were avirulent for English shorthair guinea pigs inoculated subcutaneously. These results provide evidence against the theory of origin of epidemic-equine epizootic VE virus strains that posits that epizootic virions emerge in Middle America from strains containing mixtures of enzootic and epizootic virions in enzootic habitats.

Enfermedad humana causada por el virus Nepuyo, un bunyavirus de mesoamerica transmitido por mosquitos. / Human diseases caused by Nepuyo virus, mosquito-borne bunyavirus of Middle America

En el curso de estudios sobre el virus de la encefalitis venezolana realizados en La Avellana, departamento de Santa Rosa, en la costa del Pacifico de Guatemala se contrajeron dos infecciones por virus Nepuyo, un bunyavirus del complejo del grupo C de la zona neotropical. Un caso se presento en agosto de 1972 y el otro en agosto de 1977. En ambos casos aparecio una enfermedad febril leve, parecida al dengue, con viremia, comienzo subito, cefalea, mialgias y postracion. Se encontraron anticuerpos inhibidores de la hemaglutinacion y neutralizantes, pero no anticuerpos fijadores del complemento. Probablemente los vectores selvaticos sean mosquitos del complejo Culex (Melanoconion), mientras que como huespedes vertebrados amplificadores de los arbovirus del grupo C actuan roedores, marsupiales y seres humanos. En todo caso, convendria realizar estudios ecologicos detallados

Mesenteronal infection threshold of an epizootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implication to the apparent disappearance of this virus strain from an enzootic habitat in Guatemala.

Culex (Melanoconion) taeniopus is a vector of Venezuelan encephalitis (VE) virus at a marsh focus in Guatemala and has low mesenteronal thresholds for infection by and transmission of two enzootic strains of VE virus. In contrast, samples of natural populations and subsequent F2 and F4 generations of these mosquitoes have a high mesenteronal threshold for infection by an epizootic VE strain isolated at the same marsh during the end of the 1969 VE epidemic-equine epizootic. The resistance of Cu. (Mel). taeniopus to mesenteronal infection by this VE strain probably represents a key factor in the apparent disappearance of epizootic VE virus from the marsh focus following the 1969 outbreak.

Intestinal threshold of an enzootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implications to vector competency and vertebrate amplifying hosts.

The minimal intestinal dose of an enzootic strain of Venezuelan encephalitis (VE) virus for Culex (Melanoconion) taeniopus mosquitoes caught at a marsh habitat of VE virus in Guatemala was less than five plaque forming units (pfu) of virus. Ingestion of this dose of virus in blood of viremic hamsters resulted in transmission of virus to other hamsters. This low intestinal threshold of an enzootic strain of VE virus indicates that the natural Guatemalan population of Cu. (Mel.) taeniopus can acquire VE virus from vertebrates that have viremia levels as low as 1,000-5,000 pfu/ml of blood, provided other factors do not limit virus interchange between mosquitoes and vertebrates.

Sensitivity of Toxorhynchites amboinensis mosquitoes versus chicken embryonic cell cultures for assays of Venezuelan encephalitis virus.

Toxorhynchites amboinensis mosquitoes inoculated intrathoracically with Venezuelan encephalitis virus and tested for infectious virus 12 to 19 days later by inoculation of primary chicken embryonic cell cultures yielded approximately the same titers of virus as did direct inoculation of cultures, with counting of plaques 3 days thereafter. Titers were slightly higher in T. amboinensis for three virus strains, equal for two virus strains, and slightly lower for two virus strains. Comparative titers of four strains were similar, whether virus suspensions came from infectious vertebrate cells or infected invertebrate (T. amboinensis) cells. Each of the seven strains of Venezuelan encephalitis virus reached virus concentrations of 10(4.2) to 10(5.1) plaque-forming units in individual T. amboinensis mosquitoes after extrinsic incubation periods of 12 to 19 days. No temperature-sensitive (29 versus 37 degrees C) virus was detected in T. amboinensis mosquitoes infected by six strains of Venezuelan encephalitis virus.

Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations.

Transmission of Venezuelan encephalitis virus by naturally infected Culex (Melanoconion) opisthopus.

During August 1977 two of 975 Culex (Melanoconion) opisthopus collected from an enzootic marsh habitat on the Pacific coast of Guatemala transmitted VE virus to hamsters. Eight VE strains were isolated from Cu. opisthopus. The minimal level of VE infection in this species during July-August 1977 at La Avellana, Guatemala was 1/128 (8/1,021), and the prevalence of Cu. (Mel.) opisthopus transmitting VE virus was 1/487 (2/975). This mosquito was the predominant species attacking humans at that time, suggesting that Cu. opisthopus is a vector of VE virus to man as well as a vector in enzootic cycles in Guatemala. These studies establish Cu. opisthopus as the third proven enzootic vector of VE virus.