Transmission of Venezuelan equine encephalomyelitis virus by strains of Aedes albopictus (Diptera: Culicidae) collected in North and South America

Experimental studies were undertaken to ascertain the vector potential of North American (Houston and Alsace) and South American (Sao Paulo and Santa Teresa) strains of Aedes albopictus (Skuse) for an epizootic (Trinidad donkey) strain of Venezuelan equine encephalomyelitis (VEE) virus. Infection rates were similar in all four strains of Ae. albopictus tested after ingestion of VEE virus from a viremic hamster. Virus disseminated from the midgut to the hemocoel in about 80% of infected mosquitoes, regardless of the dose ingested (10(4.6) to 10(5.7) plaque-forming units per mosquito) or the time of extrinsic incubation (7-35 d). Although all four strains of this mosquito transmitted VEE virus by bite to hamsters, transmission rates were significantly higher for the South American strains (24%, 40 of 170) than for the North American strains (5%, 9 of 165). Although VEE virus has never been isolated from Ae. albopictus, the introduction of this species into the Americas may allow it to serve as an amplification vector in areas where epizootic strains of VEE are found or introduced.     

Experimental transmission of Venezuelan equine encephalomyelitis virus by a strain of Aedes albopictus (Diptera: Culicidae) from New Orleans, Louisiana.

Experimental studies were undertaken to ascertain the vector competence of a strain of Aedes albopictus (Skuse) collected in New Orleans, LA, (Gentilly strain) for an epizootic (Trinidad donkey) strain of Venezuelan equine encephalomyelitis (VEE) virus. This strain of Ae. albopictus was significantly more susceptible to infection with VEE virus than were any of the four strains tested previously, including two from North America and two from South America. Likewise, dissemination (148 of 180) (82%) and transmission (40 of 88) (45%) rates were significantly higher in the Gentilly strain than in any of the strains previously tested. Analysis of the results of the present study along with those of a previous study with a second alphavirus, chikungunya (CHIK) virus, indicated that, although all three strains of Ae. albopictus tested were more susceptible to VEE virus than to CHIK virus, susceptibility to infection and dissemination with one alphavirus appeared to be directly related to susceptibility to infection and dissemination with the other virus and may indicate shared receptor sites for these two alphaviruses in Ae. albopictus.     

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae)

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.     

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus

Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of approximately 10(8) plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates > or = 87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates > or = 50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.     

Vector competence of three Venezuelan mosquitoes (Diptera: Culicidae) for an epizootic IC strain of Venezuelan equine encephalitis virus.

Experimental studies were undertaken to evaluate the vector competence of selected mosquito species [Aedes taeniorhynchus (Wiedemann), Culex declarator Dyar and Knab, and Mansonia titillans (Walker)] from northwestern Venezuela for the epizootic (IC) strain of Venezuelan equine encephalitis (VEE) virus that was responsible for the 1995 outbreak of VEE in this area. Ae. taeniorhynchus was highly susceptible to infection (94% of 35), and 89% had a disseminated infection. Virus-exposed Ae. taeniorhynchus that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE virus. In contrast, only 1 of 28 (4%) Cx. declarator was infected, and that individual did not develop a disseminated infection. Ma. titillans was moderately susceptible (3 of 8 infected, 38%), and 2 (25%) of these had a disseminated infection. These data indicate that Ae. taeniorhynchus was an important epizootic vector during the 1995 VEE outbreak in Columbia and Venezuela.     

A monoclonal antibody study of the receptor area of the Venezuelan encephalitis virus]

The receptor region for virus-cell interaction in Venezuelan equine encephalomyelitis (VEE) and Eastern equine encephalomyelitis (EEE) viruses was studied using a panel of 17 monoclonal antibodies (MCA). They were able to block agglutination of goose erythrocytes. The dominant role of glycoprotein E2 in the formation of viral receptor for EEE and VEE viruses was demonstrated. Competitive radioimmunoassay identified three antigenic sites in this region. These sites were also responsible for virus neutralization. MCAs to these sites protected outbred mice against lethal infection. The presence of a highly conservative region in VEE (site E2-3) and EEE (site E2a) which produced cross-reacting antibodies blocking hemagglutination of Western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya, and Pixuna viruses was established. A hypothesis is suggested concerning the existence of similar regions for the entire alphavirus genus, and the role of this region in virus-cell interaction.     

Radial haemolysis in gel used in serological studies on Venezuelan equine encephalomyelitis virus

Radial haemolysis in gel was successfully used for the detection of antibody in convalescents after laboratory infection with Venezuelan equine encephalomyelitis (VEE) virus at remote intervals after infection (up to 23 years). The antibodies reacted only with VEE antigen but not with Sindbis and Chikungunya alphavirus antigens. Radial haemolysis in gel was sensitive as the haemagglutination inhibition test but the antibody could be titrated by the diameter of the haemolysis zone omitting the serum dilutions. Slight deviations in the virus dose used for sensitization of erythrocytes or in pH values were not critical. For the test, the sera were heated at 56 degrees C for 30 min. Both sera stored for long periods and those collected on paper disks could be used.     

Vector competence of Mexican and Honduran mosquitoes (Diptera: Culicidae) for enzootic (IE) and epizootic (IC) strains of Venezuelan equine encephalomyelitis virus

Experimental studies evaluated the vector competence of Ochlerotatus taeniorhynchus (Wiedemann), Culex cancer Theobald, Culex pseudes (Dyar and Knab), Culex taeniopus Dyar and Knab, and a Culex (Culex) species, probably Culex quinquefasciatus Say, and Culex nigripalpus Theobald from Chiapas, Mexico, and Tocoa, Honduras, for epizootic (IC) and enzootic (IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. Culex pseudes was highly susceptible to infection with both the IC and IE strains of VEE (infection rates >78%). Patterns of susceptibility to VEE were similar for Oc. taeniorhynchus collected in Mexico and Honduras. Although Oc. taeniorhynchus was highly susceptible to the epizootic IC strains (infection rates > or = 95%, n = 190), this species was less susceptible to the enzootic IE strain (infection rates < or = 35%, n = 233). The Culex (Culex) species were refractory to both subtypes of VEE, and none of 166 contained evidence of a disseminated infection. Virus-exposed Cx. pseudes that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE. Although Oc. taeniorhynchus that fed on hamsters infected with the epizootic IC strain transmitted VEE efficiently, only one of six of those with a disseminated infection with the enzootic IE virus that fed on hamsters transmitted virus by bite. These data indicate that Cx. pseudes is an efficient laboratory vector of both epizootic and enzootic strains of VEE and that Oc. taeniorhynchus could be an important vector of epizootic subtypes of VEE.     

Vector competence of rural and urban strains of Aedes (Stegomyia) albopictus (Diptera: Culicidae) from São Paulo State, Brazil for IC, ID, and IF subtypes of Venezuelan equine encephalitis virus

Aedes albopictus (Skuse) is an Asiatic mosquito species that has spread and colonized all continents except Antarctica. It has major public health importance because it is a potential vector of several pathogens. The objectives of our study were to analyze the vector competence of urban and rural strains of Ae. albopictus from S?o Paulo State (Brazil) for Venezuelan equine encephalitis virus (VEE) subtypes IC, ID, and IF, and to evaluate the effect of infection with subtype IC of VEE on mosquito longevity. Both mosquito strains were susceptible to subtypes IC and ID, but the infection rate for subtype IF was low. Infection and transmission rates of Ae. albopictus for subtype IC were similar to those reported for Ochlerotatus taeniorhynchus (Wiedemann). The high infection, dissemination, and transmission rates for subtype ID reported for Oc. fulvus (Wiedemann) and Culex (Melanoconion) spp. are comparable with those found in this study. We found significant differences in the susceptibility to subtype IC between rural and urban populations of S?o Paulo. Significant survival rate differences were observed between uninfected and infected mosquitoes, but there were no differences in survival between rural and urban mosquito strains.     

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3′ terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alpha-viruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5′ and 3′ noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%,50 to 53%,and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.     

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex.

The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.     

Virulent and attenuated mutant Venezuelan equine encephalitis virus show marked differences in replication in infection in murine macrophages

One of the first target cells at the site of inoculation with an alphavirus may be monocytes or macrophages. The replication kinetics of virulent and attenuated molecularly clonedVenezuelan equine encephalitis virus(VEE) in murine macrophages were therefore compared. Infection of both quiescent and activated mouse primary peritoneal macrophages with a molecularly cloned, virulent VEE termed V3000 resulted in peak virus titres of 10⁴plaque forming units (PFU)/ml supernatant by 24 h post-infection (pi), followed by rapidly decreasing virus titres. In contrast, a molecularly cloned attenuated VEE mutant, V3032, that differs from V3000 by a single amino acid at E2 glycoprotein position 209 (glu→lys) replicated more slowly and to higher titres (10⁶PFU/ml supernatant) that peaked at 72 h pi. Replication of V3032, but not V3000, was sharply restricted by prior activation of macrophages with lipopolysaccharide or interferon-γ. These results indicate that virulent V3000 and attenuated V3032 differ in their growth kinetics in both quiescent and activated macrophages. Thus, macrophages, and their specific activation state, may play a major role in virulent and attenuated VEE replication and pathogenesis.     

Ecological characterization of the aquatic habitats of mosquitoes (Diptera: Culicidae) in enzootic foci of Venezuelan equine encephalitis virus in western Venezuela

We studied the aquatic mosquito habitats in and around enzootic foci of Venezuelan Equine Encephalitis virus (VEE) in western Venezuela. Specimens were sampled for 5 mo in three types of vegetation: tall lowland tropical forests, short inundated/secondary growth forests, and pastures/herbaceous vegetation around forests. Ground pools, flooded pastures, swamps, ponds, and canals predominated. We used a multivariate statistical approach to quantitatively assess the relationships of mosquito species with broad categories of the landscape, and with environmental variables within each aquatic habitat. Twenty-four mosquito species in the genera Aedes, Psorophora, Culex, Mansonia, and Uranotaenia were collected. Species richness was higher in the tall forests than in other types of vegetation. Discriminant Function Analysis showed a strong association between landscape category and mosquito species assemblage and identified Culex erraticus Dyar & Knab and Mansonia titillans Walker as indicator species of open areas, and Aedes serratus Theobald as an indicator of tall forests. M. titillans, Uranotaenia geometrica Theobald, Cx. erraticus, and Culex dunni Dyar were associated with unshaded, warm, vegetated waters in flooded pastures and swamps, whereas Ae. serratus, Aedes fulvus (Wiedemann), Psorophora albipes Theobald, Psorophora ferox (Humboldt), Culex caudelli Dyar & Knab, and Culex pedroi Sirivanakarn & Belkin were associated with small, shaded ground pools within the tall forests. Culex coronator Dyar & Knab was associated with partially exposed sites within short forests. These results allowed us to interpret better our previous studies on mosquito adult spread in the study area and their possible role as VEEV disseminators.     

Lymphoreticular and myeloid pathogenesis of Venezuelan equine encephalitis in hamsters.

Ultrastructural, histopathologic, and virologic studies of adult hamsters infected with virulent Venezuelan equine encelphalomyelitis (VEE) virus (Subtype I-B) demonstrated precise chronologic and topographic progression of lesions and viral replication in extraneural sites. Thymus contained the earliest lesions and the highest initial and subsequent viral titers. No particular cytotropism was observed as highly efficient viral replication and severe cytonecrosis proceded. Early cortical necrosis of splenic periarteriolar lymphocytic sheath was followed by lymphoblastoid repopulation of the peripheral zone. Massive bone marrow necrosis was accompained by ultrastructural evidence of VEE viral particle production in reticulum cells, rubricytes, myeloid cells, lymphoblastoid cells, and megakaryocytes. Speed, efficiency, destructiveness, and relative sensitivity of virtually all lymphoreticular and hematopoetic cells were hallmarks of virulent VEE infection in the hamster.     

Cross-Protection in Hamsters Immunized with Group A Arbovirus Vaccines

Cross-protection between Venezuelan, Eastern, and Western equine encephalomyelitis (VEE, EEE, WEE) viruses was studied in the hamster by using challenge responses and neutralizing antibody titers as indexes of protection. Formalin-inactivated vaccines induced only homologous protection regardless of the sequence of vaccination or the combination of vaccines employed. Use of attenuated VEE vaccine, singly, produced absolute homologous protection as well as 37 and 59% protection against WEE and EEE challenges, respectively. Neither deleterious nor enhancing interaction occurred when attenuated VEE and inactivated WEE and EEE vaccines were employed in various sequences of immunization and all possible combinations. The most rapid and simple immunization scheme eliciting excellent homologous protection consisted of a single dose of combined attenuated VEE and inactivated WEE and EEE vaccines. Studies with attenuated strains of VEE, EEE, and WEE viruses showed that all elicited excellent homologous protection when administered singly. However, use of these live strains in many combinations and sequences resulted in a significant (P < 0.05 to <0.001) decrease in the protective efficacy of the WEE or EEE strains. These results are discussed in relation to serum neutralization test data obtained on sera drawn pre- and postchallenge.     

The detection of antibodies to the Venezuelan equine encephalomyelitis virus by immunoenzyme analysis using antigen purified in a water-polymer system]

Human and animal sera were tested for the presence of antibodies to Venezuelan equine encephalomyelitis (VEE) virus by direct enzyme immunoassay (EIA). For the test, the plates were sensitized with a VEE virus preparation purified in a two-phase system of water-soluble polymers. The proposed EIA variant was as specific as that with VEE antigen obtained by fractionation on sucrose cushion, but more sensitive. The high specificity of the assay allowed the antigen purified in the water-polymer system to be used for investigation of antigen relationships among viruses of the VEE complex.     

Susceptibility of Ochlerotatus taeniorhynchus (Diptera: Culicidae) to infection with epizootic (subtype IC) and enzootic (subtype ID) Venezuelan equine encephalitis viruses: evidence for epizootic strain adaptation

To test the hypothesis that adaptation to epizootic mosquito vectors mediates emergence of Venezuelan equine encephalitis virus (VEEV) from enzootic progenitors, experimental infection studies were conducted to determine the susceptibility of Ochlerotatus taeniorhynchus (Wiedemann) to epizootic and enzootic strains. Artificial blood meals containing epizootic subtype IC strains isolated during the 1962-1964, 1992-1993, and 1995 Venezuelan/Colombian epizootics and closely related Venezuelan enzootic subtype ID strains were used to compare infectivity and transmission potential. Their greater infectivity and replication suggested that adaptation of epizootic strains to Oc. taeniorhynchus may have enhanced epizootic transmission during the 1962-1964 and 1995 IC coastal epizootics. However, strains from the small 1992-1993 Venezuelan outbreak that did not extend to coastal regions do not seem to infect this species better than closely related subtype ID strains. Adaptation of VEEV to epizootic vectors such as Oc. taeniorhynchus mosquitoes may be a determinant of some but not all VEE emergence events and may influence spread into coastal regions.     

Opsonization of alphaviruses in hamsters

Immune elimination of alphaviruses in immunized hamsters appears to involve formation of virus/antibody aggregates which are subsequently cleared from the circulation by cells of the reticuloendothelial system (RES). Virulent strains of Venezuelan (VEE) and Western equine encephalitis (WEE) viruses which were cleared slowly from the circulation of nonimmune hamsters, were cleared rapidly when inoculated into the blood of immunized hamsters. Likewise, when these viruses were mixed with specific hamster immune serum prior to inoculation, they were efficiently cleared from the circulation of nonimmune hamsters. Virus, mixed with specific immune serum, or inoculated into immunized hamsters, formed virus/antibody aggregates, as demonstrated by density gradient centrifugation, filtration through polycarbonate membranes, precipitation with Staphylococcus protein A, and electron microscopy. Cleared virus was concentrated primarily in liver and spleen, as confirmed by autoradiography. Immune clearance of virulent VEE was demonstrable within 5 to 6 days following immunization of hamsters with live attenuated VEE vaccine, strain TC-83. In these hamsters, a close association was established between formation of virus/antibody aggregates, rapid clearance, and survival of challenged hamsters. Adsorption of virus to hamster macrophages in culture was enhanced by immune serum in the presence of complement. These results are compatible with the hypothesis that immune clearance of virus in the intact hamster involves a complement-dependent interaction of virus/antibody complexes with cells which possess Fc and complement receptors. The clearance of immune complexes by the RES serves to amplify the protective effect of neutralizing antibody alone.