Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus.

An important question pertaining to the natural history of Venezuelan equine encephalitis (VEE) virus concerns the source of epizootic, equine-virulent strains. An endemic source of epizootic virus has not been identified, despite intensive surveillance. One of the theories of epizootic strain origin is that epizootic VEE viruses evolve from enzootic strains. Likely enzootic sources of VEE virus occur in Colombia and Venezuela where many of the epizootic outbreaks of VEE have occurred. We have determined the nucleotide sequences of the entire genomes of epizootic VEE subtype I-C virus, strain P676, isolated in Venezuela, and of enzootic VEE subtype I-D virus, strain 3880, isolated in Panama. VEE subtype I-D viruses are maintained in enzootic foci in Panama, Colombia, and Venezuela. The genomes of P676 and 3880 viruses differ from that of VEE subtype I-AB virus, strain Trinidad donkey (TRD), by 417 (3.6%) and 619 (5.4%) nucleotides, respectively. The translated regions of P676 and 3880 genomes differ from those of TRD virus by 54 (1.4%) and 66 (1.8%) amino acids, respectively. This study and the oligonucleotide fingerprint analyses of South American I-C and I-D viruses (Rico-Hesse, Roehrig, Trent, and Dickerman, 1988, Am. J. Trop. Med. Hyg. 38, 187-194) provide the most conclusive evidence to date suggesting that equine-virulent strains of VEE virus arise naturally from minor variants present in populations of I-D VEE virus maintained in enzootic foci in northern South America.     

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.     

Susceptibility of Psorophora confinnis (Diptera: Culicidae) to infection with epizootic (subtype IC) and enzootic (subtype ID) Venezuelan Equine encephalitis viruses

To test the hypothesis that adaptation to epizootic mosquito vectors mediates the emergence of Venezuelan equine encephalitis virus (family Togaviridae, genus Alphavirus, VEEV) from enzootic progenitors, the susceptibility of the epizootic vector Psorophora confinnis (Lynch-Arribalzaga) to epizootic versus enzootic strains was evaluated. Artificial bloodmeals containing 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 mosquito infectivity and transmission potential. Strains from the smaller 1992-1993 epizootic showed lower or equal infectivity and replication compared with enzootic viruses and to strains isolated during the larger 1962-1964 and 1995 epizootics. These experiments failed to provide evidence that Ps. confinnis selects for epizootic VEEV viruses with higher infectivity, as has been shown for Aedes (Ochlerotatus) taeniorhynchus (Wiedemann). Nonetheless, its high susceptibility, abundance in enzootic and epizootic regions, and feeding behavior suggest that Ps. confinnis is an important bridge vector for both enzootic and epizootic VEEV.     

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 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.     

Experimental Infection of Horses with an Attenuated Venezuelan Equine Encephalomyelitis Vaccine (Strain TC-83)

Ten horses (Equus caballus) were vaccinated with strain TC-83 Venezuelan equine encephalomyelitis (VEE) virus vaccine. Febrile responses and leukopenia due to a reduction of lymphocytes and neutrophils were observed in all animals. Viremias were demonstrable in eight horses, with a maximum of 10(3.5) median tissue culture infectious dose units per ml of serum in two horses. Clinical illness with depression and anorexia were observed in five horses. Neutralizing (N), hemagglutination-inhibiting, and complement-fixing antibodies to the vaccine virus were demonstrable by 5, 6.5, and 7 days, respectively, after vaccination. Differential titrations of serum to six VEE strains revealed high titers of N antibody to vaccine virus, moderate titers to the epizootic Trinidad donkey no. 1 strain (VEE antigenic subtype I, variant A) from which TC-83 was derived, and low titers to two other epizootic strains (subtype I, variants B and C) in all horses at 1 month after vaccination; some animals responded with low levels of N antibody to the enzootic viruses (subtype I, variants D and E). Fourteen months after vaccination, six animals with detectable N antibody were challenged with MF-8 (subtype I, variant B), an epidemic-epizootic strain isolated in 1969 from a man in Honduras. All horses resisted challenge with the equine pathogenic strain of VEE. Marked increases of N antibody in most horses were demonstrable to some VEE strains when tested 1 month after challenge.     

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.     

Genetic targets for the detection and identification of Venezuelan equine encephalitis viruses

Summary.  Rt-PCR probes targeted to different gene sequences of VEE (Venezuelan equine encephalitis) virus strain TC-83 were assessed for their sensitivity, specificity and non-specific cross-reactivity. A generic VEE virus amplimer (VNSP4F2/VNSP4R2), targeted against nsP4 was identified, which was sensitive (detected at least 10 pfu) and robust (worked over a wide range of salt concentrations and annealing temperatures). An E2 amplimer designed against TC-83, (VE2F/VE2R), identified VEE strains TRD (1AB), P676 (1C), 3880 (1D) Everglades (2) vRNA whilst a second E2 primer pair designed against strain 68U201, (68UF/68UR), identified all the remaining VEE viruses in the sero-complex. This would suggest that the VEE virus E2 gene can be sub-divided at the genetic level into two separate groups making it a useful target for differentiation of sero-subtypes 1 and 2 from the other VEE virus subtypes. Using a panel of amplimers targeted to different VEE genes and strains it was possible to distinguish between most of the serotypes, but most importantly, we were able to detect the epizootic strains TRD and P676 as well as other VEE viruses implicated in human disease (sero-subtypes 1D and 1E). Accepted November 13, 1997 Received August 24, 1997     

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.     

The differentiation of viruses in the Venezuelan equine encephalomyelitis complex by using monoclonal antibodies and lanthanide immunofluorescence analysis]

Potentialities of differentiation between Venezuelan equine encephalomyelitis (VEE) complex viruses by time-resolved fluoroimmunoassay and enzyme immunoassay were studied. For this, 4 test systems were used based on different combinations of native and labeled polyclonal antibodies to VEE virus, strain Trinidad, and monoclonal (MCA) antibody MAK 14-7 to protein EL of this virus. The maximal sensitivity and specificity was achieved in the test system formed from native MCA MAK 14-7 for sensitization of the solid phase and labeled polyclonal immunoglobulins for demonstration of the test results. This combination of antibodies allowed to differentiate the epidemic variant of VEF/Trinidad (IA) from epizootic variants of Mucambo (III), Pixuna (IV) and attenuated strain No. 230.     

Comparative infections of epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus in Amblyomma cajennense (Acari: Ixodidae).

To compare the potential for an enzootic or an epizootic strain of Venezuelan equine encephalomyelitis (VEE) virus to infect Amblyomma cajennense (F.), larval ticks were fed on guinea pigs (strain 13) inoculated with an enzootic viral strain of variant I-E (68U201) or on guinea pigs inoculated with an epizootic strain of variant I-A (Trinidad donkey). Peak viremias were 10(5.2) plaque-forming units (PFU)/ml and 10(7.3) PFU/ml in guinea pigs infected with enzootic and epizootic viral strains, respectively. Ticks feeding on enzootic- and epizootic-infected hosts had viral titers of 10(2.5) and 10(3.9) PFU per tick, respectively, at drop-off. Although epizootic virus was recovered from 98% (127 of 130) of larval ticks up to 16 d after drop-off, enzootic virus was recovered from 95% (19 of 20) at drop-off (mean titer, 10(2.5) PFU per tick), with recovery rates declining rapidly to 2 of 10 (mean titer, 10(1.4) PFU per tick) by 16 d after drop-off. Transstadially transmitted epizootic virus was found in 0.4% (12 of 2,950) of unfed nymphs (mean titer, 10(2.8) PFU per tick) 63 d after drop-off, 1% (5 of 521) fed nymphs 69 d after drop-off, and 1% (4 of 400) of unfed adults (mean titer, 10(3.6) PFU per tick) 106 d after drop-off. No enzootic virus was recovered from 4,600 unfed nymphs tested 63 d after drop-off.     

Use of a new synthetic-peptide-derived monoclonal antibody to differentiate between vaccine and wild-type Venezuelan equine encephalomyelitis viruses.

We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.     

Spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates.

Strains of vesicular stomatitis virus, New Jersey serotype (VSV-NJ), isolated from diseased cattle or swine were examined by genomic RNA sequencing for genetic diversity potentially leading to antigenic variations in their type-specific glycoproteins as determined by reactivity with epitope-specific monoclonal antibodies (MAbs). Seven field isolates recovered in Colorado, New Mexico, Georgia, and Mexico during the widespread 1982-1985 epizootic in the western United States resembled the prototypic 1952 Hazelhurst subtype by partial sequence homology, but amino acid reversions to the 1949 Ogden subtype occurred frequently. When studies were performed with MAbs directed to the Ogden subtype glycoprotein, relatively limited antigenic variation, and only in neutralization epitope VIII, was noted among two of five epizootic isolates from Colorado and New Mexico. However, amino acid differences in the glycoprotein of a 1983 isolate from an enzootic region of Georgia resulted in major antigenic deficiencies in epitopes V, VI, and VII as determined by Western blotting and neutralization of infectivity with epitope-specific MAbs. Quite a few genetic but no antigenic differences were noted in an enzootic 1984 isolate from Mexico, a potential origin of the United States epizootic. Marked or complete loss of epitopes VII, VI, VIII, and V can be traced to spontaneous mutations leading to amino acid substitutions at glycoprotein positions 199, 263, 275, and 317, respectively, in the enzootic Georgia isolate 07/83-GA-P and the epizootic New Mexico isolate 06/85-NM-B. By comparison, closely adjacent amino acid substitutions at glycoprotein positions 210, 268, 277, and 364 occurred in epitope-deficient mutants selected for resistance to neutralization by MAbs specific for epitopes VII, VI, VIII, and V, respectively. Two neutralization epitopes designated X and XI were found to be unique for the G protein of the 1952 Hazelhurst isolate..../52-GA-P. The epitope X-specific MAb H21, in particular, failed to neutralize the infectivity not only of the Ogden subtype..../49-UT-B but also was ineffective against all the 1982-1985 field isolates. The classical 1952 Hazelhurst strain of VSV-NJ is genetically and antigenically quite different from those viruses isolated during the 1982-1985 epizootic.     

Monoclonal antibodies capable of distinguishing epizootic from enzootic varieties of subtype 1 Venezuelan equine encephalitis viruses in a rapid indirect immunofluorescence assay.

We used previously characterized murine monoclonal antibodies to develop a panel useful in subtyping Venezuelan equine encephalitis (VEE) viruses by an indirect fluorescent antibody assay. This panel worked well with either prototype VEE viruses or a series of more recent VEE virus isolates. The panel is particularly useful for rapidly differentiating VEE viruses with epidemic-epizootic potential from other endemic varieties of this virus. Using this panel, we identified an antigenic variant of prototype VEE subtype 1E virus currently present in Mexico. This antigenic change in the E2 glycoprotein was confirmed by enzyme-linked immunosorbent assay. Because VEE virus virulence has been associated in part with the E2 glycoprotein, this observed antigenic change in the 1E virus E2 glycoprotein may explain the apparent equine virulence of this unusual VEE 1E virus.     

Newcastle disease in the European Union 2000 to 2009

Newcastle disease (ND) is a devastating disease of poultry that has to some extent been neglected by those working in the field in the past 10 to 15 years while attention has been focused on the emergence and spread of highly pathogenic avian influenza caused by a H5N1 subtype virus. During 2000 to 2009 in the European Union (EU) member states, ND viruses virulent for chickens have been detected in wild birds, domesticated pigeons and poultry. Based on these isolations it appears that the epizootic in racing pigeons caused by the variant viruses termed pigeon avian paramyxovirus type 1, which form the genetic group 4b(VIb) first seen in Europe in 1981, continued during 2000 to 2009, and the virus is probably enzootic in racing pigeons in some EU countries. This virus appears to have spread regularly to wild birds, especially those of the Columbidae family, and has been the cause of significant outbreaks in poultry. Other avian paramyxovirus type 1 viruses responsible for ND outbreaks in the EU during 2000 to 2009 have been those from genetic groups 5b(VIIb) and 5d(VIId). There is evidence that the former may well represent spread from a wild bird source and these viruses have also been isolated from wild birds, while the latter represents continuing spread from the East. Future legislation or recommendations aimed at the control and eradication of ND will need to encompass these three sources of virulent ND viruses.     

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.     

Equine arteritis virus-infected cells contain six polyadenylated virus-specific RNAs

Fluorescamine-labeled structural proteins of viruses in the Venezuelan equine encephalitis (VEE) serologic complex were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified [¹⁴C]lysine-labeled capsid protein and El and E2 envelope glycoproteins of TC-83 (serologic subtype-variant I-A) virus were mixed with the corresponding [³H]lysine-labeled structural proteins of TC-83, PTF-39 (I-B), P676 (I-C), 3880 (I-D), Mena II (I-E), Everglades Fe 3–7c (II), Mucambo BeAn 8 (III), and Pixuna BeAr 35645 (IV) viruses and codigested with trypsin. Tryptic peptides were resolved by reverse-phase high-pressure liquid chromatography. The capsid and El proteins of I-A, I-B, I-C, and I-D viruses produced identical or nearly identical tryptic peptide maps, whereas the maps of I-E, II, III, and IV capsid and El proteins were distinct from the maps of the corresponding I-A proteins. The tryptic peptide maps of the type-specific protein, E2, of the various viruses showed the most variation and correlated well with the serologic and genetic homologies determined by oligonucleotide fingerprinting of VEE 42 S RNA. Fluorescamine derivatization of viral proteins was shown to have little or no effect on the specificity of trypsin. Trypsin was shown to cleave these proteins at both lysine and arginine residues.     

A longitudinal study of velogenic Newcastle disease virus genotypes isolated in Italy between 1960 and 2000

Thirty-six representative velogenic strains of Newcastle disease virus isolated in Italy since 1960 were characterized by restriction site and partial sequence analyses of the fusion protein gene. Viruses belonging to the six known genotypes of Lomniczi et al . were found. Genotype IV, which was most probably the main epizootic group in Europe before the war, was responsible for outbreaks in the 1960s and persisted until the late 1980s in Italy. An epizootic peak in 1972 to 1974 coincided with the appearance of genotype V viruses that were present for more than a decade. Outbreaks in 1992 were caused by genotype VIIa viruses and were part of a contemporaneous epizootic of Far East origin that affected Western European countries. The Newcastle disease epizootic that commenced in Italy in May 2000 was due to a genotype VIIb virus that is indistinguishable from those causing sporadic outbreaks in Great Britain and Northern Europe in the late 1990s. Isolated cases yielded a variant of genotype VI (reference epizootic: Middle East in the late 1960s) and a group VIII virus (enzootic in South Africa).     

Spatial dispersion of adult mosquitoes (Diptera: Culicidae) in a sylvatic focus of Venezuelan equine encephalitis virus.

We studied the spatial localization of mosquitoes in sylvatic focus of Venezuelan equine encephalitis virus in western Venezuela to identify mosquito species potentially involved in the hypothesized transport of viruses out of enzootic foci. The following criteria were used to identify species with potential for virus export: (1) common in the forest and surrounding area, (2) feeding on a wide range of vertebrates; (3) long dispersal capabilities, and (4) established vectorial competence for enzootic or epizootic VEE viruses. CDC traps baited with light/CO2 were operated for four and 12-h intervals to collect mosquitoes at four stations along two forest/open area transects from September to November 1997. We collected 60,444 mosquitoes belonging to 11 genera and 34 species. The most common species were Aedes serratus (Theobald), Ae. scapularis (Rondani), Ae. fulvus (Wiedmann), Culex nigripalus Theobald, Cx, (Culex) "sp", Cx. mollis Dyar & Knab, Cx. spissipes (Theobald), Cx. pedroi Sirivanakarn and Belkin, Psorophora ferox (Humboldt), Ps. albipes (Theobald), and Ps. cingulata (F.). Very few mosquitoes were captured during the (day in the open area outside the forest, suggesting that any virus export from the forest may occur at night. The following mosquitoes seemed to be mostly restricted to the forest habitat: Ae. serratus, Ps. ferox, Ps. albipes, sabethines, Cx. spissipes, Cx. pedroi, Cx. dunni Dyar, and Ae. fulvus. The main species implicated its potential virus export were Cx. nigripalpus, Ae. scapularis, and Mansonia titillans (Walker).