Susceptibility of Peruvian mosquitoes to eastern equine encephalitis virus

Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of 4105 plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedesfulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophoraferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of approximately 10(8) PFU/ml, Cx. pedroi, Ae.II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.     

Transmission of a Venezuelan equine encephalitis complex Alphavirus by Culex (Melanoconion) gnomatos (Diptera: Culicidae) in northeastern Peru

Venezuelan equine encephalitis (VEE) complex alphaviruses are serious health threats in the Americas and regularly infect humans living in or near Amazonian rain forests. As part of a larger surveillance program, we placed six hamster-baited mosquito traps in a disturbed white sand forest of northeastern Peru for 3 d. Virus isolations from hamster serum and trapped mosquito pools demonstrated that a VEE subtype IIIC alphavirus was transmitted to a hamster by the mosquito Culex (Melanoconion) gnomatos Sallum, Hutchings & Ferreira. This species, like the other seven proven VEE complex alphavirus vectors, is a member of the Spissipes section of this subgenus. The composition of mosquitoes collected at the site over the sampling period was typical for the region.     

Seasonal distribution, biology, and human attraction patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos, Peru

This study was conducted as part of a field ecology study of arboviral activity in the Amazon Basin, Peru, to determine the taxonomy, frequency, seasonal, and vertical distributions of potential mosquito vectors. In addition, the relative efficiency of human-landing collections and dry ice-baited Centers for Disease Control (CDC)-type light traps was determined for collecting mosquitoes. A total of 70 species of mosquitoes from 14 genera were collected from June 1996 through December 1997 at a forested site near Puerto Almendras, approximately 20 km west-southwest of Iquitos, Peru. Three species [Psorophora (Janthinosoma) albigenu (Peryassu), Ochlerotatus (Ochlerotatus) fulvus (Wiedemann), and Ochlerotatus (Ochlerotatus) serratus (Theobald)] accounted for 70% of all mosquitoes captured in human-landing collections. Overall, biting activity occurred throughout the 24-h cycle but was higher during the daytime, primarily because of large populations of two day-biting species, Ps. albigenu and Oc. serratus. Oc. fulvus was active throughout the 24-h cycle but was more frequently collected during the evening. Oc. fulvus, Ps. albigenu, Culex (Melanoconion) pedroi Sirivanakarn & Belkin, and a mixture of Culex (Melaonoconion) vomerifer Komp, and Culex (Melanoconion) gnomatos Sallum, Huchings & Ferreira, accounted for 73% of the mosquitoes captured during darkness) by human collectors. In general, Ochlerotatus spp. and Psorophora spp. were more commonly captured in human-landing collections, whereas most Culex spp. were more frequently collected in the dry ice-baited CDC-type light traps. In general, mosquito populations were lowest from June through August when river levels were at their lowest. Two large population peaks occurred in November-December and in February-March as a result of "flood water" mosquito populations (e.g., Ps. albigenu). These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.     

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.     

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).     

Effect of triethylamine on the recovery of selected South American alphaviruses,flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.     

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland,Australia

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.     

Preparation and testing of vaccines prepared from the envelopes of Venezuelan, eastern, and western equine encephalomyelitis viruses.

Envelope components were separated from Venezuelan, Eastern, and Western equine encephalomyelitis viruses after treatment of the virions with detergent. Vaccines prepared from the envelope component were capable of stimulating mice to produce humoral antibodies. Protective efficacy studies were performed using mono-, di-, and trivalent vaccine combinations. These elicited varying degrees of homologous protection, and Eastern and Venezuelan equine encephalomyelitis envelope products appeared to confer protection to mice challenged with Western equine encephalomyelitis virus.     

Molecular phylogeny of the Vomerifer and Pedroi Groups in the Spissipes Section of the subgenus Culex (Melanoconion)

Members of the New World mosquito subgenus Melanoconion of the genus Culex are important vectors of many alphaviruses including eastern and Venezuelan equine encephalitis viruses (VEEV). We investigated the phylogenetic relationships among nine putative species of the Vomerifer and Pedroi Groups of the Neotropical Spissipes Section by sequencing the internal transcribed spacer 2 (ITS-2) region of ribosomal DNA and using phylogenetic analyses. Results demonstrated that, within the Spissipes Section, the Vomerifer and Pedroi Groups are monophyletic sister groups. The clade comprised by Culex adamesi and Culex ribeirensis showed a sister group relationship to the group consisting of Culex pedroi sequences. The monophyly of the Vomerifer Group corroborated previous suggestions that it is a natural group. However, our topology showed that there are two well-supported, divergent groups within a major clade consisting of Cx. pedroi sequences, suggesting the possibility of a cryptic Cx. pedroi-like species. This finding could have important epidemiological implications for VEEV transmission in Central and South America.     

Radioimmunoassay for Quantitation of Antibodies to Alphaviruses with Staphylococcal Protein A

A radioimmunoassay (RIA) procedure is described for measuring antibodies to alphaviruses in human and other mammalian sera. The test employed protein Abearing Staphylococcus aureus as a solid-phase immunoadsorbent for (3)H-labeled viruses complexed with immunoglobulin G. Using antibodies produced in humans and guinea pigs, the RIA procedure clearly differentiated among antibodies to Venezuelan, western, and eastern equine encephalomyelitis viruses. Sensitivity of the RIA depended on the concentrations of labeled viruses employed. The dilution of serum that effected binding of 50% of the (3)H-labeled virus (determined by probit analysis) was consistently higher than the neutralizing antibody titer determined by a conventional plaque reduction neutralization test using 80% plaque reduction end points. In addition, sera from 73 individuals were screened for seroconversion following live attenuated Venezuelan equine encephalomyelitis virus vaccine (strain TC-83) inoculation, by RIA using a single serum dilution (1:80); results were identical with seroconversions identified by plaque reduction neutralization test. Hyperimmune Venezuelan equine encephalomyelitis virus sera from a number of mammalian species were successfully titrated by RIA; the species tested were human, guinea pig, white rat, rabbit, burro, dog, monkey, sheep, and cotton rat. The protein A-mediated RIA is a rapid, sensitive, specific, and precise serological tool for measuring antibodies to surface antigens of alphaviruses, and should allow the subsequent development of a competitive binding RIA to measure antigenic potency of inactivated alphavirus vaccines.     

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.     

Antibody response of wild birds to natural infection with alphaviruses

From 1986 to 1990, we conducted our second longitudinal study in the central (upstate) New York (CNY) area on the wild avian hosts of eastern equine encephalomyelitis (EEE) virus. Field-collecting methods mirrored a study conducted from 1978 to 1980 at the same endemic focus. Over the 5-yr study period, we captured 6,296 birds representing 99 species and took 4,174 blood samples from representatives of 83 species. Gray catbirds, song sparrows, and veerys were the three dominant species captured and bled, accounting for 40 and 55% of birds captured and bled. Blood clots were assayed for virus and sera tested for hemagglutination-inhibition (HI) antibodies to EEE and Highlands J virus. Virus isolations from birds defined two epiornitics of EEE virus in 1988 and 1990, and an epiornitic of HJ virus in 1986. Infected birds responded with the production of HI antibodies with titers indicative of recent infection (HI > or = 1:160), and titers of sera positive during the epiornitics were significantly higher than positive sera during nonepiornitics. The 1990 EEE epiornitic extended from mid-July to the end of September, providing data to compare infection rates among species, habitats, and combinations of species with habitats. Few significant differences were found. The HJ epiornitic was only the second time this virus has occurred in CNY. Song sparrows were identified as the primary amplifying avian host of both viruses, although our capture and serological data would suggest a role for gray catbirds as the species most likely involved in yearly virus reintroduction. However, the cryptic nature of enzootic virus maintenance remains unresolved for the CNY virus foci. The appearances of HJ and EEE viruses were not epidemiologically linked, and there were no virus isolations from adults returning on site or virus isolations without concurrent isolations from mosquito vectors. Whether EEE and/or HJ virus are consistently present in or sporadically introduced into the inland foci of CNY area still has not been determined.     

Japanese encephalitis in Kerala, south India: can Mansonia (Diptera: Culicidae) play a supplemental role in transmission?

A 2-yr entomological study was carried out in Kerala, south India, to identify the mosquito vectors of Japanese encephalitis (JE) virus and to determine their seasonal abundance and infection. In total, 150,454 mosquitoes belonging to five genera and 18 species were collected from vegetation surrounding cattle sheds and pigsties in villages at dusk. Culex tritaeniorhynchus Giles (66.7%) was the most abundant species, with increases in numbers associated with rice cultivation. JE virus isolations were made from Cx. tritaeniorhynchus and Mansonia indiana Edwards. Based on high abundance and frequent JE virus infection, Cx. tritaeniorhynchus seems to be the most important vector, whereas Ma. indiana is probably a secondary vector.     

Inhibition of the infectivity of enveloped viruses while retaining the specific biological activity of the viral proteins

A method of inactivation of enveloped viruses by treatment of virus-containing material with a nonionic detergent MESK was studied. Treatment with the detergent of allantoic or culture fluids containing influenza, parainfluenza, herpes simplex, and Venezuelan equine encephalomyelitis viruses was shown to result in significant (to 10 lg ID50) decrease of the infectivity of the viruses. At that, the specific biological activity of viral proteins: the hemagglutinating and neuraminidase activities in the case of influenza and parainfluenza viruses, and the hemagglutinating activity in the case of Venezuelan equine encephalomyelitis virus, remained unchanged. Treatment of the viruses with the detergent in the presence of 2% BSA, 5% gamma-globulin, or 50% blood serum also inactivated the infectivity of virions. The inactivating effect of the MESK detergent is associated with solubilization of external glycoproteins of the virus envelope. The method of mild nondenaturating inactivation of enveloped viruses with the MESK detergent may prove to be useful in laboratory practice.     

Direct broad-range detection of alphaviruses in mosquito extracts

Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.     

Nocturnal activity of mosquitoes (Diptera: Culicidae) in a West Nile virus focus in Connecticut

Six species of mosquitoes (Diptera: Culicidae) were collected in sufficient numbers for analysis in segregating traps set at 2-h intervals by using CO2 and light as attractants in a West Nile virus (family Flaviviridae, genus Flavivirus, WNV) focus in Stratford, CT. The Kolmogorov-Smirnov one-sided test for two samples was used to analyze the data. Mosquito activity began shortly before sunset and continued until shortly after sunrise the next morning. All species had geometric means that were significantly higher during the 2-h period shortly after sunset compared with the 2-h collection before sunset. Species, known to be naturally infected with WNV, were often attracted to these traps in about equal numbers at 2-h intervals during an 8- to 10-h period commencing shortly after sunset. Differences of geometric means were not significant among the four or five 2-h collection periods commencing at sunset for Aedes vexans (Meigen), Culex salinarius Coquillett, and Aedes cinereus Meigen. Aedes cantator (Coquillett) had a significantly higher geometric mean for the 2-h period commencing at sunset, and Coquillettidia perturbans (Walker) was captured in significantly greater numbers during the 2-h period starting at sunset compared with periods commencing 6 h after sunset. Culex pipiens L. tended to have an activity pattern that was primarily nocturnal. Time of night, not meteorological conditions, was the most important factor in determining the nightly variation in the number of trapped mosquitoes. Parity rates of Cx. pipiens collected during specific periods of the night were not significant. In total, 39 isolations of WNV were made from seven species collected primarily during periods of total darkness. Humans are at risk of being bitten by infected mosquitoes throughout the night.     

Medically important arboviruses of the United States and Canada.

Of more than 500 arboviruses recognized worldwide, 5 were first isolated in Canada and 58 were first isolated in the United States. Six of these viruses are human pathogens: western equine encephalitis (WEE) and eastern equine encephalitis (EEE) viruses (family Togaviridae, genus Alphavirus), St. Louis encephalitis (SLE) and Powassan (POW) viruses (Flaviviridae, Flavivirus), LaCrosse (LAC) virus (Bunyaviridae, Bunyavirus), and Colorado tick fever (CTF) virus (Reoviridae, Coltivirus). Their scientific histories, geographic distributions, virology, epidemiology, vectors, vertebrate hosts, transmission, pathogenesis, clinical and differential diagnoses, control, treatment, and laboratory diagnosis are reviewed. In addition, mention is made of the Venezuelan equine encephalitis (VEE) complex viruses (family Togaviridae, genus Alphavirus), which periodically cause human and equine disease in North America. WEE, EEE, and SLE viruses are transmitted by mosquitoes between birds; POW and CTF viruses, between wild mammals by ticks; LAC virus, between small mammals by mosquitoes; and VEE viruses, between small or large mammals by mosquitoes. Human infections are tangential to the natural cycle. Such infections range from rare to focal but are relatively frequent where they occur. Epidemics of WEE, EEE, VEE, and SLE viruses have been recorded at periodic intervals, but prevalence of infections with LAC and CTF viruses typically are constant, related to the degree of exposure to infected vectors. Infections with POW virus appear to be rare. Adequate diagnostic tools are available, but treatment is mainly supportive, and greater efforts at educating the public and the medical community are suggested if infections are to be prevented.