• View in gallery

    Map of the Western Hemisphere showing the likely distribution of members of thePsorophora confinnis complex and trapping sites for this study.

  • View in gallery

    A, Gel showing phenotypes of the isocitrate dehydrogenase locus for Psorophora columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15 and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. B, Gel showing phenotypes of the aconitase-2 locus for Ps. columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. C, Gel showing phenotypes of the malic enzyme locus for Ps. columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15 and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. This figure appears in color at www.ajtmh.org.

  • 1

    Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC, 2004. Venezuelan equine encephalitis. Annu Rev Entomol 49 :141–174.

  • 2

    Johnson KM, Martin DH, 1974. Venezuelan equine encephalitis. Adv Vet Sci Comp Med 18 :79–116.

  • 3

    Walton TE, Grayson MA, 1989. Venezuelan equine encephalomyelitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Boca Raton, FL: CRC Press, 203–231.

  • 4

    Groot H, 1972. The health and economic impact of Venezuelan equine encephalitis (VEE). Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 7.

  • 5

    Scherer WF, 1972. History and importance of VE virus. Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 26.

  • 6

    Kubes V, 1972. Epizootic data. Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 18.

  • 7

    Kubes V, Rios FA, 1939. The causative agent of infectious equine encephalomyelitis in Venezuela. Science 90 :20–21.

  • 8

    Beck CE, Wyckoff RW, 1938. Venezuelan equine encephalomyelitis. Science 88 :530.

  • 9

    Oberste MS, Fraire M, Navarro R, Zepeda C, Zarate ML, Ludwig GV, Kondig JF, Weaver SC, Smith JF, Rico-Hesse R, 1998. Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico. Am J Trop Med Hyg 59 :100–107.

    • Search Google Scholar
    • Export Citation
  • 10

    Sudia WD, Newhouse VF, Beadle ID, Miller DL, Johnston JG Jr, Young R, Calisher CH, Maness K, 1975. Epidemic Venezuelan equine encephalitis in North America in 1971: vector studies. Am J Epidemiol 101 :17–35.

    • Search Google Scholar
    • Export Citation
  • 11

    Sudia WD, Lord RD, Newhouse VF, Miller DL, Kissling RE, 1971. Vector-host studies of an epizootic of Venezuelan equine encephalomyelitis in Guatemala, 1969. Am J Epidemiol 93 :137–143.

    • Search Google Scholar
    • Export Citation
  • 12

    Sellers RF, Bergold GH, Suarez OM, Morales A, 1965. Investigations during Venezuelan equine encephalitis outbreaks in Venezuela, 1962–1964. Am J Trop Med Hyg 14 :460–469.

    • Search Google Scholar
    • Export Citation
  • 13

    Sudia WD, Newhouse VF, Henderson BE, 1971. Experimental infection of horses with three strains of Venezuelan equine encephalomyelitis virus. II. Experimental vector studies. Am J Epidemiol 93 :206–211.

    • Search Google Scholar
    • Export Citation
  • 14

    Scherer WF, Dickerman RW, Ordonez JV, 1970. Discovery and geographic distribution of Venezuelan encephalitis virus in Guatemala, Honduras, and British Honduras during 1965–68, and its possible movement to Central America and Mexico. Am J Trop Med Hyg 19 :703–711.

    • Search Google Scholar
    • Export Citation
  • 15

    Ortiz DI, Anishchenko M, Weaver SC, 2005. Susceptibility of Psorophora confinnis (Diptera: Culicidae) to infection with epizootic (subtype IC) and enzootic (subtype ID) Venezuelan Equine encephalitis viruses. J Med Entomol 42 :857–863.

    • Search Google Scholar
    • Export Citation
  • 16

    Rico-Hesse R, Weaver SC, de Siger J, Medina G, Salas RA, 1995. Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America. Proc Natl Acad Sci U S A 92 :5278–5281.

    • Search Google Scholar
    • Export Citation
  • 17

    Rivas F, Diaz LA, Cardenas VM, Daza E, Bruzon L, Alcala A, de la Hoz O, Caceres FM, Aristizabal G, Martinez JW, Revelo D, de la Hoz F, Boshell J, Camacho T, Calderon L, Olano VA, Villarreal LI, Roselli D, Alvarez G, Ludwig G, Tsai T, 1997. Epidemic Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis 175 :828–832.

    • Search Google Scholar
    • Export Citation
  • 18

    Weaver SC, Salas R, Rico-Hesse R, Ludwig GV, Oberste MS, Boshell J, Tesh RB, 1996. Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348 :436–440.

    • Search Google Scholar
    • Export Citation
  • 19

    Howard L, Dyar H, Knab F, 1917. Mosquitoes of North and Central America and the West Indies. Washington, DC: Carnegie Institute of Washington Publication.

  • 20

    Aitken T, 1940. The genus Psorophora in California. Revista Entomol 11 :672–682.

  • 21

    Dyar H, Knab F, 1906. Diagnosis of new species of mosquitoes. Proc Biol Soc Wash 19 :133–142.

  • 22

    Carpenter S, LaCasse W, 1955. Mosquitoes of North America (North of Mexico). Berkeley, CA: University of California Press.

  • 23

    Bickley W, 1984. Notes on the Psorophora confinnis complex. Mosq Systematics 16 :162–167.

  • 24

    Ruiz-Garcia M, Ramirez D, Bello F, Alvarez D, 2003. Psorophora columbiae and Psorophora toltecum (Diptera: Culicidae) Colombian populations cannot be differentiated by isoenzymes. Genet Mol Res 2 :229–259.

    • Search Google Scholar
    • Export Citation
  • 25

    Ortiz DI, Weaver SC, 2004. 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. J Med Entomol 41 :987–993.

    • Search Google Scholar
    • Export Citation
  • 26

    Steiner W, Joslyn D, 1978. Electrophoretic techniques for the genetic study of mosquitoes. Mosq News 39 :25–54.

  • 27

    Lanzaro G, Narang S, Seawtight J, 1990. Speciation in an anopheline mosquito: enzyme polymorphism and the genetic structure of populations. Ann Entomol Soc Am 83 :578–585.

    • Search Google Scholar
    • Export Citation
  • 28

    Gargan TP 2nd, Bailey CL, Higbee GA, Gad A, El Said S, 1983. The effect of laboratory colonization on the vector-pathogen interactions of Egyptian Culex pipiens and Rift Valley fever virus. Am J Trop Med Hyg 32 :1154–1163.

    • Search Google Scholar
    • Export Citation
  • 29

    Turell MJ, Gargan TP 2nd, Bailey CL, 1984. Replication and dissemination of Rift Valley fever virus in Culex pipiens. Am J Trop Med Hyg 33 :176–181.

    • Search Google Scholar
    • Export Citation
  • 30

    Weaver SC, Scott TW, Lorenz LH, 1990. Patterns of eastern equine encephalomyelitis virus infection in Culiseta melanura (Diptera: Culicidae). J Med Entomol 27 :878–891.

    • Search Google Scholar
    • Export Citation
  • 31

    Lanzaro G, 1997. Genetics of the Mosquito Vector of Epidemic Venezuelan Equine Encephalitis. Advanced Research Program. College Station, TX: Texas A&M University.

  • 32

    Belkin J, Heinemann S, Page WA, 1970. Mosquito Studies (Diptera, Culicidae). XXI. The Culicidae of Jamaica. Contributions of the American Entomological Institute 6 :1–450.

    • Search Google Scholar
    • Export Citation
  • 33

    Clemens A, 1999. The Biology of Mosquitoes. Cambridge, United Kingdom: CABI Publishing.

  • 34

    Mitchell CJ, Monath TP, Sabattini MS, Christensen HA, Darsie RF Jr, Jakob WL, Daffner JF, 1987. Host-feeding patterns of Argentine mosquitoes (Diptera: Culicidae) collected during and after an epizootic of western equine encephalitis. J Med Entomol 24 :260–267.

    • Search Google Scholar
    • Export Citation
  • 35

    Edman JD, 1971. Host-feeding patterns of Florida mosquitoes. I. Aedes, Anopheles, Coquillettidia, Mansonia and Psorophora. J Med Entomol 8 :687–695.

    • Search Google Scholar
    • Export Citation
  • 36

    Whitehead F, 1951. Host preferences of Psorophora confinnis and Ps. discolor. J Econ Entomol 44 :1019.

  • 37

    Bishop F, 1933. Mosquitoes kill livestock. Science 77 :115–116.

  • 38

    Edman JD, Downe AER, 1964. Host-blood sources and multiple-feeding of mosquitoes in Kansas. Mosq News 24 :154–160.

  • 39

    Estrada-Franco JG, Navarro-Lopez R, Freier JE, Cordova D, Clements T, Moncayo A, Kang W, Gomez-Hernandez C, Rodriguez-Dominguez G, Ludwig GV, Weaver SC, 2004. Venezuelan equine encephalitis virus, southern Mexico. Emerg Infect Dis 10 :2113–2121.

    • Search Google Scholar
    • Export Citation
  • 40

    Turell MJ, Jones JW, Sardelis MR, Dohm DJ, Coleman RE, Watts DM, Fernandez R, Calampa C, Klein TA, 2000. Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus. J Med Entomol 37 :835–839.

    • Search Google Scholar
    • Export Citation
  • 41

    Turell MJ, Barth J, Coleman RE, 1999. Potential for Central American mosquitoes to transmit epizootic and enzootic strains of Venezuelan equine encephalitis virus. J Am Mosq Control Assoc 15 :295–298.

    • Search Google Scholar
    • Export Citation
  • 42

    Turell MJ, O’Guinn ML, Navarro R, Romero G, Estrada-Franco JG, 2003. Vector competence of Mexican and Honduran mosquitoes (Diptera: Culicidae) for enzootic (IE) and epizootic (IC) strains of Venezuelan equine encephalomyelitis virus. J Med Entomol 40 :306–310.

    • Search Google Scholar
    • Export Citation
  • 43

    Gonzalez-Salazar D, Estrada-Franco JG, Carrara AS, Aronson JF, Weaver SC, 2003. Equine amplification and virulence of subtype IE Venezuelan equine encephalitis viruses isolated during the 1993 and 1996 Mexican epizootics. Emerg Infect Dis 9 :161–168.

    • Search Google Scholar
    • Export Citation
  • 44

    Wilkerson RC, Parsons TJ, Klein TA, Gaffigan TV, Bergo E, Consolim J, 1995. Diagnosis by random amplified polymorphic DNA polymerase chain reaction of four cryptic species related to Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) from Paraguay, Argentina, and Brazil. J Med Entomol 32 :697–704.

    • Search Google Scholar
    • Export Citation
  • 45

    Tabachnick W, Black W IV, 1995. Making a case for the molecular population genetic studies of arthropod vectors. Parasitol Today 11 :27–30.

    • Search Google Scholar
    • Export Citation
  • 46

    Faran M, 1980. Mosquito Studies (Diptera: Culicidae) 34: A Revision of the albimanus Section of the Subgenus Nyssorhynchus of Anopheles. Contribution to the American Entomological Institute. Ann Arbor, MI: 215.

  • 47

    Moncada-Perez AM, Conn J, 1992. A polytene chromosome study of four populations of Anopheles aquasalis from Venezuela. Genome 35 :327–331.

    • Search Google Scholar
    • Export Citation
  • 48

    Conn J, Cockburn AF, Mitchell SE, 1993. Population differentiation of the malaria vector Anopheles aquasalis using mitochondrial DNA. J Hered 84 :248–253.

    • Search Google Scholar
    • Export Citation
  • 49

    Gimnig JE, Reisen WK, Eldridge BF, Nixon KC, Schutz SJ, 1999. Temporal and spatial genetic variation within and among populations of the mosquito Culex tarsalis (Diptera: Culicidae) from California. J Med Entomol 36 :23–29.

    • Search Google Scholar
    • Export Citation
  • 50

    Walton TE, Alvarez O Jr, Buckwalter RM, Johnson KM, 1973. Experimental infection of horses with enzootic and epizootic strains of Venezuelan equine encephalomyelitis virus. J Infect Dis 128 :271–282.

    • Search Google Scholar
    • Export Citation
  • 51

    Wang E, Bowen RA, Medina G, Powers AM, Kang W, Chandler LM, Shope RE, Weaver SC, 2001. Virulence and viremia characteristics of 1992 epizootic subtype IC Venezuelan equine encephalitis viruses and closely related enzootic subtype ID strains. Am J Trop Med Hyg 65 :64–69.

    • Search Google Scholar
    • Export Citation
  • 52

    Brault AC, Powers AM, Weaver SC, 2002. Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein. J Virol 76 :6387–6392.

    • Search Google Scholar
    • Export Citation
  • 53

    Brault AC, Powers AM, Ortiz D, Estrada-Franco JG, Navarro-Lopez R, Weaver SC, 2004. Venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope glycoprotein. Proc Natl Acad Sci U S A 101 :11344–11349.

    • Search Google Scholar
    • Export Citation
  • 54

    Kramer LD, Scherer WF, 1976. Vector competence of mosquitoes as a marker to distinguish Central American and Mexican epizootic from enzootic strains of Venezuelan encephalitis virus. Am J Trop Med Hyg 25 :336–346.

    • Search Google Scholar
    • Export Citation
  • 55

    Fernandez Z, Moncayo AC, Carrara AS, Forattini OP, Weaver SC, 2003. Vector competence of rural and urban strains of Aedes (Stegomyia) albopictus (Diptera: Culicidae) from Sao Paulo State, Brazil for IC, ID, and IF subtypes of Venezuelan equine encephalitis virus. J Med Entomol 40 :522–527.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Vector Competence of Eastern and Western Forms of Psorophora columbiae (Diptera: Culicidae) Mosquitoes for Enzootic and Epizootic Venezuelan Equine Encephalitis Virus

View More View Less
  • 1 Center for Tropical Disease and Department of Pathology, University of Texas Medical Branch, Galveston, Texas; Vector-Borne Diseases Section, Communicable and Environmental Diseases, Tennessee Department of Health, Nashville, Tennessee; Centro de Investigación de Paludismo, Instituto Nacional de Salud Publica, Tapachula, Chiapas, Mexico

Venezuelan equine encephalitis virus (VEEV) continues to circulate enzootically in Mexico with the potential to re-emerge and cause disease in equines and humans in North America. We infected two geographically distinct mosquito populations of eastern Psorophora columbiae form columbiae (Chiapas, Mexico and Texas, United States) and one mosquito population of western Psorophora columbiae form toltecum (California, United States) with epizootic and enzootic IE VEEV and epizootic IAB VEEV. We detected no differences between epizootic and enzootic IE viruses in their ability to infect any of the mosquito populations analyzed, which suggested that neither species selects for epizootic IE viruses. Psorophora columbiae f. columbiae (Texas) were significantly less susceptible to infection by epizootic IE than Ps. columbiae f. columbiae (Mexico). Psorophora columbiae f. toltecum populations were more susceptible than Ps. columbiae f. columbiae populations to epizootic IE and IAB viruses.

INTRODUCTION

Venezuelan equine encephalitis virus (VEEV) is the most important alphavirus circulating in the Americas in terms of the numbers of people and equines affected, geographic range, and duration of outbreaks.1 This virus can cause a spectrum of disease ranging from fever to acute encephalitis and death. Equine mortality rates have ranged from 19% to 83% and human neurologic disease rates are from 4% to 14%.1,2 Encephalitis can occur in all age groups, regardless of sex, but children are more likely to develop neurologic disease and fatal encephalitis.1

This virus has caused hundreds of thousands of equine and human cases in Latin America since the 1920s.36 It was first recognized as a disease of concern in 1936 in Venezuela and was isolated in 1938 after an outbreak in the Goajira Peninsula of Venezuela.7,8 From 1936 to 1968, periodic large outbreaks lasting for several years occurred in Peru, Ecuador, Columbia, Venezuela, and the Island of Trinidad. From 1969 to 1972, VEE epizoodemics began to move northward from the South American continent to North America.

Venezuelan equine encephalitis virus complex alphaviruses species are subdivided according to serotypes. The VEEV serotypes IAB and IC are thought to be responsible for the large equine and human outbreaks that have been documented. Serotypes ID, IE, II, and IIIA have been considered equine avirulent, enzootic strains that circulate among rodent reservoirs in tropical and subtropical lowland forest and swamp habitats. These enzootic strains can, however, cause fatal disease in persons that enter these habitats. In two Mexican outbreaks, the etiologic agent was found to be a IE subtype of VEEV.3 Strains from these recent outbreaks were found to be nearly identical to one another and closely related to equine avirulent strains from Guatemala and eastern Mexico.9 We refer to IE viruses involved in 1993 and 1996 outbreaks and closely related from Chiapas in 2001 as epizootic IE and those not involved in outbreaks as enzootic IE.

The entrance of VEEV into the United States resulted in a large equine and human outbreak in 1971. This outbreak was part of a regional outbreak from 1969 to 1972 that also involved Central America and Mexico. Tens of thousands of equines and people were infected during this north Central American outbreak.1 From 1973 to 1992, no VEE was documented, leading to speculation about its extinction. However, beginning in 1992, VEEV re-emerged in the Americas beginning in Trujillo State of Venezuela. In the summer of 1993, an outbreak of VEEV occurred in the state of Chiapas in southern Mexico.9 In 1996, another smaller epizootic occurred in the state of Oaxaca.

Mosquitoes of the Psorophora confinnis complex have long been regarded as epizootic vectors for VEEV.1013 Psorophora confinnis (morphologically indistinguishable from Ps. columbiae) was reported as the primary vector during the 1971 outbreak in Texas (involving VEEV subtype IAB) and during the more recent outbreaks in Mexico (involving VEEV subtype IE) and Venezuela and Colombia (involving VEEV subtype IC).1,1418 Most epidemiologic surveys during outbreaks in the Americas and post-outbreak entomologic studies have not distinguished between members of the Ps. confinnis complex.

Some entomologists have considered Ps. confinnis conspecific to Ps. columbiae, and others have considered them different species.19 The earliest separation of the Ps. confinnis complex into four species was in 1928 when Dyar distinguished among confinnis, tolteca (Dyar and Knab), jamaicensis, and columbiae. Other investigators considered confinnis as synonymous with columbiae, jamaicensis, and tolteca20 and occurring throughout the Americas and the West Indies.21 Indeed, jamaicensis can be separated morphologically from confinnis and columbiae; however confinnis and columbiae cannot be easily distinguished morphologically.22

Electron microscopy has been used to study the chorionic patterns of the Ps. confinnis complex in the United States.23 These chorionic patters can be used to distinguish among populations in California from those in Texas and states east of the Mississippi River. On the basis of morphologic studies of pectin spines in larvae, Bickley23 suggested the existence of different populations of Ps. confinnis complex in three geographic regions: California, Brazil, and other regions (Texas and the eastern United States, Mexico, Belize, Guatemala, Honduras, Nicaragua, Costa Rica, Colombia, and Venezuela). This suggestion is now interpreted as being Ps. columbiae (California), Ps. confinnis (Brazil), and Ps. columbiae f. columbiae (all other regions).24 Recent isozyme studies (Lanzaro G, unpublished data) have shown genetic variation among populations of Ps. confinnis complex in Mexico and the United States. These studies have identified the existence of two genetically distinct populations in North America, which we hereto refer to as Ps. columbiae f. columbiae and Ps. columbiae f. toltecum (which until 1948 were synonymous with each other). Psorophora columbiae f. columbiae is found along most of central and eastern Mexico including its Gulf Coast and from eastern Texas to the Atlantic Coast. Conversely, P. columbiae f. toltecum is found on the west coast of Mexico and from west Texas to California (Figure 1).

The re-emergence or introduction of arboviruses into the United States has gained renewed interest since the introduction of West Nile virus in 1999. Venezuelan equine encephalitis virus is an arbovirus that has proven capable of entering the United States and therefore must be considered as a potential future threat. Additionally, the potential use of VEEV as a biologic weapon makes the non-natural introduction of VEEV a possibility that must be considered.

In this study, we examine the vector competence of geographically distinct populations of Ps. columbiae f. columbiae (Eastern form) and Ps. columbiae f. toltecum (Western form) for epizootic IE VEEV circulating in Mexico, enzootic IE VEEV in Guatemala, and a IAB VEEV that circulated in Guatemala in 1963. Additionally, we examine the hypothesis of adaptation of enzootic/sylvatic viruses to epizoodemic vectors as a mechanism of epizoodemic virus emergence by infecting IE viruses that were involved in the 1993 and 1996 outbreaks in southern Mexico and related equine virulent IE viruses circulating this century in the same region, as well as closely related enzootic IE strains from Guatemala. Other studies have shown adaptation of ID VEEV in Ochlerotatus taeniorhynchus as a mechanism of epizootic IC VEEV emergence but have failed to demonstrate Psorophora confinnis mosquitoes from Colombia as hosts for epizootic IC emergence.15,25 We describe here infection assays of Psorophora mosquitoes from enzootic and epizootic IE viruses to test the hypothesis of enzootic progenitor adaptation in this mosquito species complex.

MATERIALS AND METHODS

Mosquitoes.

Psorophora columbiae adult females near Dorado Nuevo from the State of Chiapas in southern Mexico were collected in collaboration with the Centro de Investigación de Paludismo of the Institute of Public Health of Mexico. Female mosquitoes were collected with a large animal-baited trap. A horse was placed inside a large tent shortly before dusk with the lower edge of the tent raised 1–2 feet to enable mosquitoes to enter. Mainly Psorophora mosquitoes entered the tent and bit the horse exclusively (i.e., none of the human collectors) and subsequently rested on the inner walls of the tent. Blood fed females were aspirated and placed in pint-size containers for transport to an insectary at the University of Texas Medical Branch (UTMB) at Galveston, Texas. Previously determined populations of Ps. columbiae f. toltecum (Lanzaro G, unpublished data) were collected in Thermal, California. Blood engorged females also were transported to UTMB. Psorophora columbiae mosquitoes were collected in China, Texas, and also taken to UTMB. All field-caught females were exposed daily to Syrian hamsters for blood feeding. Eggs were collected and F1 females were used for transmission experiments. Three to seven day-old F1 females were exposed to hamsters previously infected with epizootic and enzootic strains of VEEV.

Isozyme analysis.

Prior to examining transmission potential, we used known Ps. columbiae f. columbiae (Starkville, MS) and Ps. columbiae f. toltecum populations (Tehuantepec, Chiapas) as references to compare our field populations from Dorado Nuevo (Mexico), Thermal (CA), and China (TX).

We compared 15 isozyme loci by starch gel electrophoresis (Table 1).26 Individual mosquitoes were homogenized with a pestle in 15 μL of sterile distilled water and centrifuged for 1 minute at 17,000 × g at 4°C. Samples were then fractionated by electrophoresis on 12.5% (w/v) horizontal starch gels by using standard methods.27 The enzymes and buffering systems are listed in Table 1. Loci were labeled as positive if they migrated toward the cathode and negative if the migrated toward the anode. These isozymes were used in these studies as biomarkers for these populations rather than as taxonomic identifiers

Viruses and virus assays.

We used the following virus strains for our studies. Strain 93-42124 is an IE epizootic strain isolated during the 1993 outbreak in the State of Chiapas, Mexico. This strain was isolated from horse brain and passaged once in suckling mouse and once in chick embryo cells. Strain CPA-201 is another strain of IE virus isolated from the same 1993 outbreak. This strain was passaged once in suckling mouse brains, once in rabbit kidney cells and once in baby hamster kidney cells. These two strains are virtually genetically identical, with the exception of one nucleotide in the structural E2 region at position 8920. Strain 96-32863 is an IE epizootic strain isolated during the 1996 outbreak in the State of Oaxaca, Mexico. This epizootic strain was also isolated from horse brain. Strain 68U201 is an IE enzootic strain isolated in 1968 in Guatemala. This strain was isolated from a hamster and passaged once in suckling mouse and three times in chick embryo cells. Strain 80U76 is an IE enzootic strain isolated in 1976 in Guatemala from a hamster and passaged once in suckling mouse and chick embryo cells. Strain Mex01-32 is an equine virulent IE strain isolated from Chiapas, Mexico in 2001 and passaged once in Vero cells. Strain 69Z1 is an IAB human isolate isolated in Guatemala in 1969 and passaged once in Vero cells. This subtype was included to assess the relative competence of these species for a typical epizootic VEEV strain. Specimens were tested for VEEV and titrated by 1:10 serial-fold dilution plaque assays on Vero cell monolayers.28

Vector susceptibility and transmission assays.

Adult female mosquitoes were allowed to blood feed on anesthetized Syrian hamsters at 12, 15, and 24 hours after hamsters had been inoculated subcutaneously with 104 plaque-forming units (PFU) of VEEV in 0.1 mL of 10% fetal bovine serum–minimal essential medium (10% heat-inactivated fetal bovine serum in Eagle’s minimum essential medium with antibiotics, fungizone, and sodium bicarbonate) (FBS-MEM). Serial 10-fold dilutions of hamster blood at the time of mosquito feedings were assayed for virus on Vero cell monolayers to determine feeding dose. Engorged mosquitoes were placed in a 0.5-liter cardboard container and maintained at 26°C on a 5% sucrose solution diet for 10 days prior to susceptibility and transmission assays. We considered a mosquito that had virus recovered from its body, but not its legs, to have a nondisseminated infection limited to its midgut. In contrast, if virus was recovered from both body and leg suspensions, we considered the mosquito to have a disseminated infection.29

Mosquito bodies and legs were triturated separately in 350 μL of 10% FBS-MEM solution and stored at −70°C until assayed on Vero cell monolayers as described above. Transmission potential was determined by three different experimental approaches: 1) by exposing infected mosquito cohorts to naive hamsters, 2) by plaque assays of mosquito saliva on Vero cell monolayers, and 3) by assay of saliva by injecting suckling mice brains. Naive hamsters were observed for symptoms of encephalitis and hearts from dying or recently dead hamsters were assayed for virus by observing for cytopathic effect (CPE) on Vero cell monolayers. Saliva was collected by inserting the proboscis of amputated (wings and legs removed) females into a 5-μL capillary tube containing Cargile B immersion oil (Cargile Laboratories, Cedar Grove, NJ). Mosquitoes were allowed to salivate for 1 hour or until saliva was observed in the oil.30 Saliva was assayed on Vero cell monolayers and observed for CPE. In the third set of experiments, saliva samples were tested by inoculating 20 μL intracranially into 1–3 day-old Swiss NIH suckling mice. Infection was confirmed by testing triturated brains from dead mice by plaque assay. Controls did not succumb to infection.

RESULTS

Isozyme analysis.

Isozyme isocitrate dehydrogenase (IDH-1) (Figure 2A) and aconitase-2 (ACON-2) (Figure 2B) proved to be diagnostic in distinguishing populations of the Ps. columbiae complex and we were able to identify the Dorado Nuevo, Chiapas and China, Texas, populations as Ps. co-lumbiae f. columbiae, and the Thermal, California, populations as Ps. columbiae f. toltecum. All other isozymes proved to be of variable diagnostic value or non-diagnostic. Isozymes of variable diagnostic value were acid phosphatase, glutamate oxaloacetate transaminase-1 (GOT-1), and GOT-2. Isozymes that proved non-diagnostic by our methods were ACON-1, hydroxyacid dehydrogenase, IDH-2, malic acid dehydrogenase-1 (MDH-1), MDH-2, malic enzyme (Figure 2C), 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, sorbitol dehydrogenase, trehalase-1 (TRE-1), and TRE-2 (Table 1).

Transmission assays.

Three approaches were taken to determine transmission potential of Psorophora mosquitoes to various VEEV strains: in vivo transmission to adult hamsters, in vitro Vero cell assay of collected saliva, and intracerebral inoculation of collected saliva in suckling mice. In experiments using the in vivo method, 1 of 27 to 3 of 27 (3.7–11.1%) of mosquitoes with disseminated infections were able to transmit virus (Table 2). Psorophora mosquitoes showed a high mortality rate in captivity and their ability for taking a second blood meal 10–14 days after the infectious meal in these experiments was compromised. Most did not take a full blood meal, which may explain the low transmission rates. Experiments using the in vitro assay of saliva via Vero cells, showed 32 (40%) of 80 mosquitoes with disseminated infections as being able to transmit (Tables 3 and 4). Experiments via intracerebral inoculation of saliva into suckling mice showed 100% (154 of 154) transmission by mosquitoes with disseminated infections. Suckling mice died at an average of 2.6 days after inoculation (Tables 4 and 5). Because of differences in transmission potentials resulting from our various methods, we use for the following comparisons only our infection and dissemination rates to evaluate susceptibility differences.

Comparison between epizootic and enzootic IE genotypes.

For the following comparisons, we compared mosquito cohorts that were exposed to viremic blood meals that were within one log10 PFU. Psorophora columbiae f. columbiae from Chiapas, Mexico (Table 3) infected with epizootic strains 93-42124 (108.5 PFU/mL) and 96-32863 (108.5 PFU/ mL) showed no differences in infection and dissemination rate compared with those infected with 68U201 (108.4 PFU/ mL). Infection and dissemination rates in these three cohorts were 100%. No significant differences in infection (P = 1.0) and dissemination (P = 0.555) rates were seen when mosquitoes from Chiapas were exposed to lower viremias of epizootic 96-32863 (107.3 PFU/mL) versus enzootic 68U201 (107.5 PFU/mL) and 80U76 (107.6 PFU/mL). At still lower viremias, no differences were observed when comparing epizootic 96-32863 (105.2 PFU/mL) versus enzootic 80U76 (105.3 PFU/ mL) (infection rates: P = 1.0; dissemination rates: P = 0.612).

For Ps. columbiae f. columbiae from China, Texas, (Table 5), there was a significant difference when comparing susceptibilities with epizootic strain 96-32863 (106.6 PFU/mL) and enzootic strain 68U201 (106.2 PFU/mL) (infection rates: P = 0.012; dissemination rates: P = 0.020).

No significant differences were observed between cohorts from Thermal, California, (Table 4) when infected with enzootic versus epizootic viruses. Infection rates of cohorts infected with epizootic 96-32863 (106.6 PFU/mL), Mex 01-32 (106.7 PFU/mL), and enzootic 68U201 (106.2 PFU/mL) were 100%, and differences between dissemination rates of 96-32863 versus 68U201 and Mex 01-32 versus 68U201 were not significant (P = 0.085 and P = 0.687, respectively).

Geographic comparison of susceptibility to IE viruses.

Because mosquitoes from Texas and Mexico were of the same species, we investigated possible geographic variation in susceptibility within Ps. columbiae f. columbiae. We compared cohorts from Mexico and Texas infected with epizootic genotype 96-32863 (107.3 PFU/mL versus 106.6 PFU/mL, respectively). Groups from Chiapas, Mexico, were significantly more susceptible with regards to infection (P = 0.018) and dissemination (P = 0.004).

Comparison of Ps. columbiae f. columbiae versus Ps. columbiae f. toltecum in their susceptibilities to IE viruses.

In comparing cohorts of Ps. columbiae f. columbiae and Ps. columbiae f. toltecum mosquitoes that imbibed viremic blood meals within one log10 PFU, we found no differences in their susceptibilities to epizootic and enzootic IE viruses. Comparisons were made between Ps. columbiae f. columbiae Chiapas and Ps. columbiae f. toltecum Thermal infected with epizootic 93-42124 (108.5 PFU/mL) and 93-42124 (108.6 PFU/mL) (P = 1.0), and between 96-32863 (107.3 PFU/mL) and 96-32863 (107.5 PFU/mL) (infection differences, P = 1.0; dissemination differences, P = 0.424). There also were no significant differences in infection and dissemination rates between these sibling species when infected with 68U201 (106.2 PFU/mL) (Infection differences: P = 0.274; dissemination differences: P = 0.317). When sibling species Ps. columbiae f. columbiae China and Ps. columbiae f. toltecum Thermal were infected with CPA-201 (107 PFU/mL) we did not observe differences in infection rates (P = 1.0) but we did see differences in dissemination rates (P = 0.014).

Comparison of Ps. columbiae f. columbiae versus Ps. columbiae f. toltecum in their susceptibilities to IAB virus.

Differences in susceptibilities were significant between Ps. columbiae f. columbiae China and Ps. columbiae f. toltecum Thermal infected with epizootic strain Gua69Z1 (104.5 PFU/ mL) (infection differences: P = 0.05; dissemination differences: P = 0.002) and Gua69Z1 (101.2 PFU/mL) (infection differences: P = 0.001; dissemination differences: P = 0.001). Psorophora columbiae f. toltecum was more susceptible to infection by this epizootic IAB virus strain. The low doses of the IAB strain were caused by poor replication in hamsters for the time points selected based on the IE strains. Nevertheless, mosquitoes were able to become infected and exhibited differences in susceptibility. These experiments also demonstrated that low doses of IAB virus are able to infect Psorophora mosquitoes.

DISCUSSION

The purpose of this study was 1) to investigate variation in the susceptibility of sibling species within the Ps. confinnis complex to transmit Venezuelan equine encephalitis virus, 2) to determine if there was a geographic variation to the ability of Ps. columbiae f. columbiae to transmit VEEV, and 3) to determine if there was a difference in the ability to transmit epizootic versus enzootic IE VEEV. The Ps. confinnis complex is found throughout the Americas. Psorophora. confinnis s.s. is thought to be found from southern Colombia to Argentina.24 Psorophora columbiae has a wide distribution from the United States and Mexico to southern Colombia.24 Psorophora toltecum is thought to be found from west Texas to California, and along the west coast of Mexico (Lanzaro G, unpublished data) on the western side of the Sierra Madre (the site of IE VEEV outbreaks in 1993 and 1996) and as far south as the western coast of Colombia.24 Psorophora columbiae f. columbiae appears to be present in central and eastern Mexico along the Gulf coast and from east Texas to the Atlantic Coast of the United States and as far south as the Colombian Andes. A hybrid zone for Ps. columbiae f. columbiae and Ps. columbiae f. toltecum has been observed in central Texas31 and these two species may occur sympatrically in parts of Colombia.24 Psorophora jamaicensis seems to be restricted to Jamaica.32

The larval habitat for members of this complex has been described as open grassy areas that are temporarily flooded by rain, irrigation, and flooded streams.22 The adults rest and fly in areas of open vegetation. Larval habitats and adult feeding sites can be widely separate.33 Females seek hosts large distances away from larval habitats.3436 The abundance of species within this complex can be large, so much so that Ps. columbiae has been described as being responsible for death of cattle by exsanguination caused by large populations.37

Members of this complex have a broad host range (bird, reptiles, mammals), but primarily feed on mammals such as cattle, rabbits, armadillo, raccoon.35 Members of this complex also feed on rodents in the field and experimentally.38 This ability to feed on large mammals as well as small mammals makes it potentially a good epizoodemic vector of VEEV to equines and humans. From a host preference standpoint, it also makes it a potentially good enzootic vector. With the reduced population on Culex taenipus in southern Mexico,39 enzootic virus might be circulating in rodent populations by using Ps. columbiae as a vector. It is susceptible to enzootic subytpe IE VEEV.

Psorophora columbiae have been seen in areas coinciding where VEE disease has occurred in North America. These outbreaks were caused by IAB and IE VEEV. Psorophora columbiae are found in southern Mexico where epidemics occurred in 1993 and 1996. The IE VEEV isolates continue to be found in these areas.39 Psorophora columbiae is found in areas in Texas where an IAB VEEV epidemic occurred in 1971.

Mosquitoes in the genus Psorophora (P. albigenu, P. cingulata, P. ferox) have been found to be susceptible to IAB, IC, ID and IE VEEV.40 Psorophora confinnis from Colombia appears to be a good vector of epizootic IC and enzootic ID.15 Psorophora confinnis populations from Belize are susceptible to IAB and enzootic IE VEEV but are not able to readily transmit the virus by bite.41 It is likely that it is actually Ps. columbiae that is found in Belize rather than Ps. confinnis s.s. Isozyme assays need to be conducted to verify this finding.

Aedes taeniorhynchus has been thought to be a primary vector of VEEV, but is not particularly susceptible to enzootic IE VEEV.42 Our data indicate that Ps. columbiae f. toltecum and Ps. columbiae f. columbiae populations from Mexico and the United States are potential vectors of both enzootic IE and epizootic IE. Psorophora columbiae may play an important role in both the enzootic and epizootic transmission of IE viruses. The host preference of these species on large and small mammals make them ideal bridge vectors as well as enzootic vectors.35 Gonzalez-Salazar and others43 showed that horses exposed to epizootic IE viruses do not achieve high viremia levels. Our data suggests that infection and dissemination rates generally dependent on the infectious dose. Once dissemination had occurred, transmission was generally seen to occur, which suggests a weak salivary gland barrier in Ps. columbiae for VEEV. The ability of mosquitoes to become infected after being exposed to low viremias may make them able to transmit IE viruses from viremic horses with low viremias and for these horses to serve as amplification hosts. The lowest viremias we used were 104.6 PFU/mL for IE viruses and 101.2 PFU/mL for IAB virus. It would be interesting to attempt mosquito susceptibility experiments using viremias seen in horses experimentally infected with these IE virus strains.

It is not uncommon to find members of mosquito species complexes, which are common in nature, varying in vectorial capacity.44,45 This has been observed in Anopheles aquasalis populations from Brazil, Venezuela, and Trinidad4648 and in Culex tarsalis populations.49 However, Ruiz-Garcia and others24 reported that differentiation of Ps. columbiae f. columbiae and Ps. columbiae f. toltecum was not successful by isozymes. Their study involved populations from Colombia and we used many different markers. However, the following markers were used in their studies and ours and proved to be uninformative: malic enzyme, phosphoglucose isomerase, 6-phosphogluconate dehydrogenase, and malate dehydrogenase. Enzymes we found to be diagnostic were isocitrate dehydrogenase-1 and aconitase-2. Our data indicate that these species can be distinguished on the basis of isozyme differences and that there is a difference in vector susceptibility between species of this complex with regard to epizootic IE and IAB viruses. Apparently, Ps. columbiae f. toltecum is more susceptible to these epizootic viruses than is Ps. columbiae f. columbiae. Our data indicate that Ps. columbiae f. columbiae populations from Texas were not as susceptible as Ps. columbiae f. columbiae populations from Mexico to epizootic IE viruses. Although susceptibility was significantly lower in Texas populations, epizootics have occurred in Texas. It would be interesting to know if sites of the 1971 Texas epizootics had populations of Ps. columbiae f. columbiae, Ps. columbiae f. toltecum, or an introgression of both species. Psorophora columbiae f. toltecum (Thermal) were efficient transmitters of epizootic IAB VEEV.

Adaptation by enzootic progenitors to equine hosts has been shown to occur for epizootic VEEV outbreaks to emerge.50,51 Additionally, previous studies have suggested that adaptation of enzootic VEEV to epizootic vectors may contribute to VEEV emergence.1 This hypothesis has been supported by observations that epizootic vectors such as Ochlerotatus taeniorhynchus are more susceptible to epizootic VEEV than enzootic VEEV.25,5254 However, differences in susceptibility to enzootic versus epizootic VEEV have not been documented in other vectors such as Aedes (Stegomyia) albopictus, Ps. confinnis, and other non-Culex mosquitoes.15,34,55 Our data also indicate that Ps. columbiae f. columbiae or Ps. columbiae f. toltecum do not show a greater susceptibility for epizootic IE versus enzootic IE VEEV. Therfore, our study did not suggest that adaptation of VEEV enzootic progenitors occurred in Psorophora mosquitoes from Mexico or the southern United States. The equal susceptibility for both types of viruses does suggest that Ps. columbiae can be efficient epizootic vectors and enzootic vectors.

This is the first study in which the Ps. confinnis complex was scrutinized for its ability to transmit VEEV. A closer inspection of Ps. confinnis s.l. populations in Mexico and the United States is warranted especially at historical epidemic sites. Although Ps. columbiae f. toltecum mosquitoes have been identified in Tehuantepec, State of Oaxaca, the Sierra Madre may have served as a geographic barrier between Ps. columbiae f. columbiae populations on the coast (at our collection site in Chiapas) and Ps. columbiae f. toltecum population north of the Sierra Madre where epidemics took place in Oaxaca. It is also interesting that isolates have been found along the southwestern coast of Chiapas,9 which is territory most likely populated by Ps. columbiae f. columbiae. We found that the Sierra Madre does not serve as an absolute barrier between Ps. columbiae f. columbiae and Ps. columbiae f. toltecum populations. At the southern range of these mountains in Mexico, we found large populations of Ps. columbiae f. columbiae. Therefore, the geographic distribution of Ps. columbiae f. toltecum is not as far south along the Pacific coast of Mexico as we initially suspected (Figure 1).

In central Texas, these sibling species occur sympatrically. This naturally occurring hybrid zone provides a unique opportunity to study the genetics of vector competence for a viral pathogen and to study the impact of this unusual population genetic structure on patterns of VEEV emergence and spread.

Table 1

Enzyme loci studied

EnzymeE.C. no.*AbbreviationBuffer system†Diagnostic locus
* E.C. = Enzyme Commission.
† CA-8: gel buffer = 0.074 M Tris, 0.009 M citric acid, pH 8.45 (undiluted, electrode buffer = 1.37 M Tris, 0.314 M citric acid, pH 8.1 (diluted 1:3 for the cathode and 1:4 for the anode); CA-7: gel buffer = 0.009 M-Tris, 0.003 M citric acid, pH 7.0 (undiluted), electrode buffer = 0.135 M Tris, 0.04 M citric acid, pH 7.0 (undiluted); CA-5.5: gel buffer = 0.64 M Tris, 0.026 M citric acid, pH 5.5 (diluted 1:2), electrode buffer = 0.223 M Tris, 0.093 M citric acid, pH 5.2 (diluted 3:1); C: gel buffer = 0.002 M citric acid, pH 6.0 (undiluted), electrode buffer = 0.04 M citric acid, pH 6.1 (undiluted) (pH for buffer C is adjusted with N-(3-aminopropyl-morpholine); TBE: 0.1 M Tris, 0.05 M boric acid, 0.002 M EDTA, pH 8.6 (undiluted) (gel and electrode buffer identical).
Aconitase-14.2.1.3ACON-1CA-8No
Aconitase-24.4.1.3ACON-2CA-8Variable
Acid phosphatase3.3.1.2ACPHCA-7Variable
Glutamate oxaloacetate transaminase-12.6.1.1GOT-1CA-7Yes
Glutamate oxaloacetate transaminase-22.6.1.1GOT-2CA-7Variable
Hydroxyacid dehydrogenase1.1.1.30HADCA-5.5No
Isocitrate dehydrogenase-11.1.1.42IDH-1CYes
Isocitrate dehydrogenase-21.1.1.42IDH-2CNo
Malic acid dehydrogenase-11.1.1.37MDH-1CNo
Malic acid dehydrogenase-21.1.1.37MDH-2CNo
Malic enzyme-11.1.1.40ME-1CNo
6-Phosphogluconate dehydrogenase1.1.1.446-PGDCA-7No
Phosphoglucose isomerase5.3.1.9PGITBENo
Sorbitol dehydrogenase1.1.1.14SODHCA-8No
Trehalase-13.2.1.28TRE-1CA-7No
Trehalase-23.2.1.28TRE-2CA-7No
Table 2

In vivo experiments of hamsters exposed to Psorophora columbiae f. columbiae and Ps. columbiae f. toltecum mosquitoes infected with various doses of epizootic and enzootic IE Venezuelan equine encephalitis viruses

No. with disseminated infections
Mosquito formStrainViremia*No. exposed to naive hamsterNo. refeeding% of infected†Dead hamster
* Viremia titers are expressed as log10 plaque-forming units/mL.
† Estimated dissemination rate based on dissemination rates of parallel in vitro experiment (Tables 3 and 4).
f. columbiae93-421248.5833 (100)Yes
6.31163 (45)No
4.61052–3 (50)No
96-328638.51022 (100)No
7.3511 (90)No
5.2930 (0)No
68U2018.4811 (100)No
7.5811 (91)No
80U767.61075 (73)No
5.31691 (10)No
f. toltecum93-421248.6633 (100)No
96-328637.51044 (100)No
Table 3

Infection, dissemination, and transmission rates of Psorophora columbiae f. columbiae Chiapas mosquitoes infected with infected with epizootic and enzootic strains of Venezuelan equine encephalitis virus*

GenotypeStrainBlood meal titer (log10 PFU/mL)% Infected (n)Mean body titer (log10 PFU/mL)% Infected with disseminated infection (n)Mean leg titer (log10 PFU/mL)% With saliva infection (n)Mean saliva titer (log10 PFU/mL)
* Transmission rates were determined based on plaque assays of collected salivas. PFU = plaque-forming units; NA = not available for mean leg titer and not applicable for mean saliva titer.
Epizootic IE93-421248.5100 (10)5.6100 (10)4.340 (10)2.4
6.364 (11)6.645 (11)3.127 (11)2.9
4.650 (2)5.450 (2)4.40 (2)NA
96-328638.5100 (12)6.1100 (12)4.525 (12)0.9
7.3100 (10)6.490 (10)4.560 (10)2.2
5.267 (3)2.50 (3)00 (3)NA
Enzootic IE68U2018.4100 (11)5.8100 (11)NA9 (11)1.5
7.5100 (11)5.791 (11)NA0 (11)NA
80U767.6100 (11)5.873 (11)NA9 (11)0.6
5.350 (10)4.010 (10)NA0 (1)NA
Table 4

Infection, dissemination, and transmission rates of Psorophora columbiae f. toltecum thermal mosquitoes infected with epizootic strains of Venezuelan equine encephalitis virus*

% Infected with
GenotypeStrainBlood meal titer (log10 PFU/mL)% Infected (n)Disseminated infection (n)% Potential transmission (n)†Mean ± SD time until mouse death, days
* PFU = plaque-forming units.
† Transmission rates were determined on the basis of suckling mice experiments and expressed as a percentage of those with disseminated infections.
Epizootic IE93-421248.6100 (8)100 (8)100 (8)2.5 ± 0.53
CPA-2017100 (47)89 (47)100 (47)3.1 ± 1.22
CPA-2014.371 (17)41 (17)100 (17)2.9 ± 0.38
96-328637.5100 (6)100 (6)100 (6)2.6 ± 0.53
96-328636.6100 (18)100 (18)100 (18)2.2 ± 0.83
96-328631.2100 (11)82 (11)100 (11)2.8 ± 0.83
Mex 01-326.7100 (19)79 (19)100 (19)2.1 ± 0.32
Enzootic IE68U2016.2100 (13)85 (13)100 (13)2.2 ± 0.42
68U2015.3100 (11)100 (11)100 (11)2.9 ± 0.99
Epizootic IABGua69Z14.5100 (16)100 (16)100 (16)2.1 ± 0.78
Gua69Z11.2100 (16)100 (16)100 (16)2.1 ± 0.32
Table 5

Infection, dissemination, and transmission rates of Psorophora columbiae f. columbiae China, Texas, mosquitoes infected with epizootic strains of Venezuelan equine encephalitis virus*

% Infected with
GenotypeStrainBlood meal titer (log10 PFU/mL)% Infected (n)Disseminated infection (n)% Potential transmission (n)†Mean ± SD time until mouse death, days
* Estimated overall transmission rate is based on 100% of the disseminated mosquitoes that were tested had infected saliva. Therefore, overall transmission rate = overall dissemination rate. PFU = plaque-forming units.
† Transmission rates were determined on the basis of suckling mice experiments.
Epizootic IECPA-2017100 (30)67 (30)100 (30)2.8 ± 0.62
96-328636.664 (25)36 (25)100 (25)2.83 ± 0.75
Enzootic IE68U2016.291 (23)70 (23)100 (23)3.6 ± 1.51
Epizootic IABGua69Z14.580 (34)59 (34)100 (34)2.2 ± 0.42
Gua69Z11.250 (24)33 (24)100 (24)2.5 ± 0.53
Figure 1.
Figure 1.

Map of the Western Hemisphere showing the likely distribution of members of thePsorophora confinnis complex and trapping sites for this study.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.413

Figure 2.
Figure 2.

A, Gel showing phenotypes of the isocitrate dehydrogenase locus for Psorophora columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15 and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. B, Gel showing phenotypes of the aconitase-2 locus for Ps. columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. C, Gel showing phenotypes of the malic enzyme locus for Ps. columbiae f. columbiae and Ps. columbiae f. toltecum sibling species. Samples 1–5 and 10–14 are from Dorado Nuevo, Chiapas, Mexico. Samples 6 and 7 and 15 and 16 are from Thermal, California. Samples 8 and 9 and 17 and 18 are from Starkville, Mississippi. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.413

*

Address correspondence to Abelardo C. Moncayo, Vector-Borne Diseases Section, Communicable and Environmental Diseases, Tennessee Department of Health, 630 Hart Lane, Nashville, TN 37216. E-mail: Abelardo.Moncayo@state.tn.us

Authors’ addresses: Abelardo C. Moncayo, Vector-Borne Disease Section, Communicable and Environmental Diseases, Tennessee Department of Health, 630 Hart Lane, Nashville, TN 37216, E-mail: abelardo.moncayo@state.tn.us. Gregory Lanzaro, University of California Mosquito Research Program, Department of Entomology, 396C Briggs Hall, 1 Shields Avenue, University of California, Davis, CA 95616. Wenli Kang and Scott C. Weaver, Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. Arnoldo Orozco and Armando Ulloa, Centro de Investigación de Paludismo, Instituto National de Salud Publica, Apartado Postal 537, Tapachula Chiapas 30700, Mexico. Juan Arredondo-Jiménez, Departamento de Salud Publica, Universidad de Guadalajara, Sierra Mojada 950 Col. Independencia, Guadalajara, Jalisco 44340, Mexico.

Acknowledgments: We thank Bronca Lothrop (Coachela Valley Mosquito Abatement District) for coordinating the trapping of P. columbiae f. toltecum mosquitoes and Dr. Frederick Tripet for helping with collections in California. We also thank personnel from the Centro de Investigación de Paludismo in Chiapas for their expert assistance in the capture and transport of P. columbiae mosquitoes for this project.

Financial support: This study was supported by grant AI39800 and contract AI25489 from the National Institutes of Health, the National Aeronautics and Space Administration, and the World Bank.

REFERENCES

  • 1

    Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC, 2004. Venezuelan equine encephalitis. Annu Rev Entomol 49 :141–174.

  • 2

    Johnson KM, Martin DH, 1974. Venezuelan equine encephalitis. Adv Vet Sci Comp Med 18 :79–116.

  • 3

    Walton TE, Grayson MA, 1989. Venezuelan equine encephalomyelitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Boca Raton, FL: CRC Press, 203–231.

  • 4

    Groot H, 1972. The health and economic impact of Venezuelan equine encephalitis (VEE). Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 7.

  • 5

    Scherer WF, 1972. History and importance of VE virus. Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 26.

  • 6

    Kubes V, 1972. Epizootic data. Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, DC: Pan American Health Organization, 18.

  • 7

    Kubes V, Rios FA, 1939. The causative agent of infectious equine encephalomyelitis in Venezuela. Science 90 :20–21.

  • 8

    Beck CE, Wyckoff RW, 1938. Venezuelan equine encephalomyelitis. Science 88 :530.

  • 9

    Oberste MS, Fraire M, Navarro R, Zepeda C, Zarate ML, Ludwig GV, Kondig JF, Weaver SC, Smith JF, Rico-Hesse R, 1998. Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico. Am J Trop Med Hyg 59 :100–107.

    • Search Google Scholar
    • Export Citation
  • 10

    Sudia WD, Newhouse VF, Beadle ID, Miller DL, Johnston JG Jr, Young R, Calisher CH, Maness K, 1975. Epidemic Venezuelan equine encephalitis in North America in 1971: vector studies. Am J Epidemiol 101 :17–35.

    • Search Google Scholar
    • Export Citation
  • 11

    Sudia WD, Lord RD, Newhouse VF, Miller DL, Kissling RE, 1971. Vector-host studies of an epizootic of Venezuelan equine encephalomyelitis in Guatemala, 1969. Am J Epidemiol 93 :137–143.

    • Search Google Scholar
    • Export Citation
  • 12

    Sellers RF, Bergold GH, Suarez OM, Morales A, 1965. Investigations during Venezuelan equine encephalitis outbreaks in Venezuela, 1962–1964. Am J Trop Med Hyg 14 :460–469.

    • Search Google Scholar
    • Export Citation
  • 13

    Sudia WD, Newhouse VF, Henderson BE, 1971. Experimental infection of horses with three strains of Venezuelan equine encephalomyelitis virus. II. Experimental vector studies. Am J Epidemiol 93 :206–211.

    • Search Google Scholar
    • Export Citation
  • 14

    Scherer WF, Dickerman RW, Ordonez JV, 1970. Discovery and geographic distribution of Venezuelan encephalitis virus in Guatemala, Honduras, and British Honduras during 1965–68, and its possible movement to Central America and Mexico. Am J Trop Med Hyg 19 :703–711.

    • Search Google Scholar
    • Export Citation
  • 15

    Ortiz DI, Anishchenko M, Weaver SC, 2005. Susceptibility of Psorophora confinnis (Diptera: Culicidae) to infection with epizootic (subtype IC) and enzootic (subtype ID) Venezuelan Equine encephalitis viruses. J Med Entomol 42 :857–863.

    • Search Google Scholar
    • Export Citation
  • 16

    Rico-Hesse R, Weaver SC, de Siger J, Medina G, Salas RA, 1995. Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America. Proc Natl Acad Sci U S A 92 :5278–5281.

    • Search Google Scholar
    • Export Citation
  • 17

    Rivas F, Diaz LA, Cardenas VM, Daza E, Bruzon L, Alcala A, de la Hoz O, Caceres FM, Aristizabal G, Martinez JW, Revelo D, de la Hoz F, Boshell J, Camacho T, Calderon L, Olano VA, Villarreal LI, Roselli D, Alvarez G, Ludwig G, Tsai T, 1997. Epidemic Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis 175 :828–832.

    • Search Google Scholar
    • Export Citation
  • 18

    Weaver SC, Salas R, Rico-Hesse R, Ludwig GV, Oberste MS, Boshell J, Tesh RB, 1996. Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348 :436–440.

    • Search Google Scholar
    • Export Citation
  • 19

    Howard L, Dyar H, Knab F, 1917. Mosquitoes of North and Central America and the West Indies. Washington, DC: Carnegie Institute of Washington Publication.

  • 20

    Aitken T, 1940. The genus Psorophora in California. Revista Entomol 11 :672–682.

  • 21

    Dyar H, Knab F, 1906. Diagnosis of new species of mosquitoes. Proc Biol Soc Wash 19 :133–142.

  • 22

    Carpenter S, LaCasse W, 1955. Mosquitoes of North America (North of Mexico). Berkeley, CA: University of California Press.

  • 23

    Bickley W, 1984. Notes on the Psorophora confinnis complex. Mosq Systematics 16 :162–167.

  • 24

    Ruiz-Garcia M, Ramirez D, Bello F, Alvarez D, 2003. Psorophora columbiae and Psorophora toltecum (Diptera: Culicidae) Colombian populations cannot be differentiated by isoenzymes. Genet Mol Res 2 :229–259.

    • Search Google Scholar
    • Export Citation
  • 25

    Ortiz DI, Weaver SC, 2004. 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. J Med Entomol 41 :987–993.

    • Search Google Scholar
    • Export Citation
  • 26

    Steiner W, Joslyn D, 1978. Electrophoretic techniques for the genetic study of mosquitoes. Mosq News 39 :25–54.

  • 27

    Lanzaro G, Narang S, Seawtight J, 1990. Speciation in an anopheline mosquito: enzyme polymorphism and the genetic structure of populations. Ann Entomol Soc Am 83 :578–585.

    • Search Google Scholar
    • Export Citation
  • 28

    Gargan TP 2nd, Bailey CL, Higbee GA, Gad A, El Said S, 1983. The effect of laboratory colonization on the vector-pathogen interactions of Egyptian Culex pipiens and Rift Valley fever virus. Am J Trop Med Hyg 32 :1154–1163.

    • Search Google Scholar
    • Export Citation
  • 29

    Turell MJ, Gargan TP 2nd, Bailey CL, 1984. Replication and dissemination of Rift Valley fever virus in Culex pipiens. Am J Trop Med Hyg 33 :176–181.

    • Search Google Scholar
    • Export Citation
  • 30

    Weaver SC, Scott TW, Lorenz LH, 1990. Patterns of eastern equine encephalomyelitis virus infection in Culiseta melanura (Diptera: Culicidae). J Med Entomol 27 :878–891.

    • Search Google Scholar
    • Export Citation
  • 31

    Lanzaro G, 1997. Genetics of the Mosquito Vector of Epidemic Venezuelan Equine Encephalitis. Advanced Research Program. College Station, TX: Texas A&M University.

  • 32

    Belkin J, Heinemann S, Page WA, 1970. Mosquito Studies (Diptera, Culicidae). XXI. The Culicidae of Jamaica. Contributions of the American Entomological Institute 6 :1–450.

    • Search Google Scholar
    • Export Citation
  • 33

    Clemens A, 1999. The Biology of Mosquitoes. Cambridge, United Kingdom: CABI Publishing.

  • 34

    Mitchell CJ, Monath TP, Sabattini MS, Christensen HA, Darsie RF Jr, Jakob WL, Daffner JF, 1987. Host-feeding patterns of Argentine mosquitoes (Diptera: Culicidae) collected during and after an epizootic of western equine encephalitis. J Med Entomol 24 :260–267.

    • Search Google Scholar
    • Export Citation
  • 35

    Edman JD, 1971. Host-feeding patterns of Florida mosquitoes. I. Aedes, Anopheles, Coquillettidia, Mansonia and Psorophora. J Med Entomol 8 :687–695.

    • Search Google Scholar
    • Export Citation
  • 36

    Whitehead F, 1951. Host preferences of Psorophora confinnis and Ps. discolor. J Econ Entomol 44 :1019.

  • 37

    Bishop F, 1933. Mosquitoes kill livestock. Science 77 :115–116.

  • 38

    Edman JD, Downe AER, 1964. Host-blood sources and multiple-feeding of mosquitoes in Kansas. Mosq News 24 :154–160.

  • 39

    Estrada-Franco JG, Navarro-Lopez R, Freier JE, Cordova D, Clements T, Moncayo A, Kang W, Gomez-Hernandez C, Rodriguez-Dominguez G, Ludwig GV, Weaver SC, 2004. Venezuelan equine encephalitis virus, southern Mexico. Emerg Infect Dis 10 :2113–2121.

    • Search Google Scholar
    • Export Citation
  • 40

    Turell MJ, Jones JW, Sardelis MR, Dohm DJ, Coleman RE, Watts DM, Fernandez R, Calampa C, Klein TA, 2000. Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus. J Med Entomol 37 :835–839.

    • Search Google Scholar
    • Export Citation
  • 41

    Turell MJ, Barth J, Coleman RE, 1999. Potential for Central American mosquitoes to transmit epizootic and enzootic strains of Venezuelan equine encephalitis virus. J Am Mosq Control Assoc 15 :295–298.

    • Search Google Scholar
    • Export Citation
  • 42

    Turell MJ, O’Guinn ML, Navarro R, Romero G, Estrada-Franco JG, 2003. Vector competence of Mexican and Honduran mosquitoes (Diptera: Culicidae) for enzootic (IE) and epizootic (IC) strains of Venezuelan equine encephalomyelitis virus. J Med Entomol 40 :306–310.

    • Search Google Scholar
    • Export Citation
  • 43

    Gonzalez-Salazar D, Estrada-Franco JG, Carrara AS, Aronson JF, Weaver SC, 2003. Equine amplification and virulence of subtype IE Venezuelan equine encephalitis viruses isolated during the 1993 and 1996 Mexican epizootics. Emerg Infect Dis 9 :161–168.

    • Search Google Scholar
    • Export Citation
  • 44

    Wilkerson RC, Parsons TJ, Klein TA, Gaffigan TV, Bergo E, Consolim J, 1995. Diagnosis by random amplified polymorphic DNA polymerase chain reaction of four cryptic species related to Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) from Paraguay, Argentina, and Brazil. J Med Entomol 32 :697–704.

    • Search Google Scholar
    • Export Citation
  • 45

    Tabachnick W, Black W IV, 1995. Making a case for the molecular population genetic studies of arthropod vectors. Parasitol Today 11 :27–30.

    • Search Google Scholar
    • Export Citation
  • 46

    Faran M, 1980. Mosquito Studies (Diptera: Culicidae) 34: A Revision of the albimanus Section of the Subgenus Nyssorhynchus of Anopheles. Contribution to the American Entomological Institute. Ann Arbor, MI: 215.

  • 47

    Moncada-Perez AM, Conn J, 1992. A polytene chromosome study of four populations of Anopheles aquasalis from Venezuela. Genome 35 :327–331.

    • Search Google Scholar
    • Export Citation
  • 48

    Conn J, Cockburn AF, Mitchell SE, 1993. Population differentiation of the malaria vector Anopheles aquasalis using mitochondrial DNA. J Hered 84 :248–253.

    • Search Google Scholar
    • Export Citation
  • 49

    Gimnig JE, Reisen WK, Eldridge BF, Nixon KC, Schutz SJ, 1999. Temporal and spatial genetic variation within and among populations of the mosquito Culex tarsalis (Diptera: Culicidae) from California. J Med Entomol 36 :23–29.

    • Search Google Scholar
    • Export Citation
  • 50

    Walton TE, Alvarez O Jr, Buckwalter RM, Johnson KM, 1973. Experimental infection of horses with enzootic and epizootic strains of Venezuelan equine encephalomyelitis virus. J Infect Dis 128 :271–282.

    • Search Google Scholar
    • Export Citation
  • 51

    Wang E, Bowen RA, Medina G, Powers AM, Kang W, Chandler LM, Shope RE, Weaver SC, 2001. Virulence and viremia characteristics of 1992 epizootic subtype IC Venezuelan equine encephalitis viruses and closely related enzootic subtype ID strains. Am J Trop Med Hyg 65 :64–69.

    • Search Google Scholar
    • Export Citation
  • 52

    Brault AC, Powers AM, Weaver SC, 2002. Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein. J Virol 76 :6387–6392.

    • Search Google Scholar
    • Export Citation
  • 53

    Brault AC, Powers AM, Ortiz D, Estrada-Franco JG, Navarro-Lopez R, Weaver SC, 2004. Venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope glycoprotein. Proc Natl Acad Sci U S A 101 :11344–11349.

    • Search Google Scholar
    • Export Citation
  • 54

    Kramer LD, Scherer WF, 1976. Vector competence of mosquitoes as a marker to distinguish Central American and Mexican epizootic from enzootic strains of Venezuelan encephalitis virus. Am J Trop Med Hyg 25 :336–346.

    • Search Google Scholar
    • Export Citation
  • 55

    Fernandez Z, Moncayo AC, Carrara AS, Forattini OP, Weaver SC, 2003. Vector competence of rural and urban strains of Aedes (Stegomyia) albopictus (Diptera: Culicidae) from Sao Paulo State, Brazil for IC, ID, and IF subtypes of Venezuelan equine encephalitis virus. J Med Entomol 40 :522–527.

    • Search Google Scholar
    • Export Citation
Save