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    Mortality in mice treated intraperitoneally with VEEV neutralizing antibodies (50 μL; 320 neutralizing units) or sham treated and challenged subcutaneously with 1,000 PFU of EEEV strain GML903836 (N = 5).

  • 1

    Scott TW, Weaver SC, 1989. Eastern equine encephalomyelitis virus: epidemiology and evolution of mosquito transmission. Adv Virus Res 37 :277–328.

    • Search Google Scholar
    • Export Citation
  • 2

    Weaver SC, 2001. Eastern equine encephalitis. Service MW, ed. The Encyclopedia of Arthropod-Transmitted Infections. Wallingford, UK: CAB International, 151–159.

  • 3

    Hawley RJ, Eitzen EM Jr, 2001. Biological weapons—a primer for microbiologists. Annu Rev Microbiol 55 :235–253.

  • 4

    Sabattini MS, Aviles G, Monath TP, 1998. Historical, Epidemiological and Ecological Aspects of Arboviruses in Argentina: Togaviridae, Alphavirus. An Overview of Arbovirology in Brazil and Neighboring Countries. Belem, Brazil: Instituto Evandro Chagas.

  • 5

    Alice FJ, 1956. Infeccao humana pelo virus “leste” da encefalite equina. Bol Inst Biol da Bahia (Brazil) 3 :3–9.

  • 6

    Corniou B, Ardoin P, Bartholomew C, Ince W, Massiah V, 1972. First isolation of a South American strain of Eastern Equine virus from a case of encephalitis in Trinidad. Trop Geogr Med 24 :162–167.

    • Search Google Scholar
    • Export Citation
  • 7

    Turell MJ, O’Guinn ML, Jones JW, Sardelis MR, Dohm DJ, Watts DM, Fernandez R, Travassos da Rosa A, Guzman H, Tesh R, Rossi CA, Ludwig GV, Mangiafico JA, Kondig J, Wasieloski LP Jr, Pecor J, Zyzak M, Schoeler G, Mores CN, Calampa C, Lee JS, Klein TA, 2005. Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon basin region of Peru. J Med Entomol 42 :891–898.

    • Search Google Scholar
    • Export Citation
  • 8

    Brault AC, Powers AM, Chavez CL, Lopez RN, Cachon MF, Gutierrez LF, Kang W, Tesh RB, Shope RE, Weaver SC, 1999. Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America. Am J Trop Med Hyg 61 :579–586.

    • Search Google Scholar
    • Export Citation
  • 9

    Aguilar PV, Greene IP, Coffey LL, Medina G, Moncayo AC, Anishchenko M, Ludwig GV, Turell MJ, O’Guinn ML, Lee J, Tesh RB, Watts DM, Russell KL, Hice C, Yanoviak S, Morrison AC, Klein TA, Dohm DJ, Guzman H, Travassos da Rosa AP, Guevara C, Kochel T, Olson J, Cabezas C, Weaver SC, 2004. Endemic Venezuelan equine encephalitis in northern Peru. Emerg Infect Dis 10 :880–888.

    • Search Google Scholar
    • Export Citation
  • 10

    Calisher CH, Monath TP, Mitchell CJ, Sabattini MS, Cropp CB, Kerschner J, Hunt AR, Lazuick JS, 1985. Arbovirus investigations in Argentina, 1977–1980. III. Identification and characterization of viruses isolated, including new subtypes of western and Venezuelan equine encephalitis viruses and four new bunyaviruses (Las Maloyas, Resistencia, Barranqueras, and Antequera). Am J Trop Med Hyg 34 :956–965.

    • Search Google Scholar
    • Export Citation
  • 11

    Camara A, Diaz G, Vega V, Basualdo M, Contigiani M, 2003. Seroprevalence of antibodies to Venezuelan equine encephalitis complex (subtypes IAB and VI) in humans from General Belgrano island, Formosa, Argentina. Rev Inst Med Trop Sao Paulo 45 :201–204.

    • Search Google Scholar
    • Export Citation
  • 12

    Causey CE, 1963. The role of small mammals in maintenance of arboviruses in the Brazilian Amazon forests. Microbiology 2 :119–121.

  • 13

    Gonzalez LE, Sabattini MS, Vanella JM, Bianchini NR, 1965. Primer aislamiento en la Republica Argentina de arbovirus group A, pertenecientes al complelo Mayaro-Una. I. Validez del aislamiento. Terceras sesiones cientifícas de biologia (Rosario).

  • 14

    Mitchell CJ, Monath TP, Sabattini MS, Cropp CB, Daffner JF, Calisher CH, Jakob WL, Christensen HA, 1985. Arbovirus investigations in Argentina, 1977–1980. II. Arthropod collections and virus isolations from Argentine mosquitoes. Am J Trop Med Hyg 34 :945–955.

    • Search Google Scholar
    • Export Citation
  • 15

    Oberste MS, Weaver SC, Watts DM, Smith JF, 1998. Identification and genetic analysis of Panama-genotype Venezuelan equine encephalitis virus subtype ID in Peru. Am J Trop Med Hyg 58 :41–46.

    • Search Google Scholar
    • Export Citation
  • 16

    Tesh RB, Watts DM, Russell KL, Damodaran C, Calampa C, Cabezas C, Ramirez G, Vasquez B, Hayes CG, Rossi CA, Powers AM, Hice CL, Chandler LJ, Cropp BC, Karabatsos N, Roehrig JT, Gubler DJ, 1999. Mayaro virus disease: An emerging mosquito-borne zoonosis in tropical South America. Clin Infect Dis 28 :67–73.

    • Search Google Scholar
    • Export Citation
  • 17

    Travassos da Rosa AP, Turell MJ, Watts DM, Powers AM, Vasconcelos PF, Jones JW, Klein TA, Dohm DJ, Shope RE, Degallier N, Popov VL, Russell KL, Weaver SC, Guzman H, Calampa C, Brault AC, Lemon AP, Tesh RB, 2001. Trocara virus: A newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin. Am J Trop Med Hyg 64 :93–97.

    • Search Google Scholar
    • Export Citation
  • 18

    Watts DM, Callahan J, Rossi C, Oberste MS, Roehrig JT, Wooster MT, Smith JF, Cropp CB, Gentrau EM, Karabatsos N, Gubler D, Hayes CG, 1998. Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region. Am J Trop Med Hyg 58 :35–40.

    • Search Google Scholar
    • Export Citation
  • 19

    Watts DM, Lavera V, Callahan J, Rossi C, Oberste MS, Roehrig JT, Cropp CB, Karabatsos N, Smith JF, Gubler DJ, Wooster MT, Nelson WM, Hayes CG, 1997. Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru. Am J Trop Med Hyg 56 :661–667.

    • Search Google Scholar
    • Export Citation
  • 20

    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
  • 21

    Calisher CH, Maness KS, 1975. Laboratory studies of Venezuelan equine encephalitis virus in equines, Texas, 1971. J Clin Microbiol 2 :198–205.

    • Search Google Scholar
    • Export Citation
  • 22

    Allen WP, 1962. Cross protection among group A arthropod-borne viruses. Bacteriol Proc 152.

  • 23

    Hearn HJ Jr, Rainey CT, 1963. Cross-protection in animals infected with group a arboviruses. J Immunol 90 :720–724.

  • 24

    Walton TE, Jochim MM, Barber TL, Thompson LH, 1989. Cross-protective immunity between equine encephalomyelitis viruses in equids. Am J Vet Res 50 :1442–1446.

    • Search Google Scholar
    • Export Citation
  • 25

    Byrne RJ, French GR, Yancey FS, Gochenour WS, Russell PK, Ramsburg HH, Brand OA, Scheider FG, Buescher EL, 1964. Clinical and immunologic interrelationship among Venezuelan, Eastern, and Western equine encephalomyelitis viruses in Burros. Am J Vet Res 25 :24–31.

    • Search Google Scholar
    • Export Citation
  • 26

    Shahan MS, Giltner LT, 1935. Equine encephalomyelitis studies: I. Cross-immunity tests between eastern and western types of virus. J Am Vet Med Assoc 86 :764–772.

    • Search Google Scholar
    • Export Citation
  • 27

    Beaty BJ, Calisher CH, Shope RE, 1989. Arboviruses. Schmidt NJ, Emmons RW, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. Sixth edition. Washington, DC: American Public Health Association, 797–855.

  • 28

    Jones JW, Turell MJ, Sardelis MR, Watts DM, Coleman RE, Fernandez R, Carbajal F, Pecor JE, Calampa C, Klein TA, 2004. Seasonal distribution, biology, and human attraction patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos, Peru. J Med Entomol 41 :349–360.

    • Search Google Scholar
    • Export Citation
  • 29

    Causey OR, Theiler M, 1958. Virus antibody survey on sera of residents of the Amazon Valley in Brazil. Am J Trop Med Hyg 7 :36–41.

  • 30

    Theiler M, Downs WG, 1973. The Arthropod-borne Viruses of Vertebrates. New Haven, CT: Yale University Press.

  • 31

    Scherer WF, Dickerman RW, Ordonez JV, 1979. Serologic surveys for the determination of antibodies against the Eastern, Western, California, and St. Louis encephalitis and dengue 3 arboviruses in Central America, 1961–1975. Bol Oficina Sanit Panam 87 :210–223.

    • Search Google Scholar
    • Export Citation
  • 32

    Aguilar PV, Paessler S, Carrara AS, Baron S, Poast J, Wang E, Moncayo AC, Anishchenko M, Watts D, Tesh RB, Weaver SC, 2005. Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus. J Virol 79 :11300–11310.

    • Search Google Scholar
    • Export Citation
  • 33

    Weaver SC, Frey TK, Huang HV, Kinney RM, Rice CM, Roehrig JT, Shope RE, Strauss EG, 2005. Togaviridae. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, eds. Virus Taxonomy, VIIIth Report of the ICTV. London: Elsevier/ Academic Press, 999–1008.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ENDEMIC EASTERN EQUINE ENCEPHALITIS IN THE AMAZON REGION OF PERU

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  • 1 Center for Biodefense and Emerging Infectious Diseases, Department of Microbiology and Immunology and Department of Pathology, University of Texas Medical Branch, Galveston, Texas; Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland; US Naval Medical Research Center Detachment, NMRCD/Unit 3800, American Embassy, Lima, Peru

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.

INTRODUCTION

Eastern equine encephalitis virus (EEEV; Togaviridae: Alphavirus) causes sporadic equine epizootics throughout much of the Americas.1,2 In North America (NA), EEEV has been responsible for > 180 human cases and thousands of equine cases since 1964 (http://www.cdc.gov/ncidod/dvbid/arbor/cases-eee-1964-2000.htm). In the United States, Georgia, Louisiana, Massachusetts, New Jersey, and Florida have recorded the largest number of cases. Increases in the numbers of equine EEE cases during the past several years indicate its continuing importance as an emerging arboviral threat. In addition, EEEV is a category B priority agent of the National Institute of Allergy and Infectious Disease (http://www3.niaid.nih.gov/biodefense/bandc_priority.htm) because of its virulence (30–50% mortality in human cases, 50–90% in horses), potential use as a biologic weapon, and the lack of a licensed vaccine or effective treatment of human infection.3

In South America (SA), EEEV has caused sporadic epizootics, some involving thousands of horses in Argentina, Brazil, Venezuela,4 and Colombia (S.C.W., unpublished data). Early reports from Argentina and Brazil showed serologic evidence of human infection with EEEV, with a high seroprevalence in certain areas; however, the absence of reported human encephalitis in these areas, despite active disease surveillance during equine epizootics, was remarkable. To date, there have been only two reported fatal human encephalitis cases of EEE in SA: one in Brazil in 19565 and another in Trinidad in 1970.6 In contrast, > 180 documented EEE cases have occurred in NA. In Peru, EEEV has been isolated repeatedly from Culex (Melanoconion) pedroi mosquitoes captured in the vicinity of Puerto Almendras, a rural village located near Iquitos,7 indicating active enzootic circulation. However, no human isolates have been made in this area, despite surveillance for acute febrile diseases (D.M.W. and R.M.R., unpublished data).

In NA, EEEV circulates in hardwood swamps among avian reservoir hosts, with transmission by the enzootic mosquito vector, Culiseta melanura.2 Virus strains isolated from 1933 to 1996 and from locations ranging from Mexico north to Massachusetts and east to islands in the Caribbean exhibit a high degree of sequence conservation, indicating a single major viral lineage that evolves over time.8 In SA and Central America, EEEV has been isolated from tropical humid forest and other habitats, primarily from mosquitoes in the subgenus Culex (Melanoconion). Reservoir hosts are not known but probably include small mammals and/or birds. In contrast to NA, a variety of genetically diverse EEEV lineages circulate in SA, comprising three different subtypes.8 Two of these subtypes have been isolated from mosquitoes collected near Iquitos.7

Several hypotheses could explain the paucity of human EEE in Peru and elsewhere in SA: 1) the ecology of enzootic transmission in SA limits human exposure, for example, based on vector-host preferences; 2) humans are exposed but do not develop apparent infection with EEEV (i.e., SA strains are poorly infectious for humans and do not normally induce either clinical illness or an immune response); 3) heterologous alphavirus antibodies cross-protect against EEEV infection and/or disease; 4) human cases of EEE occur, but are not diagnosed; and/or 5) apparent, febrile disease occurs without detectable viremia.

Some data and observations are inconsistent with some of the above hypotheses. For example, many EEEV isolates have been made from Cx. pedroi in the Iquitos area, and human landing collections indicate that this species is anthropophilic. Other hypotheses are supported by previous studies and epidemiologic observations indicate that, in SA, EEEV circulates concomitantly with several other alphaviruses including Venezuelan equine encephalitis (VEEV), Mucambo (VEE complex subtype IIIC and IIID strains), Mayaro (MAYV), Una (UNAV), Trocara (TROV), Western equine encephalitis (WEEV), and Aura viruses. In addition to providing opportunities for cross-reactivity, there is strong epidemiologic evidence linking many of these alphaviruses with human illness.920 In addition, laboratory and epidemiologic observations have shown some cross-protection among alphaviruses,2124 although other studies contradict those find-ings.25,26 In an attempt to resolve its role in alphaviral disease and epidemiology, we first assessed cross-protection in mice and hamsters first infected with VEEV and MAYV, the most prevalent alphaviruses infecting humans in the Amazon region of Peru, and challenged with EEEV. In addition, to examine the mechanism of cross-protection, we tested the ability of passively transferred human heterologous antibodies to protect mice against fatal EEE.

To determine more precisely whether EEEV causes human illness in Peru and to assess the possible role of alphavirus heterologous antibodies in protection, acute and convalescent sera collected in the Iquitos area from 153 febrile patients were tested for EEEV-reactive antibodies. In addition, 359 human sera, collected from healthy persons residing in the EEEV-enzootic area of Puerto Almendras, were tested for EEEV-reactive antibodies.

MATERIALS AND METHODS

Viruses.

Viruses (Table 1) were obtained from the University of Texas Medical Branch World Reference Center for Emerging Viruses and Arboviruses. The EEEV and MAYV strains were isolated in Vero cells from mosquitoes and human serum, respectively, and were chosen for these studies because of their low passage histories. Stocks were prepared by intracerebral inoculation of newborn mice, and titers were determined by plaque assay on Vero cells.

Infection of mice and hamsters and challenge protocols.

Five- to 6-week-old NIH Swiss mice and Golden Syrian hamsters from Harlan Laboratories (Indianapolis, IN) were allowed to acclimate to the laboratory conditions for 1 week before infections. Cohorts of five animals were infected subcutaneously with 1,000 PFU of VEEV vaccine strain TC-83 or MAYV strain IQT 4235. Control animals were sham infected with diluent alone. Animals were monitored daily for signs of illness. Three or 12 weeks later, animals were bled and challenged with 1,000 PFU of EEEV strain GML903836 (SA subtype III). The animals were bled 24, 48, and 72 hours post-infection (PI) for viremia determination by plaque assay and were monitored for up to 1 month for signs of illness and mortality. Animals were killed and bled by cardiac puncture on completion of the experiment. Animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch.

Passive transfer of antibodies to mice.

Five- to 6-week-old NIH Swiss mice were inoculated intraperitoneally with various dilutions of human neutralizing antisera (80, 160, and 320 neutralizing units) obtained from a human naturally infected with VEEV subtype ID. Controls included mice inoculated with alphavirus-negative sera obtained from persons who never resided in areas of known alphavirus circulation. Sera were heat-inactivated at 56°C for 30 minutes before mouse inoculations. Twenty-four hours later, mice were challenged subcutaneously with 1,000 PFU of EEEV strain GML903836 and monitored daily for signs of illness and mortality.

Plaque reduction neutralization tests.

Pre- and post-challenge sera from vaccinated animals were tested for neutralizing antibodies using a plaque reduction neutralization test (PRNT).27 In addition, to assess cross-protection between alphaviruses, samples obtained from vaccinated or naturally infected persons were tested for cross-neutralizing antibodies against heterologous alphaviruses. Sera were heat-inactivated at 56°C for 30 minutes, and 2-fold serial dilutions were mixed with 100 PFU of each virus and incubated at 4°C overnight. The virus-serum mixture was inoculated onto monolayers of Vero cells and incubated at 37°C for 1 hour before adding an overlay containing 0.4% agarose in minimum essential medium supplemented with 2% fetal bovine serum. After 48 hours, plates were stained with 0.25% crystal violet in 20% methanol, and plaques were counted. Neutralization titers were considered the highest dilution of sera that reduced plaques by ≥ 80%.

Description of the study sites in Peru.

To determine the public health importance of EEEV in Peru and to assess the potential role of alphavirus heterologous antibodies in protecting against severe EEE, epidemiologic studies were conducted in enzootic areas of EEEV transmission. Iquitos, a city of ~300,000 people on the Amazon River, was selected because EEEV has been repeatedly isolated from mosquitoes captured in the vicinity,7 and human infections by dengue, MAYV, Oropouche, Mucambo (VEE complex subtype IIID) and VEEV were previously documented in this area.9,16,18,19 The study subjects were patients who presented with an acute, undifferentiated, febrile illness and were enrolled in a study conducted by the Naval Medical Research Center Detachment, Lima, Peru, in collaboration with the Peruvian Ministry of Health. Criteria for enrollment included a fever of ≥ 38°C and ≤ 5 days in duration, accompanied by headache, myalgia, and other nonspecific signs and symptoms. Demographic and clinical data and blood samples were obtained from each patient at the time of voluntary enrollment, and convalescent samples were collected ~2–4 weeks later.

In addition, a serosurvey was conducted in the rural village of Puerto Almendras, a community of single family dwellings inhabited by hunters and gatherers, ~20 km from Iquitos.28 This location was selected because > 20 strains of EEEV were isolated from mosquitoes captured < 0.5 km from this village.7 Demographic data and blood samples were obtained from healthy volunteers.

All sera from febrile patients were tested initially at a 1:100 dilution for IgM and IgG antibodies to EEEV, MAYV, Mucambo (VEE IIID), VEEV, UNAV, and TROV by an enzyme-linked immunosorbent assay (ELISA) as described previously.18 In addition, acute samples were inoculated into Vero and mosquito cell cultures in an attempt to isolate viruses. Samples obtained from healthy volunteers were tested for IgG antibody to EEEV. All ELISA IgM- and IgG-positive samples were further tested using 80% PRNT for EEEV, VEEV, MAYV, UNAV, and TROV.

RESULTS

VEEV vaccination and cross-protection against EEEV.

Five-week-old mice were infected subcutaneously with 1,000 PFU of the VEEV vaccine strain TC-83, and 21 days later the animals were bled and challenged subcutaneously with 1,000 PFU of EEEV. Mice were bled 24, 48, and 72 hours after infection for viremia determination and monitored daily for signs of disease. Viremia levels in the vaccinated group were reduced compared with the sham vaccinated group (peak viremia at 48 hours PI: < 2 versus 4 log10 PFU/mL, respectively). None of the vaccinated animals developed signs of illness, and all survived EEEV infection; in contrast, 80% of the sham-vaccinated animals succumbed to infection 5–7 days later. Survivors were terminally bled 1 month later, and levels of neutralizing antibodies against VEEV and EEEV were determined in the sera collected pre- and post-challenge. No EEEV neutralizing antibodies were detected in samples collected before challenge. However, 1 month after EEEV challenge, all VEEV-vaccinated animals developed neutralizing antibodies against EEEV, with titers ranging from 80 to 640. Samples collected both before and after EEEV challenge had detectable levels of neutralizing antibodies against VEEV strain TC-83. Levels of neutralizing antibodies against VEEV increased after EEEV challenge, indicating an anamnestic response (Table 2).

To determine the duration of cross-protective immunity, a similar experiment was conducted; however, the animals were bled and challenged with EEEV 3 months after vaccination. As with the 21-day challenge, 100% (five of five) survival was observed in the VEEV-vaccinated group compared with 20% (one of five) survival in the untreated group (P < 0.05), indicating that vaccination with VEEV provided long-lasting protection against EEEV in most mice.

MAYV vaccination and cross-protection against EEEV.

To determine whether MAYV also induces cross-protective immunity against EEEV, 5- to 6-week-old Golden Syrian hamsters were vaccinated with MAYV and 3 weeks later challenged with EEEV. MAYV represents an alphavirus more distantly related to EEEV than VEEV. Similar to the results observed with VEEV cross-protection, all MAYV-vaccinated hamsters survived EEEV infection (five of five) and developed higher neutralizing antibodies titers against MAYV after challenge with EEEV, showing an anamnestic response. No EEEV neutralizing antibodies were detected before challenge (Table 3). To determine whether MAYV induces long-lasting immunity against EEEV, hamsters were challenged 3 months after vaccination. All MAYV-vaccinated hamsters developed central nervous system (CNS) disease and succumbed to infection like those in the control sham-vaccinated group. Thus, although infection with MAYV elicited cross-protective immunity against EEEV, protection was not long-lasting.

Cross-reactivity among alphavirus antibodies.

A possible mechanism of cross-protection among alphaviruses is that heterologous antibodies neutralize infectivity. To test this hypothesis, sera obtained from vaccinated or laboratory-infected humans were tested by PRNT against other alphaviruses. Table 4 shows that sera from VEEV-infected individuals did not neutralize EEEV, MAYV, or UNAV. Similarly, sera from VEEV/EEEV-vaccinated persons did not show any cross-reactivity with MAYV and UNAV. Combined with the results presented above, the data suggested that cross-protection among these viruses involves a mechanism other than antibody cross-neutralization.

Passive transfer of antibody to mice.

To determine whether the observed cross-protection was caused by the humoral or cellular immune response, 5- to 6-week-old mice were given various concentrations of human neutralizing antibodies (50 μL of serum) against VEEV or normal human sera administered intraperitoneally and 24 hours later were challenged with 1,000 PFU of EEEV. Despite administration of high levels of neutralizing VEEV antibodies (up to 320 neutralizing units), the mortality in the treated animals was similar to that of the control group (Figure 1). Thus, VEEV heterologous antibodies were not able to cross-protect against VEEV in vivo, strongly suggesting that the observed cross-protection was mainly caused by a cellular response.

Epidemiologic studies in Iquitos.

To assess a possible role of alphavirus heterologous antibodies in protecting people against apparent EEE in SA, we tested human sera collected from people living in EEEV-enzootic areas. Only 3 of 153 patients enrolled in the febrile illness study had EEEV-reactive IgM antibodies; however, only 2 of these 3 showed a 4-fold or greater increase in IgM titer in the convalescent sample, and 1 case was positive only in the acute sample. Although these patients had detectable IgM levels only against EEEV (none had IgM antibodies reactive against VEEV or MAYV, the most common alphaviruses causing human disease in Iquitos), EEEV-neutralizing antibodies were not detected in these samples, probably because of the poor immunogenicity of the EEEV strains circulating in this region. In all of these EEEV IgM-positive patients, there was clear evidence of pre-exposure to other alphaviruses, as indicated by VEEV, MAYV, and/or UNAV PRNT antibodies (Table 5). The most common clinical signs and symptoms in the EEEV-positive patients were fever, chills, arthralgia, muscle pain, headache, nausea, and vomiting, with no indication of neurologic manifestations.

A serosurvey in EEEV-enzootic Puerto Almendras area revealed that 9 of 359 volunteers had EEEV-reactive antibodies, indicating an overall seroprevalence of 3%. Most of the seropositive individuals had evidence of exposure to other alphaviruses such as VEEV, MAYV, or UNAV (Table 6); however, one had antibodies reactive only against EEEV. These results indicated that very few individuals were infected by EEEV, even though they resided near an enzootic focus of EEEV activity.

DISCUSSION

EEEV has been isolated in SA from horses, donkeys, sentinel hamsters, ticks, mosquitoes, but from only two fatal human cases (one each in Trinidad and Brazil).5,6 Infection of humans in the Amazon Valley was first indicated by detection of neutralizing antibodies in a Brazilian population, with 13 of 314 (4%) being sera positive.29 These seropositive people were residents living in Abaetetuba and Cameta, neighborhoods of Belem. In Cameta, 3 of 12 sera from children and 9 of 29 adult sera were EEEV positive.30 A single fatal case was reported in a 2-year-old child in Bahia, Brazil, in 1956.5 In Peru, EEEV was first isolated in 1970 in Pucallpa, and subsequent studies in Yurimaguas showed EEEV-neutralizing antibodies in horses (72%) and humans (21%).31 Despite the high seroprevalence in the area, human disease was not reported.

Recently, during ecological studies of arboviral activity in the Amazon region of Peru, EEEV was repeatedly isolated from Cx. pedroi mosquitoes captured in the area around Puerto Almendras,7 indicating enzootic circulation. However, human neurologic disease was not observed in this or any other EEEV-enzootic area near Iquitos, Peru, despite intensive surveillance. Thus, the hypothesis that EEE human cases occur but are not diagnosed or are simply not accompanied by viremia is not supported by these ongoing studies by the US Naval Medical Research Center Detachment, Lima, Peru. This surveillance program has failed to identify EEE human cases either by virus isolation or serology, despite the intensive efforts to detect febrile human disease. Similarly, during the last two EEEV horse epizootic periods in Argentina (1981 and 1989), an active human encephalitis surveillance program was established in the area; however, no human cases of EEE were observed.4

The hypothesis that there is little or no human exposure to EEEV because of the ecology of its transmission cycle in Peru is not supported by observations of Cx. pedroi biting activity. This mosquito is attracted to humans and is well represented in human landing collections in the Puerto Almendras area28 where the EEEV isolates were made.7 This species is also a competent laboratory vector of EEEV (M.J.T., unpublished data). Assuming that people are exposed to EEEV through this mosquito, our results support the hypothesis that humans rarely develop apparent infection, possibly because of a reduced virulence of the SA strains of EEEV in this area and/or cross-protection from heterologous alphavirus antibodies. However, the low proportion of individuals with EEEV antibodies, compared with antibodies reactive against other alphaviruses like VEEV and MAYV, and the high EEEV infection rates in mosquitoes known to feed on humans suggests that the EEEV strains circulating in Peru do not replicate efficiently in humans. Possible reasons include sensitivity to innate immune mechanisms such as the interferon (IFN) response.32 We recently provided evidence that strains of EEEV isolated from SA are generally more sensitive to the antiviral effect of human IFN compared with NA strains that are more resistant.32 This finding could explain the lack of human EEEV isolates and the low PRNT titers observed in the positive sera. Thus, additional methods involving viral nucleic acid amplification might also be a necessary tool for the diagnosis of human EEE infection in South America.

We evaluated the effect of heterologous alphavirus antibodies on EEEV infection of laboratory animals, and our results corroborated previous studies that found evidence of cross-protection among alphaviruses.2124 Mice and hamsters vaccinated with VEEV and MAYV survived infection with EEEV and exhibited little or no viremia. We also studied whether VEEV and MAYV induced short- or long-lasting immunity in mice and found that only VEEV induced long-lasting cross-protection, consistent with its closer genetic and antigenic relationships to EEEV.33 We also aimed to determine whether the observed cross-protection was caused by a humoral and cellular immune response to better understand the specific role of alphavirus heterologous cross-protection. Hence, VEEV neutralizing antibodies were passively transferred to mice, but failed to prevent fatal EEE. Considered together, our results strongly suggest that cross-protection of limited duration involve cellular immunity. Additional studies are needed to elucidate the protective mechanism(s).

Although our studies using animal models showed some cross-protection of VEEV and MAYV against EEEV, these data may not necessarily be applicable to humans, and we therefore do not suggest or support the cessation of human vaccination for persons working with EEEV, even if they have been already vaccinated with VEEV. Similarly, vaccination of horses against both EEEV and VEEV should be maintained in areas where both viruses circulate.

Because the results of the cross-protection experiments may not necessarily be applicable to humans and to virus circulation under field conditions, we aimed to determine whether humans in Peru with EEEV-reactive antibodies also have evidence of exposure to other alphaviruses. Our results indicated that all febrile patients with detectable EEEV IgM antibodies had neutralizing antibodies against VEEV, MAYV, and/or UNAV, suggesting that alphavirus heterologous antibodies might protect some people against EEE, including neurologic disease. Similar results were obtained for EEEV-positive samples identified in the seroprevalence study, in which most but not all of the EEEV antibody–positive samples also were positive for PRNT antibodies to VEEV, MAYV, and/or UNAV. However, one individual with EEEV PRNT antibodies was negative for all other alphavirus antibodies tested. Therefore, our results suggest a possible role of alphavirus cross-protection in limiting some infection and/or disease caused by EEEV. However, the infection of some alphavirus-nonimmune persons with EEEV occurred in the absence of apparent disease. This indicates that cross-protection alone cannot explain the nearly complete lack of neurologic disease in SA because seroprevalence against alphaviruses in Iquitos never approaches 100%. These results suggest that EEEV in Peru is probably not highly pathogenic for humans. This conclusion is also supported by the results of a febrile illness study during the 1990s that failed to reveal any evidence of human neurologic disease caused by infection with EEEV in the Iquitos region.

To our knowledge, this is the first study that provides evidence of human febrile illness caused by EEEV in Peru. We detected EEE seroconversion in two individuals with febrile illness but without neurologic signs. We also showed that very few individuals have EEEV neutralizing antibodies despite living in an enzootic focus of EEEV transmission. Additional studies are needed to fully characterize the limited febrile illness caused by EEEV in the Iquitos area and to characterize the virus strains circulating there.

Table 1

Viruses used in the study

Virus strainPlace of isolationYear of isolationPassage history
* Vaccine strain derived from Trinidad Donkey virus isolated from Trinidad in 1943.
EEEV-GML903836Panama1984Vero-2/mouse brain-1
MAYV-IQT4235Peru1997Vero-2/mouse brain -1
VEEV-TC83*1943Guinea pig heart cell-83
Table 2

Plaque reduction neutralization antibody responses in NIH Swiss mice after vaccination with VEEV strain TC-83 and challenged with EEEV

Pre-challengePost-challenge
Mouse no.VEEV PRNTEEEV PRNTVEEV PRNTEEEV PRNT
1640< 20≥ 2,56080
22,560< 20≥ 2,560640
3640< 20≥ 2,560160
480< 20≥ 2,560320
580< 20≥ 2,560320
Table 3

Plaque reduction neutralization antibody titers in Golden Syrian hamsters after vaccination with MAYV and challenged with EEEV

Pre-challengePost-challenge
HamsterMAYV titerEEEV titerMAYV titerEEEV titer
1160< 20≥ 640640
2640< 20≥ 640640
3640< 20≥ 640640
4320< 20≥ 640320
5320< 20≥ 640320
Table 4

Plaque reduction neutralization antibody responses in persons vaccinated with the TC-83 VEEV vaccine strain and formalin-inactivated EEEV

Antibody titer against
Virus vaccineHuman serum no.VEEVEEEVMAYVUNAV
VEEV120< 20< 20< 20
2640< 20< 20< 20
3640< 20< 20< 20
440< 20< 20< 20
VEEV/EEEV164040< 20< 20
216020< 20< 20
Table 5

Febrile patients with EEEV-reactive plaque reduction neutralization antibodies

Antibody titer against
Patient no.SampleEEEV IgMEEEVVEEVMAYVUNAVTROVWEEV
1AcuteNegative< 20≥ 640< 20< 20< 20< 20
Convalescent6,400< 20≥ 640< 20< 20< 20< 20
2AcuteNegative< 20≥ 640≥ 640160< 20< 20
Convalescent1,600< 20≥ 640≥ 640160< 20< 20
3Acute100< 20≥ 640< 20160< 20< 20
ConvalescentNegative< 20≥ 640< 2080< 20< 20
Table 6

Alphavirus plaque reduction neutralization antibody seroprevalence in Puerto Almendras residents

Antibody titer against
Sample no.EEEVVEEVMAYVUNAVTROV
ND, not done.
POM 015940< 20< 20< 20< 20
POM 018040< 20NDNDND
POM 021820< 20NDNDND
POM 024620< 20≥ 64020< 20
POM 066320160320< 20< 20
POM 072320< 2080160< 20
POM 074320< 20NDNDND
POM 08048020204020
IQT 865020< 20< 2040< 20
Figure 1.
Figure 1.

Mortality in mice treated intraperitoneally with VEEV neutralizing antibodies (50 μL; 320 neutralizing units) or sham treated and challenged subcutaneously with 1,000 PFU of EEEV strain GML903836 (N = 5).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 2; 10.4269/ajtmh.2007.76.293

*

Address correspondence to Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. E-mail: sweaver@utmb.edu

Authors’ addresses: Patricia V. Aguilar, Robert B. Tesh, Douglas M. Watts, and Scott C. Weaver, Departments of Microbiology and Immunology, Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, Telephone: 409-747-0758 Fax: 409-747-2415. Rebecca M. Robich, Michael J. Turell, Monica L. O’Guinn, and Terry A. Klein, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD. Alfredo Huaman, Carolina Guevara, Zonia Rios, and James Olson, US Naval Medical Research Center Detachment, NMRCD/Unit 3800, American Embassy, Lima, Peru.

Acknowledgments: The authors thank Mardelle Susman for editing the manuscript. The study protocol was approved by the Naval Medical Research Center Institutional Review Board (Protocol 31535) in compliance with all U.S. federal regulations governing the protection of human study participants. The opinions expressed in this paper are those of the authors and do not reflect the official policy of the Department of the Navy, the Department of the Army, the Department of Defense, or the U.S. Government.

Financial support: This research was supported by Grant AI049725 from the National Institutes of Health through the joint National Science Foundation/National Institutes of Health program on the Ecology of Infectious Disease, and by Work UNIT NUMBER (WUN) 847705 8200 25GB B0016. IRB Protocol Number: 31535 “Surveillance and Etiology of Acute Febrile Diseases in Peru.” PVA was supported by the James T. McLaughlin Fellowship Fund.

REFERENCES

  • 1

    Scott TW, Weaver SC, 1989. Eastern equine encephalomyelitis virus: epidemiology and evolution of mosquito transmission. Adv Virus Res 37 :277–328.

    • Search Google Scholar
    • Export Citation
  • 2

    Weaver SC, 2001. Eastern equine encephalitis. Service MW, ed. The Encyclopedia of Arthropod-Transmitted Infections. Wallingford, UK: CAB International, 151–159.

  • 3

    Hawley RJ, Eitzen EM Jr, 2001. Biological weapons—a primer for microbiologists. Annu Rev Microbiol 55 :235–253.

  • 4

    Sabattini MS, Aviles G, Monath TP, 1998. Historical, Epidemiological and Ecological Aspects of Arboviruses in Argentina: Togaviridae, Alphavirus. An Overview of Arbovirology in Brazil and Neighboring Countries. Belem, Brazil: Instituto Evandro Chagas.

  • 5

    Alice FJ, 1956. Infeccao humana pelo virus “leste” da encefalite equina. Bol Inst Biol da Bahia (Brazil) 3 :3–9.

  • 6

    Corniou B, Ardoin P, Bartholomew C, Ince W, Massiah V, 1972. First isolation of a South American strain of Eastern Equine virus from a case of encephalitis in Trinidad. Trop Geogr Med 24 :162–167.

    • Search Google Scholar
    • Export Citation
  • 7

    Turell MJ, O’Guinn ML, Jones JW, Sardelis MR, Dohm DJ, Watts DM, Fernandez R, Travassos da Rosa A, Guzman H, Tesh R, Rossi CA, Ludwig GV, Mangiafico JA, Kondig J, Wasieloski LP Jr, Pecor J, Zyzak M, Schoeler G, Mores CN, Calampa C, Lee JS, Klein TA, 2005. Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon basin region of Peru. J Med Entomol 42 :891–898.

    • Search Google Scholar
    • Export Citation
  • 8

    Brault AC, Powers AM, Chavez CL, Lopez RN, Cachon MF, Gutierrez LF, Kang W, Tesh RB, Shope RE, Weaver SC, 1999. Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America. Am J Trop Med Hyg 61 :579–586.

    • Search Google Scholar
    • Export Citation
  • 9

    Aguilar PV, Greene IP, Coffey LL, Medina G, Moncayo AC, Anishchenko M, Ludwig GV, Turell MJ, O’Guinn ML, Lee J, Tesh RB, Watts DM, Russell KL, Hice C, Yanoviak S, Morrison AC, Klein TA, Dohm DJ, Guzman H, Travassos da Rosa AP, Guevara C, Kochel T, Olson J, Cabezas C, Weaver SC, 2004. Endemic Venezuelan equine encephalitis in northern Peru. Emerg Infect Dis 10 :880–888.

    • Search Google Scholar
    • Export Citation
  • 10

    Calisher CH, Monath TP, Mitchell CJ, Sabattini MS, Cropp CB, Kerschner J, Hunt AR, Lazuick JS, 1985. Arbovirus investigations in Argentina, 1977–1980. III. Identification and characterization of viruses isolated, including new subtypes of western and Venezuelan equine encephalitis viruses and four new bunyaviruses (Las Maloyas, Resistencia, Barranqueras, and Antequera). Am J Trop Med Hyg 34 :956–965.

    • Search Google Scholar
    • Export Citation
  • 11

    Camara A, Diaz G, Vega V, Basualdo M, Contigiani M, 2003. Seroprevalence of antibodies to Venezuelan equine encephalitis complex (subtypes IAB and VI) in humans from General Belgrano island, Formosa, Argentina. Rev Inst Med Trop Sao Paulo 45 :201–204.

    • Search Google Scholar
    • Export Citation
  • 12

    Causey CE, 1963. The role of small mammals in maintenance of arboviruses in the Brazilian Amazon forests. Microbiology 2 :119–121.

  • 13

    Gonzalez LE, Sabattini MS, Vanella JM, Bianchini NR, 1965. Primer aislamiento en la Republica Argentina de arbovirus group A, pertenecientes al complelo Mayaro-Una. I. Validez del aislamiento. Terceras sesiones cientifícas de biologia (Rosario).

  • 14

    Mitchell CJ, Monath TP, Sabattini MS, Cropp CB, Daffner JF, Calisher CH, Jakob WL, Christensen HA, 1985. Arbovirus investigations in Argentina, 1977–1980. II. Arthropod collections and virus isolations from Argentine mosquitoes. Am J Trop Med Hyg 34 :945–955.

    • Search Google Scholar
    • Export Citation
  • 15

    Oberste MS, Weaver SC, Watts DM, Smith JF, 1998. Identification and genetic analysis of Panama-genotype Venezuelan equine encephalitis virus subtype ID in Peru. Am J Trop Med Hyg 58 :41–46.

    • Search Google Scholar
    • Export Citation
  • 16

    Tesh RB, Watts DM, Russell KL, Damodaran C, Calampa C, Cabezas C, Ramirez G, Vasquez B, Hayes CG, Rossi CA, Powers AM, Hice CL, Chandler LJ, Cropp BC, Karabatsos N, Roehrig JT, Gubler DJ, 1999. Mayaro virus disease: An emerging mosquito-borne zoonosis in tropical South America. Clin Infect Dis 28 :67–73.

    • Search Google Scholar
    • Export Citation
  • 17

    Travassos da Rosa AP, Turell MJ, Watts DM, Powers AM, Vasconcelos PF, Jones JW, Klein TA, Dohm DJ, Shope RE, Degallier N, Popov VL, Russell KL, Weaver SC, Guzman H, Calampa C, Brault AC, Lemon AP, Tesh RB, 2001. Trocara virus: A newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin. Am J Trop Med Hyg 64 :93–97.

    • Search Google Scholar
    • Export Citation
  • 18

    Watts DM, Callahan J, Rossi C, Oberste MS, Roehrig JT, Wooster MT, Smith JF, Cropp CB, Gentrau EM, Karabatsos N, Gubler D, Hayes CG, 1998. Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region. Am J Trop Med Hyg 58 :35–40.

    • Search Google Scholar
    • Export Citation
  • 19

    Watts DM, Lavera V, Callahan J, Rossi C, Oberste MS, Roehrig JT, Cropp CB, Karabatsos N, Smith JF, Gubler DJ, Wooster MT, Nelson WM, Hayes CG, 1997. Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru. Am J Trop Med Hyg 56 :661–667.

    • Search Google Scholar
    • Export Citation
  • 20

    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
  • 21

    Calisher CH, Maness KS, 1975. Laboratory studies of Venezuelan equine encephalitis virus in equines, Texas, 1971. J Clin Microbiol 2 :198–205.

    • Search Google Scholar
    • Export Citation
  • 22

    Allen WP, 1962. Cross protection among group A arthropod-borne viruses. Bacteriol Proc 152.

  • 23

    Hearn HJ Jr, Rainey CT, 1963. Cross-protection in animals infected with group a arboviruses. J Immunol 90 :720–724.

  • 24

    Walton TE, Jochim MM, Barber TL, Thompson LH, 1989. Cross-protective immunity between equine encephalomyelitis viruses in equids. Am J Vet Res 50 :1442–1446.

    • Search Google Scholar
    • Export Citation
  • 25

    Byrne RJ, French GR, Yancey FS, Gochenour WS, Russell PK, Ramsburg HH, Brand OA, Scheider FG, Buescher EL, 1964. Clinical and immunologic interrelationship among Venezuelan, Eastern, and Western equine encephalomyelitis viruses in Burros. Am J Vet Res 25 :24–31.

    • Search Google Scholar
    • Export Citation
  • 26

    Shahan MS, Giltner LT, 1935. Equine encephalomyelitis studies: I. Cross-immunity tests between eastern and western types of virus. J Am Vet Med Assoc 86 :764–772.

    • Search Google Scholar
    • Export Citation
  • 27

    Beaty BJ, Calisher CH, Shope RE, 1989. Arboviruses. Schmidt NJ, Emmons RW, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. Sixth edition. Washington, DC: American Public Health Association, 797–855.

  • 28

    Jones JW, Turell MJ, Sardelis MR, Watts DM, Coleman RE, Fernandez R, Carbajal F, Pecor JE, Calampa C, Klein TA, 2004. Seasonal distribution, biology, and human attraction patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos, Peru. J Med Entomol 41 :349–360.

    • Search Google Scholar
    • Export Citation
  • 29

    Causey OR, Theiler M, 1958. Virus antibody survey on sera of residents of the Amazon Valley in Brazil. Am J Trop Med Hyg 7 :36–41.

  • 30

    Theiler M, Downs WG, 1973. The Arthropod-borne Viruses of Vertebrates. New Haven, CT: Yale University Press.

  • 31

    Scherer WF, Dickerman RW, Ordonez JV, 1979. Serologic surveys for the determination of antibodies against the Eastern, Western, California, and St. Louis encephalitis and dengue 3 arboviruses in Central America, 1961–1975. Bol Oficina Sanit Panam 87 :210–223.

    • Search Google Scholar
    • Export Citation
  • 32

    Aguilar PV, Paessler S, Carrara AS, Baron S, Poast J, Wang E, Moncayo AC, Anishchenko M, Watts D, Tesh RB, Weaver SC, 2005. Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus. J Virol 79 :11300–11310.

    • Search Google Scholar
    • Export Citation
  • 33

    Weaver SC, Frey TK, Huang HV, Kinney RM, Rice CM, Roehrig JT, Shope RE, Strauss EG, 2005. Togaviridae. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, eds. Virus Taxonomy, VIIIth Report of the ICTV. London: Elsevier/ Academic Press, 999–1008.

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