Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Eastern Equine Encephalitis virus (Website 13):
      1. Common Name: EEE
      2. GenBank Taxonomy No.: 11021
      3. Description: EEE is a member of the Alphavirus genus, family Togaviridae. It is related to but antigenically distinct from a sympatric member of the western equine encephalomyelitis (WEE) virus complex, Highlands J (HJ) virus. Epidemiologically, EEE has many similarities to WEE in that both viruses cause encephalitis in horses and man, have wild avian hosts, and are transmitted from birds to mammals by mosquitoes. EEE and HJ virus are most closely related epidemiologically in that they share geographic distributions, are transmitted by the mosquito Cs. melanura, and infect a wide spectrum of wild avians, especially passerines(Morris, 1988). Venezuelan equine encephalitis, eastern equine encephalitis, and western equine encephalitis are all members of the Alphavirus genus of the family Togaviridae. As with all the alphavirus group, VEE, EEE, and WEE are transmitted in nature by mosquitoes and are maintained in cycles with various vertebrate hosts. Thus, the natural epidemiology of these viruses is controlled by environmental factors that affect the relevant mosquito and reservoir host populations and their interactions. Of the 28 viruses currently classified within this group, VEE, EEE, and WEE are the only viruses regularly associated with encephalitis. Although these encephalitic strains are restricted to the Americas, as a group, alphaviruses have worldwide distribution and include other epidemic human pathogens such as chikungunya virus (Asia and Africa), Mayaro virus (South America), O'nyong-nyong virus (Africa), Ross River virus (Australia), and Sindbis virus (Africa, Europe, and Asia). These viruses cause an acute febrile syndrome often associated with debilitating polyarthritic syndromes(Website 15). Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range(Brault et al., 1999). The North American subtype of this virus includes all isolates from all hosts from the area between Massachusetts and the Caribbean (Morris, 1988)(Morris, 1988). Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America (Brault et al., 1999)(Brault et al., 1999). The South American subtype of this virus includes all isolates from all hosts in the South American Continent(Morris, 1988).

1. EEE Virus Information
   1. Stage Information:
      1. Virion:
         • Size: The alphavirus virion is approximately 60 to 65 nm in diameter.
         • Shape: The alphavirus virion, a spherical particle approximately 60 to 65 nm in diameter is typically composed of three different structural proteins enclosing a single molecule of single-stranded RNA.
         • Picture(s):
           • Surface of an Alphavirus (Website 21)
             
             
             
             Description: This image is a computer-generated model of the surface of an alphavirus derived by cryoelectron microscopy. The spike-like structures on the virion surface are trimers composed of heterodimers of the virion surface glycoproteins E1 and E2. These spikes are used by the virus to attach to susceptible animal cells. Copyright: CDC.

1. Genome of Eastern Equine Encephalitis virus
   1. Description: Eastern equine encephalitis (EEE) is a single-stranded RNA positive strand virus and a member of the Alphavirus genus and the family Togaviridae. It has no DNA stage(Website 15).
   2. Chromosome(Website 14)
      1. GenBank Accession Number: NC_003899
      2. Size: 11675 bp(Website 14).
      3. Gene Count: 7 genes(Website 14).
      4. Description: The viral genome is a positive-stranded RNA of approximately 11,700 nucleotides and has the structural features of messenger RNA (ie, mRNA, a 5 ' methylated cap [m7GpppA] and a poly-A tract at the 3 ' end). As a complete and functional mRNA, genomic RNA purified from virions is fully infectious when artificially introduced (ie, transfected) into susceptible cells. Similarly, RNA transcribed from a full-length complementary DNA (cDNA) clone of an alphavirus is also infectious, and it is this property that allows genetic manipulation of these viruses. Mutations introduced into a cDNA clone by site-directed mutagenesis will be reflected in the RNA transcribed from the altered clone and in the virus obtained from transfected cells. These procedures are being utilized to develop improved vaccines, but conceivably could be used also to enhance specific characteristics required for weaponization(Website 15). The 5' two-thirds of the genome encodes four nonstructural proteins (nsP1 to 4) that are involved in viral replication. After virus entry into the cytoplasm of cells, a nonstructural polyprotein is translated and utilized in the production of full-length negative-sense RNA. The negative-sense RNA is used for the generation of genomic RNA as well as a subgenomic mRNA (26S) that is homologous to the 3' one-third of the genome. The subgenomic RNA is translated directly into a structural polyprotein that is proteolytically cleaved into the capsid, E2, and E1 envelope glycoproteins(Brault et al., 2002).

1. Biosafety information for Eastern Equine Encephalitis virus
   1. Level: Level 2 for diagnostic, level 3 for propagation and animal work.
   2. Precautions: Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment(Website 18). Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features. It is recognized, however, that some existing facilities may not have all the facility features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations). In this circumstance, an acceptable level of safety for the conduct of routine procedures, (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, susceptibility testing, etc.), may be achieved in a Biosafety Level 2 facility, providing 1) the exhaust air from the laboratory room is discharged to the outdoors, 2) the ventilation to the laboratory is balanced to provide directional airflow into the room, 3) access to the laboratory is restricted when work is in progress, and 4) the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director(Website 18).

1. EEE Culture Information :
   1. Description: Previously, the recovery of EEE was limited because only a few facilities had the resources to amplify the virus. Recent studies indicate excellent growth of the virus recovered from patient CSF in A549 and MRC-5 cell cultures, which are mediums that virology labs routinely use to recover adenovirus, herpes simplex virus (HSV), and enterovirus(Website 16). Virus can be isolated from CSF, blood, or CNS tissue by inoculation into newborn mice or onto a variety of tissue-culture cells, most commonly CSF or Vero cells(Griffin, 2001).

1. Outbreak Locations:
   1. Outbreaks of EEE virus have occurred in most eastern states and in southeastern Canada but have been concentrated along the eastern and Gulf coasts(Website 15).
2. Transmission Information:
   1. From: Birds(Website 15, Website 16). , To: Mosquitoes(Website 15, Website 16). , With Destination:Mosquitoes(Website 15). (Website 15, Website 16)
      Mechanism: The ability of alphaviruses to infect mosquitoes efficiently with spread to and replication in the salivary glands is essential for maintaining the natural cycle of transmission. Not all mosquitoes taking a blood meal from a viremic host will become infected, and not all infected mosquitoes develop salivary gland infection and the ability to transmit the virus(Griffin, 2001). The initial isolation of EEE virus from a bird and from Culiseta melanura mosquitoes, the two major components of the EEE natural cycle, were both reported in 1951(Website 15).
   2. From: Mosquito(Griffin, 2001). , To: Birds(Griffin, 2001). , With Destination:Bird(Griffin, 2001). (Website 15, Griffin, 2001)
      Mechanism: The primary mode of alphavirus transmission to vertebrates is through the bite of an infected mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly. Saliva virus titers are highest early after the mosquito is infected, but decline, along with transmission rates, after 1 to 2 weeks, but mosquitoes remain infected for life(Griffin, 2001). The mosquito injects the agent of EEE into the subcutaneous and cutaneous tissues of the host(Website 16).
   3. From: Mosquitoes(Griffin, 2001). , To: Human(Griffin, 2001). , With Destination:Human(Griffin, 2001). (Griffin, 2001, Website 16)
      Mechanism: The primary mode of alphavirus transmission to vertebrates is through the bite of an infected mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly. Saliva virus titers are highest early after the mosquito is infected, but decline, along with transmission rates, after 1 to 2 weeks, but mosquitoes remain infected for life(Griffin, 2001). The mosquito injects the agent of EEE into the subcutaneous and cutaneous tissues of the host(Website 16).
   4. From: Human(Griffin, 2001). , To: Mosquitoes(Griffin, 2001). , With Destination:Mosquitoes(Griffin, 2001). (Website 15, Website 16)
      Mechanism: The primary mode of alphavirus transmission to vertebrates is through the bite of an infected mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly. Saliva virus titers are highest early after the mosquito is infected, but decline, along with transmission rates, after 1 to 2 weeks, but mosquitoes remain infected for life(Griffin, 2001). The mosquito injects the agent of EEE into the subcutaneous and cutaneous tissues of the host(Website 16).
   5. From: Human(Website 16). , To: Human(Website 16). , With Destination:Human(Website 16). (Website 16)
      Mechanism: EEE is not transmitted by the aerosol route. It may cross the placenta and infect the fetus. Because of low viral titers in the donor's blood, EEE is unlikely to be transmitted by transfusion(Website 16).
   6. From: Mosquitoes(Griffin, 2001). , To: Horse(Griffin, 2001). , With Destination:Horse(Griffin, 2001). (Griffin, 2001, Website 16)
      Mechanism: The primary mode of alphavirus transmission to vertebrates is through the bite of an infected mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly. Saliva virus titers are highest early after the mosquito is infected, but decline, along with transmission rates, after 1 to 2 weeks, but mosquitoes remain infected for life (Griffin, 2001)(Griffin, 2001). The mosquito injects the agent of EEE into the subcutaneous and cutaneous tissues of the host(Website 16).
   7. From: Horse(Griffin, 2001). , To: Mosquitoes(Griffin, 2001). , With Destination:Mosquitoes(Griffin, 2001). (Website 15, Website 16)
      Mechanism: The primary mode of alphavirus transmission to vertebrates is through the bite of an infected mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly. Saliva virus titers are highest early after the mosquito is infected, but decline, along with transmission rates, after 1 to 2 weeks, but mosquitoes remain infected for life(Griffin, 2001). The mosquito injects the agent of EEE into the subcutaneous and cutaneous tissues of the host(Website 16).
3. Environmental Reservoir:
   1. Birds(Griffin, 2001):
      1. Description: Birds are the primary reservoir hosts, and many species are susceptible to infection, but often remain asymptomatic despite prolong viremia. The amplifying species for EEEV in North America are wading birds, migratory passerine birds, and starlings. Young birds in general are probably important for virus amplification because they are more susceptible to infection, have a prolonged viremia, and are less defensive towards mosquitoes(Griffin, 2001).
   2. Bats(Griffin, 2001):
      1. Description: In Central and South America, forest-dwelling rodents, bats, and marsuipials are frequently infected and may provide an additional reservoir, but these transmission cycles are not well characterized(Griffin, 2001).
   3. Rodents(Griffin, 2001):
      1. Description: In Central and South America, forest-dwelling rodents, bats, and marsuipials are frequently infected and may provide an additional reservoir, but these transmission cycles are not well characterized(Griffin, 2001).
   4. Marsuipials(Griffin, 2001):
      1. Description: In Central and South America, forest-dwelling rodents, bats, and marsuipials are frequently infected and may provide an additional reservoir, but these transmission cycles are not well characterized(Griffin, 2001).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: Although other encephalitic viruses could be considered as potential weapons (eg, the tick-borne encephalitis viruses), few possess as many of the required characteristics for strategic or tactical weapons development as the alphaviruses: These viruses can be produced in large amounts in inexpensive and unsophisticated systems; They are relatively stable and highly infectious for humans as aerosols; Strains are available that produce either incapacitating or lethal infections; and the existence of multiple serotypes of VEE and EEE viruses, as well as the inherent difficulties of inducing efficient mucosal immunity, confound defensive vaccine development(Website 15). The equine encephalomyelitis viruses remain as highly credible threats today, and intentional release as a small-particle aerosol, from a single airplane, could be expected to infect a high percentage of individuals within an area of at least 10,000 km2. As a further complication, these viruses are readily amenable to genetic manipulation by modern recombinant deoxyribonucleic acid (DNA) technology. This capability is being used to develop safer and more effective vaccines, but in theory, could also be used to increase the weaponization potential of these viruses(Website 15).
      2. Emergency Contact: At the national level, the division of Vector-borne Infectious Diseases (DVBID), Centers for Disease Control and Prevention (CDC) collects information from the states on arboviral encephalitis. Although state and federal laws do not require physicians or hospitals to report human cases, there has been good cooperation between local, state, and federal agencies in reporting cases of arboviral encephalities. Standardized report forms and electronic reporting systems are used by state epidemiologists to report cases of arboviral encephalitis. Although routine reporting of human cases of encephalitis was discontinued in 1983, many states still report cases and other relevant data on an informal basis(Website 20).
      3. Containment: Vaccination of horses is not a useful public health tool for EEE, WEE, or enzootic VEE, however, since horses are not important as amplifying hosts for these diseases. Investigational formalin-inactivated vaccines for humans are available for WEE and EEE, but they require multiple injections and are poorly immunogenic. Insecticide measures of vector control may also have an impact on ameliorating epidemic transmission(Website 15).

1. Organism Detection Test:
   1. Electron Microscopy :
      1. Description: Enveloped Toga virus particles were demonstrated by means of an electron microscopy in the brain tissues of a 3-year-old girl with acute encephalitis. Areas of demyelinization and necrosis throughout the white matter and brainstem were revealed by light microscopy. These viral particles were identified as eastern equine encephalomyelitis virus in postmortem isolation of the virus utilizing young mice and complement-fixation studies. To the authors' knowledge, this is the first demonstration of eastern equine encephalomyelitis virus particles in human tissues by electron microscopy(Bastian et al., 1975).
   2. Indirect Fluorescent Antibody :
      1. Description: Tissues were tested for virus by intracerebral inoculation of suckling mice and by examination of frozen sections and impression smears by the indirect fluorescent antibody (FA) technique. The FA technique appears useful for the rapid diagnosis of fatal eastern equine encephalomyelitis and may be applicable in laboratories not equipped for isolation of viruses(Monath et al., 1981).
   3. Inoculation of neonatal mice :
      1. Description: Isolation of alphaviruses from vertebrate sera during acute disease or from postmortem brain samples, as well as isolation from invertebrate hosts, has been accomplished most often by intracerebral inoculation of neonatal mice, an animal model that is extremely susceptible to most, if not all, alphaviruses. Because alphaviruses produce an extensive cytopathic effect in almost all common vertebrate cell cultures examined, this also is an effective means of isolation(Johnston and Peters, 1996).
2. Immunoassay Test:
   1. Hemagglutination :
      1. Description: Biochemical assays are valuable for EEE diagnosis. With early suspicion, obtain sera at 2- to 3-day intervals. A potential drawback is the inability to rapidly receive these test results(Website 16). Hemagglutination inhibition, although cross-reactive throughout the alphavirus genus, nonetheless retains sufficient specificity to define six antigen complexes or serotypes, which include the antigenic complexes of Western equine encephalitis, Venezuelan equine enchephalitis, Eastern equine encephalitis, Semilik forest, Middleburg, Nduma, and Barmah Forest(Johnston and Peters, 1996). Identification of a specific virus or subtype usually requires neutralization tests or modified hemagglutination tests. The kinetic hemagglutination inhibition test, for example, is extremely useful in differentiating subtypes of Venezuelan equine encephalitis virus as well as geographic varieties of eastern equine encephalitis and Ross river virus(Johnston and Peters, 1996).
   2. Complement Fixation :
      1. Description: Biochemical assays are valuable for EEE diagnosis. With early suspicion, obtain sera at 2- to 3-day intervals. A potential drawback is the inability to rapidly receive these test results. Complement fixation titer of at least 1:128 occurs in convalescing patients(Website 16).
   3. IgM capture ELISA :
      1. Description: IgM capture ELISA is usually sufficiently specific and gives a rapid inexpensive assessment of recent infection using a single early convalescent serum sample(Johnston and Peters, 1996).
   4. ELISA to identify EEE in Mosquitoes :
      1. Description: Surveillance of mosquito populations for virus activity is not often performed by small, vector-control districts because they do not have the financial resources to use virus isolation, or newer methods such as the polymerase chain reaction. Consequently, development and refinements of rapid, sensitive, and simple enzyme-linked immunosorbent assays (ELISAs) applicable to a wide variety of public health settings are justified. We have developed an antigen-capture ELISA for the detection of eastern equine encephalitis (EEE) virus in mosquitoes that uses both monoclonal capture and detector antibodies. The sensitivity of this assay is 4.0-5.0 log10 plaque-forming units/ml, which is comparable to previously published EEE antigen-capture assays developed with polyclonal antibody reagents. This test identifies only North American strains of EEE virus and does not react with either western equine encephalitis or Highlands J viruses. Test sensitivity was enhanced by sonicating mosquito pools, treating them with Triton X-100, and increasing the time and temperature of antigen incubation. The conversion of this ELISA to a monoclonal antibody-based format should result in a readily standardizable and transferable assay that will permit laboratories lacking virus isolation facilities to conduct EEE virus surveillance(Brown et al., 2001).
   5. ELISA Information :
      1. Description: Biochemical assays are valuable for EEE diagnosis. With early suspicion, obtain sera at 2- to 3-day intervals. A potential drawback is the inability to rapidly receive these test results (Website 16). Enzyme-linked immunosorbent assay (ELISA) detects IgM, primarily during convalescent stages or prolonged courses. ELISA may detect antiarboviral immunoglobulin G (IgG), which has similar results to the neutralization assay, used primarily as an adjunct to the IgM ELISA.IgM antibodies can be detected 0-3 days after onset of clinical symptoms and usually decline to undetectable levels within four months of onset. Measurement of IgM in CSF is test of choice when CNS disease is suspected. It has very good sensitivity(Website 16).
   6. Neutralization test :
      1. Description: Identification of a specific virus or subtype usually requires neutralization tests or modified hemagglutination tests. The neutralization test has continued to provide the highest specificity for the infecting virus species(Johnston and Peters, 1996).
   7. Enzyme immunoassay :
      1. Description: Antibody to EEE is usually measured by enzyme immunoassay (EIA) with detection of IgM in serum and CSF particularly useful(Griffin, 2001).
   8. mAB-based Epitope Blocking Assay :
      1. Time to Perform: unknown
      2. Description: An epitope blocking assay using an EEEV glycoprotein E1-expressing recombinant Sindbis virus and virus-specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut-off value of 30% inhibition for antigenic complex-specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus-, EEEV- and WEEV-complex-specific serum antibodies. As this test is based on the inhibition of binding of virus-specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation(Passler and Pfeffer, 2003).
3. Nucleic Acid Detection Test: