Transcript EEE Agent Specific Training

Eastern Equine
Encephalitis (EEE) Agent
Specific Training
Viral Isolation- Sample inoculated into
culture tube (closed) under BSC, observed
for CPE (BSL2). If CPE indicative of EEE,
tube/ flask transferred to BSL3 for
containment and further investigation.
 Molecular testing- Mosquito pools
ground in BSC, extractions performed, and
real time amplification and detection done.
 IFA and ELISA- Performed on bench
using less than 10ul/sample.
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Procedures Performed at NCSLPH
"Diagnostic and research activities involving clinical material,
infectious cultures and infected animals or arthropods should
be performed under BSL-3 practices, containment
equipment, and facilities. Due to the high risk of aerosol
infection, additional personal protective equipment, including
respiratory protection, should be considered for non-immune
personnel. Animal work with VEE virus, EEE virus and WEE
virus should be performed under BSL-3 conditions. HEPA
filtration is required on the exhaust system of laboratory and
animal facilities using VEE virus."
http://www.selectagents.gov/Containment_for_Work_with_Eastern_Equine_Encephalitis_virus.html
BMBL 5th Edition
Work Performed
PPE
Containment
Level
HEPA
Exhaust
NCSLPH
Examples
Diagnostic without
propagation
Gloves, Laboratory coat
BSL-2
Not required
Molecular
Assays
Diagnostic work with
specimens (human or
animal) with propagation
Gloves, N-95/100 or PAPR, solid front
gown, eye protection, shoe covers
BSL-3
Research with
propagation. Includes
work where aerosol
generation potential
exists.
Gloves, N-95/100 or PAPR, solid front
gown or Tyvek, eye protection, shoe
covers
BSL-3
Infected animals
Gloves, N-95/100 or PAPR, solid front
gown, scrubs or Tyvek, eye protection,
shoe covers
ABSL-3
Gloves, N-95/100, solid front gown,
scrubs or Tyvek, shoe covers
ACL-3
Gloves, N95/100 or PAPR, scrubs or
tyvek, solid front gown, eye protection,
shoe covers
BSL-3
Infected arthropods
Large Volume propagation
(4)
Not required
(1)
Viral
Isolation
Not required Culturing to
(1)
obtain stock
Required
Not required
(1)
Not required
(1)
N/A
N/A
N/A
It is recommended that entities conducting diagnostic procedures which involve the potential
for isolation and subsequent identification of EEE virus, but do not involve the manipulation of
the virus, work under BSL-3 containment. However, if BSL-3 containment is not available this
work may be authorized at BSL-2 under the following conditions:
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Strict adherence to BSL-2 safety practices, safety equipment, and facility requirements, as
described in the BMBL, must be maintained. Personnel protective equipment should, at a
minimum, consist of gloves and laboratory coat.
Work with specimens suspected of containing EEE virus must be performed in a certified
biological safety cabinet (BSC).
Centrifugation of material containing EEE virus, or suspected of containing EEE virus, must
be done in a sealed rotor that can be removed and opened in a BSC.
For assays that do not require viral replication, protocols should be developed to achieve
inactivation of the virus as an initial step to reduce the risk of exposure.
The risk assessment associated with this work should address the need for eye and/or
respiratory protection.
http://www.selectagents.gov/Containment_for_Work_with_Eastern_Equine_Encephalitis_virus.html
Further BMBL Recommendations
Manipulation- isolation from a specimen
using a testing procedure that does not
require obtaining additional EEE.
Ex: RT-PCR
Propagation- isolation from a specimen by
subculturing, or culturing to obtain
additional stock
Ex: Culture and FA
Clarification of Terms
EEE is a virus
 Human vaccination for EEE is not
currently available.
 Veterinary vaccination is available

It’s a Virus Right?
Highest potential for aerosolation of EEE
 Must use centrifuge with sealed cup
containment
 Cups should only be opened in BSC
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Centrifugation Details
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Allow aerosol to settle
Cover spill with absorbent material
Apply 10% bleach to outside and work
inward
Allow time to inactivate virus (30 min)
Discard & clean area again
Notify RO &/ ARO, supervisor & safety
officer
Document (Form 3) & ensure CDC notified
EEE Spill Cleanup