Eastern Equine Encephalitis (EEE) Agent Specific Training - PowerPoint Presentation

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Eastern Equine Encephalitis (EEE) Agent Specific Training

Procedures Performed at NCSLPH

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-

P

erformed on bench using less than 10ul/sample.

BMBL 5

th Edition

"

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

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

Not required

(1)

Viral Isolation

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

Not required

(1)

Culturing to obtain stock

Infected animals

Gloves, N-95/100 or PAPR, solid front gown, scrubs or Tyvek, eye protection, shoe covers

ABSL-3

Required

N/A

Infected arthropods

Gloves, N-95/100, solid front gown, scrubs or Tyvek, shoe covers

ACL-3

Not required

(1)

N/A

Large Volume propagation

(4)

Gloves, N95/100 or PAPR, scrubs or tyvek, solid front gown, eye protection, shoe covers

BSL-3

Not required

(1)

N/A

Further BMBL Recommendations

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:

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

Clarification of Terms

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

It’s a Virus Right?

EEE is a virus

Human vaccination

for EEE is not currently available.

Veterinary vaccination is available

Centrifugation Details

Highest potential for

aerosolation

of EEE

Must use centrifuge with sealed cup containment

Cups should only be opened in BSC

EEE Spill Cleanup

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