Variola Virus

Characteristics
Morphology	A member of the family Poxviridae, subfamily Chordopoxvirinae, genus Orthopoxvirus. Virions are shaped like bricks on electron micrographs and measure approximately 300 x 250 x 200 nm. Orthopoxviruses have an outside envelope and a second membrane underneath. Instead of a capsid, poxviruses have a nucleosome which contains DNA, and is surrounded by its own membrane. They contain single, linear, double-stranded DNA molecules of 130 to 375 kb pairs and replicate in the cell cytoplasm. Variola virus is the most complex of the orthopoxvirus genus, having many different strains.
Growth Conditions	Vero monkey kidney cells and a human fibroblast cell line (MRC5).

Characteristics

Morphology

A member of the family Poxviridae, subfamily Chordopoxvirinae, genus Orthopoxvirus. Virions are shaped like bricks on electron micrographs and measure approximately 300 x 250 x 200 nm. Orthopoxviruses have an outside envelope and a second membrane underneath. Instead of a capsid, poxviruses have a nucleosome which contains DNA, and is surrounded by its own membrane. They contain single, linear, double-stranded DNA molecules of 130 to 375 kb pairs and replicate in the cell cytoplasm. Variola virus is the most complex of the orthopoxvirus genus, having many different strains.

Growth
Conditions

Vero monkey kidney cells and a human fibroblast cell line (MRC5).

Health Hazards
Host Range	Humans and monkeys
Modes of Transmission	Transmission occurs via respiratory droplets (primary route of transmission), or via fine-particle aerosol, or skin inoculation. The conjunctiva or placenta may be occasional portals of entry.
Signs and Symptoms	Initial Symptoms (Prodrome): ~2 to 4 days. Sometimes contagious: fever, malaise, head and body aches, and sometimes vomiting. Early Rash: ~ 4 days, first as small spots on the tongue and in the mouth. Most contagious Pustular Rash: ~ 5 days. Contagious Pustules and Scabs: ~ 5 days. Contagious Resolving: ~6 days, scabs. Contagious Resolved: Scabs have fallen off. Person is no longer contagious.
Infectious Dose	Viruses in an aerosol suspension can spread widely, and infect at a very low dose (10 to 100 organisms)
Incubation Period	Can range from 7-19 days. Typically, onset of illness occurs after 10-14 days and 2-4 more days for onset of rash to occur

Medical Precautions/Treatment
Prophylaxis	None publically available
Vaccines	The vaccine consists of a live Vaccinia virus, which is a "pox"-type virus related to smallpox.
Treatment	Treatment with antiviral therapy is often prescribed. If possible, a suspected case of smallpox should be managed in a negative-pressured room, and the patient should be vaccinated, particularly in an early stage of illness. Strict respiratory and contact isolation is imperative.
Surveillance	Monitor for symptoms. Confirm using PCR, electron microscopy and histology.
GWU Requirements	Report all incidents to the Office of Risk Management as well as the Office of Research Safety (ORS) IMMEDIATELY FOLLOWING THE OCCURRENCE.

Containment
BSL-2/BSL-2+	Risk Group 4 classification is applied to Variola species. BSL-2 and ABSL-2 containment plus vaccination are recommended for work with vaccinia and other human pathogenic poxviruses. BSL-2 facilities with BSL-3 practices are advised if vaccinated personnel perform laboratory work with monkeypox virus.
BSL-3/ABSL-3	BSL-3 containment practices with vaccination are recommended for direct monkeypox virus work. ABSL-3 practices, containment equipment, and facilities are recommended for monkeypox work in experimentally or naturally infected animals.
BSL-4/ABSL-4	Research with active Variola virus is restricted to two WHO-approved BSL-4 and ABSL-4 facilities. ACTIVE VARIOLA VIRUS RESEARCH IS NOT CONDUCTED AT GEORGE WASHINGTON UNIVERSITY

Spill Procedures
Small Spills (<1 liter)	If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup. Variola exposure via aerosol transmission is common, caution should still be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against enveloped viruses (bleach, glutaraldehyde, phenolics, and Micro-Chem), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.
Large Spills	Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Spill Procedures

Small Spills (<1 liter)

If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup.

Variola exposure via aerosol transmission is common, caution should still be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against enveloped viruses (bleach, glutaraldehyde, phenolics, and Micro-Chem), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.

Large Spills

Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Exposure Procedures
Mucus Membrane	Flush eyes, mouth or nose for 15 minutes at eyewash station.
Other Exposure	Wash area with soap and water for 15 minutes.
Reporting	Report ALL injuries to the PI immediately and reported to the Office of Risk Management at [email protected] IMMEDIATELY FOLLOWING THE OCCURRENCE. Exposures that involve a bloodborne-pathogen or recombinant DNA also need to be reported to the Office of Research Safety at [email protected]. If the injury requires immediate medical attention, call GWPD at 202-994-6111 or call 911.
Medical Monitoring	Seek immediate medical evaluation, treatment, and post exposure follow-up at the Employee Health Office at GWU Hospital (900 23rd St., NW, Suite G-1090, Phone: 202-715-4275). Students should go to the Students Health Office at Marvin Center. After hours treatment can be received at the GWU hospital emergency room.

Stability
Disinfection	70% Ethanol, sodium hypochlorite (1-10% dilution of fresh bleach), 5% Micro-Chem, and quaternary ammonia compounds.
Inactivation	Inactivated by heat: autoclave cultures for 30 minutes at 121°C, 15 psi or by incineration.
Survival Outside Host	Specimens of blood, scrapings from skin lesions, saliva, pustular fluid, and crusts can be transported and stored for short periods without refrigeration. Materials from smallpox patients (dried fluid and crusts) containing virus remain infectious at room temperature for approximately 1 year. Virus has been isolated from scabs that had been sitting on a shelf for 13 years.

Personal Protective Equipment (PPE)
Minimum PPE Requirements	At minimum, personnel entering the laboratory must remove street clothing, including undergarments, and jewelry, and change into dedicated laboratory clothing and shoes, or don full coverage protective clothing. Additional PPE may be required depending on lab specific SOPs. All activities with infectious material should be conducted in a biological safety cabinet (BSC) in combination with a positive pressure suit, or within a class III BSC line.
Additional Precautions	Additional protection may be worn over laboratory clothing when infectious materials are directly handled, such as solid-front gowns with tight fitting wrists, gloves, and respiratory protection. Eye protection must be used where there is a known or potential risk of exposure to splashes

Personal Protective Equipment (PPE)

Minimum PPE Requirements

At minimum, personnel entering the laboratory must remove street clothing, including undergarments, and jewelry, and change into dedicated laboratory clothing and shoes, or don full coverage protective clothing. Additional PPE may be required depending on lab specific SOPs. All activities with infectious material should be conducted in a biological safety cabinet (BSC) in combination with a positive pressure suit, or within a class III BSC line.

Additional Precautions

Additional protection may be worn over laboratory clothing when infectious materials are directly handled, such as solid-front gowns with tight fitting wrists, gloves, and respiratory protection. Eye protection must be used where there is a known or potential risk of exposure to splashes

References
CDC (https://www.cdc.gov/smallpox/index.html) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) Canadian PHAC (https://www.canada.ca/en/public-health/services/laboratory-biosafety-bi…)