Introduction to Diagnostic Virology

Question 1

How are laboratories categorized? What are the 4 levels?

Answer 1

based on the hazard posed by the organisms being researched

• Biosafety Level 1 Lab: suitable for manipulation of a well-characterized strain of viruses, such as infectious canine hepatitis virus
• Biosafety Level 2 Lab: working with moderate-risk viruses found in the community and may be associated with some human infection, such as Cowpox, Camelpox, NDV, FMDV, Hepatitis B virus
• Biosafety Level 3 Lab: working with infectious agents that may cause severe or lethal diseases mainly transmitted by inhalation, such as encephalitis
• Biosafety Level 4 Lab: manipulation of highly contagious viruses, such as Ebola, Marburg, Lassa, and highly pathogenic Influenza viruses

Question 2

What are Containment Level 2 with Level 3 Operating Procedures Laboratories (BSL-2+)?

Answer 2

level 2 biosafety contaminants being researched and held within level 3 operating laboratories (HIV, Lentiviruses, TB, Coxsackievirus)

Question 3

What are 5 common General Laboratory Practices (GLPs)?

Answer 3

1. working personnel should wear special laboratory coats/uniforms/gowns
2. it’s contraindicated to eat, drink, smoke, or store food/drink in the lab
3. touching eyes, mouth, or nose in the lab is prohibited
4. wearing appropriate protective gloves is a must
5. hands should be washed with antiseptic, soap, and water before and after handling any biological materials

Question 4

Why aren’t mouth pipettes used?

Answer 4

avoid the incidental suction of any hazardous materials

Question 5

Where should virology work in the lab be done?

Answer 5

in a special laminar flow hood or biological biosafety cabinets

Question 6

Why is it recommended to use disposable materials for single use only?

Answer 6

minimize the possibility of contamination

Question 7

What should be done to each bench space before being worked at?

Answer 7

covered with sterile cover sheets and sterilized

Question 8

Where should gloves be worn? Shoe coverings?

Answer 8

within the lab - prohibited to wear both outside of the lab

Question 9

Where are sharps (needles, glass, etc.) thrown out? What should be done before they are picked up?

Answer 9

designated red/yellow sharps container

stored in a cold room

Question 10

What is the purpose of Biological Safety Cabinets (BSC)/Laminar flow hoods? What are the 3 classes?

Answer 10

provide protection to….
I - working personnel
II - the work itself
III - the environment

Question 11

What are the 3 major characteristics of Class III Biological Safety Cabinets?

Answer 11

1. presence of a solid tight barriers between the workers and the working materials
2. air is exhausted into the outdoors
3. uses glove boxes to manipulate the work

Question 12

What are 4 aspects of the ideal virology sample?

Answer 12

1. collected under complete aseptic conditions to avoid any bacterial/fungal contamination
2. freshly collected form the live animals or 2 hours at maximum after death
3. representative of each flock of animals
4. collected from right organs according to viral tissue tropism

Question 13

What types of samples can be taken for respiratory viruses? Enteric viruses? Vesicle-forming viruses? Skin lesions producing viruses? Nervous manifestations?

Answer 13

nasal and oropharyngeal swabs, fecal swabs, blood and serum

fecal swabs, feces, blood and serum in live animal, parts from organs in the digestive system

vesicular fluids, saliva, blood and serum

scabs from regions of the body

CSF, blood and serum, brain tissue

Question 14

What 4 precautions should be noted for sample collection?

Answer 14

1. age of animals during sample collection —> preference for virus affecting certain ages of animals
2. vaccination history of animals
3. season —> respiratory viruses are most common during the winter
4. region/location —> Rinderpest is endemic in Africa and Asia; Arboviruses are commonly found in areas with high mosquito populations

Question 15

What are the 5 different levels of sample preservation? How long can a virus be preserved in each case?

Answer 15

1. REFRIGERATOR: 24 hours
2. DEEP FREEZER (-20 to -40 degrees C): months
3. DEEP FREEZER (-80 degrees C): years
4. LIQUID NITROGEN: many years
5. LYOPHILIZATION (freeze drying): high pressure, low temperature

Question 16

What are the 3 steps taken to collect viral sample from body secretions and execrations?

Answer 16

(saliva, nasal swabs, oropharyngeal swabs)
1. clarified by centrifugation at 3000 RPM
2. supernants collected along with antimicrobial agents, like pensile/streptomycin and fungizone, to prevent contaminantion
3. samples stored at appropriate temperature until processed further

Question 17

What are the 4 steps taken to process serum samples for viruses?

Answer 17

1. blood is collected without adding any anticoagulants to obtain serum
2. tubes containing blood samples are kept at an oblique position and kept either at 4 degrees C overnight or incubated at 37 degrees C for 1 hr
3. tubes are centrifuged at 5000 RPM for 10 mins
4. serum samples are aspirated and transferred to a new, clean, and labeled tube

Question 18

What is the main way that blood samples are processed for virology work?

Answer 18

WHOLE BLOOD collected on anticoagulants, like EDTA or sodium citrates, then subjected to low speed centrifugation at 1500-2000 RPM for 10 mins

Question 19

What are the main 3 fractions of whole blood samples? Where can viruses typically be isolated from?

Answer 19

1. plasma
2. buffy coat - blood platelets and leukocytes (lymphocytes, monocytes)
3. RBCs

buffy coat

Question 20

What are the 5 main steps to processing tissue specimens for virology work?

Answer 20

1. parts from target organ are collected under aseptic conditions (1 g using a sterile scalpel or scissors)
2. tissue pieces are placed in a sterile mortar and a tissue suspension with PBS and sterile sand
3. obtained suspension is subjected to 3 cycles of freezing and thawing
4. samples undergo low-speed centrifugation at 3000 RPM for 15 mins
5. supernatant is collected and stored at appropriate temperature until use