Sampling for Virus Isolation in Animals

Study Notes

Specimens for virus isolation must meet specific criteria:
- Aseptic collection (minimal contamination)
- Fresh specimens (living or freshly dead within 2 hours)
- Contain the virus in anatomical sites of viral tropism
- Representative sample (minimum 5 samples)
General considerations during sampling:
- Clinical signs: Observe respiratory, intestinal, and nervous symptoms (e.g., suspect Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), Infectious laryngotracheitis virus (ILT), avian influenza (AI) in poultry; Bovine Rotavirus and Bovine Coronavirus in cattle).
- Age of herd/flock: Susceptibility varies by age (e.g., Marek's disease virus (MDV) affects all ages, Avian leucosis virus (ALV) affects poultry over 14 weeks).
- Morbidity and mortality rates: Calculate morbidity (infected animals/total susceptible) and mortality (dead animals/total susceptible). -Different viruses have different morbidity and mortality rates. -Newcastle Disease Virus (NDV) and Avian influenza (high morbidity and mortality) -Infectious bronchitis virus (IBV) and Infectious laryngotracheitis virus (ILT) (moderate to low morbidity and mortality)

Selecting animals:
- Live, infected, apparently healthy, or convalescing animals
- Dead/moribund animals (within 2 hours of death)
Selecting organs: Collect samples from the anatomical sites where the virus is most likely to be found (viral tropism).

Viral tropism is the ability of a virus to infect and replicate in a specific type of host cells or tissues.
Factors affecting viral tropism:
- Presence of specific cellular receptors
- Temperature (e.g., MDV replicates in feather follicles at low temperatures)
- pH (e.g., Rotavirus and Coronaviruses replicate in alkaline pH, enteroviruses replicate in acidic pH)
- Presence of certain metabolites (e.g., rabies virus replicates in tissues with catecholamines)

Specimens should be collected in sterile, labelled containers with complete history of the animal and suspected diagnosis.
Use appropriate transport media (e.g., phosphate-buffered saline (PBS), minimal essential medium (MEM), Hank's balanced salt solution (HBSS), glycerol in PBS, etc).
Transport specimens on ice or dry ice (-70°C) based on duration of transit.

Preservation temperatures depend on the virus. -Refrigerator (4°C) up to 24 hours. -Deep freezer (-20°C to -40°C) or lower, for weeks to months -Deep freezer (-70°C), for months to years -Liquid nitrogen (-196°C), for years -Lyophilization (freeze-drying).

Liquid: Sputum, urine, nasal discharge, vaginal discharge, semen, saliva, diarrhea, cerebrospinal fluid.
Solid: Liver, spleen, heart, intestine, brain, kidney, stool, trachea, joint capsule, tendon etc. -Viscous samples (e.g., viscous fluids) should be diluted with PBS.

Liquid samples: Clarify by centrifugation, collect supernatant, add antimicrobial agent (e.g., gentamycin) and incubate.
Blood samples: Anticoagulant (e.g., EDTA) required, centrifuge blood to separate the components.
Soft tissue samples: Cut sample into small pieces using forceps and scissor, add sterile sand, homogenize, dilute with PBS, apply freeze-thaw cycles, centrifuge, collect supernatant, add antimicrobial agent (gentamycin) and incubate.
Hard tissue samples: Homogenize, complete as soft tissue samples.

Freezing and thawing: Breaks cell membranes, releasing viral particles for isolation.
Antimicrobial agents: Inhibit bacterial or fungal contamination.
Degenerated samples: Cell/tissue damage may prevent virus isolation.
RP suspected samples: Glycerol-PBS not used, as glycerol/PBS prevents the inactivation of the virus and makes it possible to transport the sample for investigation.