Virology Past Paper Notes PDF

## Sec 4 Virology ### Subject: Long/Permanent Preservation: - **A) Freezing** - **Def:** Dehydration of frozen Sample at ≤ -30°C under a vacuum to convert liquid to powder and can be preserved at any temperature for a long time. - **Done by:** Lyophilizer - **B) Lyophilization (Dry-freezi...

## Sec 4 Virology ### Subject: Long/Permanent Preservation: - **A) Freezing** - **Def:** Dehydration of frozen Sample at ≤ -30°C under a vacuum to convert liquid to powder and can be preserved at any temperature for a long time. - **Done by:** Lyophilizer - **B) Lyophilization (Dry-freezing)** - We’ll talk about it later. ### Aim: (Applications - uses) - Vaccine, Serum, Antibody, Bacterial culture. ### Preparation of Sample: - Last step in sampling - Goal: Concentrate and purify the sample from residue and collect it in a small tube called an eppendorf. ### Aim of Preparing the Sample: - **1) Removal of Particulates matter that Block needle (by centrifugation)** - **2) Prepare concentrated Homogeneous suspension of the virus to be easily inoculated** ### Samples | **Sample Type** | **Liquid** | **Semi-solid** | **Solid** | |---|---|---|---| | **Examples** | Swab | Feces, sputum | Tissue (liver, spleen, kidney, skin, gizzard, proventriculus) | ### Preparation of a Swab: - **1) Vigorous shaking (manually or using a vortex machine)** - **2) Squeeze** - **3) Centrifugation (2000 - 3000 RPM) for 15 minutes at 4°C "Cool centrifuge"** - **4) Take the supernatant (that contains the virus) and put it in an eppendorf with antibiotics to avoid contamination** - The last two steps (3, 4) need to be repeated for all types of samples. ### Preparation of a Liquid: (Blood, milk, CSF, discharge, serum, urine, saliva) - **Clear** - Use the sample as it is. - It is best to centrifuge it. - **Not clear** - **Except for blood and urine:** Centrifuge. - **Blood:** - Collect with anticoagulant to isolate serum. - Collect without anticoagulant to get a serum collection. - **N.B:** Do not centrifuge blood for more than 1000-1500 RPM to avoid hemolysis. - **Urine:** (The problem is the pH and the salt content) - **1) Dilution of Salt/Toxins:** - Dilute by adding an equal amount of PBS or UTM. - For example: 2mL of urine + 2mL VTM. - **2) Adjustment of Acidic pH:** - Add NaOH (alkaline) to reach a neutral pH and measure using a phenol red indicator. - Use a phenol red indicator. - **N.B:** The urine of all animals is acidic except… - **3) Equine** - **N.B:** The urine of all animals is acidic, except equine and … - - Add HCl to make the urine neutral - **4) Centrifugation (2000-3000 RPM) for 15 min at 4°C** - **5) Add antibiotics** ### Preparation of a Semisolid: - **Feces:** - (1 gm of feces + 9 mL of VTM) <Dilution (equal amount of VTM)> - (10% Suspension) - Add 5 gm of coarse glass beads to disperse the feces - Vigorous shaking - Centrifugation (10,000 RPM) for 30 minutes at 4°C. - This is a high speed centrifugation because the sediment is very dense. - Transfer the supernatant into an eppendorf. - Add double the amount of antibiotics - 2,000 IU penicillin - 2,000 mg streptomycin - **Sputum:** - (1 gm of feces + 9 mL of VTM) <Dilution (equal amount of VTM)> - (10% Suspension) - Add 5 gm of coarse glass beads to disperse the feces - Vigorous shaking - Centrifugation (2,000 - 3,000 RPM) for 15 minutes at 4°C. - This is the standard speed. - Transfer the supernatant into an eppendorf. - Add antibiotics - 1,000 IU penicillin - 1,000 mg streptomycin ### N.B:** the parameters of centrifugation for all samples are 2,000 - 3,000 RPM for 15 minutes at 4° C, except: - Blood: 1,000 - 1,500 RPM for 15 minutes at 4°C. (To avoid hemolysis) - Feces: 10,000 RPM for 30 minutes at 4°C (High amount of debris) ### N.B:** The amount of antibiotic in all samples: - 1,000 IU pencillin + 1,000 µg streptomycin - **Except for feces (double amount)**: - 2,000 IU + 2,000µg ### Preparation of a Solid tissue: - **Soft** - **Examples:** kidney, liver, spleen - **Method:** Manual mincing/grinding using a mortar and pestle. - **1) Cutting & Cross-cutting by scalpel, scissors** - **2) Prepare (10%) solution (1 gm of tissue + 9 mL of VTM)** - **3) Use a mortar and pestle for grinding.** - Grind in an anticlockwise direction. - Add sterile sand to increase the grinding effectiveness. - **4) Centrifugation** - **5) Take the supernatant at 4°C** - **6) Add antibiotics** - **Hard** - **Examples:** skin, gizzard, proventriculus - **Method:** Automatic mincing by homogenizer. - **1) Put tissue in a homogenizer cup that’s surrounded by a cup of ice to lower the temperature during homogenization.** - **2) Prepare (10%) suspension** - **3) Centrifuge (2,000 - 3,000 RPM) for 15 minutes at 4°C** - **4) Take the supernatant in an eppendorf.** - **5) Add antibiotics** **Now the sample is ready! Now it's time to pick the technique! ** (Isolation/Serology/PCR/EM) ## Isolation of Virus “Cultivation" ### *Aim: - **In Vivo:** - **1) Cultivation and identification** - **2) Propagation of the virus for vaccine production** - Number (Quantity) - **3) Scientific Research** - **4) Titration by Infectivity Assays** - *Infective Dose (50) ID50*: The dose of infection that infects 50% of the tested organisms. - **MID50 (Mouse Infective Dose 50)**: The dose that infects 50% of the mice. - **EID50 (Egg Infective Dose 50)**: The dose that infects 50% of the eggs. - **TCID50 (Tissue Culture Infective Dose 50)**: The dose that infects 50% of the cells. - **In Vivo:** - **ECE** - **In Vitro:** - **Tissue Culture** ### Laboratory Animals: - **Ferret, Rat, mouse, Hamster, Rabbit, Guinea Pig, Birds, Monkey** ### Aim of Laboratory Animals: - **1) Cultivation of virus** - **2) Identification (Serology test)** - **3) Propagation for vaccine production** - **Example:** Mouse-Adapted Vaccine of African Horse Sickness (AHS) - **AHS** from horse viscera is viscerotropic. - Inject 70:80 of the virus into the rat by the brain route. This route can be modified with the brain route change. - It has been changed to the brain route because the virus will not have the same characteristics, requiring adaptation. - **4) Titration (MID50) Mouse-Adapted Vaccine of AHS** - **5) Study Pathogenesis** - Pathgenicity - Viremia - Study the virulence and/or pathogeneses of the virus. ### Source of RBCs (Used for serology): - **Complement** - **HI** - **CFT** ### HIS (Hyper Immune Serum): - **Def:** Serum with a high titer of antibody against a specific viral antigen. - Part of the antigen - The body learns to manufacture its own antibody - Before infection - Prophylactic - passive immunity - **How:** - **Vaccine:** Active immunity - **Serum:** Passive immunity (T therapeutic) - **Example:** - Inoculate 0.1mL intradermally (I/D) and wait a week. - Inoculate 0.3mL subcutaneously (S/C) and wait a week. - Inoculate 0.5mL intramuscularly (I/M) and wait a week. - Inoculate 1mL intravenously (I/V). - Collect the blood and serum after waiting another week. - **1) Virus Antigen** - **2) With different routes other than the natural route.** - If the virus multiplies in the respiratory system, it is more likely to infect the blood via the subcutaneous or intradermal route. - **3) Different Low Dose.** ### Uses: - **1) Therapeutic** - **Antitetanic serum (Treatment)**: Neutralizes the toxins, but the body is not protected. - **2) Diagnostic** - Serology: Detect the presence of antibodies against a specific antigen. ### Monoclonal antibodies: - They are produced by a single clone of B cells. - Polyclonal antibodies - They are produced by a mixture of B cells. ### Cell Culture (BHK21): - Baby Hamster Kidney 21 ### Studying an Immune Response: - **Differential Diagnosis (DD)** - **VSV:** - Bovine - Horses - **FMD:** - Bovine ### Advantages of laboratory animals compared to large animals: - **1) Small Ration/Size** - **2) Easily Handled** - **3) Easily Labeled** - **4) Can write the aims here again.** - **5) Show Clinical Signs that are similar to the natural host. ** - If a virus causes respiratory symptoms in a large animal, it will also cause similar respiratory symptoms in the laboratory animal. - This characteristic is very important as it doesn’t happen in eggs or cells, which do not have the same susceptibility to respiratory viruses as larger animals. ### Disadvantages of laboratory animals compared to ECE and Tissue Culture: - **① Large space** - **② Difficult Handling** - **③ Expensive** - **④ Difficult Labeling** - **⑤Septic condition cannot be applied** - This is true for urine & defecation: it will be affected by the presence of antibiotics so the results are not reliable because you get a false negative. - **⑥ Neutralizing Ab produces false-negative results** - **⑦ Latent infections:** Presence of disease without any signs. - *For example:* Herpes infection is latent and hidden in the central nervous system. This is true for diseases in which the immune system is weak. - **Problems with latent periods:** - **1) Zoonotic diseases:** Shared/transmitted between species. - **2) Interference Effects:** This is difficult to avoid, as the virus in a cell won’t show any signs. - Sometimes, the virus that is tested for does not produce any symptoms but can block the entry of other viruses. - **This leads to false negatives, so the results are not reliable.** - **3) Appearance of signs of latent infection due to stress.** - This can happen because stress weakens the immune system, so even if the animal isn’t infected with the virus, the symptoms caused by the stress can be similar to those of a latent infection. - **This leads to false positives.** **False Negative** - **Neutralizing antibody** - **Interference phenomena in Latent infection** - **False Positive** - **Appearance of signs of latent infection due to stress** ## How to overcome neutralizing antibodies and latent infections - **Gnotobiotic animal:** - **Def:** An animal raised under certain conditions, free from any pathogens with known microflora to avoid neutralizing antibodies and latent infections. - **Intrinsic variability:** - **Def:** It is useless to use two different lab animals because they will have different genomes and the results will be inconsistent and unreliable. - **To avoid it:** Inbreeding is used so the lab animals will have similar genomes. - **Welfare System (especially no stress):** - It is necessary to have permits and approvals to deal with laboratory animals and to provide them with special care (food, shelter, enrichment, veterinary care, etc.)