Lecture 18 - Viral Diseases II Flashcards
What are the viral points of entry.
- Blood viruses such as hepatitis B can enter via blood transfusion (a lot of viruses can also enter via sexual contact). It circulates through the blood but the major disease site is in the liver which is the location of the majority of the damage
- Respiratory viruses enter through the respiratory tract. It enters the lungs or other areas of the respiratory tract. Pathology entirely within the respiratory tract.
- Enters via the respiratory route however but then breachers local barriers to spread systematically such as measles.
Where the virus replicates is where the pathogenesis occurs
What are some infectious pathways.
Contact doesn’t have to be direct (Kissing sex, blood). It can be by aerosols, formites (faecal oral route) and the environment (respiratory viruses faecal oral route). These come from breath, skin etc.
How do respiratory viruses enter cells.
After entry the virus has to bind to a receptor to enter cells (proteins on the surface of cells).
In the common cold or mild flu the virus binds in the upper respiratory tract. The deeper into the lungs the virus binds the worse the pathology and symptoms. Influenza (bird flu) receptors are often in the lower respiratory tract explaining why they are so serious by why they are less transmissible as it is harder for the virus to reach.
Covid binds to ACE2 receptors which were discovered to be quite wide spread leading to pandemic
How is yellow fever transmitted via insects.
E.g. yellow fever which is found in Africa and South America. Its geographical distribution is limited by the distribution of the vector.
Yellow fever transmission cycle
* In South America - Sylvatic cycle spreads form monkey to monkey due to Haemagogus spp. If a human gets bitten it can then infect humans where it begins the human cycle as Aedes aegypti.
In Africa the sylvatic cycle is spread by Aedes africanus between monkeys if Aedes spp. Bites human it can spread to humans where it enters the urban cycle and is spread by Aedes aegypti.
How are viral diseases diagnosed
Symptoms
PCR - detects part of viral genome
Immunological techniques
* Detect viral proteins
* Detect host antibodies to viral proteins
Functional assays (mostly obsolete)
How was symptomology used to diagnose Covid-19
Symptomology
Using symptoms to diagnose Covid-19 was important in the early parts of the pandemic when there were not enough of resources. It was also important for this reason in the past where there was less technology.
Symptomology can be problematic as some symptoms may be rare.
How are plaque forming units used to diagnose viral infections.
Preparation of Sample: The sample containing the virus is typically diluted to various concentrations to ensure that there are a manageable number of viral particles to count.
Inoculation: Each dilution is then used to inoculate a monolayer of susceptible cells in a culture dish or plate.
Incubation: The culture dishes are then incubated under conditions favorable for viral replication. During this time, the viruses infect the cells and begin to replicate.
Plaque Formation: As the viruses replicate within the cells, they cause visible damage to the cell monolayer. This damage often appears as clear zones in a background of intact cells. These zones are called plaques.
Counting: After an appropriate incubation period, the plates are stained or fixed to make the plaques more visible. Then, the number of plaques on each plate is counted. Each plaque represents a single infectious viral particle that was able to infect a cell and replicate.
Calculating PFU/ml: By taking into account the dilution factor used in step 1 and the number of plaques counted in step 5, researchers can calculate the number of plaque forming units per milliliter (PFU/ml) in the original sample.
This method is particularly useful because it provides a measure of the number of infectious viral particles present in a sample, rather than just the total number of viral particles, which may include non-infectious or defective ones. It’s widely used in virology research and in the testing and development of antiviral drugs and vaccines.
How does haemagglutination inhibition assay used to diagnose viral diseases.
Haemagglutination inhibition assay
Only works with viruses that have haemagglutinin activity.
RBCs are added and mixed with various dilutions of virus. Where there is little to no virus red blood cells settle into a button indistinguishable from where there is none added. When there is sufficient virus the RBCs agglutinate and settle in a diffuse pattern. One of the rows contains no virus and is used as a control.
Allows quantification of virus as greater the concentration in initial sample the more diluted it can be and have a positive result.
How is PCR used to detect viral nucleic acid
Primer Design: Specific primers are designed to bind to unique regions of the viral genome. These primers are typically around 20 nucleotides long and are complementary to sequences flanking the target region.
Sample Preparation: Viral nucleic acids are extracted from the sample of interest. This may involve various methods depending on the sample type, such as tissue samples, blood, or swabs.
PCR Reaction: The extracted nucleic acids are mixed with the designed primers, DNA polymerase enzyme, nucleotides (A, T, C, G), and buffer solution in a PCR tube or plate. The reaction mixture goes through a series of temperature cycles:
a. Denaturation: The reaction mixture is heated to a high temperature (usually around 95°C), causing the double-stranded DNA to denature into single strands.
b. Annealing: The temperature is lowered to allow the primers to anneal (bind) to their complementary sequences in the viral DNA. This typically occurs at a temperature around 50-65°C.
c. Extension: The temperature is raised to an optimal level for DNA polymerase activity (usually around 72°C). The polymerase enzyme extends the primers by adding complementary nucleotides, thereby synthesizing new DNA strands complementary to the viral template.
These cycles are repeated typically 20-40 times, amplifying the target viral DNA exponentially.
Detection: Various methods can be used to detect the amplified DNA. One common approach involves using fluorescent dyes or probes that bind specifically to the amplified DNA. Real-time PCR machines monitor fluorescence during each cycle, allowing for the quantification of the amplified DNA in real-time.
Analysis: The presence or absence of amplification and the cycle threshold (Ct) value can be used to determine the presence and quantity of viral nucleic acid in the original sample.
What techniques are used to detect viral proteins
Serologic diagnostic test
1. A drop off blood or serum is added to the well
2. A buffer is added
3. Sample incubation - capillary action moves sample across the lateral flow test
4. Antibody-antigen recognition - antibodies with specificity bind to gold-antigen conjugates in conjugate pad
5. Sample enters testing well and antibody antigen complex binds to immobilised anti-human IgG/IgM antibodies (test line)
6. Control antibody detection - Rabbit antibody gold conjugate binds to immobilised anti-rabbit IgG antibodies
Positive = One strip in each C well and T well
Negative = one strip in C well
What techniques are used to detect host antibodies to viral proteins?
ELISA
Sandwich ELISA for antigen detection:
1. Antibody A, specific for the viral antigen being tested for, is bound to wells of a 96 well plate and excess is washed out.
2. The test sample is added and any specific antigen present binds to the antibody, everything else is washed away
3. Another antibody, B, also specific for the viral antigen but raised in a different species form antibody A is added and binds only if antigen was present at step 2; excess is washed away
4. An enzyme conjugated secondary antibody is added which has specificity for Ig from the species providing antibody B; it binds only if antibody B bound in step 3
5. A chromogenic substrate for the enzyme is added and the colour intensity after a fixed time is measured by spectrophotometry using a plate reader.
Antibody capture ELLISA
1. Microtiter plate wells are pre-coated with virus or viral antigen
2. Test samples, possibly containing specific antibody for that virus, are added to the wells in serial dilutions
3. After allowing time to bind unbound Ig is washed away and any bound antibody detected by addition of a secondary antibody to which an enzyme is coupled
A positive result is revealed by the enzyme converting its substrate to a coloured product.
Neutralisation assay
Virus A loses its infectivity after combining with A-specific antibody (it is neutralised). A specific antibody does not bind to virus B, so infectivity of virus B is unaffected. The complete test requires the reciprocal reactions
