This document discusses various practical virology techniques including viral cultivation methods, serological assays, nucleic acid detection methods, and viral genetics. It also explains the characteristics of viruses, their differences from bacteria, and the processes involved in diagnosing viral infections. Additionally, the document outlines polymerase chain reaction (PCR) techniques, their applications, and the steps involved in executing a PCR test.

1. VIROLOGY
PRACTICAL
Dr.S.Sundaresan
Assistant Professor Tagore Medical College

2. Practical Virology types
1.Viral Cultivation:
1.Cell Culture: Growing viruses in cell cultures to study their behavior and replication.
2.Embryonated Eggs: Using fertilized eggs as a medium for viral propagation.
2.Serology:
1.ELISA (Enzyme-Linked Immunosorbent Assay): Detecting viral antigens or antibodies.
2.Western Blot: Identifying specific viral proteins.
3.Nucleic Acid Detection:
1.PCR (Polymerase Chain Reaction): Amplifying viral nucleic acids for detection.
2.RT-PCR (Reverse Transcription PCR): Amplifying RNA viruses after reverse transcription to
cDNA.
3.qPCR (Quantitative PCR): Measuring the amount of viral DNA or RNA.
4.Viral Genetics:
1.Sequencing: Determining the nucleotide sequence of viral genomes.
2.Site-Directed Mutagenesis: Introducing specific mutations into viral genomes for functional
studies.

3. 1.Antiviral Viral Pathogenesis:
1.Animal Models: Studying viral infections in vivo using animals.
2.Pathological Examination: Analyzing tissues for signs of viral infection.
2.Drug Development:
1.High-Throughput Screening: Testing large numbers of compounds for antiviral activity.
2.Drug Resistance Studies: Investigating the development of resistance to antiviral drugs.
3.Vaccine Development:
1.Attenuation: Weakening a virus for use in vaccines.
2.Recombinant DNA Technology: Creating genetically engineered viruses for vaccine
production.
4.Virus Evolution and Epidemiology:
1.Phylogenetic Analysis: Studying evolutionary relationships among viruses.
2.Epidemiological Surveillance: Tracking the spread of viral infections in populations.

4. 1.Viral Immunology:
1.Immunofluorescence: Detecting viral antigens using fluorescent antibodies.
2.Neutralization Assays: Measuring the ability of antibodies to neutralize viral infectivity.
2.Bioinformatics in Virology:
1.Computational Analysis: Analyzing large datasets of viral genomes.
2.Structural Prediction: Modeling the three-dimensional structure of viral proteins.
3.Diagnostic Techniques:
1.Immunohistochemistry: Identifying viral antigens in tissue sections.
2.Electron Microscopy: Visualizing viruses at the ultrastructural level.
4.Virus Ecology:
1.Environmental Sampling: Collecting and analyzing samples from various environments for
viral presence.

5. What is the virus?
• A virus is a small infectious agent that can only replicate inside the living cells of
organisms.
• Viruses are composed of genetic material, either DNA or RNA, surrounded by a protein
coat called a capsid. Some viruses also have an outer envelope made of lipids.
Key features of viruses include:
1.Genetic Material: Viruses can have either DNA or RNA as their genetic material. The
genetic material carries the instructions for the virus to replicate and produce new viral
particles.
2.Capsid: The capsid is a protective protein coat that surrounds the genetic material. It
provides structural integrity to the virus.

7. 1.Envelope: Some viruses have an additional outer envelope, which is a lipid layer derived from the
host cell membrane. Enveloped viruses often have proteins embedded in this envelope that help the
virus attach to and enter host cells.
2.Host Dependence: Viruses cannot carry out their life cycle independently. They need to infect a host
cell and hijack its cellular machinery to replicate and produce new virus particles.
3.Cellular Parasites: Viruses are considered obligate intracellular parasites because they rely on the
host cell's machinery and resources for their replication.
4.Lack of Cellular Structure: Unlike cells, viruses lack the cellular structures such as organelles and
a cellular membrane.
5.Lack of Metabolism: Viruses do not have metabolic processes on their own. They do not carry out
cellular respiration or other metabolic activities.

8. What are the differences between the bacteria & viruses?
Cellular Structure:
• Bacteria: Bacteria are single-celled organisms with a simple cellular structure. They have cell
walls, cell membranes, cytoplasm, and a single circular strand of DNA. Some bacteria also
have flagella or pili for movement and attachment.
• Viruses: Viruses are not considered cells. They are much smaller than bacteria and consist of
genetic material (DNA or RNA) surrounded by a protein coat called a capsid. Some viruses
also have an outer lipid envelope.

9. 1.Living or Non-living:
1.Bacteria: Bacteria are considered living organisms. They can carry out independent metabolic
processes, reproduce on their own, and respond to their environment.
2.Viruses: Viruses are considered non-living entities because they lack the cellular structures and
metabolic machinery found in living cells. They can only replicate inside a host cell.
2.Reproduction:
1.Bacteria: Bacteria reproduce through binary fission, a form of asexual reproduction where one
cell divides into two identical daughter cells.
2.Viruses: Viruses cannot reproduce on their own. They need to infect a host cell and hijack its
cellular machinery to replicate and produce new virus particles.
3.Cellular Machinery:
1.Bacteria: Bacteria have their own cellular machinery, including ribosomes, to carry out protein
synthesis and other metabolic processes.
2.Viruses: Viruses lack cellular machinery. They rely on the host cell's machinery for their
replication.

10. 1.Treatment:
1.Bacteria: Bacterial infections are often treated with antibiotics, which target specific bacterial
structures or functions.
2.Viruses: Viral infections are generally more challenging to treat. Antiviral medications may be
used to manage symptoms, but vaccines are often the primary means of preventing viral
infections.
2.Host Range:
1.Bacteria: Bacteria can infect a wide range of hosts, including humans, animals, plants, and
other microorganisms.
2.Viruses: Viruses are typically more host-specific, often infecting a particular species or even a
specific cell type within that species.
3.Examples:
1.Bacteria: Examples include Escherichia coli (E. coli), Staphylococcus aureus, and
Mycobacterium tuberculosis.
2.Viruses: Examples include Influenza virus, Human Immunodeficiency Virus (HIV), and
Herpes simplex virus.

11. Viruses are too small to be seen by light microscope μ=1000nm

12. Viral classification:
Nucleic acid:
• RNA or DNA
• Single Stranded or Double Stranded.
• Segmented or Non-Segmented.
• Linear or Circular.
• If it is S.S. RNA Positive or Negative.
Virion structure:
• Symmetry (icosahedral, helical, and complex).
• Enveloped or not.
• Number of capsomeres

14. Methods of DiagnosingViral Infections
1.Virus isolation
2. Direct detection of the virus
a. Detection of the virus Ag (ELISA, latex agglutination, IHC& ICC)
b. Immune EM
c. Detection of the virus nucleic acid (PCR)
3. Indirect (Serology) … IgM, IgG (titer)

15. Methods of DiagnosingViral Infections
1.Clinical Evaluation:
1.Patient History: Inquiring about symptoms, exposure to infected individuals, recent travel,
and other relevant information.
2.Physical Examination: Assessing physical signs and symptoms associated with viral
infections.
2.Laboratory Tests:
1.Serological Tests: Detecting antibodies or antigens in the patient's blood serum.
1.ELISA (Enzyme-Linked Immunosorbent Assay)
2.Western Blot
2.Nucleic Acid Tests: Detecting viral genetic material (DNA or RNA) in samples.
1.PCR (Polymerase Chain Reaction)
2.RT-PCR (Reverse Transcription PCR)
3.qPCR (Quantitative PCR)
3.Viral Culture: Growing the virus in a laboratory setting.
4.Viral Antigen Detection: Identifying viral proteins in patient samples.
5.Viral Load Measurement: Quantifying the amount of virus in a patient's blood.

16. 1.Imaging Studies:
1.Radiography: X-rays may reveal certain viral infections, such as pneumonia.
2.CT (Computed Tomography) or MRI (Magnetic Resonance Imaging): Used for
more detailed imaging in specific cases.
2.Point-of-Care Tests:
1.Rapid Antigen Tests: Quickly detecting specific viral antigens in patient samples.
2.Rapid Molecular Tests: Rapid PCR-based tests for detecting viral genetic material.
3.Viral Genome Sequencing:
1.Next-Generation Sequencing (NGS): Determining the entire genetic sequence of a
virus for detailed analysis and tracking.
4.Electron Microscopy:
1.Visualizing Viruses: Electron microscopy allows direct visualization of viruses in
patient samples

17. 1.Viral Genome Sequencing:
1.Next-Generation Sequencing (NGS): Determining the entire genetic sequence of a virus for
detailed analysis and tracking.
2.Electron Microscopy:
1.Visualizing Viruses: Electron microscopy allows direct visualization of viruses in patient samples.
3.Cytology and Histopathology:
1.Cytological Examination: Studying cell samples for changes caused by viral infections.
2.Histopathological Examination: Analyzing tissue samples for signs of viral infection.
4.Virus Isolation:
1.Isolating the Virus: Culturing the virus from patient samples for further characterization.
5.Molecular Typing and Subtyping:
1.Genetic Analysis: Studying genetic variations within a viral species for epidemiological purposes.
6.Biosensors and Nanotechnology:
1.Innovative Technologies: Developing biosensors and nanotechnology-based methods for rapid and
sensitive detection of viral infections.

18. Isolation of the virus using three living systems:
1.Tissue Culture
2. Embryonated egg
3. Lab. Animals
Why we isolate the virus in a living system?
Viruses must replicate within the cells because they cannot generate
energy or synthesize protein

19. Polymerase Chain Reaction (PCR)
1.Conventional PCR (cPCR):
1. Standard PCR used for amplification of specific DNA sequences.
2. Requires a thermal cycler for cycling through temperature phases (denaturation, annealing, extension).
2.Reverse Transcription PCR (RT-PCR):
1. Used for the amplification of RNA, converting RNA to complementary DNA (cDNA) before PCR.
2. Essential for detecting RNA viruses.
3.Quantitative PCR (qPCR):
1. Measures the amount of DNA or RNA in real-time during the PCR reaction.
2. Provides quantitative data on the initial amount of target nucleic acid.
4.Nested PCR:
1. Involves two sets of primers in two consecutive PCR reactions.
2. Increases specificity and sensitivity by reducing non-specific amplification.
5.Multiplex PCR:
1. Amplifies multiple target sequences in a single PCR reaction.
2. Useful for detecting multiple viruses or different strains in a single sample.
6.Real-Time PCR (qPCR or RT-qPCR):
1. Monitors the amplification of DNA or cDNA in real-time using fluorescence.
2. Provides quantitative data and allows for high-throughput analysis.

20. 1. Digital PCR:
1. Divides the sample into thousands of individual reactions, allowing absolute quantification of nucleic acids.
2. Particularly useful for samples with low target concentrations.
2. In Situ PCR:
1. Amplifies nucleic acids within fixed cells or tissue sections.
2. Useful for studying the distribution of viral DNA or RNA within tissues.
3. Hot Start PCR:
1. Reduces non-specific amplification by preventing DNA polymerase activity at lower temperatures.
2. Begins PCR reaction only after an initial denaturation step.
4. Multiplex RT-PCR:
1. Detects and amplifies multiple RNA targets simultaneously.
2. Useful for identifying co-infections or differentiating between closely related viruses.
5. Quantitative Reverse Transcription PCR (RT-qPCR):
1. Combines reverse transcription and quantitative PCR to measure the amount of RNA in real-time.
2. Widely used for quantifying viral RNA loads.
6. Two-Step RT-PCR:
1. Separates the reverse transcription and PCR amplification steps.
2. Allows flexibility in optimizing each step independently.
7. Endpoint PCR:
1. Traditional PCR where the reaction is run for a predetermined number of cycles.
2. The final products are analyzed after completion of the reaction.

21. Polymerase Chain Reaction
• Polymerase: DNA polymerase
• DNA polymerase is the only enzyme used in PCR and actions through duplication
of DNA
• Chain Reaction:The product of a reaction is used to amplify the same reaction
• Results in rapid increase in the product
• PCR is a laboratory version of DNA replication in cells

22. PCR in test tube
PCR is a laboratory technique used to: amplify specific region of DNA
(gene), in order to make a huge number of copies of that gene to be adequately
tested.

25. 1) Preparation of PCR samples
A- DNA template
B- DNA Primers (for the detection of gene of interest)
Short nucleotide sequence (18-30 nucleotides)
a. Forward primer
i. Anneals to DNA anti-sense strand
b. Reverse primer
i. Anneals to DNA sense strand
C- PCR master mix

26. 2. PCR machine
What is going on inside PCR machine?Thermal Cycling
• A PCR machine controls temperature
•Typical PCR go through three steps
• Denaturation PCRThermocycler
• Annealing
• Extension

28. Agarose Gel Electrophoresis) Small
fraction of PCR product is loaded on
agarose gel together with DNA loading
dye
Agarose gel is then put on UV
transiluminator to visualise PCR product
3. Visualization of PCR product
Need visualization system to confirm the presence of the PCR
product

29. • Positive PCR product should look like this

30. PCR in test Procedure
• 1. Sample Collection:
• Collect the biological sample (e.g., blood, saliva, tissue, or swab) containing the genetic material
(DNA or RNA) of interest.
• 2. DNA or RNA Extraction:
• Isolate and extract the nucleic acids (DNA or RNA) from the collected sample. Various extraction
methods and kits are available.
• 3. Primer Design:
• Design specific primers that flank the target DNA or RNA sequence. These short, single-stranded
DNA sequences are essential for DNA polymerase to initiate the synthesis of complementary strands
• 4. PCR Reaction Setup:
• Prepare the PCR reaction mix, including:
• DNA or RNA template
• Primers
• DNA polymerase
• Nucleotide triphosphates (dNTPs)
• Buffer solution
• Cofactors (Mg2+ ions)
• Stabilizers

31. • 5. Denaturation (95–98°C):
• Heat the reaction mixture to a high temperature to denature the DNA or RNA,
separating the two strands.
• 6. Annealing (50–65°C):
• Cool the reaction mixture to allow the primers to anneal (bind) to the
complementary target sequences.
• 7. Extension (72°C):
• Raise the temperature for DNA polymerase to synthesize new DNA strands by
extending from the primers.
• 8. Cycling:
• Repeat the denaturation, annealing, and extension steps (usually 20-40 cycles) to
amplify the target DNA exponentially.
• 9. Final Extension (72°C):
• Allow final extension to ensure that any remaining single-stranded DNA is fully
extended

32. • 10. Hold (4–15°C):
• Hold the reaction at a low temperature to stabilize the newly synthesized DNA strands.
• 11. Analysis:
• Analyze the PCR products using various techniques:
• Gel Electrophoresis: Separate and visualize DNA fragments based on size.
• Real-Time PCR (qPCR): Monitor amplification in real-time for quantification.
• Fluorescence-based Detection: Use fluorescent dyes or probes for target detection.
• 12. Data Interpretation:
• Analyze the results to determine the presence or absence of the target sequence and, if
applicable, its quantity.
• 13. Controls:
• Include positive and negative controls in the PCR assay to validate the accuracy of the results
• 14. Reporting:
• Report the findings, interpreting the results based on the presence or absence of the target
sequence.