virus 3

• Detection in Clinical Samples:

  • Electron Microscopy: Visualizes virus particles directly.
  • ELISA (Enzyme-Linked Immunosorbent Assay): Detects viral antigens or virus particles.
  • Haemagglutination Assay: Determines the presence of viral particles based on their ability to agglutinate red blood cells.
• In virus-infected tissues (by taking swabs/biopsies/aspirates) • Immunoperoxidase assay • Immunofluorescence assay
•  Viral genome detection 

  • RT-PCR / PCR

   • Hybridisation
• Electron Microscope:

  • Shows virus morphology for identification.
  • Can be used on specimens directly or viruses concentrated from various sources.
  • Fast process, providing results in minutes.
  • Particularly useful for detecting viruses like rotaviruses, adenoviruses, astroviruses, and caliciviruses.
• Electron Microscope Requirements 

   • Expensive

   • Need specialise equipment 

  • Requires skilled personnel 

  • Low sensitivity 

  Need concentrated virus samples (106 particles/ml)
• Immune Electron Microscopy

   • Useful when numbers of virus particles is low 

  • Sensitivity and specificity can be improved by using virus specific antibodies 

  • Different viruses with similar morphologies can be identified 
• − Classical Immune EM 

  • Sample is mixed with antibody 

  • Negative staining of sample

   • Loaded onto EM grid and visualised
• − Solid phase Immune EM (SPIEM)

   • Grid is coated with antibody and used to capture virus particles

   • Virus sample is loaded onto antibody coated grid 

  • Negative staining etc
• ELISA: → Antibody coated well (capture Ab)→ Well must be “blocked” with excess protein→ Add sample containing Antigen (Ag)→ Wash well. Add antibody that recognizes the Antigen (detecting Ab)→ Wash well. Add conjugate labeled secondary Antibody (anti-IgG peroxidase)→ Wash well. Add substrate
• Haemagglutination Assay:• Some virus families have proteins that bind to erythrocytes (red blood cells)• Examples: Adenoviridae, Orthomyxoviridae, Paramyxoviridae• Influenza virus contains haemagglutinin, which binds to red blood cells.
• The haemagglutination assay principle:

  1. Mix red blood cells in a well.
  2. No virus: Cells form a tight pellet at the bottom.
  3. Virus present: Cells crosslink, settling as a diffuse red pattern at the well's bottom.
• Haemagglutination protocol

   • Virus sample diluted in 2 fold dilutions across plate starting with 1/10 dilution

   • Red blood cells (RBC) added to each well including control well (C = no virus)

   • Incubation at room temperature for 1 hour 

  • RBC in control well should not agglutinate but will form a button and bottom of well

   • In presence of virus, RBC will bind to each other i.e. agglutinate forming diffuse lattice that coats the well
• Immunofluorescence:

  1. Cells from the clinical specimen (e.g., nasopharyngeal aspirate) are fixed onto a glass slide.
  2. Add virus-specific antibodies.
  3. Add a labeled antibody (fluorescent dye) that binds to the virus-specific antibody.
  4. Commonly used for respiratory viruses in respiratory specimens.
• Immunoperoxidase

  → • Similar to immunofluorescence except that a peroxidase label is used
• Virus Quantification: Plaque Assay

  1. Determine levels of virus (titre) in tissues by titration.
  2. Virus spreads to adjacent cells, causing damage and death.
  3. Plaques are produced—regions with no cells, visible with the naked eye after staining.
  4. Equivalent to bacterial colonies for virologists.
  5. Used to quantify viruses by determining virus titre—the exact number of plaques.
  6. LD50 represents the dilution of virus that kills 50% of cells.
• Plaque Assay:

  1. Monolayers of cultured cells are incubated with serial dilutions of virus to allow adsorption to cells.
  2. After removal of the inoculum, cells are overlayed with semi-solid media (agar) to limit virus spread to neighboring cells.
  3. Each infectious particle produces a circular zone of infected cells, resulting in a plaque due to cell damage or death.
  4. Only viruses causing visible damage can be assayed this way.
  5. Plaques are stained with crystal violet, which stains living cells, enabling plaque counting.
• Plaque Assay (Interpretation):

  • Tenfold dilutions of the virus sample are made.
  • One milliliter of each dilution is added to columns 1 to 5 (with 4 replicates each).
  • After 4 days of incubation, cells are stained with crystal violet.
  • Plaque growth is visualized.
  • Virus titre is calculated as the number of plaques (10) divided by the number of replicates (4) and multiplied by the dilution factor.
  • Virus Titre = 2.5 × 10^7 PFU/mL
• Viral Genome Detection:

  • Polymerase Chain Reaction (PCR)
  • Nucleic acid purification (RNA or DNA)
  • Reverse transcription for RNA viruses
  • Using virus-specific primers to amplify a specific target
  • Repeated cycles of denaturation, annealing, and elongation
  • Agarose gel electrophoresis to visualize PCR products
• Indirect Methods

   • Look for signs of damage in 

  −Cells infected with virus in vitro 

  • Cytopathic effects observed in virus-infected cells 

  • Type of damage caused by some viruses can be used by virologists for diagnosis −Experimental infection of animals
• Indirect Methods

   – Detection of antibody

   • Antibody can be detected by 

  −ELISA −Immunofluorescence/Immunoperoxidase 

  −Haemagglutination inhibition assay 

  −Plaque inhibition assay
• Viral Cytopathic Effect (CPE) observed in vitro:

  • Cells are grown in vitro and infected with viruses from clinical samples
  • Some viruses kill the cells they infect, leading to visible changes that can be observed with a light microscope
  • CPE is easier to detect than the viruses themselves, which are too small to be seen directly
  • The degree of damage caused by CPE can vary from massive to no visible damage (non-cytopathic)
  • If CPE is not easily visible, viral antigens in cells can be detected using immunofluorescence or immunoperoxidase techniques.
• Sample from an infected animal is used to infect cell culture in vitro, which can be of three types:

  • Primary cell culture
  • Semi-continuous cell culture
  • Continuous cell lines
• Preparation of Primary Cell Culture:

  • Tissue fragments are cut with scissors or scalpel
  • Treated with trypsin or collagenase to obtain a cell suspension
  • Liquid media
  • Incubated in a petri dish or tissue culture flask
  • Cells attach to the solid surface and start dividing, resulting in a primary cell culture.
• primary Cell Lines:

  • Prepared directly from animal tissue
  • Subcultured only 1-2 times
  • Technically more difficult
  • Best cell culture system as it supports a wide range of viruses
  • Used when the state of cell differentiation is important but difficult to obtain a reliable supply
  • Expensive
  • Examples: Monkey kidney, Human embryonic amnion
• Continuous Cell Lines:

  • Derived from tumors or treated primary cell cultures
  • Can grow indefinitely
  • Easier to handle but limited virus range
  • Often differ from original cells:
  • Less differentiated
  • Chromosomal abnormalities
  • May cause tumors in mice
  • Examples: Hep-2, HeLa, Vero
• Normal Cell Monolayers:

  • Form a confluent monolayer
  • Attach to plastic surfaces
  • Grow in defined medium
  • Double in 24 – 48 hours
  • Survive freezing
  • Vary in virus susceptibility
• Viral cytopathic effect CPE =

  • grow cells in vitro and and infect with virus taken from clinical samples
  • viruses kill cells that they have infected and we see this in light microscope
  • Massive range from no damage ( non -cytopathic ) damage
  • IF CPE is not easily visible = you can then loom for antigens in cells by immunofluorescence of immunoperoxidase
• sample from infected animals is used to infect cells culture in vitro. we can yiuse three types of samples to infect

  • primary cell culture
  • semi continuous
  • continuous cell lines
• preparation of primary cell culture = limited lifetime survive 3 passages

  tissue fragments cut with scissors

  • treating it with trypsin or collagenase enzyme
  • single cell suspension
  • add liquid media incubated in petri dish /flask
  • cells attach to solid surface and strata dividing
  • primary culture
  • repeat
• Primary cell lines

  → Prepared directly frm animals tissue 

  → subcultured only 1-2 time s

  → technically more difficult 

  → best cell culture system as it supported wide range of viruses 

  → used when the state of cell is differentiation is important

  Difficult to obtain a reliable supply Expensive

  → examples : Monkey kidney • Human embryonic amnion
• Continuous cell lines 

  → Derived from tumours or by treatment of primary cell culture with a tumour virus or mutagenic chemical

  → Can be propagated unlimited

  → Most easy to handle

  → Range of viruses that can be grown is limited

  → Often do not resemble the original cell
• Continuous cell lines

   – Less differentiated (lost morphology and biochemical features the possessed in the organ) 

  – Often abnormal in chromosome morphology and number 

  – Can be tumourgenic (cause tumours in mice when inoculated

  → examples: 

  • Hep-2; human epithelial;

   • HeLa; Henrietta Lacks 

  – human cervical cancer

   Used to propagate polio

   • Vero; African Green monkey kidney cells
• Normal Cell monolayers

  • Confluent cell monolayer 

  • Epithelial and fibroblastic cells attach to plastic surfaces to form a monolayer

   • Cells grown in a chemically defined medium

  A) epithelial
• different types of CPE

  • Holes appear in monolayer which starts to lose
  • Grape - like cluster of rounded cells with
  • cells fuse to give multinucleate syncytia
  • Rounding up and shrinkage of cells
• what type of CPE

  A) holes appear in monolayer lose

  
• CPE ?

  A) Multinucleate syncytia

  
• CPE

  A) rounding up and shrinkage of cells

  
• CPE

  A) Grape-like cluster of rounded cells

  
• • Syncytium - fused cells containing many nuclei
• inclusion bodies = Viruses factories in nucleus or cytoplasm