Virology

• Methods of viral isolation

  • Cell culture
  • Animal inoculation
  • Embryonated egg inoculation
• Preparation of cell culture

  1. Tissue sample is packed and minced w/ mortar & pestle

  2. Put cells inside a vial/test tube containing broth medium such as buffered saline

  3. Put enzyme to separate each cell from another

  4. Store and regularly check the morphological appearance
• Virus isolation in tissue culture cell line

  1. Specimen in VTM is centrifuged @ 3000 rpm for 15-30 minutes

  2. Separate the supernatant from the sediments and add antibiotics/antifungal

  3. Inoculate supernatant into cell culture

  4. Incubate in a mesophilic environment (35-37°C)

  5. After several hours of incubation, check for possible presence of replicated viruses

  
• Cell cultures


  • PMK – primary monkey kidney
  • HDF – human diploid fibroblast
  • Hep2 – human laryngeal carcinoma cell line
  • RK – rabbit kidney
  • A549 – human lung carcinoma cell line
• Categories of cell culture

  • Primary Cell Culture
  • Cell Line/Continuous/Diploid
  • Heteroploid/Finite/Immortal
• Primary Cell Culture


  • HEK – Human Embryonic Kidney
  • RK – Rabbit Kidney
  • PMK – Primary Monkey Kidney
  • RMK – Rhesus Monkey Kidney
  • CMK – Cynomolgus Monkey Kidney
  • AGMK – African Green Monkey Kidney
• Cell Line/Continuous/Diploid


  • W1-38 and MRC-5 – derived from embryonic human lung
  • HDF (Human Diploid Fibroblast) – derived from human kidney or lung fibroblast
• Heteroploid/Finite/Immortal


  • HeLa – derived from human cervical carcinoma
  • Hep-2 – derived from human laryngeal carcinoma
  • KB – derived from nasopharyngeal carcinoma
  • A-549 – derived from human lung carcinoma
• Laboratory tests in cell culture

  • Presumptive: CPE, Hemadsorption, Interference, Decrease of acid production
  • Definitive: Complement Fixation, Neutralization, Fluorescent Antibody Assay, RIA, ELISA, Hemagglutination
• Cytopathic effect (CPE)


  Described as the formation of cytopathic effect by a second virus

  
• Hemadsorption test


  Described as the formation of cytopathic effect by a second virus

  
• Interference


  Described as the formation of cytopathic effect by a second virus

  
• Decreased acid production


  Addition of pH indicators (such as phenol red) in culture. Retention of red color: virus present. Became yellow: no virus replicated. Virus would use the cell to replicate, rendering it from normal metabolic activity and production of waste products such as acid.
• Problems with cell culture: Long period (up to 4 weeks) required for result, often very poor sensitivity, susceptible to bacterial contamination and toxic substances, many viruses will not grow in cell culture
• Viruses readily isolated by cell culture

  • Herpes simplex virus (HSV), Cytomegalovirus (CMV), Adenovirus, Polio virus, Coxsackie B virus, Entero cytopathic human orphan (ECHO) virus, Influenza virus, Parainfluenza virus, Mumps virus, Respiratory syncytial virus

  
• Less frequently isolated viruses

  • Varicella zoster virus, Measles virus, Rubella virus, Rhinovirus, Coxsackie A virus

  
• Animal inoculation


  1. Supernatant (w/ antibiotics & antifungal) containing virus is injected into test animal

  2. After several days, either extract sample or dissect test animal and use organs as specimen
• Parts of an embryonated egg

  • Egg shell
  • Shell membrane
  • Broaden part
  • Pointed part
  • Embryo
  • Amniotic fluid
  • Yolk
  • Albumen
  • Allantoic fluid

  
• Embryonated egg inoculation


  1. Prepare specimen as cell culture

  2. Disinfect egg shell with 70% alcohol

  3. Depending on type of virus to grow, inoculate area

  4. Seal hole with candle wax / sterile micropore

  5. Incubate

  6. After several days, harvest sample from egg then do further testing

  
• Embryonated egg inoculation areas

  • Chorioallantoic membrane inoculation
  • Amniotic inoculation
  • Yolk sac inoculation
  • Allantoic inoculation

  
• Microscopic identification directly in the specimen


  One of the most common approach using bright-field microscope, fluorescent microscope, and electron microscope
• Cytopathic effects seen in bright-field microscopy

  • Negri body, Owl eye, Cowdry type A
• Fluorescence microscopy


  Make use of Ag-Ab reaction by immunofluorescence. Sample suspected of viral infection is treated with antibodies tagged with fluorochrome dye. When the antigen from the virus binds with the antibody, it forms Ag-Ab complexes with fluorescing morphology.
• Electron microscopy


  Not used routinely, only for research purposes. Uses a beam of electrons to visualize minute particles. Specimen is stained with uranyl acetate. Can visualize the specific morphology of viruses. 106 virus particles per ml required for visualization, 50,000-60,000 magnification normally used.

  
• Microorganism


  Living entity not visible with the naked eye
• Specimens where viruses may be detected by electron microscopy

  • Feces – Rotavirus, Adenovirus Norwalk virus
  • Vesicle Fluid – HSV & VZV
  • Skin scrapings – papillomavirus, molluscum contagiosum virus

  
• Problems with electron microscopy: Expensive equipment, expensive maintenance, require experienced observer, sensitivity often low
• Types of microorganisms

  • Algae
  • Fungi
  • Bacteria
  • Viruses
• Serological procedures

  • Classical Techniques: Complement fixation tests (CFT), Haemagglutination inhibition tests, Immunofluorescence techniques (IF), Neutralization tests, Counter-immuno-electrophoresis
  • Newer Techniques: Radioimmunoassay (RIA), Enzyme linked immunosorbent assay (EIA), Particle agglutination, Western Blot (WB), RIBA Line immunoassay
• Antibody response to infection

  Establishing significant titer could help diagnose active viral infection by performing antibody titer from two different samples collected from the same patient and then comparing their results. Acute phase is the phase of infection when patients manifest signs and symptoms. Convalescent phase is the phase when patient recovers and symptoms diminish.

  
• Virus

  Not a cell, does not have the minimum requirements/parts to be called a cell

  
• Virus is a simple organism that only contains one nucleic acid, either DNA or RNA, have few proteins and may have a lipoprotein membrane on some but not all. Ribosomes, mitochondria and other organelles are not present but enzyme can be present in some. Additionally, they multiply in a different method.

  
• Serological tests for viral infections

  • Complement fixation tests (CFT)
  • Haemagglutination inhibition tests
  • Immunofluorescence techniques (IF)
  • Neutralization tests
  • Counter-immuno-electrophoresis
  • Radioimmunoassay (RIA)
  • Enzyme linked immunosorbent assay (EIA)
  • Particle agglutination
  • Western Blot (WB)
  • RIBA Line immunoassay

  
• Virus


  Doesn't belong to any kingdom since it is not a plant or an animal, or fungi, protist or bacteria
• Antibody titer


  Highest concentration of dilution that shows a positive reaction
• Virus


  Is an infectious agent made up of nucleic acid (DNA or RNA) wrapped in a protein coat called a capsid

  
• Acute phase


  Phase of infection when patients manifest signs and symptoms
• Virus


  Have no nucleus, organelles, cytoplasm or cell membrane - they are non-cellular

  
• Convalescent phase

  Phase when patient recovers and symptoms diminish
• Virus


  Viruses are obligate intracellular parasite