MycoViro_Lab_Finals3&4

• Methods to diagnose viral infection include:

  • Direct detection of the virus in clinical specimens
  • Nucleic acid-based detection
  • Isolation of viruses in cell cultures
  • Serologic assays to detect antibodies to virus
• Direct detection of viruses in clinical specimens offers quick results to allow rapid therapy
• Bright-field microscopy is best for detecting poxviruses electron microscopy is used to detect virions and immunofluorescence is used to detect various viral agents directly in clinical specimens
• Cytopathic effect (CPE) is produced distinctively by viruses with characteristic visual changes in infected cells and can be detected in cell scrapings from infected sites with bright-field microscopy
• Immunofluorescence allows direct visualization of virus infection, amplifies signals, and enhances sensitivity
• Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions, being less sensitive than cell cultures or nucleic acid-based tests
• Nucleic acid-based detection involves multistep procedures where the pathogen nucleic acid is extracted and purified from a biological sample, offering advantages like faster TAT, better sensitivity, and detection of inactive and active viruses
• Viral isolation methods include cell culture, animal inoculation, and embryonated eggs, with cell culture being the most commonly used method
• Cell culture can be primary, low passage, or continuous, with primary cell cultures obtained from tissue removed from an animal and finite cell cultures having limited passage generations
• Continuous cell lines examples:

  • HEp2 (derived from human laryngeal epithelial carcinoma)
  • A549 (derived from a human lung carcinoma)
  • Vero (derived from monkey kidney)

  
• Mixed/Engineered cell cultures:

  • Lines of cells containing a mixture of two different cell types or genetically modified cells for easier viral infection identification
  • Developed by combining two cell lines susceptible to certain types of viruses like respiratory or enteric viruses
  • Mixed lines have greater sensitivity to a wider range of viruses
• Cytopathic effect (CPE) on cell culture:

  • Herpes Simplex Virus (HSV) produces a focal CPE and plaques within 24 hours
  • Rapid growth, plaque formation, and growth on various cell types are presumptive evidence for HSV identification
  • Cytomegalovirus (CMV) produces an HSV-like CPE but grows more slowly and only on diploid fibroblast
• Cytopathic effect (CPE) on cell culture:

  • Varicella Zoster Virus (VSV) grows on several types of cells
  • Enteroviruses produce small, round infected cells that spread diffusely
  • Adenoviruses produce cell rounding on various cell types
• Cytopathic effect (CPE) on cell culture:

  • Respiratory Syncytial Virus (RSV) can produce syncytial formation in specific cells
  • Influenza viruses are typically cultured on PMK cells, LLC-MK2, or MDCK cells
  • Influenza viruses do not produce a CPE, so a hemagglutination or hemadsorption test is done to detect them
• Centrifugation-Enhanced Shell Vial Culture:

  • More rapidly identifies viruses than traditional cell culture method
  • Cells grown on a round coverslip in a shell vial, inoculated with the clinical sample, centrifuged for viral absorption, then incubated for 24 to 48 hours
• Animal Inoculation:

  • Ideal animal host for virus study is the natural host
  • Laboratory animals like mice, rats, hamsters, rabbits, guinea pigs are practical for virus studies
• Embryonated Eggs:

  • Many viruses are cultivated in embryonated eggs for rapid isolation or vaccine production
  • More economical and convenient than animal inoculation
  • Eggs infected by various routes depending on tissue support for virus replication
• SeroLogic Assays:

  • Detect circulating antibodies to viruses after exposure
  • Provides limited information and has inherent problems like measuring host response instead of directly detecting the virus
• DNA replication involves the parental DNA as a double helix with labeled 5' and 3' ends, an origin of replication, a primer complementary to the parental DNA, and DNA polymerase adding nucleotides to the 3' end of the primer

  
• PCR (Polymerase Chain Reaction) is a nucleic acid amplification technique used in diagnostic clinical laboratories to determine and confirm the presence of DNA or RNA, and to characterize viruses, cells, or organisms by determining the DNA/RNA sequence of an amplified target

  
• Reverse Transcription Polymerase Chain Reaction (RT-PCR) involves the conversion of RNA into complementary DNA (cDNA) and is crucial in analyzing gene transcripts; it can be used to estimate gene copy numbers and validate gene expression

  
• Dideoxynucleotides (ddNTPs) lack a 3'-hydroxyl group, preventing their incorporation into DNA, while deoxynucleotides (dNTPs) can be incorporated into DNA; this difference is crucial for DNA sequencing where ddNTPs are added in small amounts and dNTPs in abundant amounts

  
• In DNA sequencing, ddNTPs are added in small amounts as they terminate DNA synthesis due to the lack of a 3'-hydroxyl group, while dNTPs are added in abundant amounts as they allow DNA synthesis to continue

  
• The Sanger DNA sequencing method involves the use of dideoxynucleotides (ddNTPs) that terminate DNA synthesis, resulting in fragments of varying lengths that can be separated and analyzed on a DNA sequencing gel

  
• Polymerase Chain Reaction (PCR) components include:

  • DNA template containing the target region of interest
  • Primer pair (forward and reverse)
  • Thermostable polymerase like Taq polymerase
  • Magnesium (Mg2+) as a cofactor for polymerase activity
  • Deoxynucleotides triphosphates (dNTPs) as equimolar building blocks of DNA
• PCR thermal steps:

  1. Heat denaturation to separate DNA duplexes

  2. Primer annealing for primer-template hybridization

  3. Primer extension at optimal temperature for polymerase activity
• Whole genome sequencing provides a comprehensive analysis of entire genomes, capturing small and large variants that might be missed with targeted approaches; it is used to identify potential causative variants for further studies of gene expression and regulation mechanisms
• Polyacrylamide Gel Electrophoresis (PGE) separates DNA products according to their molecular size
• Smaller DNA fragments tend to migrate at the bottom portion of the gel, while larger fragments migrate at the top portion
• Reading of the gel is done from the bottom portion to the top portion
• A new DNA strand is sequenced during the process
• Reverse transcriptases are RNA-dependent DNA polymerases that convert RNA templates to their complementary DNA sequence in the presence of dNTPs and other essential cofactors
• PCR Components:

  • DNA template contains the target region of interest
  • Primer pair: Forward locates the replication start point along the template's sense strand, while Reverse marks a second replication start point along the antisense, downstream of the forward primer
• PCR Thermal Steps:

  1. Heat denaturation disrupts and separates DNA duplexes

  2. Primer annealing occurs at 3.5 degrees Celsius below temperature

  3. Primer extension happens at the optimal temperature for polymerase activity
• Embryonated eggs are used for the rapid isolation of viruses and for vaccine or antigen production
• Animal cell culture:

  • Primary cell cultures are obtained from tissue removed from an animal and are used for minimal cell division
  • Finite cell cultures (diploid) can divide but are limited to about 50 generations
• Viral Isolation methods in diagnostic virology:

  • Cell culture is the most commonly used method
  • Animal inoculation is costly and used as a special resource or in reference/research
  • Embryonated eggs are rarely used
• Examples of nucleic acid-based assays include:

  • Hybridization assay
  • Polymerase Chain Reaction (PCR)
  • Reverse Transcription Polymerase Chain Reaction (rtPCR)
  • Branched DNA assay
  • Nucleic acid sequence-based amplification
  • Combination of PCR and flow cytometry (Luminex system)