Lecture 10 Virological Diagnostics PDF

Chapter 10: Virological Diagnostics Yap Wei Boon, Ph.D Email: [email protected] Learning outcomes In the end if the lecture, students are able to Apply viral structural analysis; Apply analysis of virally infected cells; Apply molecular virological diagnostics;...

Chapter 10: Virological Diagnostics Yap Wei Boon, Ph.D Email: [email protected] Learning outcomes In the end if the lecture, students are able to Apply viral structural analysis; Apply analysis of virally infected cells; Apply molecular virological diagnostics; for detecting virus infections. Part 1: Observation of Viral Structures Structural investigation tools: Electron microscopy Investigation of structure of a virion or of virus-infected cells. Samples are negative-stained with potassium phosphotungstate or ammonium molybdate. Allows estimation of shape and size of a virion. X-ray crystallography Virions are condensed and dehydrated with alcohols and chemicals until crystals are formed. Molecules and atoms of the virus crystals can be determined using X-ray. Cryo-electron microscopy the three-dimentional (3D) images of the frozen virus structural are analyzed with a computer program. Cultivation and isolation of virus Animal cell culture is used to culture viruses from samples. Eg. Vero, MDCK, HepG2, Calu-3, A549 cell lines. The effects of virus infections in host cells are observed using an inverted light microscopy detects cytopathic effects in virus-infected cells. [i.e. shrinking and rounding up]. Some virus infections cause fusion of cells to form multinucleated giant cells (syncytia), such as Nipah, measles, parainfluenza virus. CPEs are indications of cell damages, loss of normal cellular functions. To more precisely confirm the presence of viruses, confocal (fluorescent) light microscope can be used. Detection of fluorescently tagged antibody that binds to virus antigens in infected cells (nucleus or cytoplasm or membrane-bound). Laboratory animals such as nonhuman primates and mice are developed into virus infection models. Embryonated eggs – cultivation of viruses for vaccine production (herpes, varicella-zoster, influenza). Isolation and Purification of virus To isolate pure virus particles from infected cells. Plaques assay : Plaques formed at the areas where cells are infected and killed by viruses. Virus particles are isolated from the middle of a plaque and inoculated onto monolayer cells to amplify the virus particles : plaque purification. If a virus replicates poorly in cells in the laboratory, it can be passaged a few cycles to increase its replication in the cells (laboratory strain). Part 2: Molecular Diagnostics for Virus Infections A. Detection of virus nucleic acids Hybridization [Southern (DNA) or Northern blotting (RNA)] specific nucleic acid probes bind to the complementary virus genes. RNA or ssDNA Radioactive-tagged probes dsDNA Polymerase Chain Reaction (PCR) Virus DNA can be amplified using a pair of oligonucleotide primers that are specific to a virus gene. Viral RNA can be converted to cDNA using a reverse transcriptase and then amplified using a pair of primers (Reverse transcriptase (RT)- PCR. Amplified genes are separated using agarose gel electrophoresis and stained with ethidium bromide or non-toxic stains such as Gel Red. The sizes of virus genes are compared to molecular weight markers. Real-time PCR Determines a gene copy number/virus titer in a virus infected sample by absolute or relative quantification. The gene amplification is monitored from the beginning to the end of the process It uses fluorescent dyes (eg. Sybergreen). If the amount of virus in a sample is higher than another sample, the fluorescent signal shows sooner and vice versa. Genome sequencing Amplified viral genes/whole viral genome can be sequenced using Sanger sequencing method and/or Next-Gene Sequencing (NGS). Sequence similarity is compared to databases using computer tools (Artemis and BLAST). Information such as sequences of open reading frames (ORF), promoters, enhancers, virus strains causing a viral outbreak can be deduced. Construct a phylogenetic tree. B. Detection of virus antigens Western blotting : detects virus-specific antibodies or virus antigens. Positive results are indicated by the presence of a label (enzyme, fluorescence, gold, radio-active) which is linked to the primary or secondary antibody. Enzyme-linked Immunosorbent Assay (ELISA) Detects virus particles/virus proteins/virus antigens in the microplate format. Uses virus-specific antibodies. High sensitivity and reproducibility. THANK YOU