Viral Structure Herpes virus. Pathology of viral disease. Viral Structure. Topics for the first lecture. Virus size
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1 Pathology of viral disease Viral Structure Herpes virus Ila Singh, MD, PhD Department of Pathology P & S is132@columbia.edu Envelope Tegument Spikes Nucleocapsid Genome Principles of Virology: Molecular Biology, Pathogenesis, and Control S. J. Flint, L. W. Enquist, V. R. Racaniello, A. M. Skalka Topics for the first lecture. Viral Structure General virology Viral lifecycle Viral pathogenesis Laboratory diagnosis Flu Ebola Rhino 14 Vaccinia Polio-TEM Polio Virus size Classification schemes for animal RNA viruses QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Principles of Virology: Molecular Biology, Pathogenesis, and Control, S. J. Flint, L. W. Enquist, V. R. Racaniello, A. M. Skalka 1
2 Some useful terms Viral assembly and release Plaque pfu MOI Particle to infectivity ratio Neutralizing Abs Cytopathic effect Protein synthesis Budding Assembly Replication of genome Nuclear transport ER Methods of diagnosis for viral diseases Viral life cycle Serology Cytology or Histology Viral growth in cell culture Detection of viral genome E C Entry into cells I. Serology Binding Internalization Look for viral antigens or anti-viral antibodies Uncoating Low-pH induced conformational change A four fold or greater rise in titer between two serum specimens provides a positive diagnosis. Paired sera, the first taken as early as possible in the illness and the second 10 to 14 days after the onset of symptoms. Nuclear transport Fusion 2
3 Serology Methods ELISA ELISA Rapid tests for Flu, RSV Hep B, Hep C etc etc Western Blots HIV antigens - from virus or recombinant proteins or synthetic peptides are immobilized on microtitre plates Incubate test serum. Wash Enzyme-labeled antibody specific for hu- IgG. Wash. Substrate changes color Serology: ELISA Serology: Western blot Principles of Virology: Molecular Biology, Pathogenesis, and Control, S. J. Flint, L. W. Enquist, V. R. Racaniello, A. M. Skalka EIA for RSV Western blot % sensitivity and 90-97% specificity when compared to tissue culture - results in about 6 minutes - room temperature storage of kit 3
4 env Western Blot + +w - Inclusion bodies CA Adenovirus: cytomegaly, multinucleate cells, inclusions II. Histology and cytology Inclusion bodies Inclusion bodies Syncitia Tzanck test for VZV and HSV Negri bodies in rabies Adenovirus: cytomegaly, multinucleate cells, inclusions Inclusion bodies CMV: owl eyed nucleus HSV Tzanck smear HSV, VZV: multinucleated giant cells 4
5 Negri bodies Primary human fibroblasts HSV Adenovirus: ells, inclusions TEM NIH3T3 Cell culture in virology HeLa Electron microscopy Cytopathic effect HSV Adenovirus: ells, inclusions Rota Syncytia CMV: owl eyed nucleus Corona Principles of Virology: Molecular Biology, Pathogenesis, and Control, S. J. Flint, L. W. Enquist, V. R. Racaniello, A. M. Skalka III. Grow virus in culture Cytopathic effect Look for cytopathic effects (CPE) in culture Detect viral antigens by Shell vial culture Identify virus by type of cell it grows in, time to detection of CPE and morphology of CPE Rounding, syncitia, vacuoles etc Confirm with fluorescent-labeled antibodies Results in days to weeks 5
6 Cytopathic effect Cell culture plus IF Grow virus in culture Detect viral antigens by Shell vial culture Inoculate specimen into many vials (one for each virus to be tested) Stain with specific antibody Results in 1-2 days Cell and Tissue-types for culture Screening cells Monoclonal antibodies (commercially available and FDA approved) Rhesus Monkey Kidney (1 ) Myxo-, Paramyxoviruses etc Human Embryonic Kidney (1 ) Very sensitive for adenovirus and important for lung transplants MRC-5 (human embryonic lungs) CMV, VZV, HSV HSV 1 and 2 VZV CMV Flu A and B Parainfluenza 1, 2 & 3 RSV Adeno Mumps Measles Some enteroviruses Chlamydia Cell-types for culture African Green Monkey Kidney Rubella grows only on these Hep-2 RSV Vero HSV Primary rabbit kidney HSV, enteroviruses Detect and analyze viral genomes PCR RT-PCR Quantitative PCR to detect viral load Branched DNA Hybridization, using microarrays Genotyping Phenotyping? 6
7 Polymerase chain reaction Southern blot slot blot Detect and analyze viral genomes Hybridization with microarrays PCR RT-PCR Quantitative PCR to detect viral load Branched DNA Southern blots Hybridization, using microarrays Genotyping Phenotyping? Southern blot Sensitivity of NAT Combination of PCR/Southern blot: 95% confidence intervals HAV, 5-9 copies/ml HBV, 1-2 copies/ml HCV, 3-5 copies/ml Reduce risk of HCV transmission From 1:100,000 to 1:500,000-1:1,000,000 Data from National Genetics Institute, Labcorp 7
8 Pool testing CDC 8 X 8 X 8 = 512 samples Small pox, Hantavirus, Ebola etc Usually via the State labs Other labs State Department of Health lab Centers for Disease Control Other commercial labs What specimen to collect? When? NYDOH lab for viral encephalitis Herpes Simplex Varicella Zoster Cytomegalovirus Epstein-Barr Virus Enteroviruses St. Louis Encephalitis (SLE) Eastern Equine Encephalitis (EEE) California Encephalitis Powassan (POW) Rabies West Nile Virus Tests include: 1) PCR, and 2) ELISA. Freeze leftover CSF at -70 C in the event that PCR testing becomes necessary. QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Viremia Principles of Virology: Molecular Biology, Pathogenesis, and Control, S. J. Flint, L. W. Enquist, V. R. Racaniello, A. M. Skalka 8
9 HIV infection What specimen to collect? When? Genital HSV, vulvar swab (not endocervical) in last month of pregnancy Buffy coats CMV (fresh specimen, <1hr) Bronchial and BAL wash RSV, Flu, Adeno-, CMV etc Other Biopsy, autopsy specimens What specimen to collect? When? Throat first presentation with fever (measles, mumps, rubella, also viral meningitis caused by enteroviruses and neonatal HSV). Vigorous swab, because you need cells. Nasopharyngeal swab or wash Flu, RSV, Rhino-, CMV (if lots of virus) Rectal entero- and adenoviruses (meningitis), rotavirus Urine Adenovirus (hemorrhagic cystitis) MMR, after cleared from throat or sometimes concomitant CMV and HSV (rare) Transport to lab Since we still depend on viral growth for diagnosis, rapid transport to lab is essential Specimen on ice Refrigerate if delay inevitable, DO NOT FREEZE If need to store for more than 6 days, freeze at -70 C Transport and store in viral transport medium Enteroviruses more stable and will tolerate some delay Hand delivery encouraged (also for better communication: viruses suspected, source of material) What specimen to collect? When? CSF PCR for HSV, VZV, CMV, adeno or flu Rarely can grow coxsackie or echo Lesion VZV, CMV, measles (scrape for cells) HSV, Tzanck smear Conjunctival Web resources get a free electronic MMWR subscription HIV database: hiv-web.lanl.gov All the Virology on the WWW: Pan-American Society for Clinical Virology: 9
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