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Morphologic changes in viral infections

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  • 1. MorphologicalChanges in Viral Infections Shridhan Patil 25 June 2012
  • 2. "The Father of Modern Vaccines." Nobel laureate John Franklin Enders • Known for polio viruses • Classified viruses on the basis of morphologic effects on cells.
  • 3. Virus NOT considered as cells• Small, obligate intracellular parasites• Strucure – DNA/RNA and protein coat• Mobile genetic elements• Virion - Function
  • 4. Classification• Size and shape• Chemical composition• Structure of genome• Modes of replication
  • 5. Cytopathic effect(CPE)• Morphological changes in cells caused by viral infections; the responsible virus is said to be cytopathogenic• Degree of CPE depends on o Virus and host type o Multiplicity of infection (MOI) o Some viruses do not cause CPE – Interference/ hemadsorption• Application- Virus identification, unknown virus
  • 6. How virus infects• Live inside cells- avoid immune response• Attachment receptors- DOCKING• PENETRATION- cytoplasm• BIOSYNTHESIS of viral components• MATURATION• RELEASE
  • 7. Sampling• What types of cases): stool, tissue, specimens are • Gastrointestinal tract saliva, brain biopsy, collected to diagnose? infections: cerebrospinal fluid stool and rectal swabs• Respiratory tract • Genital infections: infections: • Vesicular rash: vesicle vesicle fluid or Nasal fluid, skin Swab and bronchial Scrapingswashings, throat • Urinary tract infections:and nasal swabs, sputum • Maculopapular rash: urine throat, stool,• Eye infections: throat and rectal swabs • Bloodborne infections: and blood• conjunctival • CNS (encephalitis and swab/scraping meningitis
  • 8. Techniques of virus study• Direct 1. Electron microscopy/ Immuno-electroon microscopy 2. Light microscopy 3. Ag detection, molecular methods etc..• Indirect 1. Cell culture 2. Egg pocks on chorio-allantoic membrane 3. Animal disease/death
  • 9. • Find suitable host – primary, semi-continuous, continuous cells• Host grown as MONOLAYER on a solid surface• Infect cell with virus of interest- ONE VIRION/cell• Circle of death- easily counted and quantified
  • 10. Cytopathic effects• Clinical signs, symptoms- morphologic- physiologic- biochemical- immunologic effects of viruses• Unfixed, unstained smears- 10x- some CPE directly observed• Translucent cells- condenser down- iris diaphragm partly closed• Inclusion bodies fixation and staining
  • 11. • Requires experience to use CPE as diagnostic tool• Virus may not conform to norm for its family• CPE best viewed by daily observation of cultures admixed with low MOI (<0.1)• Normal cell ageing to be distinguished• Rate of CPE appearance- characteristic• General rule : Slow virus- 4-5 days• Rapid – 1-2 days for CPE to appear
  • 12. Types 1) Total destruction o Of all monolayer- most severe o Cells shrink rapidly, become pyknotic o Detach from glass within 72 hrs o Typical of ENTEROVIRUSES 20x objective within 24 hrs Bovine fetal spleen cells
  • 13. 2) SubtotalBovine fetalspleen cells 2 destructiondays • Some cellspostinfection dead/detachedRhabdovirus • Togavirus • Some picornavirus • Paramyxovirus 20x objective
  • 14. 3) Focal degeneration • Herpesvirus, Poxvirus • Localised foci of infection • D/t cell to cell virus trasnfer rather than extracellularly • Enlarged, rounded, refractile cellsBovine fetal spleen cells 2 days postinfection, Herpesvirus • Strangling of cytoplasmBlack arrows - cell rounding in a focal pattern. • Cell fusionBlue arrows -cytoplasmic stranding.20x objective . IMP: view at a low MOI
  • 15. 4) Swelling and clumping • Adenovirus • Cells greatly enlarge and clump together in ‘grape-like’ clustersBovine fetal spleen cells 4 days postinfection with a high MOIof the bovine adenovirus, an Adenovirus, showing cellrounding and small amounts of clumping.10X objective
  • 16. 5) Foamy degeneratioon • d/t large/numerous cytoplasmic vacuoles • Retrovirus • Paramyxovirus • Flavivirus • Difficult to see withoutGiemsa-stained bovine fetal spleen cells 1 day stainingFlavivirus, showing vacuoles (arrow).40X objective
  • 17. 6) Cell fusion • Syncytium/polykaryon • Plasma membranes of 4 or more cells fuse • Enlarged cell with 4 or more nuclei • Easily seen when stained • To be distinguished from cellGiemsa-stained bovine fetal clumping/clusteringspleen cells 4 days • Paramyxoviruspostinfection with the • Herpesvirusbovine respiratory syncytialvirus, a Paramyxovirus,showing syncytia (arrows)and faint basophiliccytoplasmic inclusionbodies (dashed arrows).20x objective
  • 18. 7) Inclusion bodies • Areas of altered cell staining • CANNOT be seen in live cell cultures • Single or multiple- depends on virus • Large/small • Round/irregular • Intranuclear/intracytoplasmic • Eosinophilic/basophilic • Chromatin margination • Indicate areas of viral protein synthesis or nucleic acid synthesis or virion assembly • Some cases no virus is present Herpesvirus, 40x. Viral scarring
  • 19. Cytoplasmic stranding Adenovirus, 20x.Intranuclear inclusion Rough-edged nuclear inclusion bodies
  • 20. Poxvirus, showing pink eosinophilic cytoplasmic Feline nasal turbinate primary cell culture infectedinclusion bodies (arrows) and cell swelling near the with feline herpes virus-1, a Herpesvirus, showingtop of the field, 20x. chromatin margination (dark ring around the edge of the nucleus).
  • 21. CPE of Common Viruses • MEASLES o dilated skin vessels, edema o Lymph nodes, lungs o Warthin-Finkeldey cells
  • 22. • MUMPS o Parotitis o Orchitis o Pancreas o Rarely encephalitisMumps parotitisEnlarged glands, C/S reddish brown,Glistening.Macrophage, lymphocyte infiltration.Oedematous
  • 23. • HERPES SIMPLEX o Fever blisters o Gingivostomatitis o Genital herpes o Herpes epithelial keratitis o Herpes stromal keratitis o Cowdry type A inclusion bodies • Pink to purple intranuclear inclusions Glassy intranuclear herpes simplexinclusion bodies
  • 24. • Varicella-Zoster Virus o Chickenpox • Intranuclear inclusions o Shingles/ Herpes zoster • Reactivates in dorsal nerve root ganglia • Infects keratinocytes • Vesicular lesions
  • 25. • Cytomegalo virus (CMV) o Intranuclear basophilic inclusions o Clear halo o Enlarged cells (40 microns) o “Owl eyes” o Cellular and nuclear pleomorphism
  • 26. • Epstein-Barr virus (EBV) o causes infectious mononucleosis o Absolute lymphocytosis (T cells) o 5-80% are atypical, large o Multiple clear vaculations in cytoplasm o Express CD8 o RS like cell may be seen
  • 27. • HPV- LSIL o Multinucleation o Perinuclear halos o Nuclear enlargement o Hyperchromasia (KOILOCYTES)
  • 28. • HPV Thickened spuamous epithelium (Acanthotic)
  • 29. • RABIES o Negri bodies o Eosinophilic , sharply outlined o 2-10 microns diameter o Cytoplasmic o RIBONUCLEAR PROTEINS of Rabies virus. o Found in • Pyramidal cells of Ammon’s horn • Purkunje cells of cerebellum
  • 30. 1. References Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7627/2. http://www.microbelibrary.org/component/resource/laboratory-test/2875-cytopathic- effects-of-viruses-protocols3. http://classes.midlandstech.edu/carterp/Courses/bio225/chap15/lecture4.htm4. http://en.wikipedia.org/wiki/John_Franklin_Enders5. Thomas C. Wright, Jr.; Pathology of HPV infection at the cytologic and histologic levels: Basis for a 2-tiered morphologic classification system; International Journal of Gynecology and Obstetrics (2006) 94 (Supplement 1), S22---S316. Robbins & Cotran; Pathologic Basis of Disease; 8th edition