Virology Review

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Virology Review

  1. 1. Virology Review Just the basics!
  2. 2. Diagnostic techniques used in the Virology Laboratory
  3. 3. Laboratory diagnosis  Direct antigen detection from lesions  Direct Fluorescent antibody (DFA) stain  Collect cells from base of vesicular lesion  Stain with Fl antibody specific for HSV and/or VZV  Look for fluorescent cells using fluorescence microscope  Can provide a HSV and VZV diagnosis  More sensitive and specific than Tzanck prep (DFA 80% vs. Tzanck 50%)  Tzanck prep= Giemsa stain/examine for multinucleated giant cells of Herpes virus Tzanck
  4. 4. Rapid detection of viral antigens  Enzyme immunoassay –  Antigen/antibody complex formed – then combined with color forming compound  Detection of non-culturable viruses – such as Rotavirus  Detection of Influenza A and B , and Respiratory syncytial virus (RSV)  Membrane EIA Liquid/well EIA
  5. 5. Molecular Detection  Amplification of DNA or RNA  Rapid results  Exceed sensitivity of culture  Standard of practice for detecting respiratory viruses  Standard of practice for HSV and Enterovirus detection in CSF  Culture <=20% PCR >=90%  CMV quantitative assays in transplantation  Hepatitis B and C detection and viral load  HIV viral load  Test of diagnosis not cure – can retain DNA/RNA for 7 – 30 days after initial diagnosis
  6. 6. Viral Cell Culture  Viral cell culture  Inner wall coated with monolayer of cells covered with liquid maintenance media  Primary cell lines – directly from animal into tube (Rhesus monkey kidney-RMK)  Diploid cell lines– Can survive 20 – 50 passes into new vials – fibroblast cells MRC-5-(Microbiology Research Council 5) human diploid fibroblasts  Continuous cell lines – can survive continuous passage into new vials, usually of tumor lineage, HEp-2 and HeLa
  7. 7. Viral Cell culture  Cytopathic effect – CPE  Appearance of cell monolayer after being infected with a virus  Specific for each virus type
  8. 8. Spin Down Shell Vial Method Designed to speed up virus recovery Cells are on the round coverslip Specimen put into vial Centrifugation to induce virus invasion Incubate 24 - 72 hours DFA stain cells on coverslip with early antigen for virus of interest Cover slip
  9. 9. Specimen collection and transport  Viral transport media- Hanks balanced salt solution with antibiotics, needed for the transport of lesions, mucous membranes and throats to the laboratory I
  10. 10. Which viruses will survive the trip to the laboratory?  Most likely viable - HSV  Intermediate  Adenovirus  Influenza A and B  Enterovirus  Least likely to survive  Respiratory Syncytial Virus (RSV)  Cytomegalovirus (CMV)  Varicella Zoster virus (VZV)  Amplification preferred due to survival issue
  11. 11. Which viruses grow the fastest in conventional cell culture?  Fast (>=24 hours)  HSV  Intermediate (5 -7 days)  Adenovirus Enterovirus  Influenzae VZV  Slow (10 - 14 days)  RSV  Slowest (14 - 21 days)  CMV  Molecular superior for slow growers
  12. 12. Herpesviridae
  13. 13. Herpes Viruses  Double stranded DNA virus  Eight human Herpes viruses  Herpes simplex 1  Herpes simplex 2  Varicella Zoster  Epstein Barr  Cytomegalovirus  Human Herpes 6, 7, and 8  Latent infection with recurrent disease is the hallmark of the Herpes viruses  Latency occurs within small numbers of specific kinds of cells, the cell type is different for each Herpes virus
  14. 14. Herpes simplex virus 1 and 2  Transmission: direct contact/secretions  Latency: dorsal root ganglia *  Disease –  Gingivostomatitis  Herpes labialis  Ocular  Encephalitis  Neonatal  Disseminated in immune suppressed  Therapy – Acyclovir, Valacyclovir
  15. 15. Herpes virus diagnosis Herpes 1 & 2 do well in culture Grow within 24-48 hrs in Human diploid fibroblast cells (MRC-5) / Observe for characteristic CPE  Antigen detection by direct fluorescent staining of cells obtained from vesicular lesions  Amplification methods for diagnosis  Cytology/Histology - intra nuclear inclusions, multinucleated giant cells  Serology – Most helpful to detect past infection
  16. 16. Negative fibroblast cell Culture -uninfected cells HSV infected monolayer Rounded cells throughout the monolayer in cell culture Multinucleated Giant Cells of Herpes Simplex in tissue histology
  17. 17. Varicella Zoster Virus  Transmission: close contact  Latency: dorsal root ganglia  Diseases:  Chickenpox (varicella)  Shingles (zoster – latent infection)  Chicken pox +/- eliminated due to effective vaccine program – most serious disease occurs in immune suppressed or adult patient – can progress to pneumonia and encephalitis  Histology – Herpes simplex multi-nucleated giant cells like those of  Serology useful for immune status check  Amplification useful for disease diagnosis
  18. 18. Varicella-Zoster Diagnosis In cell culture – Limited # of Foci 5- 7 days to develop Sandpaper look to the Background Scattered rounded cells Younger wet vesicular lesions area the best for culture and/or molecular testing
  19. 19. Cytomegalovirus (CMV)  Transmitted by blood transfusion , vertical and horizontal transmission to fetus, close contact  Latency: Macrophages  Disease: Asymptomatic in most individuals infected  Congenital – most common cause  Perinatal  Immunocompromised – Primary disease most serious  Diagnosis: Cell culture CPE (Human diploid fibroblast , PCR and quantitative PCR  Histopathology: Intranuclear and intracytoplasmic inclusions “Owl Eye” Inclusions
  20. 20. CMV pneumonia with viral inclusions CMV infected fibroblast monolayer - Focal grape like clusters of rounded cells
  21. 21. Epstein Barr virus (EBV)  Transmission - close contact, saliva  Latency - B lymphocytes  Diseases include:  Infectious mononucleosis  Lymphoreticular disease  Oral hairy leukoplakia  Burkitt’s lymphoma  Nasopharyngeal Carcinoma  1/3 Hodgkin’s lymphoma  Unable to grow in cell culture  Serology and PCR methods for diagnosis
  22. 22. EBV Serodiagnosis using the Heterophile Antibody  Heterophile antibodies (HA) react with antigens phylogenetically unrelated to the antigenic determinants against which they were raised  HA secondary to EBV are detected by the ability to react with horse or cattle rbcs (theory of the Monospot test)  HA rise in the first 2 - 3 weeks of EBV infection, then rapidly fall at @ 4 weeks  Cannot be used in children < 4 years of age
  23. 23. VCA = viral capsid antibody EBNA = Epstein Barr nuclear antigen EA = early antigen
  24. 24. Human Herpes virus 6, 7 & 8  HH6  Roseola [sixth disease]  6m-2yr high fever & rash  HH7  CMV like Disease  HH8  Kaposi’s sarcoma  Castleman’s disease Onion skin of Castleman disease
  25. 25. Adenovirus
  26. 26. Adenovirus     DNA - non enveloped/ icosahedral virus Latent: lymphoid tissue Transmission: Respiratory and fecal-oral route Diseases:  Adenovirus type 14 – new virulent respiratory strain / pneumonia  Pharyngitis (year round epidemics)  Gastroenteritis in children  Adenovirus types 40 & 41  Keratoconjuctivitis  Disseminated infection in transplant patients  Hemorrhagic cystitis in immune suppressed
  27. 27. Adenovirus Round cells with stranding  Diagnosis  Conventional cell culture (CPE)  2-5 days with round cells connected by strands – Grows best in Heteroploid continuous passage cell lines (HeLA, Hep-2)  Amplification  Histology - Intranuclear inclusions / smudge cells  Stool EIA for enteric infections  Antigen detection – staining respiratory cells by DFA for Respiratory infections  PCR – has become the standard of practice  Supportive treatment – no specific viral therapy
  28. 28. Smudge cells- Adenovirus
  29. 29. Parvoviridae – Parvovirus The smallest known viruses!
  30. 30. Parvovirus  DNA virus  Parvovirus B19 Slapped face appearance of fifth disease  erythema infectiosum (Fifth disease)  fetal infection and stillbirths  aplastic crisis in patients with chronic hemolytic anemia and AIDS  Histology - virus effects mitotically active erythroid precursor cells in bone marrow  Molecular and Serology methods for diagnosis
  31. 31. Papovaviridae Papillomavirus Polyomavirus Infectious and oncogenic or potentially oncogenic DNA viruses
  32. 32. Pap smear Papillomavirus  Diseases:          skin and anogenital warts, benign head and neck tumors, cervical and anal intraepithelial neoplasia and cancer HPV types 16 and 18 = 70% Cervical CA HPV types 6 and 11 = 90% Genital warts Pap Smear for detection Hybrid capture DNA probe for detection and typing PCR – FDA approved platforms for detection/typing Guardasil vaccine = To guard against HPV 6,11,16,18
  33. 33. Polyomavirus Giant Glial Cells of JCV  JC virus [John Cunningham]  Cause of Progressive multifocal leukoencephalopathy   Encephalitis of immune suppressed Destroys oligodendrocytes in brain  BK virus  Causes latent virus infection in kidney  Progression due to immune suppression  Hemorrhagic cystitis  Histology/PCR for diagnosis
  34. 34. Hepadnavirus Hepatitis B
  35. 35. Hepatitis B virus     Enveloped DNA – Hepadna virus Virion called Dane particle Surface Ag (HBsAg)- Australian Ag Clinical Disease  90% acute  1% fulminant  9% chronic  Carrier state …….Hepatic cell carcinoma
  36. 36. Hepatitis B Serology  Surface Antigen Positive  Active Hepatitis B or Chronic Carrier  Do Hep B Quantitation  Do Hep e antigen – Chronic and “bad”  Core Antibody Positive  Immune due to prior infection, acute infection or chronic carrier  Surface Antibody Positive  Immune due to prior infection or vaccine
  37. 37. Flaviviridae RNA Viruses Hepacivirus Hepatitis C Flavivirus West Nile Dengue Yellow Fever
  38. 38. Hepatitis C virus  Spread parenterally - drug abuse, blood products, poorly sterilized medical equipment, sexual  Effects only humans and chimpanzees  Seven major genotypes  Acute self limited disease to start with progressive disease that mainly affects the liver  Infection persists in 80%  20 - 30 % develop cirrhosis  Associated with hepatocellular CA  Require liver transplantation
  39. 39. Hepatitis C  Diagnosis:  Hep C antibody test  If antibody positive do:  RNA qualitative or quantitative assay for viral load  Requires Genotyping for proper therapy  Type 1 most common  No vaccine – Antivirals currently in clinical trials that can cure a large % of infected
  40. 40. Flaviviruses – Mosquito borne  St. Louis  Dengue – breakbone fever  Yellow fever  West Nile Fever, Headache, Muscle weakness Various species Mosquitoes  Serology / PCR
  41. 41. Picornaviridae Enteroviruses Hepatitis A
  42. 42. Enteroviruses  Diverse group of > 60 viruses  infections occur most often in summer and fall  Polio virus - paralysis         Salk vaccine Inactive Polio Vaccine (IPV)**  Sabine vaccine Live Attenuated Vaccine (OPV) Coxsackie A – Herpangina Coxsackie B – Pericarditis/Myocarditis Enterovirus – Aseptic meningitis in children, hemorrhagic conjunctivitis Echovirus – various infections, intestine Rhinoviruses – common cold Grow in cell culture (Diploid mixed cell – Primary Monkey Kidney) PCR superior for diagnosis of meningitis (CSF)
  43. 43. CPE of Enterovirus Teardrop and kite like cells in Rhesus Monkey Kidney cell culture
  44. 44. Hepatitis A  Fecal – oral transmission  Can be cultured but not reliably  Usually – short incubation, abrupt onset, low mortality, no carrier state  Travel  Diagnosis – serology, IgM positive in early infection  Vaccine available
  45. 45. Orthomyxoviruses Influenza virus A Influenza virus B
  46. 46. Influenza A  Segmented RNA genome  Hemagglutinin and Neuraminidase glycoproteins spikes on outside of viral capsid  Give Influenza A the H and N designations – such as H1N1 and H3N2  Antigenic drift - minor change in the amino acids of either the H or N glycoprotein  Cross antibody protection will still exist so an epidemic will not occur  Antigenic shift - genome re assortment with a “new” virus created/usually from a bird or animal/ this could create a potential pandemic  H5N1 = Avian Influenza  H1N1 = 2009 Influenza A
  47. 47. Influenzae A Disease: fever, malaise …. death Diagnosis  Cell culture obsolete [RMK]  Enzyme immunoassay on paper membrane can be used in outpatient setting – Rapid but low sensitivity (60%) and can have specificity issues in off season.  Amplification (PCR) gold standard for Influenza Detection  Treatment: Amantadine and Tamiflu  Seasonal variation in susceptibility  Vaccinate to prevent  Influenza B   Milder form of Influenza like illness Usually <=10% of cases /year
  48. 48. Paramyxoviruses – SS RNA Measles Parainfluenza 1,2,3,4 Mumps Respiratory Syncytial Virus Human Metapneumovirus
  49. 49. Measles  Measles  Fever, Rash, Dry Cough, Runny Nose, Sore throat, inflamed eyes (photosensitive)  Respiratory spread - very contagious  Koplik’s spots – bluish discoloration inner lining of the cheek  Subacute sclerosing panencephalitis [SSPE]  Rare chronic degenerative neurological disease  Persistent infection with mutated measles virus due to lack of immune response  Diagnosis: Clinical symptoms and Serology  Vaccinate – MMR (Measles, Mumps, Rubella) vaccine  Treatment: Immune globulin, vitamin A
  50. 50. Parainfluenzae  Types 1,2,3, and 4  Person to person spread  Disease:  Upper respiratory tract infection in adults – more serious in immune suppressed  Croup, bronchiolitis and pneumonia in children  Heteroploid cell lines (Hep-2) for culture  PCR methods are the gold standard  Supportive therapy
  51. 51. Mumps  Person to person contact  Classic infection is Parotitis, but can cause infections in other sites: T
  52. 52. Respiratory Syncytial Virus  Transmission:  Hand contact and respiratory droplets  Respiratory disease - from common cold to pneumonia, bronchiolitis to croup, serious disease in immune suppressed  Classic disease:  Young infant with bronchiolitis  Specimen: Naso-phayrngeal, nasal swab, nasal lavage  Diagnosis: EIA, cell culture (heteroploid cell lines), PCR is standard practice  Treatment: Supportive, ribavirin
  53. 53. Classic CPE = Syncytium formation In heteroploid cell line Respiratory syncytial virus CPE Histology
  54. 54. Human Metapneumovirus  1st discovered in 2001 – community acquired respiratory tract disease in the winter  Common in young children – but can be seen in all age groups  @95% of cases in children <6 years of age  Upper respiratory tract disease  2nd only to RSV in the cause of bronchiolitis  Will not grow in cell culture  Amplification (PCR) for detection  Specimen: Nasal swab or NP  Treatment: Supportive
  55. 55. Reoviridae Rotavirus
  56. 56. Rotavirus  Winter - spring season  6m-2 yrs of age,  Gastroenteritis with vomiting and fluid loss – most common cause of severe diarrhea in children  Fecal – oral spread  Major cause of death in 3rd world  Diagnosis – cannot grow in cell culture  Enzyme immunoassay, PCR  Vaccine available
  57. 57. Calciviruses Norovirus
  58. 58. Norovirus  Spread by contaminated food and water, feces & vomitus – takes <=20 virus particles to cause infection – so highly contagious  Tagged the “Cruise line virus” – numerous reported food borne epidemics on land and sea  Leading cause of epidemic gastroenteritis – more virulent GII.4 Sydney since spring 2012  Fluid loss from vomiting  Disease course usually limited, 24-48 hours  PCR for diagnosis  Cannot be grown in cell culture
  59. 59. Retrovirus RNA Virus/Reverse Transcriptase Enzyme Human Immunodeficiency Virus HIV
  60. 60. Human Immunodeficiency virus  CD4 primary receptor to gain entry for HIV into the lymphocyte  Reverse transcriptase enzyme converts genomic RNA into DNA  Transmission - sexual, blood and blood product exposure, perinatal  Non infectious complications:  Lymphoma, KS, Anal cell CA, non Hodgkins Lymphoma
  61. 61. HIV  Diagnosis  Antibody EIA with Western Blot confirmation  Antibody test alone is NOT sufficient  Western blot detects gp160/gp120 (envelope proteins), p 24 (core), and p41(reverse trans)  Must have at least 2 bands on Western blot to confirm the diagnosis of HIV  Positive patients require additional testing  HIV RNA/DNA quantitation >= 100 copies  Resistance Testing – report subtype  Most isolates in USA type B  Monitor CD4 counts for infection severity
  62. 62. HIV infectious complications  Non-compliant patients or newly diagnosed        Pneumocystis C. neoformans and Histoplasma TB/Mycobacterium avium complex Microsporidia and Cryptosporidium Hepatitis B Hepatitis C STD’s – Syphilis, GC, Chlamydia  Syphilis rate high (mucosal contact)
  63. 63. Togaviridae RNA Virus Rubella
  64. 64. Rubella  Respiratory transmission  Known as the “Three day measles” – German measles  Congenital rubella – occurs in a developing fetus of a pregnant women who has contracted Rubella, highest % in the first trimester pregnancy  Diagnosis - Serology in combination with clinical symptoms – Rash, low fever, cervical lymphadenopathy  Live attenuated vaccine (MMR) to prevent
  65. 65. Bunyaviridae enveloped RNA viruses Hantavirus
  66. 66. Hantavirus  USA outbreak in four corners (NM,AZ,CO,UT) Indian reservation in 1993 brought attention to this virus  Source - Urine and secretions of wild field mice  Deer mouse and cotton rat  Myalgia, headache, cough and respiratory failure  Found in states west of the Mississippi River  Diagnosis by serology/ no therapy
  67. 67. Poxviruses Smallpox virus (Variola virus) Vaccinia virus
  68. 68. Smallpox  Smallpox virus is also known as the Variola virus  Vaccinia virus = vaccine strain used in Smallpox vaccine, it is immunologically related to smallpox, Vaccinia can cause disease in the immune suppressed, which prevents vaccinated this population  Last case of Smallpox - Somalia in 1977  Disease begins as maculopapular rash and progresses to vesicular rash - all lesions in same stage on a body area – rash moves from central body outward  Potential agent of Bioterrorism  Any potential cases directly reported to public health department – they will investigate and diagnose
  69. 69. Chickenpox vs Smallpox lesions Chicken pox – Lesions of different stage of development Smallpox – all lesions same stage of development
  70. 70. Rhabdoviruses bullet shaped RNA virus Rabies virus
  71. 71. Rabies  Worldwide in animal populations  Bat and raccoons primary reservoir in US  Dogs in 3rd world countries  Post exposure shots PRIOR to the development of symptoms prevent infection  Rabies is a neurologic disease – classic sympton is salivation, due to paralysis of throat muscles  Detection of viral particles in the brain by Histologic staining known as Negri bodies  Public health department should be contacted to assist with diagnosis
  72. 72. Rabies virus particles EM showing the bullet shaped virus Negri bodies – intracytoplasmic

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