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Virology 2014

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Virology 2014

  1. 1. Virology Review Margie Morgan, PhD, MT(ASCP), D(ABMM) microbeswithmorgan.com
  2. 2. Diagnostic techniques used in the Virology Laboratory 1.Direct Staining for Antigen 2. Enzyme Immunoassay 3. Molecular Amplification 4. Viral Cell Culture
  3. 3. Detect Antigen in lesion 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  Fluorescent cells = viral infected cells  More sensitive & specific method than Tzanck prep (DFA 80% vs. Tzanck 50%)  Tzanck prep= Giemsa stain of lesion cells/examine for multinucleated giant cells of Herpes virus Tzanck Tzanck DFA
  4. 4. Detection of Viral Antigens by EIA  Enzyme immunoassay –  Antigen/antibody complex formed – then bound to a color producing substrate  Used most for detection of non-culturable viruses – such as Rotavirus from stool  Detect Influenza A and B , & Respiratory syncytial virus (RSV) from nasal/NP swab  Membrane EIA Liquid/well EIA
  5. 5. Molecular Amplification  Molecular Amplification (DNA or RNA)  Rapid/Sensitive/Specific for numerous viruses  Exceeds sensitivity of culture/replacing culture  Standard of practice for detecting respiratory viruses  Standard of practice for HSV and Enterovirus detection from CSF  Culture <=20% PCR >=90%  Quantitative assays in transplantation - CMV  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  Inner wall coated with monolayer of cells lines covered with liquid maintenance media  Three basic types of cell lines:  Primary cell lines – directly from animal organ into culture tube (Rhesus monkey kidney-RMK)  Diploid cell lines– Can survive 20 – 50 passes into new vials – human diploid fibroblast cells, example: MRC-5-Microbiology Research Council 5  Continuous cell lines – can survive continuous passage into new vials, usually of tumor lineage, HEp-2 and HeLa
  7. 7. Viral Cell culture  Tubes/flasks read under microscope for Cytopathic effect/ CPE  Appearance of cells in the monolayer after being infected with a virus  Destruction is specific for each virus type
  8. 8. Spin Down Shell Vial Virus Culture - • Designed to speed up virus recovery • Cells are on the round coverslip • Specimen inoculated into vial • Centrifuge vial to induce virus invasion into cell monolayer • Incubate @ 35*C, 24-72 hours • Direct fluorescent antibody stain to stain cells on coverslip – target early • antigens for virus of interest Cover slip
  9. 9. Specimen collection and transport  Viral transport media (VTM) - Hanks balanced salt solution with antibiotics,  Also known as Universal Transport Media  needed for the transport of lesions, mucous membranes and throats to the laboratory  It is cell protective, protect the cell / protect the virus  Short term transport storage 4˚C  Long term transport(>72hours) storage-70˚C  VTM specimens filtered (45nm filter) to eliminate bacteria in specimen prior to being placed on cell monolayer
  10. 10. Which viruses will survive the trip to the laboratory?  Most likely to survive - HSV  Intermediate  Adenovirus  Influenza A and B  Enterovirus  Least likely to survive  Respiratory Syncytial Virus (RSV)  Cytomegalovirus (CMV)  Varicella Zoster virus (VZV)  Amplification preferred for these viruses due to survival issues
  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  Amplification methods are 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 (nucleoside analogs)
  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 available for detection from lesions and bodily fluids  Cytology/Histology - intra nuclear inclusions, multinucleated giant cells  Serology – More helpful to detect past infection HSV1 and HSV2 can x-react in serology
  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 disease has decreased due to effective vaccine program – most serious disease occurs in immune suppressed or adult patients which progresses to pneumonia and encephalitis  Histology – multi-nucleated giant cells like those of Herpes simplex  Serology useful for immune status check  Amplification useful for disease diagnosis
  18. 18. Varicella-Zoster Diagnosis In cell culture – Limited # of Foci in monolayer Require 5- 7 days to develop Sandpaper look to the Monolayer background with scattered rounded cells - diploid fibroblast monolayer 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, also by close contact  Latency: Macrophages  Disease: Infection asymptomatic in most individuals  Congenital – most common cause of TORCH  Perinatal  Immunocompromised – Primary disease most serious  Laboratory Diagnosis:  Cell culture CPE (Human diploid fibroblast)  PCR and quantitative PCR (best method)  Histopathology: Intranuclear and intracytoplasmic inclusions “Owl Eye” Inclusions Treatment:ganciclovir, foscarnet, cidofovir
  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 available for diagnosis EBV infection with B cell transformatin
  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 to diagnose 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 – virulent respiratory strain / pneumonia  Pharyngitis (year round epidemics)  Gastroenteritis in children  Adenovirus types 40 & 41  Keratoconjuctivitis – very red eyes @ 2 wks  Disseminated infection in transplant patients  Hemorrhagic cystitis in immune suppressed
  27. 27. Adenovirus  Diagnosis  Conventional cell culture (CPE) Round cells with stranding  2-5 days with round cells connected by strands – Grows best in Heteroploid continuous passage cell lines (HeLA, Hep-2)  Amplification (PCR) is best for respiratory infection  Histology - Intranuclear inclusions / smudge cells  Stool EIA for enteric infections  Antigen detection – staining respiratory cells by DFA for Respiratory infections  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  Erythema infectiosum (Fifth disease)  Cause fetal infection and stillbirths  Aplastic crisis in patients with chronic hemolytic anemia and AIDS  Histology - virus infects mitotically active erythroid precursor cells in bone marrow  Molecular and Serology methods for diagnosis Slapped face appearance of fifth disease
  31. 31. Papovaviridae Papillomavirus Polyomavirus Infectious and oncogenic or potentially oncogenic DNA viruses
  32. 32. Papillomavirus  Diseases: Pap smear  skin and anogenital warts,  benign head and neck tumors,  cervical and anal intraepithelial neoplasia and cancer  HPV types 16, 18, & 45 = 94% Cervical CA  HPV types 6 and 11 = 90% Genital warts  Pap Smear for detection  Hybrid capture DNA probe for detection and typing  PCR – FDA cleared platforms for detection/typing  Gardasil vaccine = To guard against HPV 6,11,16,18
  33. 33. Polyomavirus  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 Giant Glial Cells of JCV
  34. 34. Hepadnavirus Hepatitis B
  35. 35. Hepatitis B virus  Enveloped DNA – Hepadna virus  Hepatitis B clinical disease  90% acute  1% fulminant  9% chronic  Carrier state can lead to cirrhosis and hepatic cell carcinoma  newer therapies – stops disease progression  Vaccinate to prevent
  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, and Yellow Fever
  38. 38. Hepatitis C virus  Spread parenterally - drug abuse, blood products or organ transplants (prior to 1992), poorly sterilized medical equipment, sexual (low risk)  Effects only humans and chimpanzees  Approx 3.2 mil persons in USA have chronic Hep C  Seven major genotypes (1-7)  Acute self limited disease that progresses to a disease that mainly affects the liver  Infection persists in @ 75-85%/ no symptoms  5 - 20 % develop cirrhosis  1-5 % associated with hepatocellular CA  Require liver transplantation
  39. 39. Hepatitis C  Diagnosis:  Hepatitis C antibody test  If antibody positive do:  RNA qualitative or quantitative assay for viral load  Requires Genotyping for proper therapy  Type 1 Hep C most common in USA  No vaccine – Antivirals currently in clinical trials and/ or FDA cleared that can cure >= 85% of infected with Hepatitis C
  40. 40. Flaviviruses – Mosquito borne  Dengue – “breakbone fever”  Aedes mosquito / Asia and the Pacific  Fever, severe joint pain, rash  Small % progress to hemorragic fever  West Nile Common across the US, Bird primary reservoir  Fever, Headache, Muscle weakness, 80% asymptomatic. Small % progress to encephalitis. Meningitis, flaccid paralysis  Mosquitoes – Aedes & Culex  Immunoassays for Antibody & PCR  Serum and CSF
  41. 41. Alpha virus – Mosquito borne  Chikungunya virus – RNA virus  Mosquito borne – Aedes mosquito  Origin in Asia and African continents  Recent migration to the Caribbean and SE USA with mosquito migration  Travel advisory to the Caribbean  Acute febrile illness with rash followed by extreme joint pain, less fatalities than Dengue / no hemorrhagic phase  Diagnosis – Serology(IgM, IgG) and PCR
  42. 42. Ebola Virus  >20 outbreaks since discovery in 1976  current outbreak Dec 2013 - West Africa  Prolonged due to area effected is high population with limited medical facilities  Transmission direct contact with bodily fluids – fatality rate 55%  Animal reservoir (?) fruit bats  Asymptomatic are not contagious  Fever, weakness, myalgias, headache, travel history  Consider malaria and typhoid  Susceptible to hospital disinfectants  Testing (EIA, PCR) at CDC – pos >= 4 days of illness
  43. 43. Coronovirus/SARS  Severe Acute Respiratory Syndrome (SARS)  Outbreak in China 2003 – spread to 29 countries  Incubation period of 2-10 days  2-7 days by dry cough and/or shortness of breath  Development of radiographically confirmed pneumonia by day 7-10 of illness Lymphopenia in most cases  Laboratory testing for SARS-CoV available at state public health laboratories. Available tests include antibody testing enzyme immunoassay (EIA) and reverse transcription polymerase chain reaction (RT-PCR) tests for respiratory, blood, and stool specimens. In the absence of person-to-person transmission of SARS-CoV, the positive predictive value of a diagnostic test is extremely low.
  44. 44. MERS CoV- Middle East Respiratory Syndrome Coronavirus  Isolated to Arabian peninsula (2012)  Close human to human contact can spread infection – no outbreaks  2 unrelated cases in US from travel  Fever, rhinorrhea, cough, and malaise followed by shortness of breath – 30% fatality rate  NP, Lower respiratory specimen and serum for PCR at CDC
  45. 45. Picornaviridae Enteroviruses Hepatitis A
  46. 46. Enteroviruses  Diverse group of > 60 viruses – SS RNA  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) and more rapid and sensitive for all sites
  47. 47. CPE of Enterovirus Teardrop and kite like cells in Rhesus Monkey Kidney cell culture
  48. 48. 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
  49. 49. Orthomyxoviruses Influenza virus A Influenza virus B
  50. 50. 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
  51. 51. Influenzae A Disease: fever, malaise …. death Diagnosis  Cell culture obsolete [RMK]  Enzyme immunoassay (EIA) 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 (Oseltamivir)  Seasonal variation in susceptibility  Vaccinate to prevent  Influenza B  Milder form of Influenza like illness  Usually <=10% of cases /year
  52. 52. Paramyxoviruses – SS RNA Measles Parainfluenza 1,2,3,4 Mumps Respiratory Syncytial Virus Human Metapneumovirus
  53. 53. Measles  Measles Measles syncytium  Fever, Rash, Dry Cough, Runny Nose, Sore throat, inflamed eyes (photosensitive)  Can invade lung (see HE of Lung)  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
  54. 54. 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
  55. 55. Mumps  Person to person contact  Classic infection is Parotitis, but can cause infections in other sites: Testes/ovaries, Eye, Inner ear, CNS  Diagnosis: clinical symptoms, serology available  Prevention: MMR vaccine  No specific therapy, supportive
  56. 56. 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
  57. 57. Classic CPE = Syncytium formation In heteroploid cell line Respiratory syncytial virus CPE Histology
  58. 58. 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
  59. 59. Reoviridae Rotavirus
  60. 60. 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
  61. 61. Calciviruses Norovirus
  62. 62. 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 can be debilitating  Disease course usually limited, 24-48 hours  PCR for diagnosis  Cannot be grown in cell culture
  63. 63. Retrovirus RNA Virus/Reverse Transcriptase Enzyme Human Immunodeficiency Virus HIV
  64. 64. 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
  65. 65. HIV Laboratory Diagnosis Antibody EIA with Western Blot confirmation (old way)  Antibody test alone is NOT sufficient – all positive must be confirmed with a western blot test  Western blot detects gp160/gp120 (envelope proteins), p 24 (core), and p41(reverse trans)  Must have at least 2 solid bands on Western blot to confirm as a positive result New test - Antigen/antibody combination (4th generation) immunoassay* that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen to screen for established infection with HIV- 1 or HIV-2 and for acute infection Positive patients on either test 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
  66. 66. HIV infectious complications  Non-compliant patients or newly diagnosed  Pneumocystis  Cryptococcus neoformans & Histoplasma (disseminated)  TB/Mycobacterium avium complex (disseminated)  Microsporidia and Cryptosporidium (stool)  Hepatitis B  Hepatitis C  STD’s – Syphilis, GC, Chlamydia  Syphilis rate high (mucosal contact)
  67. 67. Togaviridae RNA Virus Rubella
  68. 68. Rubella  Known as the “Three day measles” – German measles  Congenital rubella – occurs in a developing fetus of a pregnant women who has contracted Rubella, highest % (50%) in the first trimester pregnancy  Deafness, eye abnormalities, congenital heart disease  Respirastory transmission  Diagnosis - Serology in combination with clinical symptoms – Rash, low fever, cervical lymphadenopathy  Live attenuated vaccine (MMR) to prevent
  69. 69. Bunyaviridae enveloped RNA viruses Hantavirus
  70. 70. 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 most implicated  Myalgia, headache, cough and respiratory failure  Found in states west of the Mississippi River  Diagnosis by serology/ no therapy
  71. 71. Poxviruses Smallpox virus (Variola virus) Vaccinia virus
  72. 72. Smallpox  Smallpox virus is also known as the Variola virus  Vaccinia virus is the strain used in Smallpox vaccine, it is immunologically related to smallpox, Vaccinia can cause disease in the immune suppressed, which prevents vaccination of this population  Last case of Smallpox - Somalia in 1977  Disease begins as maculopapular rash and progresses to vesicular rash - all lesions in same stage of developemnt in body area – rash moves from central body outward  Category A Bioterrorism agent (can maim or kill)  Requires BSL4 laboratory (self contained lab)  Any potential cases directly reported to public health department – they will investigate and diagnose
  73. 73. Chickenpox vs Smallpox lesions Chicken pox – Lesions in different stage of development Smallpox – all lesions same stage of development
  74. 74. Rhabdoviruses bullet shaped RNA virus Rabies virus
  75. 75. 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 is diagnostic  Public health department should be contacted to assist with diagnosis
  76. 76. Rabies virus particles EM showing the bullet shaped virus Negri bodies – Intracytoplasmic brain biopsy specimen

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