1. Viral Diagnostics Jonathan Gubbay Ontario Agency for Health Protection and Promotion Public Health Laboratory-Toronto

2. Overview Clinical virology lab can provide significant benefit to patient care Traditionally epidemiologic and academic role Current rapid assays impact on therapeutic and public health decisions. –Change largely due to molecular methods

3. Impact of PCR on virology Recent identification of several respiratory viruses –Metapneumovirus –Multiple coronaviruses: SARS, 229E, NL63, OC43, HKU1. –Human bocavirus –Polyomaviruses KI, WU

4. Why Expanding Role for Diagnostic Virology Lab Increased pool of immunocompromised Increasing antiviral agents Results in increasing demand for rapid methods, viral load testing, antiviral susceptibility, genotyping.

5. Methods in use in virology. Detecting Active Infection: –Electron Microscopy –Viral culture –Detection of viral antigens –Detection of viral nucleic acid. –Histopathology Assessing virus-specific immune response –Serologic testing (won’t cover today)

6. Specimen choice and collection Specimen quality limits test quality Pathogen detection depends on: –Appropriate collection site. –Proper timing of specimen collection. –Effective and timely processing of sufficient specimen.

7. Specimen storage and transport Keep specimens other than blood at 4ºC If delay >24hrs, freeze at 70ºC or below. Avoid any storage at -20ºC: greater loss in infectivity Nonenveloped viruses (adenovirus, enteroviruses) more stable than enveloped (e.g. RSV, VZV, CMV).

8. Viral Transport Medium Salt solution – ensures proper ionic concentrations Buffer - maintains pH Protein - for virus stability Antibiotics or antifungals – to prevent contamination

9. Cell Culture Viruses are obligate intracellular organisms – require living cells for virus isolation Advantages: –Relatively sensitive and specific –Can detect many different viruses –Provides a viral isolate for further characterization (serotyping, genotyping, susceptibility)

10. Cell culture –limitations Certain viruses don’t grow or grow slowly Other techniques for detecting viral infection more cost effective Successful culture depends on viability of virus in specimen

11. Standard Cell Cultures Originally used animals and embryonated eggs. Monolayer cell culture techniques (1933) Roller tube cell cultures (1940)

12. Standard Cell Cultures Primary cells –1-2 passages Diploid (semicontinuous) cells –20-50 passages Heteroploid cells. –Indefinite passages

13. Cytopathic Effect Monitor tube cultures daily initially Monitor for 10-21 days Compare to uninoculated controls from same batch Rounding, refractile cells, syncytium formation, cell destruction

14. Cell culture – clues to virus causing CPE Type of specimen Cell line displaying CPE Type of CPE

15. Hemadsorbing viruses Orthomyxoviruses (influenza) and some paramyxoviruses (parainfluenza, measles, mumps) Insert viral glycoproteins (haemaglutinin) into host cell membrane. Promotes attachment of RBC of certain species (e.g guinea pig) to cell membrane.

16. Interference Rubella virus growing in monkey kidney cells inhibits infection with echovirus 2.

17. Adenovirus CPE

18. RSV - syncytia

19. Cell culture – definitive identification Reaction with monoclonal antibodies. Antibodies chemically conjugated to a fluorochrome. Indirect or direct immunofluorescence. Neutralization – use monospecific or pooled antisera to prevent infection of susceptible cells (enterovirus serotyping)

20. Shell Vial Culture System which detects viral infection prior to CPE developing. Low speed centrifugation enhances infection. First used for CMV –MRC5 cells, stain for early antigen protein after 18- 48hr incubation Also developed for other viruses: VZV, HSV, adenovirus, respiratory viruses

21. CMV early antigen staining

22. Mixed Cell Culture Shell vial culture with mixed cell lines Allows detection of wider range of viruses. R-Mix TM : mink lung + A549 –RSV, parainfluenza 1-3, influenza, adenoviruses R-Mix Too: MDCK + A549 –Above plus metapneumovirus E-Mix: BGMK + A549

23. Recent R-Mix Too vs Rhesus Monkey Kidney at PHL-Toronto 257 stored primary samples; 194 positive R-Mix shell vials stained at 1,2 and 5d Tube culture monitored for 10d R-Mix detected 67.5% of and tube culture 45.9% of previously positives Of pos R-Mix, 73.3% and 95.5% detected after 1 and 2 days. Of pos TC, 2%, 25% and 60% isolated by day 1,2 and 7; 36% took 10 days to be isolated.

24. Genetically Engineered Cell Lines HSV –Baby hamster kidney (BHK-21) transformed with an HSV-inducible promoter (UL39 gene) –UL39 attached to functional E Coli β-galactosidase gene. –β-galactosidase activity induced by HSV 1 or 2 infection. –Addition of substrate (X-Gal) for this enzyme results in coloured product in HSV-infected cells. –Commercially available as enzyme-linked virus- inducible system (ELVIS HSV ID).

25. Genetically Engineered Cell Lines Rapid detection after overnight incubation.

26. Antiviral Susceptibility Testing May do for herpesviruses when: –HSV or VZV cutaneous lesions fail to resolve or appearance of new ones while on oral therapy. –Progressive retinal or visceral CMV disease while patient on therapy.

27. Ganciclovir-resistant CMV First reported ganciclovir R CMV among HIV-infected with CD4 <50 x 10 9 / Emerging problem in HSCT and SOT patients. R testing now recommended for HIV treatment failure; many do routinely at diagnosis

28. CDC Issues Interim Recommendations for the Use of Influenza Antiviral Medications in the Setting of Oseltamivir Resistance among Circulating Influenza A (H1N1) Viruses, 2008-09 Influenza Season Friday, December 19, 2008, 11:50 EST (11:50 AM EST) http://www2a.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?AlertNum=00279 Resistance testing now important in influenza therapy

29. Phenotypic assays Measure effect of antiviral drug on growth of a virus. Measured by infectivity (plaque reduction), viral antigen or viral nucleic acid production, enzyme activity. Directly measure and quantify effects of antivirals on growth. Slow, labour intensive, difficult to standardise

30. Phenotypic assays Expressed as drug concentration that inhibits 50% or 90% of viral growth (IC 50 and IC 90 ) relative to a no drug control. Phenotypic susceptibility assays in use for herpesviruses (CMV, HSV, VZV), influenza and HIV-1. Plaque reduction assay for HSV susceptibility approved by CLSI.

31. Plaque Reduction Assay Proposed breakpoints for HSV and CMV S, I, R based on IC 50. Many variables effect results –Cell line –Viral inoculum –Incubation period

32. Plaque Reduction Assay http://pathmicro.med.sc.edu/mhunt/plaque.jpg

33. Phenotypic susceptibility to neuraminidase inhibitors Directly measure NA activity Viral NA incubated with different concentrations of NA inhibitors. Fluorescent or chemiluminescent substrate added and quantitated.

34. Phenotypic assays for HIV susceptibility Traditionally measured p-24 antigen in cell culture by EIA in presence of antiviral drug. Recombinant virus phenotypic assays –Insertion of RT and polymerase from patient into a vector consisting of a rapidly replicating viral strain and a reporter gene (luciferase). –Measure viral growth in presence of drug compared to wild type virus. (Phenosense, Antivirogram)

35. Genotypic Assays Allow rapid detection of genetic mutations associated with antiviral drug resistance. 1. Nucleic acid amplification 2. Sequencing of amplified product 3. Compare amplicon sequence to reference strain

36. Genotypic Assays Most useful when a discrete number of known resistance mutations –CMV mutations causing ganciclovir resistance. UL 97 (protein kinase) – ganciclovir UL54 (DNA polymerase) – ganciclovir, foscarnet, cidofivir UL54 (DNA polymerase) – ganciclovir, foscarnet, cidofivir –Antiretroviral-refractory HIV infections. Amplification and sequencing of RT and polymerase genes

37. Genotypic Assays –Lamivudine R in Hep B –M2 mutations in amantadane R Influenza A. –NA (H275Y) and/or hemagluttinin mutations in Influenza A associated with NA inhibitor R.

38. Direct Detection of Virus or Viral Antigen: Electron Microscopy Superseded by other methods in most labs Still important for rapid detection of viruses in clinical samples. Able to detect multiple pathogens

39. Direct Detection of Virus or Viral Antigen: Electron Microscopy Specimen absorbed onto thin plastic/carbon film. Applied to surface of EM grid before staining. Can use positive or negative stain (most common – phosphotungstic acid) –Penetrates virion and provides contrast for visualization of cell surface.

40. Direct Detection of Virus or Viral Antigen: Electron Microscopy Quick Looks for many viruses Useful if unknown pathogen Less prone to cross contamination vs molecular. Expensive equipment, need expertise to read Not well suited to screening large numbers of samples. Low sensitivity – need 10 5 -10 8 viral particles/ml to detect.

41. Adenovirus http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Safrica/adeno3.htm

42. Human Rotavirus

43. Paramyxovirus (Parainfluenza)

44. Histopathology/Cytology Some viruses produce characteristic cytologic/histologic changes Not enough time to go into detail now….

45. Direct Detection of Viruses: Immunoassays Utilize antibodies (monoclonal or polyclonal) against specific viral antigen/antigens. Ag/Ab complexes detected by: –Direct visualization –Solid Phase Enzyme immunoassays –Enzyme Linked immunoassays

46. Direct Visualization: direct method Direct or indirect staining methods Direct: antibody conjugated with either an enzyme (e.g horseradish peroxidase) or fluorescent label (e.g FITC). Substrated added to Ag/Ab-Conjugate on glass slide, resulting in colour. Ag/Ab-FITC visualized using fluorescent microscope.

47. Direct visualization: indirect method An unlabelled (e.g mouse) antibody is added to bind to the antigen of interest A second antibody labelled with conjugate is added. Visualized using substrate as with direct method Indirect allows amplification of signal – many more labelled antibodies can bind to the intermediate unlabelled one.

48. Direct and indirect immunofluorescence DFA – direct immunofluoresence. IFA – indirect immunofluoresence. Widely used in virology Cheap, easy to do. Monoclonal abodies give high specificity. Pooling of monoclonals can detect multiple viruses – e.g resp virus DFA Used for CMV pp65 antigenemia quantification

49. DFA RSV

50. CMV pp65 antigen http://ibmi.mf.uni-lj.si/acta-apa/acta-apa-00-3/Marin.html

51. ELISAs/EIAs Surface of solid phase (microtitre plate) coated with antibody Antigen of interest binds if present. Second enzyme-conjugated antibody added Substrate added and colour generated/read by spectrophotometer.

52. http://www.dshs.state.tx.us/lab/images/eia_1.jpg

53. ELISAs/EIAs Sometimes second antibody is unlabelled and use 3 rd labelled antibody. Most give qualitative result, some quantitative. Newer EIAs use microbeads as the solid phase to increase surface area of contact. –Decreases reaction time to 30min. Sensitive – detect viral antigens at pico to nanomolar (10 -12 to 10 -9 mol/litre)

54. ELISAs Can be noncompetitive “sandwich” as described previously Competitive – Solid phase is coated with antigen to be detected. –Patients sample is mixed with detecting antibody, which is then applied to solid phase. –If sample contained antigen, less antibody is free to bind to the solid phase with prebound antigen. –Rest of test run as before. Intensity of colour formation inversely proportional to amount of antigen in sample.

55. Nucleic Acid Detection Short length of viral genome makes them ideal candidate for nucleic-acid based diagnosis PCR –conventional PCR – agarose gel detection of product –Real-time PCR- products detected using probes or intercalating dyes within the reaction. –Microarrays – chip or bead based.

56. PCR Each cycle of PCR doubles the number of copies (amplicons) Over 1 million amplicons after 20 cycles. Primers determine sensitivity and specificity of PCR reactions.

57. ©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2 5’ Exonuclease Probes

58. ©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2

59. Molecular Beacons

60. FRET probes

61. Multiplex PCR Multiple viruses can cause same clinical syndrome –Respiratory infections Can perform multiplex PCR assays to detect multiple viruses in one reaction. Commercial assays to detect up to 18 respiratory viruses in 1 test.

62. Seeplex® RV/PB18 ASE Detection Multiplex–PCR System for the detection of 13 Respiratory Viruses (Influenza A/B virus, RSV A/B, Rhinovirus, Coronavirus OC43/HKU1, coronavirus 229E/NL63, adenovirus, parainfluenza virus 1-3m bocavirus, enterovirus 5 Pneumonia causing bacteria (Mycoplasma pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila).

63. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay J. Mahony, et al. Department of Pathology and Molecular Medicine, McMaster University, and St. Joseph’s Healthcare, Hamilton, Ontario, Canada,1 and TmBioscience Corporation, Toronto, Ontario, Canada2 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 2965–2970 Vol. 45, No. 9 Detects 20 different respiratory viruses

64. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay Reverse transcriptase PCR Target specific primer extension – –containing both a virus-specific oligonucleotide sequence and a tag oligonucleotide that hybridizes to a complementary anti-tag oligonucleotide bound to 21 spectrofluorometrically labeled microspheres

65. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay J. Mahony, et al. J Clin Micro; Sept. 2007, p. 2965–2970 Vol. 45, No. 9 TSPE: Target specific primer extension

66. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay Biotin labelled TSPE product reacts with streptavadin/phycoerythrin Flow cell with 2 lasers –Red laser detects specific bead (1 per test target) –If virus was present and TSPE occurred, green laser detects presence of phycoerythrin and gives positive signal.

67. Non-PCR-based Nucleic Acid Amplification Systems Strand Displacement Amplification Ligase Chain Reaction Nucleic Acid Sequence-Based Amplification Hybridization –Based Assays Quantitative NATs

68. Summary Viral diagnostics is a dynamic field New applications of molecular technology being introduced continuously Increased therapeutic options for viral infections have increased the clinical relevance of making a viral diagnosis.