Ethiological Diagnosis of Viral Diseases

1. Ethiological Diagnosis of Viral Diseases

2. Virological diagnosis - direct Visualisation of the virus and its activities - in the biological sample - replication, isolation and detection of the virus and its activities • Microscopy – elektron, imunofluorescence • Antigen detection - ELISA, Latex • Detection of genetic information of the virus - (PCR, genetic probes) • Detection of cell or tissue damages (Cytopathic effect, histological staining) - indirect • Detection of specific antibodies in serum (serological reactions)

3. Detection of virus in the biological sample • Rapid method – • - cytology of the sample – characteristic CPE – transformation of the cell, lysis, vacuolisation, formation of syncitia (morbili) or inclusions (Negri body – rabies) • - elektron microscopy, imunoelektron mi. • - detection of antigen (latex agglutination – rotaviruses) • - detection of NA in the tissue by genetic probes – (HPV)

4. Histological stainning for specific changes of the tissue caused by viral infection Specific microscopical picture in the tissue infected vith virus: multinuclear cells in the lung of the patient with morbilli pneumonia

5. Histological staining for specific tissue changes caused by viral infection Negri´s bodies caused by rabies virus infection of CNS cells

6. Imunofluorescence localises neurons infected by herpes virus infikované

7. Virological diagnosis - direct replication of the virus • Correct identification, sampling and transport of biological sample 1) phase of the replication of the virus Víruses are not cultivable on artificial media Replication is performed in the living cell – embroynated egg – tissue cultures – cell lines 2) phase of detection of virus, its antigen or activities (ELISA, IFA, serological methods, microscopy, staining, CPE, interference)

8. Systems for replication of viruses • Annimals: laboratory mouses, rats, suckling mouses • Embryonated egg • Organ or tissue cultures: - cultures of desintegrated organs, - primary tissue and cell cultures, - cell lines – diploid, tumor and immortalised cell lines – HeLa cells (cells that lost the ability of programmated cell death - apoptosis – and are able to multiply indefinitely)

9. Replication of the virus in the embryonated egg and the detection of the virus in it • With the aid of extremly thin needle we innoculate to the egg the suspension derived from the biological material and let it cultivate for 14 days. • If the sample contained the virus, it will replicate inside the egg resulting in the • Embryo death or - formation of specific cell changes in the egg The existence and identification of the virus replicated in the egg can be performed by detection method: (fluorescence, ELISA, PCR, elektron microscopy)

10. Replication and idnetification of the virus in the tissue cultures • Suspension of the tested biological sample is innoculate to th isolated annimal or fetal organs – monkey kidneys, fetal liver • Virus is replicated • Replication and the type of virus is identified by:– cytopathic effect (CPE) - elektron microscopy, - detection of antigen (Latex, ELISA), - detection of specific genetic infomation (PCR), - neutralistion test – after innoculation of the specific antibodie to the tissue culture the virus is not replicating), - Detection of interference of the growth – several viruses are not replicating in the presence of others in the tussue culture

11. cell lines • Tested tissue or the suspension of it is innoculated on the cell line model • Cell lines: cancer cells, that are able of uncontroled growth on the surface of artificial materials – laboratory glass, HeLa cells... • Virus replicates and is identified • – cytopathic effect (CPE) - elektron microscopy, - detection of antigen (Latex, ELISA), - detection of specific genetic infomation (PCR), - neutralistion test – after innoculation of the specific antibodie to the tissue culture the virus is not replicating), - Detection of interference of the growth – several viruses are not replicating in the presence of others in the tussue culture

12. Detection of antigen by ELISA These tests are prepared in such a way that the specific antibody against the virus/antigen we want to detect in the sample is anchored on the solid phase (surface of the PE well of microtitration plate, surface of the slide). • When the sample is added the antigen is bound on the antibody and the complex Ab+Ag results • Another anticomplex antibody (labelled with enzym) is added • The last step consist of addition of the chromoge substrate that is subsequently dissolved by the enzyme that result in the appearance of the color

13. ELISA test for antigendetection • specific antivirus antibodies are bound on the surface of the well • sample containing/or not the virus is added, • unbound antigen is washed • 3. Antiviral antibodis are added to bind with the antigen. • 4. Antibodies against the complex AGAB conjugated with enzym are added • Chromogen substrat that was added is lysed by enzym • what results in color reacttion

14. Detection of antigen by immunofluorescence Antigen in the tissue can be detected by fluorochrom labeled antibodies or indirectly by the help of antiviral antibodies anchored on the solid surface and by the fluorescein labeled anti agabcomplex imunoglobulin

15. Imunofluorescence

16. Elektron microscopy

17. PCR • Every organism – microorganism too, and virus equally – contain specific sequence of aminoacids • Thanks to enzymes (transcriptase) such a sequence can be multiplied in the sample by the help of specific primer. • Copies of genetic code is than identified in the sample by the help of enzymatic or fluorescenc detection method the method is very sensitive (in can find the needle in the hay), but is requiring the high specificity of the primer

18. PCR polymeras chain reaction – is a rapid method for multiplication – amplification of the known DNA. The sample is mixed with the termostable DNA polymerase, DN-trifosfates and 2 DNA specific molecules - primers, that are complementar to the ends of target sequence. Mixture is heated for denaturation and cooled for hybridisation – binding of the primer on the target DNA sequence Cycles are repeated 32 times. After the first amplification only the sequences lysed by the added primer are lamplified In this way the genetic information of the virus can be multiplied to the detectable level in the sample within 6 hours

19. Genetic probes • This method is based on the similar principle. • Directly in the sample (swab of the cervix uteri.....) the virus can be identified with the help of the anticodon of specific sequences labeled with ensym of fluorescence

20. Detection of the virus infected cell by the help of DNA probe – With virus infected cells can be localised in the biological sample. DNA probe containing app. 9 nucleotides is added to the sample and heated for denaturaltion of DNA and cooled for hybridisation of the complementary sequeces. The whole system is visualised by the enzym-substrat reaction (similar to ELISA)

21. In situ hybrodosatopm – localising CMV infection with the help of genetic probe. CMV infection of the kidney tubules is localised with labeled specific CMV DNA probe and visualised similarly to ELISA teste

22. Histological staining for detection of cell damages in the virus infected cells

23. cytopatic effects • Cell death – round cells, degeneration, aggregation, lost of capacity of binding on the substrat • Characteristic histological changes: Inclusion bodies in the nucleus or cytoplasma, margination of the chromatin • Formation of syncitia: multinucleated giant cells resulting from cell fusion induced by virus • Changes of the surface of cell: expresion of antigen, hemadsorbtion

24. Cytopatic effect of HSV B HSV-1 infected Vero cells – round multinucleated cells and lost of one layer organisation of the cell culture . A not infected Vero cells

25. HSV induction of CPE – A:In bioptic sample eosinofil inclusions are seen with Halo and the ring of chromatin in the nuclear membrane B: Infection of cell – resulting in small condensed pycnotic nucleus

26. Cytopatic effect

27. Indirect diagnosis • Detection of antibodies in the serum • ELISA,IF - imunofluorescencia, RIA, • Neutralisation, agglutination, hemagglutination, latexagglutination, KFR (complement fixating reaction), immunodiffusion • Western blot

28. Serological methods • Antigen + antibody in the serum Komerčný hľadaná protilátka Type of the reaction is done by the characteristic of antigen and the environment in which the reaction is performed (in FR - agglutination, Ag bound on latex - latexagglutination, on ery - hemagglutination, dissolved in agar - diffussion, bound on PE plate - ELISA, na nitrocelulose - WB, na fluorochrom - IF, radionuclid - RIA)

29. Antigen • Substance able to start the reaction of immune system – immunity reaction – specific to eliminate the antigen and reacting with products of such a reaction (antibodies)

30. Antibodies Blood serum of immunised annimals contain specific antibodies that are able to bind the antigen, that evocated their production (Behring, Kitaso 19th century) Serum– Plazma– antiserum Function of antibodies – specific binding of antigen, neutralisation of its function by other reactions (activation of complement, macrophages). Reakcia antigAccording to the differenct qualities of antigen *corpuscular antigens = microorganism, erytrocyte – agglutination *soluble antigens = small immunocomplexes – in solution, big are precipitating

31. Dynamicity of antibody formation after contact of antigen

32. Role of specific antibodies in detection of viral infections • Viruses are foreign antigens – stimulate the production of specific antibodies • IgM are detected after 7-14 days , MX in serum after 3 mnths, eliminated 3-6 mnths after stimulation - acute • IgG detected after 14-21 days, their decrease is slow – protective, longlasting • Total (IgM,IgG,IgA...) double sampling – fourfold rising of antibody titer in the interval of 14 days , or changes from seronegativity to seropositivity are significant for acute infections

33. SEROLOGICAL REACTIONS Agglutination – antigen + diluted serum – macroscopically visible agglutinates Latexagglutination – Ab bound on latex + Ag in the sample – big agglutinates Precipitation – Ag soluble + Ab – in liquid medium – ring, precipitate, Imunodiffussion – in agar – 2 wells , Ag and Ab are diffunding in opposite direction, in the place of contact – line of precipitation Neutralisation – serum containing Ab neutralises activity of Virus or toxin in annimal model or in tissue model Hemagglutination – Ag bound on erytrocyty KFR – Ag + Ab +C´ - no lysis of erytrocytes

34. Aglutinácia • diluted serum + corpuscular antigen = agglutination • the higher the concentration in serum, the higher is the dilution, where agglitation still occures • dilution is geometrical 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128....... TITER = 1, 2, 4, 8, 16 significant rise is 4 fold rise fo geometrical titer (1-8, 2-16, 4-32.....)

35. Latex aglutinácia

36. Sérologické reakcie • known antigen antigén + unknown presence of antibodies (in serum) • quality of antigen • environment • quantity of antibodies (prosoone phenomen)

37. hemagglutination and phenomenon of prozoone

38. Precipitácia • test CRP in capilarie • radial imunodiffusion sc Mancini – • Imunodiffusion sc Ouchterlony (ELEK) • Imunoelektroforesis

39. Imunodiffusion

40. Neutralisation • Vírus + serum with antibodies = inhibition of vital function of virus

41. Komplement fixation reaction KFR ˇ antigen + antibody = komplex resulting in consumption of complement hemolytical system is added ˇ ery + complement = hemolysis IF complemet was consumpted in the reaction with present ab, ther is no more complement for hemolysis REACTION IS POSITIVE FOR SPECIFIC ANTIBODIES IF NO HEMOLYSIS there are not specific antibodies, complement was not consumpted in the first phase and it is present to lyse erytrocytes – REACTION IS NEGATIVE FOR AB IF HEMOLYSIS IS PRESENT

42. ELISA • the same as for antigen detection but • antigen is bound on the solid phase that reacts with antibodies present in the serum sample

43. Imunofluorescencia IFT • for detection of antibodies in serum, the antigen is anchored on the surface of the slide • the serum with antibodies is added • the second antikomplex antibody labeled with fluorochrome is added

44. Western blot • On the filtrate paper the whole cell is devided by electrophoresis to different antigens • application of serum will allow binding of specific antibodies to different unique antigenic molecules of the virus and production of several „bind complexes“ that are visualise by chromogen. • Method enable detection of specific antigenic molecules and increases the specificity of detection of different serotypes

45. Limits of serological reaction and serological diagnosis • Detection of antibodies gives the picture on the history of infections of the patient • information on the course of the disease, the stage, the chronicity, the prognosis • specificity and sensitivity of tests • false positive (pregnancy, autoimmunity) • false negative (anergy, immunosupression, AIDS) • cross reacting antibodies • interpretation of the result – treatment of the patient not of serological chifers -- in close correlation with clinical symptoms, history and epidemiological characteristic