Medical laboratory course Viral diagnosis.ppt

Medical laboratory virology

1. V iral diagnosis FS(AP)…

2. Specimen collection for virus 1-4 days after symptom are most likely to contain recov erable virus Sterility and quick transportation to the laboratory H eat liability and cant resist drying Transport media contains protein , a buffer at neutral pH, and antibiotics to kill or suppress the growth of bacteria and fungi

3. Viruses vary in their ability to survive ambient temperatures Specimens for virus isolation should always be transported to the diagnostic laboratory on ice and, unless a delay of more than 4 days is anticipated, specimens should be held at 4◦C and not frozen If specimens are frozen, they should be kept at —70◦C since conventional freezer temperatures (—10◦C to —20◦C) are detrimental to infectivity of many viruses

4. The site of specimen collection should correlate with the clinical presentation and local epidemiology pattern However, if there is deep or generalized disease (e.g., non-vesicular rash, meningitis, fever or unknown origin, congenital infection), it is advisable to sample multiple sites

5. Syndrome Common Uncommon Specimens Aseptic meningitis Enteroviruses (Coxsackie, echo), mumps Polio, , HSV- 2, adenovirus NP- throat, CSF, urine, stool Encephalitis Arboviruses a , HSV- 1, - 2 Mumps, measles, influenza, rubella, VZV, rabies, EBV a , enteroviruses NP- throat, stool, CSF, brain biopsy (if herpes suspected), serum URI, bronchitis, ‘‘flu’’ Rhinovirus, parainfluenza, influenza, adenovirus, enterovirus, RSV Measles, coronavirus (NL,HK), bocavirus NP- throat, nasal aspirate Croup Parainfluenza RSV, adenovirus, influenza NP- throat, nasal aspirate Pneumonia RSV, adenovirus, influenza, metapneumovirus Parainfluenza, CMV, rhinovirus, measles, rubella, HSV, VZV, enterovirus NP- throat, stool, tracheal, aspirate, nasal aspirate, urine, serum Bronchiolitis RSV, influenza Influenza, adenovirus, rhinovirus NP- throat, nasal aspirate

6. Rashes – vesicular VZV, HSV- 1, - 2 Vaccinia, enterovirus Vesicular fluid, NP- throat, stool (for enterovirus), serum Rashes – non- vesicular Measles, rubella, enterovirus EBV a , Hepatitis B virus NP- throat, stool (for enterovirus), serum Congenital infection CMV, HSV- 2, rubella Parvovirus B19 NP- throat, stool, pleural fluid, pericardial fluid, serum Pleurodynia, pericarditis Enterovirus (Coxsackie and echo) Polio virus, mumps, influenza, adenovirus NP- throat, stool, pleural fluid, pericardial fluid, serum Eye lesions (keratitis, keratoconjunctivitis, conjunctivitis) HSV- 1, - 2, adenovirus Measles Eye swabs, NP- throat, nasal washing Gastroenteritis Rotavirus norovirus a , adenovirus Enterovirus (newborns), influenza Stool, NP- throat, urine Hepatitis Hepatitis A, B, C a , D, EBV a , CMV, VZV Enterovirus, adenovirus, HSV- 1, - 2 Serum, NP- throat, stool, urine Parotitis Mumps parainfluenza, influenza Adenovirus, LCMV, EBV a , enterovirus NP- throat, urine, nasal aspirate, serum

7. Viral culture viruses are obligate intracellular parasites so they depend on host for their survival They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication The primary purposes of virus cultivation is: To isolate and identify viruses in clinical samples. To do research on viral structure, replication, genetics and effects on host cell. To prepare viruses for vaccine production .

8. Isolation of virus is always considered as a gold standard for establishing viral etiology of a disease Most of the viruses can be cultivated in Experimental animals Embryonated eggs Tissue culture

9. Animal inoculation Mouse is most frequently used but rabbits , hamsters, newborn or suckling rodents are also used. Experimental animals are rarely used for cultivation of viruses but play an essential role in study of pathogenesis of viral infections and that of viral oncogenesis . Intracerebral , subcutaneous, intraperitoneal , or intranasal routes are various routes of inoculation. After inoculation, the animals are observed for signs of disease or death. The infected animals are then sacrificed and infected tissues are examined for the presence of viruses by various tests, and also for inclusion bodies in infected tissues .

10. Embryonated Eggs Embryonated chick egg was used first for cultivation of viruses by Goodpasture in 1931 Usually , 8–11 days’ old chick eggs are used for culture of viruses The viruses are isolated in different sites of the egg, such as yolk sac, amniotic cavity, and allantoic cavity, and chorioallantoic membrane (CAM )

11. Many of these viruses cause well-defined and characteristic foci, providing a method for identification, quantification, or assessing virus pathogenicity The embryonated egg is also used for growing higher titre stocks of some viruses in research laboratories and for vaccine production.

12. Yolk sac: for cultivation of West Nile virus Amniotic cavity: for primary isolation of influenza virus Allantoic cavity: influenza virus, yellow fever (17D strain), and rabies ( Flury strain) viruses for preparation of vaccines For production of rabies virus, duck eggs were used due to their bigger size than that of hen’s egg

13. Chorioallantoic membrane: Inoculation of some viruses on CAM produced visible lesions known as pocks Each infectious virus particle produces one pock The pox viruses, such as variola or vaccinia Nowadays, chick embryo inoculation has been replaced by cell cultures for routine isolation of viruses.

14. Tissue Culture Cell culture is most widely used in diagnostic virology for cultivation and assays of viruses Different types of tissue cultures are used to grow viruses Tissue culture can be of three different types as follows:

15. Organ Culture This was used earlier for the isolation of some viruses, which appear to show affinity for certain tissue organs For example, coronavirus, a respiratory pathogen, was isolated in the tracheal ring organ culture S mall bits of the organs are maintained in vitro for days and weeks preserving their original morphology and function Nowadays , organ culture is not used

16. Explant Culture In this method, components of minced tissue are grown as explants embedded in plasma clots Earlier , adenoid tissue explant cultures were used for isolation of adenoviruses. This method is now seldom used in virology.

17. Primary Culture T he initial culture of cells derived directly from a tissue or organ Culture of normal cells obtained freshly from the original tissues that have been cultivated in vitro for the first time and that have not been subcultured Cells are typically obtained by disaggregating or dissociating the tissue using enzymatic digestion or mechanical methods

18. Contain a mixture of cell types native to the tissue Primary cultures are used to study the properties and behavior of cells in their native environment and are often employed for short-term experiments H ave the normal diploid chromosomal number and are capable of only limited growth (5–10 divisions) in culture. cannot be maintained in serial culture, but can be subcultured to obtain large number of cells

19. Monkey kidney, human embryonic kidney, and chick embryo cell culture Primary monkey kidney cell cultures are highly useful for the primary isolation of myxovirus , paramyxovirus , many enteroviruses , and some adenoviruses

20. Secondary Culture S ubculture, propagation of cells from a primary culture After confluence and passaged into new culture vessels to promote further growth and expansion The detached cells are then reseeded into fresh culture vessels at a lower density to allow for continued growth and proliferation M ay be subjected to multiple rounds of passaging to generate large numbers of cells E xhibit differences in growth kinetics, morphology, and behavior compared to primary cultures due to adaptation to the in vitro environment

21. Cell culture In this method, tissues are dissociated into component cells by treatment with proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking The cells are then washed, counted, and suspended in a growth medium containing essential amino acids and vitamins, salts, glucose, and a buffering system. This medium is supplemented by up to 5% of fetal calf serum and antibiotics The cell suspension is dispensed in glass or plastic bottles, tables, or Petri dishes .

22. On incubation, the cells adhere to the glass surfaces and divide to form a confluent monolayer sheet of cells covering the surface within a week The cell culture may be incubated either as a stationery culture or as a roller drum culture The latter is useful for growth of some fastidious viruses due to better aeration by rolling of the culture bottle in special roller drums.

23. Diploid cell strains : S ingle cell type that retains their original diploid chromosome number and karyotype T hey have specific characteristics and compositions and are usually composed of one basic cell type They are usually fibroblasts and can be cultured for maximum 50 serial passages before they undergo senescence (die off) or undergo a significant change in their characteristics

24. Diploid cells derived from human fibroblasts are useful for isolation of some fastidious viruses They are also used for production of vaccines WI-38 human embryonic, lung cell stem is used for the cultivation of fixed rabies virus H uman fetal diploid cells for isolation of adenovirus, picornaviruses , HSV, CMV, and VZV.

25. Continuous cell lines : Continuous or immortal cell lines are cells of a single type, which are derived from cancerous tissue and are capable of continuous serial cultivation indefinitely without senescing U sually derived from diploid cell lines or from malignant tissues and have altered and irregular number of chromosomes .

26. Immortalization may occur spontaneously or by chemical mutagens, tumorigenic viruses, or oncogens Hep-2 , HeLa , and KB derived from human carcinoma cervix, human epithelioma of larynx, and human carcinoma of nasopharynx The type of cell line used for virus culture depends on the sensitivity of the cells to a particular virus; for example, Hep-2 cell line is excellent for the recovery of respiratory syncytial viruses, adenoviruses, and HSV.

27. Growth of viruses in cell cultures can be detected by the following methods : Cytopathic effect observation of the morphological changes in the cultured cells in which they replicate The CPE produced by different types differ Nuclear shrinking, vacuoles in the cytoplasm, syncytia formation, rounding up, and detachment

28. Hemadsorption A dsorption of erythrocytes to the surfaces of infected cells which serves as an indirect measurement of viral protein synthesis U sed to detect infection with noncytocidal viruses E.g. influenza virus, parainfluenza virus, mumps virus, and togavirus These hemagglutinins bind some erythrocytes to the infected cell surface.

29. Heterologous interference U sed to detect viruses that do not produce classic CPEs in the cell lines In this method, the growth of non-CPE-producing virus in cell culture can be tested by subsequent challenge with a virus known to produce CPEs The growth of the first virus will inhibit infection by the cytopathic challenge virus by interference. For example, rubella virus usually does not produce any CPE, but prevents the replication of picornaviruses , which is inoculated as a cytopathic challenge virus.

30. Transformation Oncogenic viruses that are associated with formation of tumors induce cell transformation and loss of contact inhibition in the infected cell lines This leads to surface growth that appears in a piled-up fashion producing microtumors H erpes viruses, adenoviruses, hepadanoviruses , papovavirus , and retroviruses

31. Light microscopy : S taining virus-infected cells of tissue sections with specific viral antibody conjugated with horseradish peroxidase This is followed by addition of hydrogen peroxide along with a benzidine derivative substance In a positive reaction, a red insoluble precipitate is deposited on the cell line, under ordinary light microscope Tzank preparation used to diagnose herpes virus infections stained with Giemsa , Wright, or Papanicolaou stain.

32. Electron Microscopy T he rapid detection of viruses in clinical specimens This technique relies on the identification of viruses by their characteristic morphology One limitation is virus must be present in sufficient quantity The most potent usefulness lies in detecting viruses in fecal contents EM is not used widely for routine diagnosis because it is expensive, cumbersome, and insensitive

33. Immunofluorescence R apid , precise, and sensitive but it requires expertize and must be neat Staining may be direct, using a specific antimicrobial antibody with attached fluorescence dye, or indirect, using an unlabeled specific antimicrobial antibody followed by fluorescein-labeled antibody directed against the initial antibody. The indirect test may be more sensitive than the direct test, used to detect and distinguish herpes simplex and varicella-zoster viruses in vesicle fluids.

34. T he preferred method for rapidly diagnosing (a) herpes simplex encephalitis in brain biopsies and (b) rabies in animal brain Immunofluorescence has the following advantages: One can prepare a slide and stain for a number of different organisms at a single time; the adequacy of the specimen can be determined, and slides may be made and sent to the reference laboratory for reading IF is more sensitive than cultures since it does not require intact viable viruses; as a result, a specimen may be positive by IF in the face of negative cultures.

35. Reading assignment ELISA PCR Nucleic acid hybridization Serology microarray