1. DIAGNOSTIC METHODS IN VIROLOGY

2. Definition Viruses = non cellular organisms
genomes - nucleic acid,
- protected by a protein shell
- must replicate inside host cells

3. cannot be grown on sterile media
require the presence of specific host cells
Embrionated egg
Cell culture
Laboratory animals F

6. DIAGNOSTIC METHODS IN VIROLOGY
3 categories:
(1) direct detection,
(2) virus isolation,
(3) serology.

7. 1. Direct Examination of Specimen
clinical specimen - examined directly for the presence of
virus particles
virus antigen
viral nucleic acids.
Electron Microscopy morphology / immune electron microscopy
Light microscopy histological appearance - e.g. inclusion bodies
Antigen detection immunofluorescence, ELISA etc.
Molecular techniques for the direct detection of viral genomes

8. 2. Indirect Examination = Virus isolation
Cell Culture
cytopathic effect,
haemadsorption,
confirmation by neutralization, interference,
immunofluorescence etc.
Eggs pocks - haemagglutination, inclusion bodies
Animals disease or death confirmation by neutralization

9. detection of IgM in primary infection. Classical Techniques
3. Serology
rising titres of antibody between acute and convalescent stages of infection,
detection of IgM in primary infection.
Classical Techniques
1. COMPLEMENT FIXATION TESTS (CFT)
2. HAEMAGGLUTINATION INHIBITION TESTS
3. IMMUNOFLUORESCENCE TECHNIQUES (IF)
4. NEUTRALIZATION TESTS
5. SINGLE RADIAL HAEMOLYSIS
Newer Techniques
1. RADIOIMMUNOASSAY (RIA)
2. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
3. PARTICLE AGGLUTINATION
4. WESTERN BLOT (WB)

10. 1. Direct Examination rapid diagnostic Result => the same/next day.
Obs:
virus isolation
serological methods
May sometimes give a rapid result. !

11. Examples – IF testing of :
1.1. Antigen Detection
Examples – IF testing of :
nasopharyngeal aspirates for respiratory viruses
e.g.. RSV, flu A, flu B, and adenoviruses,
detection of rotavirus antigen in faeces,
detection of HSV and VZV in skin scrappings,
detection of HBsAg in serum.
Usually considered as a serological test!

12. 1.1. Antigen Detection
main advantage: rapid (hours)
BUT technique - often tedious
- result difficult to read and interpret,
- sensitivity and specificity poor.
quality of the specimen - most important in order for the test to work properly.

14. 1.2. Electron Microscopy (EM)
basis = morphology.
magnification - 50,000 X
mainly used for viral gastroenteritis
detecting viruses in feces:
rotavirus, - calicivirus
adenovirus, - Norwalk-like viruses
astrovirus,
Occasionally used for detection of viruses in skin lesions (e.g. vesicles)
herpesviruses
papillomaviruses.

15. 1.2. Electron Microscopy (EM)
sensitivity and specificity enhanced by immune electron microscopy:
virus specific antibody used to agglutinate virus particles =>easier to recognize.
Problems with EM:
expense - purchasing and maintaining the facility.
sensitivity - often poor (106 v.particles/ml in sample required for visualization)
observer - highly skilled.

16. Electronmicrographs from patients suffering from gastroenteritis
Electronmicrographs from patients suffering from gastroenteritis. From left to right: rotavirus, adenovirus, (Courtesy of Linda M. Stannard, University of Cape Town,

17. Animal virus classification: DNA Viruses
mil y P o x H er p e s A d e n o P a p o v a P a rv o H e p a dn a
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l ds D NA G e n o m e C a ps i d C om p l e x
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sah e d ra l - -- - -- - -- - -- - -- - -- - - - -- - -- - -- - -- - -- - -- - -- - -- - - - -- -
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> Y e s V ac c i n i a v i r us H er p e s si m p le x Hu ma n P a p i l l om a
Adeno-
Associated H e p a t i t i s B e. g . v ir
us 2 a d e n o v i r us
Molluscum
Contagiosum

18. Plus Sense RNA Viruses

19. Minus Sense RNA Viruses

20. histology serves as an adjunct test.
1.3. Light Microscopy
Replicating virus => histological changes (characteristic or non-specific) in infected cells.
Viral inclusion bodies = collections of replicating virus particles (in nucleus or cytoplasm).
Examples
Babeş-Negri bodies in rabies
cytomegalic inclusion bodies in CMV infections
histology serves as an adjunct test.

21. RABIES VIRUS
Babeş-Negri bodies in rabies

22. Rabies

23. Rabies virus Babes –Negri bodies

24. INTRANUCLEAR INCLUSIONS IN HERPESVIRUS INFECTIONS

25. INTRACITOPLASMATIC INCLUSIONS
REOVIRIDAE
INTRACITOPLASMATIC INCLUSIONS

26. INTRACITOPLASMATIC INCLUSIONS
V.EBOLA HEPATOCITES

27. 1.4. Viral Genome Detection
molecular methods
future direction of viral diagnosis
Classical molecular techniques
(dot-blot and Southern-blot)
use of specific DNA/RNA probes for hybridization
specificity depends on the conditions used for hybridization.
quantification of DNA/RNA present in the specimen
sensitivity of these techniques ~ conventional viral diagnostic methods.

28. 1.4. Viral Genome Detection
b) Newer molecular techniques:
POLYMERASE CHAIN REACTION (PCR)
LIGASE CHAIN REACTION (LCR)
NUCLEIC ACID BASED AMPLIFICATION (NASBA)
BRANCHED DNA (BDNA)
PCR
extremely sensitive : 1 DNA molecule in a clinical specimen.
PCR – problems:
contamination,
positive PCR result - difficult to interpret
latent viruses such as CMV !

29. Specimen - inoculated into cell culture, eggs or animals
2. Virus Isolation
Specimen - inoculated into cell culture, eggs or animals
Eggs and animals - difficult to handle
most viral diagnostic laboratories - cell culture only.
3 types of cell cultures:
Primary cells
Semi-continuous cells
Continuous cells

30. Primary cells culture - e.g. Monkey Kidney.
2.1. Types of cell cultures
Primary cells culture - e.g. Monkey Kidney.
= normal cells obtained from freshly killed adult animals.
(+)
best cell culture systems available
support the widest range of viruses
(-)
can only be passaged once or twice.
very expensive
difficult to obtain a reliable supply.

31. b. Semi-continuous cells
2.1. Types of cell cultures
b. Semi-continuous cells
Human embryonic kidney
skin fibroblasts.
cells taken from embryonic tissue
passaged up to 50 times.
c. Continuous cells
HeLa, Vero, Hep2, LLC-MK2, BGM.
immortalized cells i.e. tumour cell lines
passaged indefinitely.
most easy to handle
range of viruses supported is often limited.

32. 2.2. Identification of growing virus
a. Cytopathic Effect (CPE) - specific or non-specific
HSV and CMV produces a specific CPE,
enteroviruses do not produces a specific CPE.
b. Haemadsorption
cells acquire the ability to stick to mammalian red blood cells.
mainly used for the detection of
influenza
parainfluenzaviruses.

33. Left to Right: Cytopathic effect of HSV, enterovirus 71 (ballooning), and RSV in cell culture (syncytia formation) (Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)

34. 2.3 Problems with cell culture
long period for a result (up to 4 weeks)
sensitivity - often poor and depends on many factors,
condition of the specimen,
condition of the cell sheet.
very susceptible to
bacterial contamination
toxic substances in the specimen.
many viruses - not grow in cell culture at all :
Hepatitis B and C,
Diarrhoeal viruses,
parvovirus etc.

35. CELL CULTURE CONTAMINATION

36. CELL CULTURE INCUBATOR

37. 2.4 Rapid Culture Techniques
viral antigens - detected 2 to 4 days after inoculation.
Examples: shell vial cultures and the CMV DEAFF test.
CMV DEAFF test
cell sheet grown on individual cover slips in a plastic bottle.
then bottle is spun at a low speed for one hour (to speed up the adsorption of the virus)
incubated for 2 to 4 days.
cover slip  taken out
examined for the presence of CMV early antigens by IF (immunofluorescence)

38. Left: Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus. Also note the presence of syncytia (indistinguishable from that of RSV) (Courtesy of Linda Stannard, University of Cape Town). Right: Positive CMV DEAFF test. (Virology Laboratory, Yale-New Haven Hospital)

39. CELL CULTURE RECIPIENTS CELL CULTURE PH CHECKING

40. 3. Serology = the mainstay of viral diagnosis
IgM - first antibody to appear,
IgG – follow, much higher titer
Techniques:
EIA and RIA
specifically for IgM or IgG,
most sensitive tests available
CFT, HAI => detect total antibody (comprises mainly IgG)
EIAs
better sensitivity, specificity and reproducibility than classical techniques ( CFT and HAI. )

41. 3. Serology 3.1. Criteria for Primary Infection
a. Significant rise in titre of IgG/total antibody
acute - convalescent sera
CFT and HAI: 4 X or greater
Problem: dg. = retrospective
b. Presence of IgM
- EIA, RIA, and IF
rapid
Problems:
interference by rheumatoid factor,
re-infection by the virus,
unexplained persistence
Seroconversion = changing from a previously antibody negative state to a positive state.
HIV following a needle-stick injury,
antirubella following contact with a known case.

42. 3.2. Criteria for diagnosing re-infection/re-activation
difficult to differentiate re-infection/re-activation
important under certain situations
rubella or toxoplasma infection
first trimester of pregnancy:
primary infection - high risk of fetal damage
re-infection not associated with a high risk of fetal damage
sharp large rise in antibody titres found in re-infection
IgM usually low or absent in cases of re-infection/re-activation.

43. Serological events in primary infection and reinfection.
Reinfection: IgM absent or only present transiently at a low level.

44. Rubella and hepatitis A:
3.3. Limitations
Rubella and hepatitis A:
onset of clinical symptoms - development of antibodies
=> detection of IgM or rising titres of IgG = active disease.
Many viruses (respiratory and diarrhoeal):
clinical disease before the appearance of antibodies => serological diagnosis = retrospective
Some viruses (HIV and rabies):
clinical disease months or years after seroconversion => the mere presence of antibody is sufficient to make a definitive diagnosis.

45. CFT in Microtiter Plate.
Rows 1 and 2: acute and convalescent phase serum specimens, respectively.
The observed 4-fold increase is significant and indicates infection.

46. Microplate ELISA: coloured wells indicate reactivity
darker the colour, higher the reactivity

47. healthy person - little or no antibodies in CSF.
3.4. Antibody in the CSF
healthy person - little or no antibodies in CSF.
viral meningitis or encephalitis,
antibodies - produced by lymphocytes
finding of antibodies in the CSF
significant when ratio between the titre of antibody in the serum and that in the CSF is less than 100 (depend on an intact blood-brain barrier !)