support for 2nd year medical students

1. Viruses: general characters;
laboratory diagnosis

2. General characters of viruses
• Small size (20-300 nm) / bacteria (size measured in µm)
• Viral genome – single type of nucleic acid:
– DNA – dezoxyriboviruses
– RNA - riboviruses
• Totally dependant on living cells for all metabolic
processes
• May infect humans, animals, plants, bacteria, fungi,
parasites, insects
• Virion = viral corpuscle = elementary, infectious unit

3. Morphology of viruses - SIZE
• measured in nanometers
• Clinically relevant viruses:
– 20-30 nm (picornaviruses)
– 300 nm (poxviruses)
• Only visible by electron microscopy

4. Morphology of viruses - SHAPE
• Under Electron Microscopy:
– spherical e.g. influenza viruses, adenoviruses
– parallelipipedic e.g. poxviruses
– bullet e.g. rabies virus
– comma e.g. some bacteriophages

5. Structure of viruses
• Nucleocapsid:
– Core – contains viral genome (DNA or RNA)
– Capsid = proteic shell
• ± Peplos = external lipo-proteic envelope (derived from
the citoplasmatic membrane of host/infected cell)
↓
Classification:
- Enveloped (nucleocapsid + peplos)
- Non-enveloped (just nucleocapsid)

6. Structure of viruses - continued
VIRAL GENOME – DNA or RNA – never both!
• Contains the entire genetic information for viral replication
(multiplication); support for viral infectivity
• Single stranded (ss): all RNA viruses except reoviruses
• Double stranded (ds): all DNA viruses except parvoviruses
Size of viral genome – correlated to size of capside
• large viruses – large genome (hundreds of genes encoding for
large number of proteins e.g. Poxviruses, Herpesviruses
• small viruses – small genome (3-4 genes encoding for small
number of proteins)

7. Structure of viruses - continued
VIRAL CAPSID – proteic
shell
• composed of capsomeres
(proteic units)
Classification:
• Simetrical structures:
– helical – tubular aspect
– icoshaedral – spherical
aspect
• Non-simetrical structures:
– binar (icoshaedral-helical)
– Complex structures

8. Viruses:
Classification & characterisation criteria
• Capsid shape
• Envelope (+/-)
• Nucleic acid (DNA/RNA)
• Disease(s)
E.g.
– Adenoviruses: icoshaedral, non-enveloped, DNA
viruses causing respiratory diseases
– Hepadnaviruses: icoshaedral, enveloped, DNA
viruses causing hepatitis B

9. Laboratory diagnosis of viral infections
Methods:
• A. Cytology
• B. Electron microscopy
• C. Cultivation
• D. Detection of viral proteins
• E. Serology
• F. Molecular diagnosis (detection of
genetic material i.e. nucleic acids)

10. Laboratory diagnosis of viral infections
- Cytology -
• Rapid method; involves detection of
effects on cell structures
• Only applicable in viral infections which
produce such effects:
– morphologic changes, multiple nuclei
– cell lysis
– vacuolisation
– syncitia (cell fusion)
– inclusions (intranuclear / intracytoplasmatic)

11. Laboratory diagnosis of viral infections
- Electron microscopy -
• Detection and
identification of virions in
clinical specimens
Influenza virus →
______________________
Ebola virus →

12. Laboratory diagnosis of viral infections
- Cultivation -
Cell cultures
• Primary – obtained from animal organs; cells obtained by enzymatic
lysis and cultured in monolayer + growth factors (calf serum)
• Secondary – dissociation of primary cell cultures (tripsin) followed
by successive transfers
• Cell lines – tumor cells, ”immortalised” cells – artificially induced
continuous multiplication for experimental purposes; may support an
infinite number of transfers
e.g.
– Primary culture of simian kidney cells - orthomyxoviruses,
paramyxoviruses, some enteroviruses, adenoviruses
– Diploid fetal cell cultures - herpesviruses, picornaviruses, adenoviruses
Embryonated chicken eggs
• Influenza viruses

13. Laboratory diagnosis of viral infections
- Serology -
Useful for:
– non-cultivable viruses,
– slow evolving infections and
– assessment of immune response
Main serological tests:
• 1. Complement fixation
• 2. Inhibition of hemagglutination
• 3. Neutralisation
• 4. Direct and indirect immunofluorescence
• 5. Latex-agglutination; passive hemagglutination
• 6. ELISA - "enzyme-linked immunosorbent assay“; ELFA – „enzyme
linked immunofluorescent assay”; Western blot
• 7. Radioimune assay (RIA)

14. ELISA (Enzyme-linked Immunosorbent
Assay)
immune reaction (Ag-Ab)
linked to
enzymatic reaction (Enzyme-Substrate)
ELISA types:
- Direct
- Indirect
- ”Sandwich”

15. Solid support for ELISA:
96 microwell plastic plate

16. Direct ELISA
• Add serum on solid support (plastic
microwell plate); adherence to solid
support by charge interactions
• Add CONJUGATE: Ab conjugated
to enzyme
• Add SUBSTRATE of enzyme
• COLOUR = Ag present in serum

17. Indirect ELISA
• Solid support pre-coated with
Ag
• Add Patient serum
• if Ab in serum→formation of
Ag-Ab complex
• Add CONJUGATE: Ab anti-
human Ab+Enzyme
• Formation of Complex: Ag-Ab-
Ab anti-human Ab+Enzyme
• Add substrate of enzyme →
COLOUR develops as a
result of enzyme-substrate
reaction → presence of Ab
in patient serum (”primary
antibody”)

18. ”Sandwich” ELISA
• Solid support pre-coated with
”capture” Ab (speciffic for the Ag
tested for)
• Add patient serum; if Ag is
present, the Ab on plate
capture it
• Add ”detection” Ab which will
bind Ag (Ag bound between 2
Antibodies: sandwich)
• Add CONJUGATE: Ab
anti-”detection” Ab conjugated
to Enzyme
• Add SUBSTRATE of enzyme
• Colour develops

19. Western blot
• Confirmatory test to detect antibodies in ELISA-positive serum
samples e.g. HIV
• Proteins from known infected cells are separated by electroforesis
and blotted on a nitrocellulose strip
• Serum applied to strip (primary antibody incubation step)
• if specific Ab are present in serum they will bind to the nitrocelulose
strip
• Add secondary anti-human antibody conjugated with enzyme signal
• stained bands will indicate the presence of Ab in patient serum

20. Western blot

21. Laboratory diagnosis of viral infections
- Molecular methods -
Relevant definitions
• Nucleic acids
• Nucleotides
• Nucleobases (nitrogenous bases)
• Base pairing
• Polymerase
• Primers
• Denaturation
• Annealing
• Extension (elongation)

22. Nucleic acids
- long, linear macromolecules = polymers which
carry genetic information
- composed of linked nucleotides = monomers
- Each nucleotide has 3 components:
- a 5 carbon sugar = pentose:
- dezoxiribose in DNA or
- ribose in RNA
- a phosphate group
- a nitrogenous base (nucleobase)

23. Nucleobases (nitrogenous bases)
• Nitrogen containing biological compounds found in the
structure of nucleotides
• Primary nucleobases:
– Cytosine (C) (in DNA and RNA)
– Guanine (G) (idem)
– Adenine (A) (idem)
– Thymine (T) (only in DNA)
– Uracil (U) (only in RNA)

24. Base pairing
• Base pairs - formed between specific nucleobases due
to complementarity i.e.
– A with T
– C with G
• ensures the DNA double helix → folded structure of both
DNA and RNA
• DNA structure of each species depends on nucleotide
sequence = succession on DNA strand (basis of the
genetic code)

25. RNA and DNA

26. Polymerases
• DNA-, RNA-polymerase, reverse-transcriptase =
enzymes that catalyze the formation of
DNA or RNA using an existing strand of DNA or RNA as
a template

27. Semiconservative DNA replication
1. DNA strands separated
2. New complimentary DNA
strands synthesized by base
pairing
3. RESULT:
• 2 identical copies (all biological
information from ”parental”
DNA)
• ”daughter” DNA molecules are
"Half old" and "Half new“ = Half
of parental DNA is saved
(conserved) in each daughter
DNA = semi-conservative
replication

28. Primer
• strand of nucleic acid that serves as a
starting point for DNA synthesis under the
action of a polymerase

29. Probe
• labeled segment of DNA or RNA used to find a specific
sequence of nucleotides in a DNA molecule
• synthesized with a specific sequence complementary to
a target DNA sequence (i.e. of the suspected virus)

30. Laboratory diagnosis of viral infections
- Molecular methods -
a) DNA probes – sensitive and specific detection of viral genomes; based upon
complementarity between the sequence of the DNA probe and a specific
section of viral genome; DNA probes are labeled with radioactively or
chemically treated nucleotides
b) In situ hybridization – detection of specific viral genome sequences in
tissue biopsies by DNA probes
c) ”Dot blot” hybridization techniques
– "Southern blot" – DNA-DNA hybridization: viral DNA is separated by
elecrtoforesis, transfered onto nitrocellulose filtre and identified by electroforetic
mobility and by hybridization with labeled DNA probe
– "Northern blot" – RNA-DNA hybridization: viral RNA is electroforetically
separated, transfered onto nitrocellulose filter and detected by specific labeled
DNA probe
d) Polymerase chain reaction (PCR) – see next slides

31. Polymerase chain reaction (PCR)
• Based upon semiconservative DNA replication
• Purpose in microbiological diagnosis:
– to obtain a huge number of copies of nucleic acid of a certain
microorganism (amplification) e.g. bacteria, viruses
– to detect and identify the amplified product

32. PCR – preparatory steps
1. Extract nucleic acid (NA) from biological product e.g.
nasopharyngeal exudate – bacterial / viral NA:
• cell lysis
• elution
• membrane filtration
2. Prepare ”reaction mix”:
• Specific primers (sequence depends on NA to be detected =
target NA)
• Polymerase
• Other components to favour future steps
3. Add extracted NA to ”reaction mix”

33. PCR – the cycling reactions
• Performed in thermal cyclers (PCR machines) =
instruments that employ precise temperature control and
rapid temperature changes
• Thermal block where PCR tubes are placed in
• Thermal prophile is defined:
– number of cycles
– temperature and duration for each cycle

34. Strip with 8 PCR tubes containing reaction
mix

35. PCR thermal cycler

36. PCR – the cycling reactions (30-40 cycles)
1. Denaturation (around 94 C):
• DNA double strand opens → single stranded DNA
• Annealing (around 54 - 64 C):
- Primers in the reaction mix find complementary nucleobase
sequences on each DNA strand and bind in the respective
positions (A with T; C with G)
- Extension (around 72 C):
- Polymerase in the reaction mix catalyzes the synthesis of the
2 new DNA strands

37. PCR: annealing and extension

38. PCR – exponential amplification

41. PCR: detection and identification of
amplified product
Conventional end-point
PCR:
• gel electroforesis of
amplified products
• Visualise sample
migration under UV
light
• Compare bands of
samples with bands of
positive control

42. PCR: detection and identification of
amplified product (2)
Real time PCR
• Fluorescence-based detection; compare cycle threshold
(Ct) of sample with Ct of positive control