Viruses: general characters;

laboratory diagnosis

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

Morphology of viruses - SIZE

• measured in nanometers

• Clinically relevant viruses:– 20-30 nm (picornaviruses)– 300 nm (poxviruses)

• Only visible by electron microscopy

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

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)

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)

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

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

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)

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)

Laboratory diagnosis of viral infections- Electron microscopy -

• Detection and identification of virions in clinical specimens

Influenza virus →

______________________

Ebola virus →

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

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)

ELISA (Enzyme-linked Immunosorbent Assay)

immune reaction (Ag-Ab)

linked to

enzymatic reaction (Enzyme-Substrate)

ELISA types:

- Direct- Indirect

- ”Sandwich”

Solid support for ELISA: 96 microwell plastic plate

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

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”)

”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

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

Western blot

Laboratory diagnosis of viral infections- Molecular methods -

Relevant definitions

• Nucleic acids• Nucleotides• Nucleobases (nitrogenous bases)• Base pairing• Polymerase• Primers• Denaturation• Annealing• Extension (elongation)

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)

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)

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)

RNA and DNA

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

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

Primer

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

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)

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

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

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”

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

Strip with 8 PCR tubes containing reaction mix

PCR thermal cycler

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

PCR: annealing and extension

PCR – exponential amplification

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

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