Mol Virology Techniques

virus: 20 -30 nm

Immuno-electron microscopy is used to localize molecules at the ultrastructural level by labeling them with specific antibodies. The antibodies are visualized by electron-opaque markers (colloidal gold particles) attached to them.

befruchtete Hühnereier

• Viruses are inoculated into chick embryo of 7-12 days old.

   The egg used for cultivation must be sterile and the shell

  should be intact and healthy.

   For inoculation, eggs are first prepared for cultivation, the shell

  surface is first disinfected with iodine and penetrated with a

  small sterile drill.

   After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days.

   After incubation, the egg is broken and virus is isolated from tissue of egg.

   Viral growth and multiplication in the egg embryo is indicated

  by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes

   Viruses can be cultivated in various parts of egg like

  chorioallantoic membrane, allantoic cavity, amniotic sac and yolk sac.

avian flu = Vogelgrippe

foci (→ focus-forming units (FFU))

Cellular transformation: loss of contact inhibition causes cells to pile up…

Direct detection

Restriction fragment length polymorphism (abbreviated RFLP) refers to differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes. Such variation results in different sized (or length) DNA fragments produced by digesting the DNA with a restriction enzyme.

Real-time PCR, also known as quantitative or qPCR, determines the actual amount of PCR product present at a given cycle. By using a fluorescent report in the PCR reaction, this process allows you to measure DNA generation in the qPCR assay. There are two methods of qPCR: SYBR Green and probe-based.

Reverse transcription PCR (RT-PCR / RT-qPCR)

Detection (and quantification) of RNA

- cDNA construction

- RNA-sequencing

- Preparation of a sequencing library (adaptor ligation)

- PCR amplification step required

- “Short” reads

- No (PCR) amplification step required

- “Longer” reads

• Antigen capture ELISA (Sandwich ELISA)

—> Corona

indirect detection

• Seroconversion is the transition from the point of viral infection to when antibodies of the virus become present in the blood.

• Antibody serology tests check for the presence or level of specific antibodies in the blood.

• the concentration of an antibody

• The complement fixation test is one of the most established serology methods. This relies on the formation of specific antibody-antigen complex that activate the complement system. Serum for antibody testing is mixed with known target antigen. Any antibodies present will react with antigens, make immune complex and use up any complement that has been deliberately added. Complement use is measured by adding red cells treated with red cell antibody. Residual complement, left over from binding specific antibody-antigen complex, completes the reaction that lyses the red cells

- Detection of antibodies specific for viral proteins

- Higher specificity than antibody ELISA (protein band of distinct size)

• lateral flow assay (dipstick)

• Western-blotting (for antibody detection)

• Hemagglutination inhibition test (HIT)

• Complement fixation test CFT

• (virus) Neutralization test

• IgM capture ELISA (µ capture ELISA)

• Antibody ELISA (detection of virus-specific IgG (or IgM) antibodies)

• Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an "infectious clone”

• Reverse genetic systems are available for (almost) all virus families

• The limiting factor is the availability of a cell line that is “permissive” (geeignet) for a certain virus

• The permissive cell line might contain trans-complementing (viral) factors

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