Lab Dx of viral infections

Question 1

List 2 types of inclusion bodies produced by viruses and state how these are produced.

Answer 1

• Intracytoplasmic, intranuclear or both
• Aggregation of mature viral particles
• Degenerative changes due to infection
• Altered staining patterns at sights of virus synthesis

Question 2

Explain the difference between direct and indirect immunofluorescence.

Answer 2

Direct: Indicator-labelled capture Aby bind to Ag
Indirect: indicator-labelledreporter Aby bind to capture Aby

Question 3

What types of serological assays can be used to detect viruses and viral antibodies?

Answer 3

ELISA
Haemagglutination
LPA
> detect Aby produced due to viral infection

Question 4

What is “CPE”?

Answer 4

Cytopathic effect: is the effect of viral replication on host cell. It occurs since host cell’s function will change = detectable

Question 5

What are the advantages and disadvantages of cell culture?

Answer 5

Adv: isolate virus, can detect new viruses
Dis: require highly equipped facilities, long turnover for results (~15days), sterility, continous monitoring

Question 6

What are the steps in a PCR

Answer 6

1. Denature @ 94-95C
2. Anneal @ 50-60C
3. Extension @72C

Question 7

Explain what Real Time –PCR is

Answer 7

• fluorescent probe & quencher on 3’ end of target DNA
• Polymerase extends primer & eventually reach fluorescent probe => displace probe & Endonucleolytic activity = break 5’ of probe (fluorescent) = release signal

Question 8

Explain the principle behind MassTagPCR

Answer 8

• label primers w/ Masstag (each w/ diff MW)]
• detected by MS

Question 9

What are the main advantages and disadvantages of MassTagPCR for virus detection?

Answer 9

Adv: Multiplexing (unlimited tags); high throughput (~2000 test per 6-8hrs run); Reduce false pos; potential for virus discovery
Dis: Less Sn than RT-PCR (analytical Sn matters in virology)

Question 10

What are the differences between standard PCR and isothermal amplification?

Answer 10

Iso thermal: no changes in temp; Enz used to separate DNA strands while amplifying
PCR: changes in temp. DNA pol extend on ssDNA

Question 11

Explain the principles behind Microarray and how it can be used to detect viruses.

Answer 11

• Oligonucleotide probes attach to chip - complimentary to specific V sequnece (DNA or RNA)
• detection by labelled reagent: fluorophore, chemiluminescence

Question 12

If a new virus infected a population causing disease, what steps could be done to identify it?

Answer 12

Cell culture?*

Question 13

what would be important in designing a PCR to detect a virus?

Answer 13

1. primer design: primer length; product size; post PCR manipulations
2. samples: DNA extraction; inhibitors
3. the information required: not/detected / quantification / clinical significance (Sn?)

Question 14

what are the advantages of real time PCR over standard PCR for virus detection?**

Answer 14

Use specific probes to ID sequence by hybridisation

Question 15

Difference b/w these 2 Cell types/lines
- Primary/Diploid
- Continous

Answer 15

• Diploid: Normal cells (human/animal tiss). Mix of differentiated cells. Limited passage (/#replications?). Broad growth spectrum for viruses. e.g. human fibroblast =HSV, primary monkey kidney (PMK) = influ.
• Continous: Malignant (human/animal). Use of particular cell lines useful. Indefinite passage. e.g. Vero cells, HeLa (cervical Ca) = HSV

Question 16

What’s the Hayflick limit

Answer 16

telomeres don’t shorten = cells always replicate

Question 17

Briefly describe these target amplification methods:
Polymerase Chain Reaction (PCR)
a) Multiplex PCR
b) Nested PCR
c) RVS transcriptase PCR for RNA viruses
Real time PCR
MassTag

Answer 17

a) Do 1+ PCR rxn in 1 tube. * possible interference b/w 2
b) 1st round PCR = template -> transferred to 2nd round = primes. *possible contamination
c) RNA -> DNA -> PCR