Lecture 9: Applied: Molecular detection of novel viruses & Coronaviruses and coronavirus reinfections

Question 1

What is viromics and what is it used for?

Answer 1

Enrich for viruses and sequence all nucleic acids via Next Generation Sequencing. It is ‘the best’ technique to find unknown viruses.

Question 2

What is VIDISCA?

Answer 2

A viromics typing method used in the AMC: Virus discovery cDNA-AFLP. Its main characteristics is restriction enzyme digestion for fragmentation to create a library.

Question 3

What is a condition you want in enriching for viruses and how do you optimize it?

Answer 3

Low host DNA, low in ribosomal RNA (contaminants you don’t want to see). You optimize it by size selection to filter out all but viruses, DNase to digest loose DNA from host for example, and reverse transcription for ribosomal RNA.

Question 4

How do you perform viromics?

Answer 4

You first enrich for viruses, then create a library, then sequence all viruses and analyse the reads. Then there will be follow up research to know more about the virus.

Question 5

How do they prepare the library?

Answer 5

With VIDISCA. They use frequent cutting restriction enzymes to cut viral DNA or cDNA (synthesized from viral RNA).

Question 6

Which restriction enzymes do they use in VIDISCA?

Answer 6

MSE1 which recognizes TTAA. 4 nucleotide recognition cuts in every piece of 256 bp. This is ideal for sequencing.

Question 7

What are the Koch’s psotulates?

Answer 7

• The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
• The microorganism must be isolated from a diseased organism and grown in pure culture.
• The cultured microorganism should cause disease when introduced into a healthy organism.
• The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Question 8

What is the cytopathic effect?

Answer 8

You see this in culture, infected host cells structure is different (can be rounding, clustering etc.) than normal cells.

Question 9

What does the human seasonal coronavirus cause?

Answer 9

Respiratory infections.

Question 10

When do you get your first infection with a seasonal coronavirus?

Answer 10

Early in life, before the age of 4 years.

Question 11

Can you be reinfected by coronaviruses?

Answer 11

Yes, the capacity to re-infect seems a general characteristic of coronaviruses. Reinfection occurs frequently 1 year post-infection, sometimes shorter.