Lecture 2 Flashcards

Question

In what class do ambisense ssRNA genomes fall?

Answer 1

- Can be considered like (-) ssRNA or dsRNA since it needs to carry RdRp because it cannot be read by ribosomes as partially negative

Answer 2

Before we had refrigerators, cell cultures and all that jazz, viral stocks were kept by passage from animal to animal. This was done by infecting animals with extracts from infected animals, but sometimes it selected for viruses with unexpected properties (+ expensive and inconvenient). For example, polio used to be passed through fecal-oral route but was was passed in brains, so the virus developed a neurotropism.

Answer 3

Yes, although we can study infections on plates, it is often best to infect an animal to assess the nature of the infection, the symptoms it causes, how it interacts with immune cells, organs and metabolism (not possible in dishes).

Answer 4

People used to use embryonate chicken eggs. After 5-14 days of fertilization, the virus was injected through a hole into the appropriate site for replication. This was useful for vaccine production (1 egg per vaccine for influenza). Examples: chorioallantoic membrane inoculation (HSV, Poxvirus, Rous sarcoma virus), amniotic inoculation (influenza virus, mumps virus), Yolk sac inoculation (HSV), allantoic inoculation (Influenza virus, mumps virus, NDV queen, avian adenovirus)

Answer 5

- 1949 - Enders, Weller and Robbins - Primary cell cultures: from animal tissue, limited life span (5-20 divisions) like baby foreskin since babies are born all the time - Continuous cell lines: single cell type that can live forever (tumour tissue, HeLa cells are cervical cancer cells Henrietta Lacks, immortalized primary cells, however they start to look different after many infections and life cycles

Answer 6

We look for cytopathic effects (CPE): rounding up of cells, detachment, cell lysis, syncitium formation (cell fusion so multinucleate cells common in measles), nuclear shrinking/swelling, accumulation of virions or viral proteins, membrane alterations and even cell death (like in the case of poliovirus).

Answer 7

We can use plaque assays to determine virus titer (concentration of virus in sample). Here we count plaque forming units (PFUs) where a bacteriophage has cleared bacteria in a little circle you know. It is important to note that we count the number of infectious particles with high reproducibility and accuracy. EMPHASIS ON THE INFECTIOUS.

Answer 8

The cells are purple because they are stained with crystal violet. We do serial dilutions multiply by dilution factor and volume to know initial concentration all that jazz then pour it into the cultures which are semi-solid because we do not want viruses to float to far away when they pop out of cells. Plus do three times for statistical power.

Answer 9

- We do a focus-forming unit (FFU) assay and count foci. In this case, cells are permeabilized and stained with an antibody against a viral protein so we can count foci using fluorescence microscopy. - Another assay is the endpoint dilutions assay where we observe for cytopathic effects and clearing although the plaque is not perfectly clear like in plaque assays. We usually use TCID50 which is the dilution where 50% of the thing shows clearing.

Answer 10

- Never equal to 1 - The higher it is, the less likely it is to get infected on first contact (if that makes sense) - A single particle can initiate e the infection, it is simply an estimate/probability thing - Not all virus particles are successful - WHYYY? because of damaged particles, mutations, "empty" particles (no genome), complexity of viral life cycles, strong antiviral defence mechanisms, multicomponent viruses that need multiple genomes to come together for successful infection

Answer 11

- Number of infectious particles added per cell not physical particles - Not the number of infectious particles each cell receives - If you add 10 to the 7 PFUs to 10 to the 6 cells (MOI=10), each cell does NOT receive 10 virions - Infection depends on random collision between cell and virus - When cells are susceptible, they can receive 0, 1, 2, 3 particles maybe more, all random - Distribution of virus particles received per cell is best described by the Poisson distribution

Answer 12

36.8% of cells are uninfected, 36.8% of cells receive 1 particle, and 26.4% of cells receive more than 1 particle (so total 63.2% of cells are infected). At MOI = 8 nearly 100% of cells are infected.

Answer 13

- EM - Hemagluttination assay (check how much hemagluttination and estimate virus titer, takes 30 minutes)

Answer 14

- Serology techniques like immunostaining (fluorescence) or ELISA - Viral nucleic acids with PCR, Microarray and NGS

Answer 15

Plaque assays allow us to select viruses (animal virus genetics) with altered specific properties and purify them by plaquing. We can engineer mutations into viral genomes using infectious DNA clones (modern validation of Hershey Chase experiment. We can put insertions, deletions, substitutions, nonsense, missense, all that jazz.

Answer 16

Transfection comes from transformation-infection. This term was used to describe the production of infectious viral particles after the transformation of cells by viral DNA or RNA (first done with bacteriophage lamda). Unfortunately, now the term has become synonymous with introduction of any RNA or DNA into cells.

Answer 17

One of the first cDNA produced with virus, modern validation of H-C experiment, since the genome (DNA) alone suffices to replicate and make an infectious viral particle. (do it through direct virus infection, transfection of viral RNA, cDNA transfection and + strand RNA transcript from cDNA all gave virus)

Answer 18

Reverse genetics are possible to infect cells through cDNA and all that lovely in vitro stuff (like for Influenza virus)

Answer 19

False, usually the full virus is needed to be infectious, cannot travel in monkey meat. HELLO

Lecture 2 Flashcards

(43 cards)