Intro to virology + genetics

Question 1

What are some reasons to study viruses?

Answer 1

1) Viruses are important pathogens as they are able to shape populations

2) Viruses infect ALL living organisms

3) They are the most ABUNDANT biological entities on Earth; have important effects on ECOSYSTEM DYNAMICS

4) Everything we eat, breathe, and are covered by billions of virus particles

5) We CARRY VIRAL GENOMES as part of our genetic materials

Question 2

(T/F) Viruses can infect a wide range of cells, but not all.

Answer 2

False! If there is a living cell, a virus will be able to infect it!

Question 3

What is the first virus to be discovered?

Answer 3

The tobacco mosaic virus

Question 4

What does the ICTV stand for?

Answer 4

The International Committee on Taxonomy of Viruses

*they have developed a universal system for naming viruses

Question 5

Match the following terms to their definitions:

1) Order
2) Family
3) Subfamily
4) Genus
5) Species

A) -viridae
B) -virus
C) -virales
D) usually the form of [disease] virus
E) -virinae

Answer 5

Order: -virales

Family: -viridae

Subfamily: -virinae

Genus: -virus

Species: usually the form of [disease] virus

Question 6

A virus is an _________, ___________ _________ parasite comprising genetic material surrounded by a ________ coat and sometimes a membrane.

Answer 6

infectious; obligate intracellular; coat

Question 7

What is the common strategy for viral propagation?

Answer 7

All viral genomes are PACKAGED inside particles that mediate their transmission from host to host.

The viral genome contains the information for initiating and completing an INFECTIOUS CYCLE with a host cell.

All successful viruses are able to ESTABLISH THEMSELVES IN A HOST population so that viral propagation is ensured.

Question 8

(T/F) If viruses exist today, they were successful in propagating.

Answer 8

True!

Question 9

What are the six steps of virus infectious cycle?

Answer 9

1) Attachment and entry
2) Production of viral mRNA and viral protein synthesis (using host cell mechanisms)
3) Genome replication
4) Assembly of viral particles
5) Release
6) Maturation (doesn’t happen 2 all)

Question 10

What kind of host cell does a productive infection require?

Answer 10

1) Susceptible: allows ENTRY of genetic material at the site of replication

2) Permissive: REPLICATION + PRODUCTION of infectious progeny virions

Question 11

What are the two steps of breaking and entering of a virus into a host cell?

Answer 11

Step 1: Binding susceptible cells (virus bind to attachment factors or receptors at the cell surface)

Step 2: Delivering the genome to the site of replication (direct entry or through endocytosis)

Question 12

What are the two steps of the virus expressing and multiplying?

Answer 12

Step 1: Making viral proteins (by making mRNA that the host machinery requires)

Step 2: Replicating the genome (replication doesn’t occur everywhere on the cell; they’re very concentrated at specific sites of the cell)

Question 13

What is building?

Answer 13

The synthesis of viral proteins

Question 14

What mechanisms do the two different viruses use to escape the host cell at the end of their infectious cycle?

Answer 14

Enveloped viruses: BUDDING (allows virus to live longer in the cell)

Non-enveloped viruses: CELL LYSIS (much more inflammatory) and SPECIAL MECHANISMS (non-lytic mechanisms such as EXOSOMES or other membrane-wrapping mechanisms)

Question 15

(T/F) By growing viruses in animals (eggs), we can produce vaccines.

Answer 15

True!

That is how influenza vaccine is produced

Question 16

Match the following types of cultured cells to their definitions:

A) Primary human fibroblasts
B) Mouse fibroblasts
C) Human epithelial cells

1) CONTINUOUS CELL LINE, IMMORTALIZED and TRANSFORMED. Cancerous cells; high metabolism.

2) LIMITED LIFE SPAN, non-cancerous; new cells required to make new viruses. High genetic variability as each batch is different.

3) CONTINUOUS CELL LINE and IMMORTALIZED (telomerase is inactive)

Answer 16

A) Primary human fibroblasts: LIMITED LIFE SPAN, non-cancerous; new cells required to make new viruses. High genetic variability as each batch is different.

B) Mouse fibroblasts: CONTINUOUS CELL LINE and IMMORTALIZED (telomerase is inactive)

C) Human epithelial cells: CONTINUOUS CELL LINE, IMMORTALIZED and TRANSFORMED. Cancerous cells; high metabolism.

Question 17

(T/F) Limited life span cells are better to grow viruses than continous cell lines.

Answer 17

False!

Question 18

Define cytopathic effects.

Answer 18

Signs that cells are infected and producing virions.

(phenotypes displayed during viral infection)

Question 19

Define syncytium formation.

Answer 19

Cells that fuse together (is done in SARS-CoV-2 infection)

Question 20

What are the two ways viral titer (quantity of viruses in a given volume) can be expressed as?

Answer 20

1) INFECTIVITY ASSAYS (infectious viruses per volume)

2) PHYSICAL MEASUREMENTS (viral particles and their components per volume)

Question 21

Match the two forms of infectivity assays to their definitions:

1) Plaque assay
2) Endpoint dilution assay

A) Dilutions of a virus stock are prepared and inoculated. The number of cell cultures that are infected is then determined for each virus dilution, by looking for cytopathic effect. Used if virus can’t make plaques.

B) Adherent monolayer of cells mixed with a VIRUS and covered by a SEMI-SOLID AGAR, restricting viruses from being released. This results in infection of neighbouring cells, causing plaques. Plaques are counted and multiplied by the dilution factor.

Answer 21

Plaque assay: Adherent monolayer of cells mixed with a VIRUS and covered by a SEMI-SOLID AGAR, restricting viruses from being released. This results in infection of neighbouring cells, causing plaques. Plaques are counted and multiplied by the dilution factor.

Endpoint dilution assay: Dilutions of a virus stock are prepared and inoculated. The number of cell cultures that are infected is then determined for each virus dilution, by looking for cytopathic effect. Used if virus can’t make plaques.

Question 22

What is the difference between PFU and TCID50?

Answer 22

PFU: Plaque Forming Unit

TCID50: 50% Tissues Culture Infectious Dose

Question 23

(T/F) All viral particles are infectious.

Answer 23

False! Not all viral particles are infectious. These non-infectious particles are also known as DEFECTIVE PARTICLES.

Question 24

What are the different types of physical measurements of virus particles?

Answer 24

1) Hemagglutination
2) Electron microscopy
3) Viral enzymes (e.g., polymerase)
4) Nucleic acids (presences of the genome*)
5) Detection of viral antigens

*just because the genome is present does not mean it is infectious; physical measurement!

Question 25

Briefly describe how a COVID-19 Diagnostic Test works through RT-PCR.

Answer 25

1) Nasopharyngeal swab
2) Collected specimen; stored at 2-8˚C for up to 72 hours.
3) RNA extraction; purified RNA extracted from deactivated virus
4) RT-qPCR; purified RNA is reverse transcribed to cDNA and amplified by qPCR.
5) Test results; positive tests cross the threshold line within 40 cycles.

*a form of physical measurements using nucleic acids

Question 26

What is ELISA?

Answer 26

Enzyme-Linked Immunosorbent Assay.

It is used to detect viral antigens or antibodies.

To detect antigens: A captured antibody binds a viral antigen and this complex binds a second antibody with an indicator.

To detect antibodies: A viral antigen is bound to the solid support and an antibody in the sample binds to it. Finally, a second antibody with an indicator binds to the complex.

While a measurement of viral antigens is a physical measurement of virus particles, detection of current or previous infection (done by measurement of antibodies) is not a measurement of viral particles.

Question 27

What do viral genomes encode?

Answer 27

They encode gene products and regulatory signals required for:

1) Replication of the genome
2) Efficient expression of the genome
3) Assembly and packaging of the genome
4) Regulation and timing of the reproduction cycle
5) Modulation of host defenses
6) Spread to other cells and hosts

Question 28

What do viral genomes NOT encode?

Answer 28

1) Genes encoding a complete protein synthesis machinery (no virus do this; they make mRNA that can be read by host machinery)

2) Genes encoding proteins of energy metabolism or membrane biosynthesis (E is provided by host cell)

3) Telomeres or centromeres

Question 29

(T/F) CMV, a virus, encodes genes that change the way a cell makes lipids, making long chain phospholipids. However, they do not make those lipids themselves.

Answer 29

True! Viruses do not encode a complete protein synthesis machinery.

Question 30

How are viral genomes structurally diverse?

Answer 30

They can be:
1) linear, circular, or segmented
2) DNA or RNA
3) can have covalently attached proteins
4) single, double-stranded, or gapped
5) cross-linked ends of dsDNA
6) DNA with covalently attached RNA
7) can be +, -, or ambisense

Question 31

The length of viral genomes can vary from ______kbp to ______kbp.

Answer 31

1.7 to 2,500

Question 32

Fill in the blanks:

1) mRNA is defined as the _______ strand because it can be translated.

2) The RNA or DNA complement of the positive strand is called the _______ strand (antisense).

Answer 32

1) Positive

2) Negative

Question 33

What are the seven groups that viral genomes can be classified into?

What is a characteristic they all share?

Answer 33

1) dsDNA
2) gapped dsDNA
3) ssDNA
4) dsRNA
5) +ssRNA
6) +ssRNA with dsDNA intermediate (Retrovirus)
7) -ssRNA

All viral genomes must be copied to produce mRNA that can be read by host ribosomes.

Question 34

For viruses with dsDNA genomes, some require host DNA-dependent DNA and RNA polymerases, while others don’t.

Briefly describe which ones do and which ones don’t and why.

Answer 34

Small dsDNA viruses do not have enough room in their genome to encode for enzymes to replicate their genome and make protein.

Large dsDNA viruses have the room to encode their own enzymes.

Question 35

Small dsDNA viruses replicate in the host _______, wile the large dsDNA viruses replicate in the host _______.

Answer 35

Nucleus; cytoplasm

Question 36

For gapped dsDNA genomes, the gap must be filled to generate a complete dsDNA molecule for the production of _______, done by ______ polymerase.

The gapped DNA genome is generated from an RNA template by the ______-encoded _________ __________ (this will assemble as proteins).

Answer 36

mRNA; host

virus; reverse transcriptase

*when the gapped dsDNA enters the host nucleus, it is detected as host’s DNA being damaged and gets repaired by host enzymes to make dsDNA.

Question 37

How do ssDNA genomes make mRNA?

Answer 37

They can’t do anything as a single strand, thus they make a dsDNA intermediate using HOST DNA polymerase.

This dsDNA makes mRNA (using host RNA polymerase) and proteins, and also makes ssDNA to assemble into viral particles.

Question 38

Because cells have no RNA-dependent RNA polymerases that can replicate the genomes of RNA viruses or make mRNA from RNA templates, what are the two strategies RNA viruses use to cause infection?

Answer 38

1) Virus-encoded RNA-dependent RNA polymerase (RdRp)

2) Virus-encoded reverse transcriptase (RT) to generate dsDNA from the RNA genome. This dsDNA can be transcribed by the host RNA polymerase.

Question 39

How do viruses with dsRNA genomes replicate themselves?

Answer 39

Since the ribosome can’t use dsRNA, it cannot be used as mRNA directly.

Thus, the (-) strand of the dsRNA is used to make (+) mRNA using RNA dependent RNA polymerase.

This newly synthesized (+) strand is encapsidated and copied to produce dsRNA.

**dsRNA VIRUSES MUST HAVE A RdRp as VIRAL PARTICLES (NOT JUST BE ENCODED) to make mRNA!!

Question 40

Briefly answer the questions regarding viruses with (+) ssRNA genomes.

1) Why are they normally infectious if introduced in cells?

2) Why don’t they need to carry RdRp as a viral particle but do need to encode it?

3) Give an example of this virus.

Answer 40

1) The (+) RNA genome can usually be translated directly into proteins by host ribosomes.

2) Because the genome can be directly translated, they do not need to make RNA using RdRp. However, they do need this enzyme later on to make (-) RNA which is copied back to (+) strand, so the (+) RNA can be encapsulated with newly synthesized proteins.

3) SARS-CoV-2

Question 41

In some cases of (+) ssRNA genomes, a ________ mRNA is produced.

Answer 41

subgenomic (deletions at the 5’ end)

Question 42

For retroviruses [(+) ssRNA genomes with a dsDNA intermediate], _________ __________ and an ________ must be in the viral particles.

Why?

Answer 42

Reverse transcriptase; integrase

Reverse transcriptase converts the (+) RNA genome into a dsDNA.

Then, the dsDNA gets integrated into the host cell genome by the integrase, which is used as a template for viral mRNA and genome RNA synthesis by cellular enzymes.

Question 43

What enzyme do the (-) ssRNA viruses require in their viral particles? Why?

Answer 43

RNA Dependent RNA polymerase;

RdRp copies the (-) genome into a (+) strand mRNA.

The (-) genome is also the template for the synthesis of full-length (+) strands, which are copied to produce the (-) strand genomes.

Question 44

(-) ssRNA genomes can be a single molecule (________) or _________.

Some are also ________: they can contain both (+) and (-) strand information on a single strand of RNA (but contains the viral RdRp in the particles).

Answer 44

Mononegavirales; segmented

Ambisense

Question 45

How can you generate mutations in RNA viruses and in DNA viruses to determine which genes are responsible for the mutations?

Answer 45

RNA viruses: Replication usually generate enough mutants. You can also incubate cells infected with virus with antivirals, resulting in resistant mutants.

DNA viruses: Due to cellular/viral DNA polymerases having a high fidelity, replication can not generate enough mutants. Thus, random mutagenesis by exposure to a chemical mutagens or UV light produces mutations.

*Antiviral drugs do not general mutations in DNA viruses; one drug can treat them!
*After achieving mutations, you can study the mutants and determine the gene responsible

Question 46

What is an infectious DNA clone?

Answer 46

a dsDNA copy of the viral genome that is carried on a bacterial vector such as a plasmid.

Question 47

Why is an infectious DNA clone useful? (List 3 reasons)

Answer 47

1) The infectious DNA clones, or in vitro transcripts from them, can be introduced into cultured cells to produce replicative virus (these clones have everything needed to produce viruses in a permissive cell)

2) Recombinant DNA techniques allow easy manipulation of the infectious DNA clone (to introduce new mutations and new genes)

3) Infectious DNA clones provide a stable repository of viral genome (dsDNA is highly stable)

Question 48

(T/F) To recover an infectious virus from an infectious DNA clone, you need to extract and purify plasmids that contain a copy of the viral genome. Then you can either transfect the plasmid into cultured cells, or do in vitro transcription to produce an mRNA which you can transfect into the cells.

Answer 48

True!

Question 49

dsDNA copies of DNA genomes are generally ___________, as they make use of cellular enzymes.

However, ________ DNA are not infectious because the viral _______ cannot be recognized by cellular DNA-dependent RNA polymerases. The viral polymerase and proteins need to be provided by a _______ virus.

Answer 49

Infectious

Poxviruses; promoters; helper

Question 50

What are the four ways a (+) ssRNA virus can make an infectious virus in cultured cells?

Answer 50

1) Infection; the virus can directly infect the cells

2) Transfection of the viral RNA into the cells

3) The viral RNA can undergo cDNA synthesis and cloning to make the dsDNA (infectious DNA clone), which can be directly transfected or;

4) The infectious DNA clone can undergo in vitro RNA synthesis, generating a (+) strand RNA transcript, which can be transfected

for #3 if u have the ds clone, w a good promoter, the cell will just make the mRNA and infectious cycle starts.

Question 51

(T/F) You need to produce and transfect both the (+) and the (-) strand of a dsRNA virus infectious DNA clone to cultured cells to produce the virus.

Answer 51

False!

You only need the (+) strand!

Question 52

What do you need to produce and transfect into a cultured cells to produce an infectious (-) ssRNA virus?

Answer 52

1) You need to produce the (+) strand
2) You need to produce the plasmids encoding the NUCLEOCAPSID, PHOSPHOPROTEIN and POLYMERASE to form the RdRP

Question 53

(T/F) Once we have the DNA infectious clone of a virus, it can be manipulated to a lot of things. For example, we can engineer viral vectors to deliver genes to treat diseases or to fight cancer or to make vaccines.

Answer 53

True!

*learning how viruses replicate allows us to manipulate the genome!

Question 54

Viruses that fight cancer are called _________ viruses.

Cancer cells are also more _______ to viruses.

Answer 54

Oncolytic

Permissive

Question 55

We can manipulate viral genomes to make vaccines.

Briefly describe how the ebola vaccine (Zebov) works and how it was made.

Answer 55

A negative RNA virus aka Vesicular Stomatitis Virus (VSV) was used to make Zebov. The fusion protein (glycoprotein) of VSV was replaced by that of the ebola virus.

This allowed VSV to look like ebola. However, VSV is a very safe virus and our immune system also can stop its infection. This allows us to have immune response to the glycoprotein of ebola!

Question 56

The Covid vaccine is a _______________ viral vector. Thus, multiple shots are needed.

Answer 56

Non-replicating