Week 6 Module 3

Question 1

What is the function of the Liver?

Answer 1

-detoxification
-carbohydrate, lipid, protein synthesis
-produces bile (goes to intestine)
-filters bacteria etc. etc.

Question 2

What is Hepatitis C?

Answer 2

-Liver infection caused by the hepatitis C virus.
-Viral transfer can occur through any contact with infected blood (sharing of infected needles, birth from infected mother, piercings, razors, blood transfusions), sexual transmission

Question 3

What is Acute Hepatitis C?

Answer 3

-Occurs within the first 6 months after someone is exposed to the hepatitis C virus; 85% of acute infections lead to chronic infections.

Question 4

What is Chronic Hepatitis C?

Answer 4

-Can be lifelong infection if left untreated.
Chronic hepatitis C can cause serious health problems:
-liver damage or cirrhosis (scarring of the liver) – 20%,
-liver cancer – 3 to 5%; latent period can be decades
-death

Question 5

What is the classification of Hepatitis C virus (HCV)?

Answer 5

Hepatatis C Virus (HCV )is a flavivirus – linear ssRNA ‘+’ Strand
-Other flaviviruses include: zika, west nile, dengue fever, and yellow fever
-HCV genome is 9.6 kb

Question 6

How do transcription and translation occur in Hepatitis C virus?

Answer 6

-Flavivirus genome used also as mRNA since it is ssRNA (+)
-HCV genome is one long ORF
-translation gives 3000 amino acid polyprotein cleaved by viral
and host proteases to give 10 viral proteins
-one of proteins is RdRp (RNA dependent RNA polymerase) can use RNA genome to make more copies of RNA genome

Question 7

What is the danger of Hepatitis C virus?

Answer 7

-The RNA dependent RNA polymerase RdRp is very error prone and so variants that are resistant to the human immune system and drugs can arise
-infected person will make antibodies but fast evolution means
antibodies aren’t protective for long

Question 8

What is the HCV replication cycle?

Answer 8

-HCV has multiple cellular receptors in liver including CD81, tight junction proteins etc.

-virus enters liver cells
through receptor-mediated
endocytosis

-HCV particle fuses with
acidic endosome and RNA
genome release

-genome replicated, viral proteins made, viral assembly and virion

Question 9

How does the vesicle allow the virion to be released?

Answer 9

Two vesicles eventually fuse. Vesicle is a membrane compartment that’s holding your virus, virus needs to escape, acidification helps virus to break down. Another vesicle in cell called acidic endosome, and it has a low pH, this fuses with regular vesicle, and acidifies the pH and allows virus to come out, capsid opens and virion is released

Question 10

What results from RdRp error prone activity?

Answer 10

-Get new genomes with slight changes, and proteins have slight changes because of RdRp error prone activity

Question 11

How can immunoassays be used against HCV?

Answer 11

Use an HCV antibody test
-serological test for antibodies against HCV
-packaged tests exist
-requires minimum threshold antibody titer
-might indicate past infection and not current infection
-also western blotting and immunofluorescence tests exist

Question 12

What is HCV RNA test?

Answer 12

-usually done after HCV immunoassay
-HCV genome is RNA so this is RT-PCR test (RT= reverse transcriptase)
-also real-time PCR is used meaning it’s quantitative (qPCR)
-indicates active infection

Question 13

What are some treatments for Hepatitis C virus (HCV)?

Answer 13

-Treatments include: Interferons + ribavirin, Direct acting antivirals (DAAs), and CRISPR Cas 9 is a potential treatment for the future.

Question 14

Interferons + ribavirin?

Answer 14

-older treatment
-antiviral response induced in immune cells; Complete mechanism
not understood

Question 15

Direct acting antivirals?

Answer 15

-more current
-four classes: NS3/4A Protease inhibitors, Nucleoside and Nucleotide NSSB polymerase inhibitors, NSSA inhibitors, and Non-nucleoside NSSB Polymerase Inhibitors

-DAAs are expensive

Question 16

CRISPR Cas 9 in regards to Hepatitis C virus?

Answer 16

-possible treatment of the future?
-in 2015 group used Cas9 enzyme from bacterium F. novicida and targeted it to RNA genome from HCV using a guide (rgRNA plasmid) with complementarity to 5’ or 3’ UTR (untranslated region)
– introduced to hepatocellular carcinoma cells (Huh cells) in culture
- HCV viral protein levels were significantly reduced
-CRISPR CAS 9 normally used to make DSB in DNA, but in HCV they can use it to target an RNA genome

Question 17

Vaccination with viral vectors

Answer 17

-viral vectors deliver genetic material designed to express
recombinant protein that will trigger immunological memory

-AstraZeneca and Johnson & Johnson used adenovirus as the vector to express the spike protein from SARS-CoV-2 for the Covid-19 vaccine.
-Vaccinia virus (part of the poxvirus family) is also used as a viral vector. Other viruses that can be used as vectors include: retrovirus, adenovirus, lentivirus etc.
-Viral vector will never cause an infection, harmless version of spike protein for example

Question 18

What is Phage Therapy?

Answer 18

-use lytic phages to lyse bacteria
-most lysis mechanisms use two groups of phage proteins:
i) holins – perforate (make holes in) host cell membrane
ii) endolysins – degrade peptidoglycan layer from within causing osmotic pressure & lysis
-either use entire phage or infective phage that carry important genes encoding lysis proteins but not all packaging genes

Question 19

What are the Advantages of Phage Therapy?

Answer 19

-antibiotic resistance of a bacteria doesn’t affect phage
-targeting will be specific (not disrupt normal microflora), can disrupt biofilms, and low toxicity to host
-single phage can kill bacterium if MOI (multiplicity of infection) good so low doses needed
-frequency of phage-resistant bacterial strains arising seems low
-lower environmental impact
-relatively low cost to manufacture

Question 20

What are the Disadvantages of Phage Therapy?

Answer 20

-phage with possible lysogenic life cycle could be dangerous
-low virulence, poor adsorption (and therefore low m.o.i), poor
pharmacokinetics could be issues
-Transduction may allow transfer of genes between bacteria
leading to unexpected bacterial evolution
-likely to have narrow host range vs. broad spectrum antibiotic
-once bacteria lysed e.g. gram ‘-’ bacteria, toxins might be
released e.g. LPS layer
-human immune system may attack therapeutic phages and
inactivate them

Question 21

Why was Phage therapy used in Eastern European countries?

Answer 21

-Because they are cheaper than antibiotics

Question 22

What is Oncolytic Virotherapy?

Answer 22

use of viruses to kill cancer cells

Question 23

What are Oncolytic viruses?

Answer 23

– naturally occurring or genetically
engineered viruses that selectively kill cancer cells.

Question 24

What is the history of oncolytic virotherapy?

Answer 24

-viral anti-tumour activity first shown definitively in late 1940s & early 1950s; used natural viruses
-not without complications; Erratic or short-lived response and/or normal tissue damage and virulence in non-cancer cells couldn’t be controlled
-major interest renewed in 1991 when Science report showed that
modified HSV-1 improved intracerebral glioma in mouse model

Question 25

Why are cancer cells more likely to be sensitive to viral infection and lysis?

Answer 25

-because they have increased metabolic activity; Good for
viral replication
-also, there is an impaired antiviral response by immune system in cancer tissue compared to normal tissue

Question 26

What is the goal or strategy when using oncolytic virotherapy?

Answer 26

-use virus (natural or engineered) that cannot replicate in normal cells but replicates very well in cancer cells
-deliver virus either directly to tumour site(s) or by intravenous (IV) delivery
-ideal results: i) viral replication and lysis kill cancer cells in initial
tumour
ii) Increase immune response by exposing cancer antigens

Question 27

If oncolytic virotherapy is successful and lyses cancer cell what is the benefit of this?

Answer 27

-If virotherapy is successful and lyses cancer cell, it can expose cancer cellular debris and your body could make antibodies to cancer antigens which is beneficial

Question 28

Oncolytic virotherapy stages

Answer 28

-Initial stage of infection, cell killing, and Immune cells recruited; development of anti-tumour immunity