SARS-CoV-2

Question 1

Transmission of viruses

Answer 1

Expelled on large droplets or fine aerosols
Airborne transmission - particle cut off size 30-100 micrometres
Affected by environment, temperature, humidity, air flow

Question 2

Short range transmission of viruses (primary transmission)

Answer 2

Droplets - > 5 micrometres that fall on the ground
Aerosols - 5 micrometers suspended in the air
Direct (physical) contact
Indirect contact (fomite (inatimate objects))

Question 3

Long-range transmission of viruses (primary transmission)

Answer 3

-Aerosol
-Indirect contact (fomite - inatimate objects)

Question 4

How to prevent transmission

Answer 4

Viral RNA decays slowly over time - sensitive to heat (highly stable at low temperatures).
Droplet transmission is more important that aerosol transmission - good ventilation is cruical to lower secondary transmission

Question 5

Main sources of transmission from humans

Answer 5

Directly from respiratory tract
Poor hand hygiene

Question 6

Vertical Transmission (from mother to offspring)

Answer 6

IgM does not cross the placenta due to its size -seen in fetuses as a result of fetal immunologic response to pathogens, an ability that is acquired early in the first trimester of pregnancy..?
Breast milk can contain vRNA – no reported cases
Very Rarely occurs

Question 7

Faecal-Oral/ Faecal Aerosol transmission

Answer 7

High number of ACE2 receptors in the bowel
vRNA is in faeces but it is not infectious
No evidence in humans

Question 8

Sexual Transmission

Answer 8

Semen can contain vRNA
Vaginal fluids negative for vRNA except in very rare cases
Not infectious

Question 9

Bloodborne transmission

Answer 9

Virus is in the periphery..?perhiperal nervous system???
No documented cases of bloodborne transmission

Question 10

Pets and Animals transmission

Answer 10

Reports of cats, ferrets and dogs
No documented case of transmission from pet to humans

Question 11

Non-Alcohol based Hand-sanitiser - Example and how it works

Answer 11

-Benzalkonium chloride – commonly used disinfectant

-Disrupts bacterial cell membranes

-Hydrogen peroxide kills bacterial spores

Question 12

Alcohol based Hand-sanitiser - examples and mechanism

Answer 12

Isopropyl alcohol and ethanol

Dissolves viral envelope proteins and the nucleocapsid

Exposes the vRNA/ genetic material for degradation

Disrupts viral entry and transmission

Ethanol is very effective against enveloped viruses

Question 13

Virology of Coronavirus

Answer 13

Family: Coronviridae
Subfamily: Coronavirinae
Four genera:
– Alphacoronavirus, Betacoronavirus (humans and mammals)
– Gammacoronavirus and Deltacoronavirus (birds and fish, some mammals)

Alphacoronavirus (α-CoV 229E and NL63)
Betacoronavirus (β-CoV OC43 and HKU1)

Highly pathogenic and virulent subtypes: Betacoronavirus
SARS-CoV-1, (10 % mortality)
MERS (36 % mortality)
SARS-CoV-2 (3.8 % mortality globally) – 79 – 82 % genome sequence identity

Non-Structural protein (NSP1) is identified in Alpha and Beta.

Question 14

Components of SARS-CoV-2 Virus

Answer 14

Structural proteins:
Nucleocapsid , Membrane Protein, Envelope Protein , Spike Glycoprotein (24-40 proteins)

Positive sense single stranded RNA of 30,000 nt

13-15 open reading frames

12 expressed proteins

ORF NS proteins 1a = replicase
ORF NS proteins 1b = protease – 96 % similarity

Question 15

Mechanism of SARS-CoV-2

Answer 15

Virus prevents cellular mRNA from exiting the nucleus – blocking the immune response system – NSp1 prevents interferon from being activated

Question 16

Mechanism of SARS-CoV-2 - role of spike glycoprotein

Answer 16

• Glycosylated Type I membrane protein
• Trimeric pre-fusion form
• Host furin protease – cleaves the S protein into S1 and S2 units
• N Terminal S1 contains receptor binding domain (RBD)
• Host - Angiotensin converting enzyme 2 receptor (ACE2) – 2-4 x strength
• Further cleavage by host serine protease TMPRSS2 of S2 unit triggers entry
• In vitro – exists as pre and post fusion forms
  -ACE2 - enzyme cleaves angiotensin I into 1-9 and angiotensin II into 1-7
  ~ 600 plasma and sera samples identified majority nAb to the RBD

Question 17

TMPRSS2 role

Answer 17

Causes further viral cleavage of S2 to trigger viral entry.
Highly expressed in several tissues
-Co-expressed with ACE2 in nasal epithelial cells, lungs and bronchial branches
-Cathepsin L and Furin involved alongside TMPRSS2
-SARS-CoV-2 receptor RBD

Question 18

What happens inside a host cell after viral entry for viral replication inside host

Answer 18

Viral entry triggers the translation of ORF1a and ORF1b
Produces pp1a and pp1ab - forms the replication complex

Exocytosis is through Golgi pathway or lysosomes – slow and inefficient but persistent

Exit includes five aa – proline, arginine, arginine, alanine and arginine – host furin clips it – essential for lung cell entry

Question 19

SARS-CoV-2 RdRp

Answer 19

RdRp Used for replication of its genome and the transcription of its genes.
Composed of a catalytic subunit known as NSP 12 - Accessory units are NSP8 and NSP7

Unique RdRp contains long protruding extended RNA regions in NSP8

NSP12 subunit binds to the vRNA between fingers and thumb

Active site is on the palm

The vRNA exits as a duplex from the NSP12

It is unknown how vRNA strands are separated during transcription of viral genes

Question 20

Why do we see variants?

Answer 20

Poor public health infrastructure

Poor education and understanding of infection and disease

Increased infections of other infectious diseases

Lower vaccination rate

Anti-vaxxers and mis-information

Immunocompromised individuals - Weak immune protection More susceptible to infection Chronic infections

Question 21

Immunodominance -

Answer 21

Is the consistent feature of the human immune response against a wide array of small and complex pathogens

Study of 650 individuals - 90 % of plasma and serum Ab target the RBD (receptor binding domain)

-nAb block viral protein conformational changes or interactions with entry receptors

-S2 subunit is protected by glycan shielding and immunogenicity is not fully understood

Four classes of RBD-binding neutralising antibodies (nAb)
Class 1 – bind the spike protein in open conformation
Class 2 – ACE2 blocking antibodies – bind RBD in open and closed formation
Class 3 – do not block ACE2 – bind the RBD in open and closed conformation
Class 4 – bind outside the ACE2 site and only in RBD open conformation

Question 22

Virus escape mutants -

Answer 22

specific mutations helping to achieve the process of immune escape.

Question 23

Virus escape mutants - Antigenic change examples

Answer 23

Amino acid substitutions – altering the viral epitope
Substitutions in the receptor binding domain (RBD) – increases ACE2 affinity
Glycosylation – the glycan shield – masking
Deletions and insertions – alters the epitope conformation – blocking ab
Allosteric effects outside the epitope – alters the protein confirmation

Question 24

Virus escape mutants - Antigenic change examples in Cov- 2

Answer 24

two mutations per month in the global population between Dec 2019 and october 2020:Amino acid substitutions within the spike receptor binding motif - Enhancing binding of the ACE2 receptor and reduceing neutralising antibodies.

-B.1.1.298 lineage has the Y453F mutation and a Δ69-70 deletion – alters the conformation of an exposed amino terminal domain loop – increases infectivity

Question 25

Variants of Concern (VOC)

Answer 25

Alpha B.1.1.7
Beta B.1.351
Gamma p.1
Delta B.1.617.2
Omicron B.1.1.529

Question 26

Variants of Concern (VOC) are associated with one or more

Answer 26

Increase in transmission

Detrimental change in epidemiology

Increase in virulence

Change in disease presentation

Decrease in effectiveness of public health and social health

Decrease in diagnostics, vaccines and therapeutics

Question 27

Omicron: EG.5 and EG.5.1 (Eris

Answer 27

Delta variant – the most dominant variant

Estimated to be 3 – 6 x more infectious in the same time frame than Delta

Previous infection immunity and vaccine protection - questionable

Question 28

Omicron – B.1.1.529

Answer 28

Has 32 mutations as of december 2021 within the spike protein - delta had 16.
Clade identfier (grouping that includes a common ancestor and all the descendants (living and extinct) of that ancestor.) - 21K and 21L
Not considered to be of concern but it cant be ignored.

Question 29

Omicron - B.1.1.529 - Mutations

Answer 29

Mutations include: NSP 12 RdRp and NSP 14 Exo

Spike target failure due to 69-70 deletion

Mutations and deletions known to:
Increase transmission
Higher viral binding affinity
Higher antibody escape

Other mutations currently of unknown purpose

Question 30

Why is Omicron concerning?

Answer 30

Mutations are linked with faster transmission = faster spread internally

High replication fitness leading to a higher viral load

Possible higher viral shedding – in turn increases transmission

Production of more variants

Omicron wave has a doubling time higher than any of the previous waves - more transmissible, quickly became the most dominant variant after delta

Mutation near the Furin cleavage site - cell entry

Deaths occur when health care systems are overloaded

Clinical presentation suggests – infects the younger population

Emerged when vaccine protection is increasing – unknown if it has developed vaccine escape