Coronaviruses (12-14)

Question 1

What class are coronaviruses in?

Answer 1

Baltimore class 4: +ve ssRNA genome → can be used directly as mRNA
Order: Nidovirales, Family: Coronaviridae, Genera: Alpha-, Beta-, Gamma-, Delta-coronaviruses
Alpha and beta coronaviruses of bat origin (include human and mouse hepatitis (MHV))
Gamma and delta coronaviruses of avian origin
Betacoronaviruses → SARS-CoV, SARS-CoV-2, MERS-CoV

Question 2

What is the history of human coronaviruses?

Answer 2

1960s → HCoV-229E and HCoV-OC43
2002 → original SARS-CoV - high case fatality, less transmissible, outbreak controlled in 2003
2004/5 → HCoV-NL43 and HCoV-HKU1
2012 → MERS-CoV - now only sporadic cases, emerged from camels, didn’t spread vastly
2019 → SARS-CoV-2 - global pandemic, highly transmissible

Human coronaviruses have varying pathogenicity
→ mild upper respiratory tract infections: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1
→ severe lower respiratory tract infections: SARS-CoV, MERS-CoV, SARS-CoV-2

Question 3

What is the structure of coronavirus?

Answer 3

Enveloped spherical particle (125nm diameter) with crown-like surface proteins
→ lipid membrane embedded with spike proteins, M proteins, hemagglutinin-esterase dimer (HE) and E protein
→ spike protein - trimer, part that interacts with receptors to allow entry
→ within is the genome - +ve ss RNA wrapped in nucleoprotein

Question 4

What does the coronavirus genome made up of?

Answer 4

30 kb +ve sense ssRNA (longest viral RNA genome), 13 ORFs

1st half of genome encodes poly proteins by frame-shifting - genes translated from viral RNA genome
→ the viral genome RNA itself has a 5’ cap and poly A tail so can act as an mRNA encoding ORF1a/b

3’ end half of genome has a different type of gene expression
→ discontinuous translation producing subgeneric RNAs

Question 5

Does coronavirus utilise proof reading?

Answer 5

Yes - COVID replication has proof reading activity (transcriptase/replicase complex) → reduces rapid evolution, replication doesn’t produce as many errors
→ the many variants are seen due to its high transmissibility - the sheer scale of cases

Most RNA virus polymerases don’t have any proof reading activity
→ error prone polymerases produce many mutations leading to rapid evolution

Question 6

What are the stages of the coronavirus replication cycle?

Answer 6

→ attachment by S (spike) protein to receptor
→ entry by endocytosis into cytoplasm
→ uncoating, from nucleoprotein, of +ve sense ssRNA genome
→ translation of genome to produce transcriptase, replicase complex (the viral polymerase)
→ replication and transcription of viral RNAs
→ translation of structural proteins
→ assembly of viral particles in ER/Golgi vesicles
→ exit by exocytosis

Question 7

What are the two stages of gene expression in the coronavirus replication cycle?

Answer 7

Initial → translation of incoming genome to produce the transcriptase/replicase complex (doesn’t bring own polymerase in - can translate it upon entry)
Later → replication of genome and transcription of viral RNAs

Question 8

What is involved in SARS-CoV attachment and entry?

Answer 8

Trimer of spike protein binds to angiotensin-converting enzyme 2 (ACE2) on human epithelial cells
→ binding triggers endocytosis and viral particle enters cell in an endosome

Spike protein has 2 domains → S1: interacts with receptor, S2: fusion peptide
→ there needs to be cleavage between these domains to release fusion peptide
→ proteases in the cell membrane (e.g. TMPRSS2) and within the endosome (e.g. cathepsins) release the fusion peptide within the S2 domain
→ has 2 activation sites - 2nd allows pre-activation step which contributes to high transmissibility
→ insertion of fusion peptide into endosomal membrane leads to fusion of viral and endosome membrane releasing genome into cytoplasm

Question 9

What is the difference between the protease cleavage of SARS-CoV and influenza?

Answer 9

SARS-CoV spike cleavage → carried out by proteases in the cell membrane of cell its infecting and within endosome
→ late-stage process
Influenza HA cleavage → occurs early when viral particles being made

Question 10

How can the 2 conformations of SARS-CoV S1 domains influence its transmissibility?

Answer 10

Standing up → higher affinity for receptor but many epitopes on show - very visible to the immune system
Lying down → less epitopes presented, hides receptor binding domain - better immune evasion

One of the protease cleavage sites is for enzyme farin → found in the respiratory tract
→ this cleavage by farin makes spike protein more accessible for the cleavage that releases the fusion peptide (S2 domain more accessible) - enhancing entry of virus into epithelial cells

Question 11

How do the features of viral entry differ between SARS-CoV and SARS-CoV-2?

Answer 11

Frequency of receptor binding domain standing up → SARS-CoV: high, SARS-CoV-2: low allows for immune evasion (hidden RBD)
Human ACE2-binding affinity by RBD → SARS-CoV: low, SARS-CoV-2: high allows for enhances entry (compensation for hidden RBD)
Pre-activation by furin → SARS-CoV: no, SARS-CoV-2: yes allows for enhanced entry into some types of cells (compensation for hidden RBD)

Question 12

What is produced by the translation of coronavirus genomic RNA?

Answer 12

The genome +ve sense ssRNA has a 5’ cap (ribosomes can bind) and poly A tail → can act directly as mRNA
→ ribosomes translate this to produce polyproteins pp1a and pp1ab - by ribosomal frame shifting (pause over slippery sequence required caused by pseudo knot structure in RNA)
→ pp1a and pp1ab encode components of the transcriptase/replicase complex
→ protease within pp1a self-cleaves out and is able to cleave rest of the poly proteins
→ components of pp1a and pp1ab assemble into transcriptase/replicase complex on the ER membrane

Question 13

What does the transcriptase/replicase complex of coronavirus consist of?

Answer 13

Includes: the polymerase, RNA helicase, capping enzyme, exoribonuclease (proofreading)
Function: genomic RNA is used as a template to make 2 types of -ve sense RNA
→ full length -ve sense RNA - template for producing more genomic +ve sense RNA - to be packaged into new viral particles
→ subgenomic -ve sense RNAs - templates for producing subgenomic mRNAs that encode viral structural proteins by discontinuous translation

(different to influenza - doesn’t have its own capping enzyme so steals caps from cellular mRNAs, while coronavirus has bigger genome - encodes more proteins, including capping enzyme)

Question 14

How does the transcription/replicase complex of coronavirus transcribe genomic RNA?

Answer 14

Once the transcription/replicase complex is made - binds to genome at polyA tail
Makes full length -ve antigene RNA, complementary to the full length genome
→ can be used to make more genome copies
Can also copy the genome in discontinuous manner
→ complex binds at 3’ polyA tail, starts copying then at TRS (transcription-regulating sequences) can dissociate then re-associate at leader sequence
→ produces panel of different subgenomic RNAs - all contain poly U and leader sequence - messages that encode the other structural proteins of the virus

Question 15

What do the subgenomic RNAs (sgRNAs) of coronavirus encode for?

Answer 15

sgRNAs all contain the same leader region and same 3’ end (nested) - all capped and polyadenylated

They encode structural proteins including: S (spike), M (matrix), E (envelope) and N (nucleocapsid)
→ S, M and E are co-translationally inserted into ER membranes
→ S and M highly glycosylated in ER then Golgi
→ N is translated on free ribosomes and bonds to new genomic RNA as it is synthesised - as RNA is made it wraps it up to protect it

Question 16

What is involved in coronavirus assembly and release?

Answer 16

Coronavirus particles assembly occurs at ER-Golgi intermediate compartment (ERGIC) membranes
→ particle assembly driven by M protein (they way M proteins embeds in membranes causes membrane to curve - starts to encompass genome), but also requires E
→ new particles bud into the lumen of ERGIC
→ transported out of cell by exocytosis into cargo vesicles

Question 17

How is coronavirus transmitted?

Answer 17

Droplet transmission → sneezing, coughing, talking
Contact transmission → skin-to-skin contact (direct), via contaminated object (indirect)
Aerosol → though small particles suspended in the air

Question 18

What are the symptoms of SARS-CoV-2?

Answer 18

Symptoms: fever, fatigue, respiratory symptoms, loss of smell/taste, rash, severe cases: pneumonia, organ failure, death
→ some people may be asymptomatic
→ majority of young adults experience mild disease
→ 80% recover without needing special treatment
→ 20% will be critically ill: elderly, chronic respiratory disease, obesity, diabetes, high BP, heart disease, cancer

Question 19

What is the immune response to SARS-CoV-2?

Answer 19

Once physical barriers of immune system breached → innate immune system responds with inflammation recruiting immune cells to site of infection (macrophages, NKs, dentritics cells)
Adaptive immune response involving lymphocytes and cytokines
→ damaged lung cells, from inflammation, are repaired, the individual will recover and acquire protection from infection on future encounter with the virus

Question 20

What leads to pneumonia in SARS-CoV-2 patients?

Answer 20

Pulmonary destruction → viral persistance causes an excessive immune response/inflammation
→ results in damage to healthy tissue - more cells die and slough off into the lungs causing pneumonia

Question 21

What is involved in the immune sensing of SARS-CoV-2?

Answer 21

PRRs recognise PAMPs detect viruses
→ toll-like receptors (TLR) including TLR3, TLR7, TLR8 recognise viral components of SARS-CoV-2
→ RIG-I (retinoid acid-inducible gene I), MDA5 (melanoma differentiation-associated protein 5) and PKR detect viral SARS-CoV-2 RNA in cytoplasm of infected cells
STING (stimulator of interferon genes) pathway activated → induced by cGAS

Leads to activation of interferon genes via TBK1 and IRF3 - IFN signal transduction
→ stimulates production of type I and III IFN, leaves cell
→ binds to IFNAR2, inhibits IFNAR1stimulating JAK-STAT pathway
→ IRF9 inducing interferon stimulated genes - induces antiviral proteins

INF response if induced early and was restricted to the site of infection properly localised, can effectively limit CoV infection

Question 22

How can SARS-CoV-2 evade immune sensing?

Answer 22

Inhibit sensing by PRRs
→ inhibits cytokine production
→ inhibits INF signal transduction

Mild and moderate infection associated with powerful type I IFN response
→ IFN not produced in 20% of patients - patients with severe infection exhibit lower IFN response
→ absence or delayed IFN response may allow viral replication to continue (viral persistence) leading to severe respiratory infection during first week of illness
→ timing of IFN-I is key as IFN is protective early in disease (but later becomes pathologic)

Question 23

What does the clinical outcome of COVID-19 depend on?

Answer 23

The fine balance between the immune responses and viral replication/infection
→ a clear understanding of the antiviral and inflammatory innate immune programs essential for developing effective biomarkers and therapeutics for disease

Question 24

What are the types of viral diagnostic methods?

Answer 24

Direct detection:
→ testing for presence of virus, viral proteins (usually as antigens), viral nucleic acid
→ may not be detectable in all samples if virus not systemic - risk of false negatives
Indirect detection:
→ testing for the presence of specific antibodies
→ doesn’t indicate current infection as antibodies persist after infection is gone - risk of false positives

Question 25

What are cyto pathic effects (CPEs) of viral infections?

Answer 25

Distinct observable cell abnormalities/changes in the cells due to viral infection
e.g. destruction, shrinking - poliovirus
syncytium (multinucleated giant cells) - herpesviruses

Question 26

What are cell culture caveats?

Answer 26

Cell culture as a tool for diagnosis looking for cyto pathic effects
→ relatively slow (1-3 days HSV, 3> wks CMV)
→ low sensitivity
→ successful culture depends on the viability of the virus in the specimen (collection and transport)
→ cell culture is not applicable to a number of viruses - hepatitis B and C, provirus, papillomavirus
Uses electron microscopy → time consuming, expensive requires skilled personnel

Question 27

What is viral detection based on antibody:antigen interactions?

Answer 27

Relies on the specific binding of antibodies to viral antigens present in clinical samples
→ antibodies are proteins produces in response to foreign substances i.e. viral antigens - they interact through highly specific binding
Types of antibodies used:
→ monoclonal antibodies - produced from a single clone of B cells and recognise a single epitope on a viral antigen - high specificity and reproducibility
→ polyclonal antibodies → derives from multiple clones of B cells and recognise multiple epitopes on a viral antigen - less specific but cover a broader range
Detection methods: ELISA, immunofluorescence assay, western blotting, lateral flow assays

Question 28

What is ELISA?

Answer 28

Enzyme-linked immunosorbent assay
→ uses an enzyme, such as alkaline phosphatase, as label
→ either antigen or antibody immobilised (fixed) onto a support, (e.g. 96-well plates) and specific antibodies labeled with an enzyme are added
→ after washing away unbound antibodies the enzyme substrate is added - regulating colour change measures to quantify amount of viral antigen
Useful tool for determining specific antibodies in serum to micro-organism

Question 29

What are lateral flow immunoassays (LFIs)?

Answer 29

Rapid diagnostic test that use antibodies labeled with colloidal gold nanoparticles to detect viral antigens in clinical samples
→ provide qualitative results within minutes

Question 30

What is haemagglutination (HA) assay?

Answer 30

Some viruses (e.g. influenza) can bind to red blood cells via surface proteins called haemagglutinins
→ they cross link the erythrocytes - sialic acid receptors
Assay used to detect presence of virus based on their ability to agglutinate (clump together) RBC
→ prepare a series of serial dilutions of the sample, add fixed amount of red blood cells, incubated wait 45mins, plate examined visually with microscope to observe agglutination
→ the tire of the virus in the sample is determined by the highest dilution that still causes visible agglutination of RBCs

Question 31

What is nucleic acid detection using PCR?

Answer 31

Qualitative nucleic acid amplification by polymerase chain reaction (after RT for RNA detect)

Quantitative PCR (qPCR) → perform PCR, after each cycle number of DNA copies doubles amplifying target sequence, progress of the PCR reaction and the accumulation of amplifies DNA are monitored in real-time using fluorescent dyes of probes that specifically bind
→ the presence of the target sequence is detected by analysing the flouresecne signal which increases with each cycle amplification
→ the cycle threshold (Ct) value representing the cycle number at which the fluorescence signal crosses a predefined threshold, is used to quantify the amount to target nucleic acid in the sample
→ can use very sensitive DNA-binding dye (SYBR green fluoresce when donut to dsDNA) - needs light-cycler PCR machine

Question 32

What are the 3 main methods for laboratory diagnosis of SARS-CoV-2?

Answer 32

→ detection of viral nucleus acid - RT-PCR
→ detection of viral antigens
→ detection of viral antibodies

Question 33

What is involved in the detection of viral nucleic acid using RT-PCR?

Answer 33

Specimen collection
RNA extraction
Reverse transcription
cDNA ampliation

Question 34

What is involved in specimen collection for RT-PCR

Answer 34

Most commonly performed on upper respirator samples, mainly nasopharyngeal (NP), oropharyngeal (OP)
→ swab specimens should be place into universal transport medium (UTM) immediately after collection - preserve viral RNA
→ global swab shortage, discomfort associate with NP collection and need for trained healthcare personnel - interest in alternatives with saliva being the leading candidate

Question 35

Why must RNA isolation follow optimised protocols?

Answer 35

To minimise degradation at each step
→ unlike DNA, RNA is highly susceptible to degradation
→ sample storage, handling and RNA isolation must follow optimised protocols
→ critical for the assay’s reproducibility and biological relevance

Question 36

What occurs after RNA purification in RT-PCR?

Answer 36

RT conducted using different primers
→ SARS-CoV-2 viral RNA is reverse transcribe to produce cDNA that can be used for qPCr

one-step RT-PCR → one reaction tube, RT and PCR consolidated into one reaction, single buffer
→ rapid, reproducible, suitable for high-throughput diagnosis, reduce risk of contamination, reduce human error
two-step RT-PCR → RT and PCR carried out in two reaction tubes, reactions are done sequentially - independently optimised buffers
→ more time consuming, more sensitive, offers lower detection limits

Question 37

Which genes should be targeted by RT-PCR?

Answer 37

Highly concerned and abundantly expressed genes should be targeted by the RT-PCR primers
→ recommended that assays detect viral nucleocapsid genes N
Positive control - for quality assurance
→ previously validates positive samples, spiked synthetic SARS-CoV-2 RNA

Question 38

Does detection of viral RNA by RT-PCR demonstrate the presence of infectious virus?

Answer 38

Not really → patients who have recovered still show PCR +ve but are not infectious
→ confusion for quarantine and control
→ cell culture more accurate indicator of contagiousness - but must be perfumed in biosafety level 3 (not routine)

Question 39

How is virus detected with viral antigen lateral flow assay?

Answer 39

Based on the specific binding between viral antigens and antibodies immobilised on a nitrocellulose membrane
Consist of: sample pad, conjugate pad, nitrocellulose membrane, absorbent pad
The viral antigens present in the patient sample bind to specific antibodies conjugated to Tag (colloidal gold) to form antigen-antibody complexes
→ as the sample flows through the nitrocellulose membrane the antigen-antibody complexes encounter a test line containing antibodies specific to the viral antigen of interest
→ if the viral antigens are present they bind to the test line forming a visible line
→ excess continue to migrate and are captured by a control line - serves as an internal procedural control indicating that the assay is functioning correctly

Question 40

What improvements can be made to lateral flow assays for SARS-CoV-2 to improve its specificity?

Answer 40

Lower specificity due to high nucleotide sequence similarity with SARS-CoV - may lead to cross-reactivity
→ target unique and conserved domains of proteins in SARS-CoV-2
→ use of monoclonal antibodies to different epitopes of the antigen being detected

Question 41

How are viruses detected using antibodies?

Answer 41

Detection of antibodies in patient’s serum
→ based on principles of immunochromatography, chemiluminescence, or ELISA to detect IgG or IgM in serum
→ many commercially available serological kits developed to detect antibodies against SARS-CoV-2
→ interference from other antibodies produces against other related viruses is to be evaluated
→ consists of: sample pad, conjugated pad, NC membrane, absorbent pad, test line, control line

Question 42

How many SARS-CoV-2 vaccines are there?

Answer 42

251 in development, 61 in clinical testing, 11 in use
→ all SARS-CoV-2 vaccines (except inactivated attenuated and AstraZeneca) are based on the spike protein

Question 43

What are the SARS-CoV-2 vaccination strategies?

Answer 43

Inactivated vaccines → contain SARS-CoV-2 that is grown in cell culture and then chemically inactivated
Live attenuated vaccines → made of genetically weakened versions of SARS-CoV-2 that is grown in cell culture
Recombinant spike-protein based vaccines
Recombinant RBD-based vaccines
Virus like particles → carry no genome but display the spike protein on their surface
Replication-incompetent vector vaccines → cannot propagate in the cells of vaccinated individual but express the spike protein
Replication-competent vector vaccines → can propagate to some extent in the cells of the vaccinated individual and express the spike protein
Inactivated virus vector vaccines → carry copies of the spike protein on their surface but have been chemically inactivated
DNA vaccines → consist of plasmid DNA encoding the spike gene under a mammalian promoter
RNA vaccines → consist of RNA encoding the spike protein and typically packages in LNPs

Question 44

What do the BioNTech/Pfizer vaccines for SARS-CoV-2 consist of?

Answer 44

30ug mRNA encoding the full length SARS-CoV-2 spike protein
→ spike protein is the primary target of the immune response and is responsible for facilitating viral entry
→ mRNA encapsulated in lipid nanoparticles to protect from degradation and facilitate entry
→ no adjuvants, no immune system recognises the spike protein as foreign and mounts an immune response producing antibodies - also stimulates activation of memory T cells providing long term immunity
→ 95% vaccine efficacy