Lecture 13. Coronavirus Disease and Laboratory Diagnosis of Viral Diseases

Question 1

What are coronaviruses?

Answer 1

+ve ssRNA enveloped viruses
Corona = crown

Question 2

What infections do coronaviruses cause?

Answer 2

They cause mild respiratory infections restricted to the upper respiratory tract
But rarer forms can cause Infections that extend to the lower respiratory tract
SARS-CoV , MERS-CoV, SARS-CoV-2

Question 3

How can coronaviruses be transmitted?

Answer 3

Droplet transmission (coughing and sneezing)
Contact transmission (touching contaminates surfaces)
Aerosol transmission (inhaling contaminated aerosols)

Question 4

What are the symptoms of SARS-CoV-2?

Answer 4

Very mild to severe (some may be asymptomatic)
Majority of young individuals experience mild disease
fever, fatigue, respiratory symptoms (cough, shortness of breath), loss of smell/taste, rash
In more severe cases there could be pneumonia, organ failure or death

Question 5

How many people recover from SARS-CoV-2 infection without needing special treatment?

Answer 5

80%

Question 6

Who is most at risk of being critically ill from SARS-CoV-2 infection?

Answer 6

Older people and/or those with chronic respiratory disease, obesity, diabetes, high blood pressure, heart disease, cancer

Question 7

What happens in the immune response to SARS-CoV-2?

Answer 7

The immune system recognises infectious agent and develops immune response in the form of antibodies, macrophages and lymphocytes
Causes inflammation as immune response tries to clear virus from lungs

Question 8

What happens to the patient after recovering from SARS-CoV-2 infection?

Answer 8

Damaged lung cells are repaired, the individual will recover and acquire protection from infection on future encounter with the virus

Question 9

What happens in 20% of people who get SARS-CoV-2 infection?

Answer 9

Excessive immune response
Normally immune response inhibited after virus is cleaved from the cell to protect the healthy cells
20% of people fail to cleave the virus, causing viral persistence, resulting in excessive immune response (cytokine storm) which results in damage to healthy tissue
More cells die and slough off into the lungs causing pneumonia

Question 10

How is SARS-CoV-2 recognised by the immune system?

Answer 10

Toll-like receptors (TLRs) 7 and 8 are the key components that recognise SARS-CoV-2 RNA, which triggers downstream signals that lead to the production of IFN types I and III

Question 11

What is the role of IFN I and III in limiting CoV infection?

Answer 11

Trigger interferon stimulated genes which induce antiviral proteins to limit CoV infection

Question 12

How does SARS-CoV-2 inhibit sensing by pattern recognition receptors (PRRs)?

Answer 12

Inhibit cytokines production
Inhibit IFN signal transduction
This allows the virus to persist, so immune response continues

Question 13

What do patients with COVID-19 show reduced production in?

Answer 13

Patients with severe COVID-19 show remarkable reduced production and activity of IFN-I as compared to mild or moderate cases

Question 14

What were mild and moderate SARS-CoV-2 infections associated with?

Answer 14

Powerful type I IFN response
Type I IFN is detected in plasma samples collected from COVID-19 patients during the first week of disease

Question 15

What does the absence of or the delay of an IFN response allow?

Answer 15

Viral replication to continue (viral persistence) leading to severe respiratory infection during the first week of illness

Question 16

Why is the timing of IFN-1 key?

Answer 16

As IFN is protective early in disease but later becomes pathologic

Question 17

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

Answer 17

The fine balance between the immune responses and viral replication/infection
So a clear understanding of the delicate balance between antiviral and inflammatory innate immune programs is essential in developing effective biomarkers and therapeutics for COVID-19

Question 18

What are ways of getting a sample to test for the presence of a virus?

Answer 18

CSF (cerebral spinal fluid), blood, biopsies, nasal swab, saliva, urine, faeces

Question 19

What are viral diagnostic methods that result in direct detection of the virus?

Answer 19

Testing for the presence of virus
Testing for viral proteins (usually as antigens)
Testing for viral nucleic acid
May not be detectable in all samples if virus is not systemic (risks false-negatives)

Question 20

What are viral diagnostic methods that result in indirect detection of the virus?

Answer 20

Testing for the presence of specific antibodies
Doesn’t indicate current infection as antibodies persist after infection is gone (risks false-positives) - cannot be used to see if someone is still infectious

Question 21

What are cytopathic effects (CPEs) of viral infections?

Answer 21

Distinct observable cell abnormalities/changes in the cells due to viral infection (e.g infected Hela cells in those with poliovirus)

Question 22

What are the caveats of using cell cultures?

Answer 22

Relatively slow: 1-3 days (HSV) to 3 > wks (CMV) Herpes simplex viruses and sometimes
enteroviruses can produce CPE within one day of inoculation
Low sensitivity
Successful culture depends on the viability of the virus in the specimen (collection and transport of specimen)
Cell culture is not applicable to a number of viruses: e.g hepatitis B and C, parvovirus, papillomavirus
Must be preserved properly to prevent infection

Question 23

What are the downsides for using election microcopy?

Answer 23

Time consuming (hard to prepare)
Expensive
Requires skilled personnel
Not all viruses can be differentiated by shapes
Used for characterising viruses rather than diagnosis

Question 24

How do viral detection based antibody tests function?

Answer 24

Detects antigens/components of antigens instead of the virus itself
Based on specific interaction between antibodies and antigens

Question 25

What is an enzyme immunoassay (EIA)?

Answer 25

Enzyme immunoassay (EIA) uses as an enzyme such as alkaline phosphatase and horseradish peroxidase as label

Question 26

What is an enzyme linked immunosorbent assay (ELISA)?

Answer 26

Where either the antigen or antibody is immobilised (fixed) onto a support, mostly the surface of a 96-well microtiter plates

Question 27

What is ELISA used for?

Answer 27

Is a useful tool for determining specific antibodies in serum to microorganisms (e.g specific antibodies to the hepatitis B virus, HIV or West Nile virus)
Also used to determine cytokines, hormones, tumour markers, allergens

Question 28

What is a haemagglutination (HA) assay?

Answer 28

Some viruses (Influenza) can bind to red blood cells (Haemagglutinin)
They cross link the erythrocytes (sialic acid receptors)

Question 29

How do HA assays function?

Answer 29

Assaying virus stock with 1 in 2 dilutions whilst using the same amount of erythrocytes (whilst the number of virus particles decreases through the assay)

Question 30

How can specific nucleic acid be detected/targeted?

Answer 30

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

Question 31

What is quantitative PCR (qPCR)?

Answer 31

Use very sensitive DNA-binding dye (SYBR green) to quantify DNA made in the PCR as it proceeds
Needs Light-Cycler PCR machine
SYBR green fluorescence when bound to dsDNA
Each time a new dsDNA is produced/amplified you will get a signal

Question 32

in qPCR, what is the cycle threshold (CT) value?

Answer 32

The number of PCR cycles that is needed to pass for a signal to pass a defined threshold