Coronaviruses (12-14) Flashcards

Question

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

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

Specimen collection RNA extraction Reverse transcription cDNA ampliation

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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)

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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