Virology Exam 1
Define latent infection.
-Does not produce infectious virions immediately but has potential to later (reactivation) -Can still detect genome, few mRNA, few proteins, no virions
What are the major routes of viral transmission? Give at least one example of virus transmitted by each route.
-Respiratory: Rhinovirus, Coronavirus, Influenza A & B -GI tract (oral-fecal route): Norovirus, Poliovirus, Hepatitis A & E -Genital tract: Papillomaviruses, Ebola, Zika -Conjunctiva: Conjunctivitis sometimes caused by Enterovirus type 70, Ebola, Zika -Blood (parenteral): HIV, Hepatitis B (also vertical transmission viruses) -Vector bite: Yellow fever, Dengue, West Nile
What is the main cause of continued measles activity in the developed world?
-Unvaccinated US residents contracting it and traveling to/from affected regions -Andrew Wakefield: MMR vaccine and autism
Define chronic infection.
Acute --> clear --> stagnant.
Define productive infection and abortive infection.
Produces infections virions (with genome, mRNA, proteins); does not infectious virions ever.
How SARS-CoV-2 (virus) transmitted? What are the risk factors of COVID-19 (disease)?
How virus is transmitted: -Person-to-person -Close contact -Through respiratory droplets during coughs, sneezes, or talking -Droplets land in mouth or nose of people nearby, or inhaled into lungs -Can be transmitted if asymptomatic Disease risk factors: -Elderly -Cardiovascular disease -Diabetes -Chronic respiratory disease -HTN -Cancer -Seems to be higher rate of infection in old men than in woman and children
Briefly describe current diagnostic strategies for influenza, HIV, HPV, and measles.
Influenza: -Rapid test: ID nucleic acid without amplification (sensitivity down but specificity still good) -Most people use PCR -For flu, rapid influenza test (chromatographic assay) -Add substrate then look if binding has happened -Not very accurate HIV: -Rapid test or ELISA to screen, Western blot to confirm -OraQuick: collect sample and insert device into buffer to detect Ab in 20 min HPV: -Use PCR -DNA virus so looking for viral DNA Measles: -Use EIA -Specific IgM right after rash onset
What are structural proteins? What are nonstructural proteins? Why retroviruses need to carry their reverse transcriptase (RT) enzyme in their virions? Why negative-sense single-stranded RNA viruses need to carry their RdRP in their virions?
Structural proteins: -Viral proteins in virions (+ in virus-infected cells) -Envelope, glycoprotein, capsid, matrix, etc. -Some enzymes (RT: retrovirus, RdRp: -ssRNA, dsRNA) Nonstructural proteins: -Viral proteins not in virions (only in virus-infected cells) -Most enzymes Retroviruses need to carry their reverse transcriptase (RT) enzyme in their virions because: - Normally, RNA would be generated from DNA. Retroviruses, however, have +ssRNA genomes that use dsDNA as an intermediate, which means DNA would be generated from RNA. The host cell does not have the enzyme to do this so the virus would need to carry its own RT. (-)ssRNA viruses need to carry their RdRp in their virions because: -They have genomes that act as a template from which mRNA is synthesized. The host cell does not have the pol to do this, so the virus has to use its own RdRp to do this. This is unlike (+)ssRNA viruses whose genomes are mRNA that can be directly translated into proteins.
Describe how PCR is used in viral diagnosis. Give examples. Describe advantages of using PCR in viral diagnosis.
Uses in viral diagnosis/examples: Detection of viral genome (marker of infection): nucleic acid saves you from any risk of growing virus (collect in chemical, kill virus, but still stabilize genome) -Hard to detect by other methods: HBV, HCV, papilloma, HIV, norwalk, SARS-CoV-2 -Small samples: ocular, amniotic, CSF-HSV, VZ, rubella -When Ab testing fails (window period/immunosuppressed): HIV, HCV -Exclude infectivity: blood Quantification of viral genome (viral load): can quantify how much genome is there -Diagnostic marker: CMV -Prognostic marker: HIV (CD4 count/short term risk and viral load/short term risk but long term progression risk) -Therapeutic marker: monitor efficacy of anti-viral or predict tx failure: HIV, HCV, HBV -Infectivity: risk of transmission: HIV risk of vertical transmission Characterization of viral genome (viral genotyping, mutation): can determine if virus mutating, PCR can sequence and know every mutation site -Prognostic: HIV, HCV -Epidemiology: chain or infection -Therapeutic monitoring: emergence of resistant mutant: HIV, HBV Advantages of using PCR: -Every virus has specific nucleic acid; useful to detect that nucleus; don't need to culture the whole virus; very sensitive (unlike other tests where there's certain amount of virus); even tiny amount of RNA can be amplified through PCR; detect a lot more sample than you could with any other technique that only detects what's there and does not amplify -How to know if patient's recovering: PCR -Many viruses mutate: PCR can determine new strains/whether it's mutating -Reasonably quick -Only limitation is high positive (contamination) and high false negative (quality control)
Describe principles of sample collection, storage, and transport for diagnosing viral diseases.
*****different principles for DETECTION, not DIAGNOSIS***** Collection/Storage: -Collect specimens ASAP after onset of symptoms. Viral recovery is best during first 3 days after symptom onset (not necessarily after infection) and is greatly reduced after 5 days. Collect autopsy samples as soon after death as possible. -Collect specimens aseptically (prevent bacteria contamination). -Place each specimen into separate container labeled with patient's name, other necessary info. -Obtain complete patient history (date of symptom onset, clinical findings, recent exposure/travel, etc.). Transport/Storage: -Prevent specimen from drying (otherwise, nucleic acid will degrade). -Help maintain viral viability or specimen integrity. -Retard growth of microbial contaminants. -Transport specimens to the lab as soon after collection as possible. If not possible, to slow loss of viability, refrigerate (2 to 8 C). Do not keep at room temperature but also do not freeze (will kill virus - cytomegalovirus and some respiratory viruses are inactivated by even one freeze-thaw cycle). -Transport specimens for respiratory syncytial virus culture immediately for prompt inoculation into cell culture. Cytomegalovirus and varicella-zoster virus specimens may lose infectivity rapidly. -VTM (viral transport medium) is gelatin (protects infectivity) and antimicrobial agents in buffered salt solution. Examples of media include Richards viral transport, Bartel's viral transport, and virocult.
List each part of a virus' structure. Describe its composition and its function.
-Capsid, nucleocapsid, envelope, and virion (all viruses will have nucleic acid and capsid, some will have envelope (with or without spikes) -Capsid: protein shell surrounding nucleic acid core; icosahedral, helical, or complex shapes; made up of stable proteins -Nucleocapsid: protein shell + nucleic acid (genome) -Envelope: lipid bilayer stolen from host cell + viral membrane glycoprotein (spikes), lipid unstable but less immunogenic compared to protein; glycoprotein spikes help virus attach to cellular receptors, determine tropism (cell/tissue + species preference), and are targeted by immune system -Virion: complete virus particle that contains everything
Explain in detail how viruses are classified by ICTV? Describe current nonsystemic, polythetic and hierarchical system.
-Classified based on physical and chemical properties -Order (-virales), family (-viridae), subfamily (-virinae), genus (-virus), species -Nonsystemic: no fixed list of properties (unlike Baltimore system) -Polythetic: sharing common properties (not one property is essential): virion morphology (size, shape, capsid, symmetry, envelope); virion physical properties (genome structure, sensitivity to physical/chemical); insults, specific viral lipids (carbohydrates, (non)structural proteins); antigenic properties (serology); biologic properties (replication strategies, host range, transmission, pathogenicity) -Hierarchical: see picture (strains are different isolates of same virus, variants differ from original WT strain)
List drugs in clinical trials for the treatment/management of COVID-19 illness (include mechanism of action for each drug).
-Hydroxychloroquine: ---Anti-parasitic drug -Favipiravir: ---Useless ---Broad spectrum RdRp inhibitor of RNA viruses -Remdesivir: ---Adenosine nucleotide analogue pro-drug having broad-spectrum antiviral activity against RNA viruses ---Was made for Ebola -Chloroquine: ---Anti-parasitic drug -Oseltamivir: ---Antiviral that inhibits viral neuraminidase enzyme -Thalidomide: ---Immunomodulator, suppress TNF-alpha production -Tocilizumab: ---A mAb competitive inhibitor of IL-6 and IL-6R binding (inversely related to number of T-cells) -Anti-SARS-CoV-2 convalescent plasma: ---Infuse neutralizing Abs from SARS-CoV-2 infected persons recovered from COVID-19 to control viremia and symptoms -Recombinant interferon alfa 2b (Nasalferon) -CytoSorb for patients with cytokine storm -EU: single-dose bevacizumab for ARDS -Azithromycin
Describe icosahedral symmetry and helical symmetry. Define protein subunit, protomer, and capsomere.
-Icosahedral: capsomers in regular geometric pattern; most efficient of possible arrangements for subunits in close-shell; smallest units to build shell of fixed size; closed-shell symmetries of higher order than icosahedral symmetry not possible; pentagonal at vertices and hexagonal at faces; always 12 pentons but number of hexons vary; spherical; common -Helical: single type of subunit stacked around central axis to form helical structure TMV; repeating identical subunits (capsomeres); u: #unit/turn, p: axial rise/unit, P: pitch of helix, P = u*p, rod-like -Protein subunit: single polypeptide chain -Protomer: structural unit of oligomeric protein -Capsomere: structural units of capsid, made of protomers, seen under EM as regularly spaced rings with central hole, used to refer to morphological units on surface of virus shell, best for when all capsomeres are identical and hence represent an oligomer
Describe SARS-CoV-2 detection methods. List advantages (BLUE) and disadvantages (YELLOW) of each method.
-RT-PCR: ----Amplifying (more sensitivity) ----But more expensive -ELISA -Plaque Assay -Lateral flow assay: ----Same as PCR but don't amplify ----Faster and cheaper ----But less sensitivity Culture (direct detection of virus): -Highly sensitive and selective -Viable -Expensive -Slow (days) -Not robust (reagents need refrigeration) -Not user friendly -Needs equipment -Not deliverable to end-users (needs lab) -No self-testing -Multiplexing but needs equipment -Quantitative but takes days Nucleic acid (direct detection of virus): -Highly sensitive -Rapid (<15 min) -Expensive -Needs equipment -Not deliverable to end-users (needs space, electricity) -No self-testing -Not quantitative (only yes/no) -Not viable -Highly selective but needs right primers -May/may not be robust (some reagents need refrigeration) -Only new systems are user-friendly -Multiplexing but expensive Antigen capture (direct detection of virus): -Cheap -Rapid (<15 min) -User-friendly -No equipment needed -Deliverable to end-users -Self-testing -Not robust b/c depends on antibody stability and may need refrigeration -Not viable -Not quantitative -Sensitivity and selectivity depend on QC of antibodies and manufacturer -Multiplexing but expensive Detection of antibodies: -Cheap -Rapid (<15 min) -User-friendly -No equipment needed -Deliverable to end-users -Self-testing -Not robust b/c depends on antibody stability and may need refrigeration -Not quantitative -Sensitivity and selectivity depend on QC of antibodies and manufacturer -Multiplexing but expensive Detection of inflammatory biomarkers: -Expensive (needs clinical lab) -Slow (24-48 hours) -Not robust (reagents need refrigeration) -Not user-friendly -Needs equipment -Not deliverable to end-users (needs lab) -No self-testing -Sensitivity and selectivity depend on QC of antibodies and manufacturer -Quantitative but slow -Multiplexing but needs equipment
What is Transmission EM? Describe its usefulness in virology.
-Virus is made thin and stained with metal/gold -Electrons transmit through sample -2D image of interior -Allows us to visualize viruses
Define CPE (cytopathic effects).
-Virus-caused effects in host -Light microscopy shows: changes in cell morphology (size, shape, detached, etc.) -Cell-cell fusion (syncytia, multinucleated cells) -Aggregations -Cell lysis (death)
What is Baltimore's classification system? List all the classes of viruses in David Baltimore's classification criteria.
-Viruses in 7 groups (Roman numerals) based on replication and genome -Most viruses belong to groups 1, 4, and 5 -I: dsDNA II: ssDNA III: dsRNA IV: +ssRNA V: -ssRNA VI: reverse transcribing ssRNA VII: reverse transcribing dsRNA
Define acute infection.
-Within a few days of getting infected, it goes away. -Can become latent.
List 6 virus replication steps. Define attachment factor, receptor, co-receptor. Give examples. How do you experimentally prove a newly identified protein is in a functional receptor of a virus?
1. virus attachment (adsorption) to host cell membrane 2. entry into host cells 3. uncoating (genome released from coating/capsid) 4. genome replication + gene expression 5. assembly 6. virion release (to infect other cells) *****cell surface receptor: (glyco)proteins, carbohydrates, glycolipids***** -Attachment factor: cell surface molecule (mostly carbohydrates) that binds virus (not highly selective), enhances entry, does not actively promote entry or mediate signaling -Receptor: cell surface molecule (cell membrane protein) that binds virus (highly selective), promotes entry by inducing conformational changes of virus surface proteins, transmitting signals, and guiding bound virus to endocytic pathway -Co-receptor: 2nd or subsequent receptor (cell membrane proteins); if only co-receptor, then infection not as bad; monocyte: co-receptor essential vs. T-cell: co-receptor just enhances -Example: Attachment factor: heparan sulfate (a GAG or glycosaminoglycan) Receptor: CD4 (T-cells) Co-receptor: CCR5, CXCR4 -How to prove protein is functional receptor of virus? (A) binding between virus surface protein + receptor (Co-IP, IP-WB, ELISA, flow cytometry) but...binding does not mean it's the receptor so go to... (B) blocking experiments using (1) soluble receptor (2) anti-receptor Ab, polyclonal or MAb (3) natural ligand of receptor (C) genetic experiments: non-permissive: express receptor to see if virus infects, permissive: KO receptor to see if non-permissive
List 7 human coronaviruses. Describe the structure and genome of the coronavirus. Describe classic CoV pathogenesis (mostly for non-SARS CoV).
7 human coronaviruses: 1. Respiratory pathogen of animals 2. Upper respiratory disease in humans -Group 1: HCoV-229E -Group 2: HCoV-OC43 3. SARS-CoV-1 (2003: 1st big outbreak b/c went to lungs) 4. HCoV-NL63 5. HCoV-HKU1 6. MERS-CoV 7. SARS-CoV-2 Structure and genome: -Linear (+)ssRNA -Has huge genome compared to other RNA viruses -Envelope (E) with spikes (S), membrane proteins (M) -S protein important in fusion of viral and cell membrane CoV pathogenesis (mostly non-SARS): -Group 1 and Group 2 coronaviruses isolated from bronchitis, PNA in elderly (10% of adults) and from sore throat, bronchiolitis, PNA in kids (5% of kids with colds) -Winter-spring seasonality -Universal seropositivity by age 6 (almost everyone becomes infected) -Mostly upper respiratory (by droplet spread), not lower -COVID-19: 3-10 day period is dangerous, after that it drops (antibodies will drop viral load but not effective (not long lasting) = re-infection common)
Define permissive cells and non-permissive cells.
Can support productive infection; cannot.
List clinical similarities and epidemiologic differences between SARS-CoV-1 and MERS.
Clinical similarities: -High mortality rate (10-50% for SARS, 43% for MERS) -Short incubation period (~5 days) -Severe respiratory illness worse in aged, immunocompromised -Rapid progression to lung failure + death -Lungs + respiratory epithelium most affected -1/3 patients with diarrhea Epidemiologic differences: -SARS: rapidly progressive epidemic with spread from single focus to mult. countries and spread w/in those countries -MERS: slowly progressive epidemic with limited international spread from single focus -SARS: affected healthcare workers while MERS didn't -Both are nevertheless very lethal
List various serological assays used for viral diagnosis. Describe how ELISAs, Immunofluorescence, and Latex Agglutination Test work.
List of serological assays (study of host response to virus/detection of Ab; for dx + nature of infection i.e. acute/chronic, primary/reinfection): -Complement fixation -Hemagglutination inhibition -Neutralization -Immunofluorescence -Latex agglutination -Enzyme immunoassay (EIA)/ELISA -Radioimmunoassay ELISA: -Detection of soluble viral antigens or antiviral antibodies in clinical samples/culture -Useful for detecting: ---Presence of antibody or antigen in a sample ---Serum antibody concentrations (HIV, West Nile) -Can be used anywhere -Need 2 antibodies: ---One is specific to target antigen (or vice-versa) ---Second is "enzyme-linked" and causes chromogenic or fluorgenic substrate to produce signal (if enzyme active, add substrate, catalyze substrate, color produced) -Need HIV antigen (envelope, capsid, etc.) -Add patient's serum (which may have the antibodies) -Let them bind -Wash to get rid of stuff not bound -Add secondary antibody linked with enzyme (inactive until binds with it) -HRP: common enzyme to use -Washing after every step -Add substrate for enzyme (if enzyme still there after all that washing, enzyme will catalyze substrate, color will be produced) -Radioimmunoassays have been replaced by ELISAs. They were similar to ELISAs in that they were specific, expensive. Difference was that it used radioisotope (can give off light) rather than enzyme to detect Ag. Immunofluorescence: -Detection of viral antigens on cell surface or within the cells in clinical samples/culture -2 basic methods: ---Direct: antiviral Ab conjugated with indicator ---Indirect: primary anti-viral Ab, secondary anti-species Ab labeled with indicator -Used for some viruses (not often b/c laborious) -Antibody conjugated with color -Indirect: primary antibody bound to antigen then secondary has color -Wash a lot to make sure binding is specific and certain -Antigen injected into rabbit, will produce antibody -Must add secondary antibody (binds to primary antibody only if anti-rabbit) -Some rabbit antigen and put in goat, mouse, chicken = can produce antibody against rabbit -Primary: against antigen -Secondary: against species (rabbit) to be able to bind to primary -We can synthesize antibody against any antigen -Cells stained with green; nucleus stained with blue; red (JCV) is virus (on nucleus, not cytoplasm) -One cell = mult. nuclei (common for DNA viruses) -2 primary antibodies: one used to stain cell itself and used to stain virus (both produced in different host, cannot be produced in same host) Latex Agglutination Test: -Can be used to detect antibody or soluble antigen in bodily fluids such as saliva, urine, CSF or blood -Antibody or antigen is attached to latex beads. If corresponding antigen or antibody is present in sample, latex beads agglutinate when mixed with sample -Easy to perform, fast (CMV test), semi-quantitative -Just gives you yes or no answer really though -Basically if antigen and antibody bind together, will aggregate and can see aggregation (much faster than using any dye, substrate, enzyme)
List commonly used methods for diagnosis of viral infection (major approaches): YELLOW. For each assay, describe what component is being detected (virus, antigen, nucleic acid, antibody): BLUE.
Major approaches: -Detection and demonstration of virus itself: animal or in-vitro culture, EM: expensive, labor-intensive, often unavailable -Detection of virus-induced CPEs (to see if there's virus or not): ---Syncytia (mult. cells together, one huge cell with mult. nuclei) formation: Paramyxovirus, HSV, VZV, HIV ---Inclusion bodies (unique shape seen under microscope): ------Nuclear owls eye inclusion bodies: CMV ------Cowdry type A inclusions: HSV, VZV ------Negri bodies: Rabies ---Plaque assay/plaque-forming units (most commonly used, quantify how many cells virus is killing) -Detection of viral components (antigen, enzymes/protein, genome): ---Antigen-antibody complex: Ab highly specific to Ag; agglutination, ELISA, complement fixation and radio immuno assays, hemagglutination, immunofluorescence ---Viral protein: immunostaining, fluorescence microscopy ---Viral genome: molecular techniques (PCR) -Study of host's response to that virus: detection of antibody: ELISA, hemagglutination inhibition assay, etc.
What animal models can be used to study SARS-CoV-2? List advantages (BLUE) and disadvantages (YELLOW) of each model.
Mice: -Good for large scale studies of viruses due to low cost, small size, easy operation and high reproducibility -Showed lack of clinical disease and early clearance of virus when infected with SARS-CoV-2 -SARS-CoV is human pathogen, not mouse pathogen; switch from mouse to human receptor = mouse die Transgenic mice: -Expressing hACE2 supports high SARS-CoV-2 replication in the lungs and brain -Most commonly used for SARS-CoV research -Disease in these mice usually really severe (not seen in humans) Ferrets: -Conflicting data reported: one observed clinical illness while other did not Syrian hamster: -Showed SARS-CoV-2 symptoms -Virus cleared after 7 days of infection -Doesn't show as much disease as mice Non-human primates: -Showed some replication in the lung -But not practical and widely applicable as a small-animal model
List the signs and symptoms of infectious diseases caused by SARS-CoV-1 and MERS.
SARS-CoV-1: -fever -join pain + malaise -headache -diarrhea -sore throat -SOB (PNA) -low white cell count -illness mild in children -while adults have severe fever, cough, most have PNA -fatality rate goes from 10% to 50% when age is above 60 MERS: -fever -muscle pain -cough, SOB -vomiting, diarrhea, abd pain -more severe sx -not as many neurologic sx like SARS
What are the common intracellular replication site for DNA viruses and RNA viruses? Any exception?
RNA viruses usually replicate in the cytoplasm, while DNA viruses usually replicate in the nucleus. Exceptions for RNA viruses are: HIV and orthomyxovirus, which replicate in the nucleus. Exception for DNA virus is poxvirus, which replicates in the cytoplasm.
Describe pathogenesis of SARS-CoV-1 and MERS. Why SARS-CoV-1 and MERS are so deadly?
SARS pathogenesis: -ACE = Angiotensin-Converting Enzyme -Virus can bind to receptor ACE2 for both SARS-CoV-1 and 2 to enter the respiratory and intestinal cell, causing down regulation of Renin-Angiotensin System which is vital for lung and kidney function -Other receptors like D-SIGN + L-SIGN can be used by SARS -Inoculation of URT as with classic HCoVs -Lungs and bronchi by airborne spread -Tropism for Type I pneumocytes in alevoli -Viremia with virus reaching lungs, kidney, GI tract, brain, liver, spleen, thyroid gland, muscle -Lung: diffuse alveolar damage, hyaline membranes, leading to consolidation organization and fibrosis, vascular injury, virus signal within alveolar cells, lymphocytes and macrophages -Incubation period is 3-10 days MERS pathogenesis: -Inoculation of URT as with classic HCoVs -Lungs ad bronchi by airborne spread -DPP4 is virus receptor on cell membrane -Tropism for Type II alveolar pneumocyte and non-ciliated bronchial epithelium -Don't have many cases but can continue to shed virus for very long (up to month) Why are they so deadly? SARS: -SARS is the most infectious of the human CoVs -Airborne spread -Viral load peaks at day 10, detection in lung tissue up to day 51 -Risk with crowding, aerosols, lack of effective masks, poor hand washing, health personnel working while ill -Most only get URI and most recovery, but could go to LRT to affect lungs (that's when downregulation of ACE2 and inflammation happens) -Expressive of ACE2 likely proportional to infection i.e. lungs, kidneys, brain have high ACE2 expression while spleen and liver have lower expression -When virus binds, there is downregulation of ACE2 = body doesn't have enough of it for normal purposes but if we give ACE2, then just enhancing the virus also -No other human CoV uses ACE2 like this -"Super spreader" phenomenon -Adults get it severe (10% fatality) and when they get to be over 60 years, fatality goes up to 50% -If progresses to respiratory failure, need ICU/intubation/ventilation MERS: -Mortality rate is 43%
How SARS-CoV-1 and MERS CoV transmitted? Describe initial transmission kinetics and timeline of SARS-CoV-1 and MERS CoV outbreaks.
SARS-CoV-1 (2003) transmission: -SL-CoV in bats does not use ACE2 receptor (unlike humans) -Initial infections in wild animal markets in China -"Super spreader" Dr. X from China infected while staying at M hotel and infected 143 ppl + healthworkers -Infected ppl from the hotel traveled all over the world (most cases in China, Hong Kong, Canada, Singapore, etc.) -Airborne -2002: epidemic of severe PNA in Guangdong -2003: epidemic of PNA in Guangzhou -3/2003: WHO notified of 303 cases 5 deaths, issues travel advisory; Singapore + Toronto cases; SARS CoV ID'd -4/2003: genome of SARS CoV completed -6/2003: SARS CoV isolated from animals in China -7/2003: end of pandemic transmissions, last cases in Taiwan -9/2003 + 2/2004: lab accident transmission MERS-CoV (2012) transmission: -Originated from camels most likely -Bats could've eaten fruit and contaminated the water -Camels could've been bitten by other animals -Humans could've inhaled it, had contact with meat/milk/urine, etc. -Transmitted through close contact -6/2012: Saudi man with PNA, rapidly progressed to lung + kidney issues then death -9/2012: Qatari man from Saudi Arabia with PNA transferred to London hospital (but no spread to caregivers) -9/2013: HCoV isolated in Netherlands medical center; first 3 cases in London + Saudi Arabia (all men with life-threatening PNA from Middle East) -1/2015: widespread mostly in Saudi Arabia + UAE; 969 documented infections all with Middle East origin -Transmission seems limited (not many healthcare workers affected) though bc doesn't replicate much in URT (more in LRT) -Mortality rate is much higher (43%) than SARS-CoV-1: worse if old, immunocompromised, with kidney disease or DM -Single person who went to hospital led to huge outbreak in Korea but was well-controlled -Outbreak still exists
Describe the structure, genome, and life cycle of a Coronavirus.
Structure: -Enveloped 120-160nm in diameter -S, E, and M proteins -S proteins help fuse viral and cell membranes Genome/life cycle: -Largest RNA genome (27-32 kb) -5' end (20-22 kb): carries replicase gene which encodes for mult. enzymes -Replicase gene products encoded within ORF1a + ORF1b -(+)ssRNA virus (5'-->3') -Also has additional ORFs (areas of mRNA that get translated) that encode 2-4 NSPs of unknown function -Genome has: ---Structural region: encodes for structural proteins: S, E, M, N (nucleocapsid) proteins ---Replicate: mRNA region that encodes for NSPs (every virus has different #NSPs, big genome = big #) -NSPs (pol) then SPs: S, E, M, N -ORF1a + ORF1b encode NSPs which form replicase-transcriptase complex which directs genome replication and subgenomic RNA synthesis -Rest of coding region codes for structural proteins -NSPs for downregulating immune response -NSP 12: RNA pol; target of redesivir, favi for antivirals -NSP 3+5: protease (Pro); can cleave at different regions to make individual proteins; can cleave pol + helicase (NSPs involved in virus replication) -NSP 1+2: can bind to cellular RNA required for immune response and degrade it -Helicase: important for replication of nucleic acid -Coronavirus: subgenomic RNA (serves as mRNA; makes proteins faster and easier which is needed since RNA so big) -5' end with cap, 3' end with poly A tail -5' end of each gene with common intergenic sequence of ~7 bases which is important for subgenomic RNA synthesis -Ribosomes translating ORF1a need to make a frameshift since ORF1b is one codon off -PP1a + PP1ab are proteins that result from translation -3' end is initiation site for pol to start copying genome and making complementary minor strand (-) which can be used to produce more (+) strands -Template RNA not only copied into genomic minor strands but also used to make subgenomic minor strand RNAs -Body of subgenomic RNA copied from 3' end of template; then transcription complex jumps to 5' end where complement is added to (-) subgenomic RNA at 3' end -8 different subgenomic (-) RNAs produced -Infected cells have mRNAs with common 3' end -Each subgenomic mRNA and viral mRNA (both can serve as mRNA) is translated to yield only protein encoded by 5' gene on mRNA -Generally, only 5' located ORF of each mRNA can be translated into proteins -Only first reading frame translated into subgenomic RNAs -E proteins translated on ER then travel through ER-Golgi transportation system (exocytosis) to plasma membrane -Newly made genome RNA encapsidated by N protein -Budding: N interacts with E proteins in pre-Golgi membranes -While wrapping itself in piece of host cell memb., N buds into lumen in exocytosis to become enveloped and go to plasma membrane where it matures -Fusion releases new virus particles to infect more cells -Main receptor that virus uses: ACE2 (binds to spike protein and virus enters) -Basically, spike protein binds to receptor = entry = +mRNA = mRNA translated into proteins = make RdRp available = more transcription for subgenomic RNA = more translation = more protein then assembly in ER + Golgi then leave by budding
Describe two major routes (mechanisms) of virus entry and virus release. Give examples of viruses that enter and exit cells through each route.
The 2 major routes of virus entry are endocytosis and fusion. The 2 major routes of virus release are lysis and budding. Endocytosis: -Receptor-mediated -Taken into vacuoles (nucleocapsid in vesicle then genome released from capsid) -Used by most viruses -Clathrin-mediated (most common, fast, high capacity, proteins transiently assembled on plasma membrane to help protect and migrate genome and shape membrane into vesicle) or non-clathrin-mediated -Influenza (enveloped) + adenovirus (naked) -Influenza A virus: clathrin or non-clathrin endocytosis vs. HIV-1: fusion or endoctyosis Fusion: -Membrane of envelope (spikes not required) fuses with cytoplasmic membrane of host -Receptor-binding + membrane fusion mediated by 1 or 2 glycoproteins (gp) -Fusion proteins: ---Class I (spikes): Ebola, SARS-CoV, Paramyxovirus, Influenza, HIV-1 ---Class II (envelope but no spikes; fusion domain inside envelope, virus binds, conformational change, outside): DENV, TBEV, SFV ---Class III: Herpes, Rhabdovirus; Other: Bunyavirus -Fusion protein for flu: HA2 (fusion binding domain HA1) -Fusion protein for HIV-1: gp41 (fusion binding domain gp120 which binds to CD4 or CCR5) -Retrovirus Lysis: -Kills cell or disrupts membrane (cytolytic virus) -Not common because will trigger huge immune response -Non-envelope usually -Smallpox Budding: -Most efficient -Envelope -Cytopathic virus -Influenza A (assembly at cell membrane), Ebola https://www.youtube.com/watch?v=xqIxZruKpm0
What are routinely used methods for virus culture? Describe cell culture and plaque assay. List the pros (BLUE) and cons (YELLOW) of growing virus in the laboratory.
Viruses are obligate intracellular parasites + cannot be grown on inanimate culture medium. -Animal inoculation: mostly for lab experiments, limited use in dx, use in pathogenesis, immune response + epidemiological study; but very expensive and takes long (weeks) -Chick embryo: pox, herpes simplex, influenza; egg will turn yellow (plaque) when blood with virus on egg -Cell culture: ---Primary cell culture: rhesus monkey kidney cell culture (influenza, parmyxo, entero, adeno), human amnion cell culture, chick embryo fibroblast cell culture; not all primary cells grow in culture (must grow in animal); primary cell from human will be more relevant for COVID (primary = relevancy) ---Diploid cell culture: many divisions in culture, fibroblastic cells: support many viruses: HSV, VZV, CMV, adeno, picorna ---Continuous cell line: immortalized cancerous cells: HeLa cells (human carcinoma of cervix cell line), HEP 2 (human epithelioma of larynx cell line): RSV, HSV, adeno; use cancer cells if you just want to grow/mutate virus or something convenient Cell culture: -Grow cells in flask -If cells grow nicely, will make connections -Cells attached to bottom of flask, make connections, start dividing -Some grow, some don't -Will detach if overgrow -Basically comparing healthy vs. not (CPEs) -Media has phenol red in it (along with salts, sugars, AAs, vitamins). If red, everything is fine. As cells grow (use nutrients), pH lowers, so color changes. Must add more phenol red before it becomes too acidic (continuous change of media very important). If color doesn't change, it means your cells aren't growing. -But cells easy to contaminate Plaque assay: -Serial dilutions of viruses plated on monolayers -Cells stained with dye -Number of plaques = plaque-forming units (PFU) -When virus infects, will kill cells -Indirect way of quantifying virus (looking at cell death) -But takes long (5 days) General viral culture limitations: -slow -expensive to maintain culture -only limited viruses are cultivable: HSV, VZ, CMV, entero, rhino, RSV, adeno, influenza, etc. -isolation of virus may not be related to patient's disease
Why COVID-19 is deadly and difficult to treat? Describe immunopathogenesis of coronavirus infection.
Why is it deadly? -People have been having more than just respiratory symptoms (PNA (fluid in lungs), ARDS (autoimmune disease), fatal lung damage) -Also affects organs such as: ---Heart: inflammation/damage; heart attacks ---Blood vessels: clotting in arteries/veins; clots break off + stop blood flow to organs ---Kidneys: damage requiring dialysis ---Brain: strokes + seizures; confusion + delirium; CNS issue (loss of smell) ---Intestines: diarrhea Immunopathogenesis: -Viral replication in the lung-->activation of alveolar macrophages-->local inflammation-->increased vascular permeability-->amplification of inflammation due to local cell damage and activation of migrated leukocyte-->attract more neutrophils and monocytes from nearby capillaries-->fluid accumulation in alveoli-->SOB + PNA-->ARDS-->death -Basically, damage of alveolar epithelial cells (AECs)-->innate immune receptors induce inflammatory cytokines -CD8+T cells cause cytotoxic clearance of infected cells -Rise in inflammatory cytokines drives depletion of T-cells
Briefly describe replication strategies of DNA viruses.
ssDNA: -Must convert linear ssDNA --> dsDNA (virus has to carry enzyme to do this because host does not have DNA-dependent RNA pol to convert ssDNA --> mRNA) dsDNA: -Uses cell nucleus for DNA replication + RNA transcription -Will use DNA pol in our nucleus to make more DNA, then use RNA pol to make more RNA, then make protein
For single-stranded RNA virus, what is positive sense? Negative sense? Briefly describe replication strategies of RNA viruses (include +sense, -sense, dsRNA, and retroviruses).
ssRNA: -Positive sense: serves as mRNA (does not need to carry own enzyme) -Negative sense: is mRNA template (must carry own enzyme) Replication strategies of RNA viruses: -(+)ssRNA: have genomes that can act as mRNA so can be translated into viral proteins right away by host cell ribosome. (+)ssRNA viruses encode RdRp (doesn't have to carry own RdRp but carries it in genome - when genome translated, one of the proteins is RdRp) which is used for replication of the genome to synthesize (-)antigenome that is then used to create new (+)viral genome. (+)-->(-)-->(+). -(-)ssRNA: have genomes that act as complementary strands from which mRNA is synthesized by viral enzyme RdRp (must carry own). During replication of viral genome, RdRp synthesizes (+)antigenome that is then used to create new (-) viral genome. (-)-->(+)-->(-). -(+)ssRNA genomes using dsDNA intermediate: retroviruses; cDNA; RNA --> DNA (nucleus, explains why HIV is chronic) --> more RNA -dsRNA: must carry own RNA-dependent RNA pol within virion
