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Microbio > Viruses > Flashcards

Viruses Flashcards

1
Q

Viruses

A
  • Cannot multiple on their own or generate energy
  • Multiple within host while utilizing the cellular machineries
  • Infect almost every species on the earth including animals, plants and bacteria
  • Harm host cells, i.e. viruses are parasites
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2
Q

Viral genome

A
  • Viral genome is either DNA or RNA
  • May be linear, circular, of fragmented
  • May be continuous or segmented
  • May be single or double-stranded
  • Most viruses are 20-300 nanometers, Very small - not visible under light microscope, Electron
    microscope(EM) uses electrons
    instead of visible light –up to 10
    million times magnification
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3
Q

Nucleocapsid

A

Nucleocapsid
- All viruses have capsids - protein coats that enclose and protect their viral genome
- Each capsid is constructed from identical subunits called capsomers made of protein
- The capsid and viral genome form the nucleoscapsid
Enveloped/naked
- Some viruses have an external covering are called enveloped
- Those lacking an envelope are called naked

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4
Q

Envelope

A

Enveloped/naked
- Some viruses have an external covering are called enveloped
- Those lacking an envelope are called
naked

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5
Q

types of symmetry for capsid

A
  • Helical capsid: forms a cylinder with the nucleic acid contained in the hollow center
  • Icosahedral capsid: consists of 20 triangular faces arranged around the surface of a sphere
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6
Q

Enveloped Viruses

A
  1. Coat is a lipid membrane acquired from the host cell during viral assembly/budding. Embedded in this membrane are proteins from the virus (so called “structural proteins”)
  2. Some of the structural proteins of virus are embedded in the coat. They are called surface proteins and are mainly responsible for viral attachment to new cells
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7
Q

Host Genome

A
  • Viral genome is much smaller than host’s
  • Human genome – 3 billion basepairs
  • Estimated 25,000 human proteins
  • Viral genome - 2 kilobases to thousands of kilo bases
  • Most viruses have less than 20 proteins
  • Very big DNA viruses have a few hundreds of proteins
  • Viral genome is too small to encode all the machineries needed in its life cycle
  • Virus cannot multiple on their own or generate energy
  • Virus must multiple within host while utilizing the cellular machineries
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8
Q

Viral Components

A

Viral components
* Viral genome (RNA or DNA) is used to produce the 2 types of proteins
* Structural proteins (including capsid and surface proteins) – use to package viral
genome and assemble into new virus
* Non-structural proteins – use to make more viral genome, only expressed in infected cells

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9
Q

Attachment

A
  • Involves two proteins
  • Viral surface protein & Cell receptor
  • Some viruses attach to many types of tissues, some viruses attach to one single tissue
  • Dependent on the presence of the receptors on host cell
  • One contributor to “tissue tropism” but replication is also dependent on other host factors
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10
Q

Recptors

A
  • Found on the outer surface of plasma membrane
  • Usually a transmembrane
    protein

Non-protein cell receptors
* Big organic molecules like sialic acid

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11
Q

Tissue/Species tropism

A
  • Type of tissues supporting the life-cycle of a virus
  • “Attachment” is a major factor
  • Cell receptors may be found only in one tissue of the body, e.g. hepatitis virus, respiratory virus
  • Cell receptors found in one or multiple species – species tropism & zoonotic transmission
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12
Q

Lower vs Upper Respiratory Tract

A
  • Influenza A virus in birds preferentially binds α2-3 sialic acid receptors found in birds
  • Seasonal influenza A virus binds α2-6 sialic acid receptors found in human (upper) respiratory tract
  • α2-3 sialic acid receptors are found in human lung (bronchioles and alveoli)
  • Pigs have both α2-3 and α2-6 receptors – “mixing vessel” when two types of virus infect the same pig
  • H1N1 spreads easier(Upper), H5N1 more deadly(Lower)
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13
Q

Virology of SARS-CoV-2

A
  • Betacoronavirus (lineage B)
  • Enveloped, single‐stranded positive‐sense RNA virus
  • Large genome size of ~30kb
  • Encodes for 16 non‐structural (ORF1ab), 4 structural proteins and 6 (putative) accessory proteins
  • Binding of spike protein of virus to ACE2 receptor in host cells
  • Difficult to predict cell receptor for a virus
  • Has to be determined experimentally
  • Different coronaviruses bind to different cell receptors because spike protein sequence is different
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14
Q

Penetration

A
  • Cell membrane is penetrated by the virus through:
    – Fusion – viral membrane merges directly with cell membrane
    – Endocytosis – entire virus is engulfed and enclosed in a vacuole or vesicle in the cell
    – Endocytosis and fusion
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15
Q

Endocytosis + fusion

A
  1. Virus binds receptor on host cell and enters by endocytosis.
  2. Virus enters the acidic endosome and the viral envelope protein undergoes a confirmation change
  3. Viral membrane fuses with endosome membrane
  4. Nucleocapsid released into the cell
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16
Q

Immunity

A

Immunity: Body’s ability to eliminate
potentially harmful foreign materials or abnormal cells (innate and adaptive)

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17
Q

Vaccination

A

Vaccination
* Adaptive immunity can be achieved by vaccination
* Most vaccines produce antibodies that block viral attachment
* Protects against future infection by the same virus

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18
Q

Types of Vaccine

A

Inactivated Vaccine
- Viral structural protein(s) responsible for binding to cell receptors

Subunit Vaccine
- Containing only certain viral proteins, usually the capsid/surface protein which is responsible for viral attachment
- Both HPV and HBV do not grow easily in laboratory – cannot make inactivated vaccine
* For subunit vaccine, only requires viral sequence and knowledge on “attachment”
* L1 capsid protein for HPV, non-enveloped
* Hepatitis B surface antigen for HBV,
enveloped virus

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19
Q

VLP Vaccines

A

Virus-like particles (VLPs)
* Expression of viral structural proteins can result in the self-assembly of virus like particles (VLPs).
* VLPs are non-infectious because they do not contain any viral genetic material.
* Can use either mammalian or non-mammalian systems for VLP production

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20
Q

Viruses with Vaccines

A

HPV and vaccine
* Non-enveloped, circular, double-stranded DNA viral genome
* Causes cervical cancer
* Cervarix (GlaxoSmithKline) - insect cells
* Gardasil (Merck) – yeast cells
* VLP contains L1 capsid protein of two or more HPV types

HBV and vaccine
* Enveloped, circular, DNA viral genome
* Causes liver cancer
* Vaccine contains one of the viral surface proteins, hepatitis B surface antigen (HBsAg)
* VLP produced in yeast cells (since 1986)

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21
Q

Multivalent vaccines

A
  • Vaccine can be based on single strain of virus or multiple strains
  • Dengue – requirement to have antibodies against 4 serotypes of virus
  • Influenza – requirement to have 2 strains of influenza A virus (A(H1N1) and A(H3N2)) & 1 or 2 strains of influenza B virus (B/Yamagata and B/Victoria)
22
Q

Intracellular Trafficking

A

Intracellular trafficking
* Virus is at least 1000 times smaller than cell
* Virus components (nucleic acids and proteins) are even smaller
* Cannot come together by passive diffusion
* Transported between compartments by cellular transport pathways

23
Q

main cellular transport pathways

A
  • Receptor-mediated endocytosis: moving across the plasma membrane
  • Virus genome enters cytoplasm directly (RNA virus) OR Remains as nucleocapsid in cytoplasm until transport to nucleus (DNA virus)
  • Microtubule-Dependent Trafficking: directional movement within the cytoplasm
  • Motor proteins (dynein, kinesin-1) transport cargos along cytoskeleton (actin, microtubule, intermediate filaments)
  • Dynein and kinesin-1 move in opposite direction along microtubule
  • Nuclear transport: import and export through the nuclear membrane
  • Nuclear localization signal (NLS) is needed for import into nucleus
  • Nuclear export signal (NES) is needed for export out of nucleus
24
Q

Viral RNA production

A

Virus proteins produced by host’s translational machinery (ribosome)
- Messenger RNA (mRNA) is positive sense, so virus must produce positive sense single stranded RNA
- For single-stranded (ss) DNA virus :
ssDNA -> dsDNA -> mRNA(+)
- -ve sense RNA requires a virus-encoded enzyme called RNA-dependent RNA polymerase (RdRp)
* All RNA virus encodes its own protein called RNA-dependent RNA polymerase (RdRp). For negative sense RNA virus, virus RdRp is incorporated in the virion –
i.e. a structural protein
* New RdRp can only be produced after –ve sense is converted to +ve RNA
* Replication of RNA does not use host polymerase and most (but not all) RNA viruses replicate in cytoplasm
* More RdRp will be synthesized inside host cell and is required for multiplication of virus genome before assembly
* RdRp lacks proof-reading ability = high mutation rate because RdRp cannot repair any error in new RNA produced

25
Q

DNA Virus

A

DNA virus
- For double-stranded (ds) DNA virus, protein synthesis is same as host protein mechanism
* Most DNA viruses replicate in nucleus
* Require host cell polymerases for replication
* Highly dependent on host status
* Virus DNA can integrate into chromosomes
* Low chance of virus mutation
- Host’s DNA polymerase can correct error in the newly synthesized virus DNA

26
Q

Types of Drugs that block sythesis

A
  • Inhibits RdRp
  • Inhibits new virus genome formation
  • Inhbits viral protein processing like cleavage by viral protease
27
Q

RdRp of influenza A virus (ssRNA(-))

A
  • RdRp consists of three subunits:
    polymerase basic protein1 (PB1)[Favipiravir], polymerase basic protein 2 (PB2)[pimodivir], and polymerase acidic protein (PA)[baloxavir marboxil]
28
Q

Ribavarin

A

Ribavirin - nucleoside drug
* Use to treat various virus infection
* Hepatitis C virus (with interferon alpha)
* Respiratory syncytial virus and Lassa fever virus
* Interfere with the synthesis of new virus genome

  • Ribavirin resembles ATP and GTP
  • Can be incorporated when new RNA genome is made -> mutations can be lethal to virus
  • May affect host gene transcription but this rate is not as fast as virus’s
29
Q

RNA virus Assembly

A

RNA virus assembly
- Vesicular stomatitis virus(VSV) is an enveloped, negative-sense RNA virus

  • All components of the virion must be transported to site of assembly
  • Need to use host’s transport system
  • Each virus assembly in one cellular compartment
  • Surface protein is derived from virus genome
  • Virus’s RNA/DNA protected by nucleocapsid(N) protein (RdRp can be incorporated)
  • Coat is obtained from host’s membrane
30
Q

DNA virus assembly

A

Example of DNA virus assembly
- Virus proteins are transported across the nuclear membrane
- Assembly occurs in the nucleus
- Adenovirus is a naked DNA virus.

31
Q

Blocking Assembly

A

Blocking assembly
* Potential drugs being developed:
- Inhibit the function of structural proteins
- Cause formation of wrong type of virion
- Capsid assembly modulators (CAM) : Misdirects Assembly of Hepatitis B Virus Capsids
- CAM in clinical trials but not approved for human use yet

32
Q

Release

A

Release
* Assembled viruses can leave host cell in one of these manners:
– Budding : “Budding” through the cell envelope involves using the cell’s membrane for the virus itself. Usually for enveloped viruses.
– Cell death or lysis : By forcing the cell to undergo apoptosis or other forms of cell death, viruses are released into the extracellular space.
– Exocytosis : The host cell’s transport system is used to enclose vacuoles of viruses, which are then released via exocytosis.

Coronavirus
- enveloped, positive-sense RNA virus
- replicates & assemble in cytoplasm
- Exit the cell via exoctyosis

33
Q

Neuraminidase (NA) of influenza A virus

A
  • Removes sialic acid from the cell surface receptors
  • Allows newly-made viruses to be released
  • Drugs can be used to block activity of NA
    – treatment for influenza

Tamiflu (oseltamivir), a drug that inhibits NA activity, binds to a pocket in the NA protein of influenza A virus.

34
Q

Antivirals

A

Antiviral therapy
* Target one of the steps
* Drugs bind to virus protein
* No effect on host proteins
* Relatively few side-effects

Drug resistance
* Therapy works only for certain strains
* Drug resistance due to virus mutation
* Particularly for RNA virus

35
Q

Challenges of Antivirals

A
  • Therapy works only for certain strains
  • Drug resistance due to virus mutation
  • Particularly for RNA virus
  • Combination therapy – already in use
  • New drugs targeting virus-host interaction
  • Destroy virus genome inside host cell
36
Q

Antiviral Therapies

A

HIV : 2 nucleoside RTIs, 1 other(NNRTI, fusion inhibitors, protease inhibitors)

Evolution of SARS-CoV-2 reduces protection from neutralization antibodies from vaccine
Neutralizing antibodies (Abs) are produced upon vaccination & match the spike gene used in vaccine – spike gene changes with time!
- mRNA-1273: contains spike of original virus
- Updated mRNA-1273.214: contains two spike genes (original plus Omicron (BA.1) viruses)

37
Q

Features of Detection Methods

A
  • Sensitivity: test must be able to detect very small amounts of the microbe even in the presence of other molecules including other microbes
  • Specificity: test yields a positive result only when the microbe is present
  • Other factors are equipment/manpower requirement, cost and speed of test. Ease of sample collection.
38
Q

Phenotypic Methods

A

Growing of virus in the lab
* Need to have a lab-made “host”
* Use immortalized cell lines
* Use embryonated eggs
* Amplify virus from small sample taken
* Larger amount of virus generated in lab can be used for identification

Continuous cell line
Cells have mutation (from cancer patient) or genetically modified such that they won’t die. They then grow continuously, i.e. become immortalized

Appearance of CPE does not yield virus identity -> need to harvest secreted virus particle for EM and other assays

Electron microscopy identify virus family but not exact virus identity

39
Q

Limitations of Phenotypic analysis

A

1 Virus does not grow in the cells available in lab. May not have enough virus for EM.

Limitations for culturing method & microscopy

Advantage for culturing method
- Grow large amount of virus for many experiments, e.g. to make inactivated vaccine, develop diagnostic assays and screen for antiviral drugs

40
Q

Antigen Detection

A

Virus antigen detection tests
* Use a lab-made antibody specific for a virus to detect the virus protein synthesized by the infected cells via immunofluorescence (earlier)
* If the virus protein is secreted out of cell as a protein or as structural proteins in the virus particles after assembly, then sandwich ELISA can be used.
* BUT two lab-made antibodies are required.
* More difficult to get these but assay can be very specific as both antibodies have to bind to “virus”
- Antigen Rapid Test (ART) for COVID-19 is a “sandwich ELISA” detecting nucleocapsid protein

41
Q

Serological Test

A

Use lab-made virus proteins to detect
antibodies found in patients

42
Q

Advantages of POC

A
  • Easy sampling like a single drop of whole blood, urine or saliva
  • No elaborate processing
  • No special equipment
  • No special skills
  • Easy & fast readout
  • Can be conducted in GP clinics or at home.
  • Advances in microfluidic engineering
43
Q

Techniques used for SARS-CoV-2 detection

A
  • Phenotypic methods: Virus grows in Vero E6 cell line -> cytopathic effect when patient sample is cultured on cells
  • Detect viral RNA: RT-PCR, next-generation sequencing (can detect new variants)
  • Detect viral protein – ART POC
  • Detect antibodies against viral proteins
44
Q

Different isotypes of antibodies are
produced by our bodies

A
  • For most viruses, IgM appeared much earlier than IgA and IgG.
  • In this practical, we measured IgG because volunteers have recovered.

1 st week: Real-time PCR (viral RNA) and virus isolation (whole virion) are positive
Antigen detection detects virus protein in 1-5 days if virus load is HIGH. Less sensitive than virus isolation/PCR. Virus culture and PCR have amplification steps in the lab.
Serological test (IgG isotype) remains positive for a few months after recovery – cannot distinguish between recent and past infection (or vaccination with killed vaccine). IgA and IgM drop faster with time.

45
Q

Recommendations for Use of Antibody

A

Tests for COVID-19
* Not suitable to detect early phase of infection because
antibodies take >1 week to become high
* Determine if a person has COVID-19 antibodies, which suggests past infection or vaccination (depending on viral protein used in assay & vaccine type)
* Aid in the diagnosis of multisystem inflammatory syndrome in children (MIS-C) and in adults (MIS-A).
* Serology used in contact tracing, surveillance or epidemiology - assess disease prevalence in a population

46
Q

Uses of serology tests for other viral
infections:

A
  • Some people have asymptomatic infection
  • Some viruses persist in hosts for years, e.g. HBV causes chronic infection
  • Transmission of virus to other people can happen during asymptomatic/chronic infection
47
Q

Combine assays to detect chronic
infection in blood donors

A
  • Hepatitis B virus (HBV) Hepatitis B surface antigen (HBsAg) detection
  • Hepatitis B core antibody (anti-HBc) detection
  • Nucleic acid amplification testing (NAT) for HBV
  • HIV : dual Ab Ag Alere HIV combo detection kit
48
Q

Similarity in experimental design
for both ELISA and IFA

A
  • Negative control – expect low reading/signal
  • Positive control – expect high reading/signal
  • Above must be working as expected
  • If not, there is problem with assay or execution of experiment -> Repeat!
49
Q

Differences between IFA & ELISA

A
  • Amount of sample required (i.e. sensitivity)
  • How many proteins are used in assay?
  • Amount of protein used – too high can cause non-specific binding of un-related abs
  • Diluted serum to avoid unspecific binding
  • Are we measuring all the (virus specific) antibodies in the blood sample?
  • Readout – qualitative vs quantitative
50
Q

Challenges in diagnosis

A
  • Low specificity can lead to false positive
  • Low sensitivity can lead to false negative
  • Conducting two different assays will be ideal
  • Require extensive knowledge on virus, eg:
  • Design of primers in PCR
  • Prepare made-in-lab protein for serological assay
  • Generation of lab-made antibody for antigen detection (need a pair of antibodies for sandwich)
  • Level of virus at different stage of infection
51
Q

Difficult to identify new virus
(zoonotic transmission)

A

Sequences of viruses are highly divergent
New virus that crosses the species barrier can differ significantly from any other sequence in existing reference databases
PCR failed -> Existing primers for known viruses do not match!
No suitable antibodies to detect viral proteins

Microbio (5 decks)

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