Introduction to Virology Flashcards
What is the biomass of bacterial viruses on the planet?
Bacteriophage weighs about 1 femtogram = 10^-15 gm
10^30 total bacteriophage particles
So biomass = (10^-15 * 10^30)
= space for 200 million light years if arranged head to tail
Prokaryotes (bacteria + archaea) represent = _____ % of the biomass and ___% nucleic acid containing particles
90%, 10%
Viruses represent = _____ % of the biomass and ___% nucleic acid containing particles
5%, 94%
Endogenous viruses represent ___% of the human genome
8%
Innate immunity
- Mucus barrier breached -> Host Pattern Recognition Receptor (PRR) recognizes Pathogen Associated Molecular Pattern (PAMP) ->Activation of various transcription factors releasing cytokines and chemokines by dendritic cells, monocytes, macrophages, neutrophils -> Pro-inflammatory cytokines and chemokines stimulate NK cells -> NK cells kill virus infected cells directly throudh degranulation/receptor mediated apoptosis.
- PRR recognition -> Interferon regulatory factors (IRF) activated -> Travel to nucleus -> Promotion of transcription of type I IFN -> IFN released -> bind to IFN receptors on cells -> JAK/STAT signaling pathway -> Interferon stimulated genes activated and transcribed -> Increase cell’s ability to resist viral replication
Adaptive Immunity
Type I IFN -> Matures DC and Macrophages into APCs -> APCs process viral proteins and present them on MHC molecules (Class I - CD8, Class II - CD4) -> APC migrate to lymph -> If naive T cells recognize the molecules, become activated helper T cells or cytotoxic T cells -> CD4+ release cytokines activating other immune cells like B lymphocytes and CD8+ -> B cells recognize through their own receptors plus become activated with the help of T follicular helper cells -> B cells become plasma cells -> produce antibodies -> Some B and T cells become memory cells
- CD8+ kill through releasing perforin and granzymes
Good virus example
Dichantelium lanuginosum (Panic grass) [Found in geothermal soils in Yellowstone National Park, USA, grows at >50 degree C] -> Curvularia protuberata (Fungus) -> Curvularia thermal tolerance virus (CThTV)
Good virus example 2
IMLYGIC (talimogene laherparepvec) - Weakened HSV1; oncolytic virus
What is a virus?
An infectious, obligate, intracellular parasite comprised of genetic material (DNA/RNA) surrounded by a protein coat called capsid and/or an envelope derived from a host cell membrane
Unique Virus features
- Do not divide by binary fission
- Contain either DNA/RNA
- Do not contain muramic acid
- Not sensitive to antibiotics
- Do not grow on artificial media
- Do not contain protein synthesis machinery
Steps of life cycle
Attachment -> Entry (Endocytosis) -> Uncoating -> Replication -> Viral mRNA used to make viral proteins -> Assembly -> Release
What begins the next infectious cycle?
Disassembly of the virion in the next host cell/organism
One of the smallest viruses
Poliovirus (30nm) - Ribosomes (20nm)
One of the largest viruses
Smallpox viruses (250nm) - Approximate size of the smallest bacteria Chlamydia
Herpesvirus
200 nm
Why can viruses not be seen with light microscope?
Viruses range from 20-300 nm; light microscope have a resolution limit of 200nm due to wavelength of visible light; cannot see lower than that.
How many viruses can fit on the head of a pin?
500 million rhinoviruses.
head of a pin = 2mm = 2000 microns
Pandoravirus salinus
Giant virus
Genome approx 2.5 million base pairs
Drop foot syndrome
Characteristic of polio
Variolation
Inoculation of healthy individuals with materials from a smallpox pustule
Who introduced variolation?
Lady Montagu
Vaccination
Edward Jenner, England, 1790s
Concept of microorganisms
Leeuwenhoek, Pasteur, Koch
Dimitri Ivanowsky
Studied the tobacco mosaic disease (TMD) and defined it as filterable virus (virus = poison), 1892; Virus discovery
Martinus Beijerinck
TMV= responsible for TMD, contagious, living liquid, inactivated by boiling, 1898
Loeffler and Frosch
Agent of foot and mouth disease (1898)
Filterable, 0.2 um; replicate only in host, not in broth
1901
First human virus (yellow fever virus)
1903
Rabies
1906
Variola virus
1908
Chicken leukaemia virus, poliovirus
1911
Rous sarcoma virus
1915
Bacteriophages
1933
Influenza virus
1930
Electron microscope; 100,000 fold magnifying power; direct visualization of virus particles
T4
Complex, nonenveloped, intricate tail and head
TMV
Nonenveloped, helical
Rhabdovirus, vesicular stomatitis virus
Enveloped
Rotavirus
Nonenveloped, icosahedral
Order-
Family-
Genus-
(Species)
Order - Viriales (8)
Family - Viridae (125) (12 from Antarctica)
Genus - Virus (677)
Species - 2618 (10000 species from Lake Limnopolar, Antarctica)
Why study viruses?
- They outnumber cellular life by at least 10:1
- They drive global cycles
- Comprise the greatest biodiversity on earth
- Source of new pathogens
Two simple facts about viruses
- Obligate intracellular parasites; only function after they replicate in a host cell
- must make mRNA translated by host ribosome; parasites of host protein synthesis machinery
Why do we use cell cultures rather than animals to conduct studies on viruses?
Cell cultures provide a much simpler and more homogenous experimental system.
Susceptible cell
Has a receptor for the given virus
Resistant cell
Does not have a receptor for the given virus
Permissive cell
Has the capacity to replicate virus
Which cell can take up virus and replicate in it?
Susceptible + Permissive
Suckling mice
- Experimental laboratory animal
- Relatively easy, inexpensive to raise
- Most animal viruses are able to replicate
Embryonated chicken egg
- Animal viruses grow
- Reduced both the time and expense of virus assays
Enders, Weller, Robbins
No animal viruses could grow in cell cultures until 1949; propagated poliovirus in human cell culture- primary cultures of embryonic tissues.
Nobel prize in 1954: Discovery of the ability of poliomyelitis virus to grow in cultures of various types of tissue.
Primary Cell Line
- Derived from: Human foreskin fibroblast, monkey kidneys, human embryonic amnion, human embryonic kidneys, chicken or mouse embryos
- Prepared from animal tissues
- Limited life span (5-20 cell divisions)
- Live attenuated poliovirus vaccine strains may be propagated in primary monkey kidney cells
- Include several cell types
- Used for experimental virology when state of cell differentiation is important
- Mandated for human vaccines to avoid contamination from potential oncogenic DNA of continuous cell lines
Established Cell Line (Continuous 1)
- Mouse fibroblasts 3T3; Continuous;
- Artificial mutation (Treating a primary cell culture or diploid strain with mutagenic chemical or tumor virus)
Transformed Cell Line (Continuous 2)
- HeLa Cells; Continuous; Loss of contact inhibition
- Consist of a single cell type
- Propagated indefinitely in a culture system
- Derived from tumor issue
- Often DO NOT resemble the cell of origin, less differentiated
Diploid Cell Strains
- WI-38, Human embryonic lung
- Homogenous population of a single type
- 100 cell divisions lifespan
HeLa Cell Line
- Most studied continuous cell line
- Derived from Henrietta Lacks in 1951
- Used to propagate poliovirus; poliovirus vaccine development
- Biomedical ethics
How can you detect and quantify viruses?
Two ways:
Biological (Infectivity)
Physical (Viral particles and their components)
How can you identify viral growth in cell culture?
Cytopathic effects: Structural changes in host cell that are caused by viral invasion.
Can be seen with a simple light or phase-contrast microscope at low power, without fixing or staining the cells.
1. Rounding up and detachment of cells from culture dish
2. Cell lysis
3. Swelling of nuclei
4. Formation of a group of fused cells: Syncytium (Viral fusion protein used)
Plaque Assay
Used to measure the infectious titer of a virus suspension (PFU/mL)
1930s: Used to study multiplication of bacteriophages
1952: Renato Dulbecco developed for animal viruses
1975: Nobel Prize
Plaque counting range: 30-300
Aliquots from the last dilution transferred to four different petri dish covered in semi-solid agar medium; such as low melting point agar or carboxymethyl cellulose
Why is carboxymethyl cellulose/low melting point agar added in the petri dishes for plaque assay?
to prevent virus diffusion after lysis of infected cells
Titer determination
Number of plaques on a plate*Factor by which the original virus suspension was diluted before an aliquot was applied to the plate/aliquot transferred (mL)
Endpoint Dilution Assay
TCID (Tissue Culture Infectious Dose 50)
The dilution of virus at which 50% of the cell cultures are infected.
At low dilutions, all cell cultures are infected. (Cytopathic effects seen)
At high dilutions, none of the cell cultures are infected.
What is particle to PFU ratio? Why this is high for some viruses?
Number of virus particles in a sample/ Number of infectious particles
Ratio of physical virus particles to infectious particles can be much greater than 1.
For example, reovirus: 10
Low infectivity, high particle to PFU ratio because:
1. Not all virus particles may be intact
2. Some may contain defective genomes
3. High number of empty capsids
4. Host defense system (Antiviral)
Viral replication cycle (Mouse polyoma virus)
8-10 hours: early mRNA; T-antigens shortly after
12-15 hours: Viral DNA replication; late mRNA copied from a different set of viral genes-> Viral capsid proteins
18-20 hours -> New progeny virus
For the next 24 hours -> Virus titer increase slowly, most infected mouse dead
Multiplicity of infection (MOI)
Ratio of infectious virus particles to the number of target cells in a culture.
(Infection depends on the random collision of virions and cells)
High MOI- Ensures synchronous infection; key to one step growth cycle. As it would more likely result in infection of all the cells.
Distribution of virus particles per cell is best described by
Poisson distribution
Steps of virus life cycle
- Attachment
- Penetration
- Uncoating
- Replication
- Assembly
- Release
Physical measurements of virus particles
- Hemagglutination
- Serology
- Nucleic acids
- Viral enzymes
- Electron microscopy
Hemagglutination Assay
1941
GK Hirst
-First rapid quantitative assay for eukaryotic viruses
-Enveloped viruses have hemagglutinin
-When no virus, RBC settle at the bottom of the well due to gravity forming a red dot
-The highest dilution where the clumping still happens gives you the HA titer. This titer indicates the conc of virus particles.
Hemagglutination Inhibition Assay
-The highest dilution of the serum where red dot is still visible (clumping does not occur) is the titer of the antibody against the virus.
-Known amount of virus added to serial dilutions of the serum, and then RBC added to mixture.
Immunostaining
Direct - Antigen -> Antibody with indicator (fluorescent)
Indirect -> Antigen-> Antibody (Murine)-> Secondary antibody (Anti- mouse Ab) with indicator
Localization
ELISA (Enzyme linked immunosorbent assay)
Antigen detection:
Solid support - Ab -> Ag in sample -> Secondary antibody with HRP + Substrate added (color change)
Ab detection->
Solid support- Ag -> Ab in sample -> Anti-IgG antibody with HRP -> Substrate added
Immunoblotting (Western blot analysis)
Separate based on size and charge (SDS Page? Separation gel) -> Nitrocellulose membrane -> Immunostaining of blot with labeled antibodies -> Visualize bands through autoradiography
RDT (Rapid diagnostic tests)
based on colorimetric lateral flow of immunoassay
-Abs migrate across an adhesive pad (nitrocellulose)
PCR application
Industry
Research
Diagnosis
Denaturation - 95
Annealing - 61
Elongation - 72
Hershey-Chase Experiment
1952
Phage T2
Experiment 1: 32-P, DNA: Radioactivity in pellet of cells
Experiment 2: 35-S, Protein: Radioactivity in supernatant
Original baltimore classification
Gapped DNA of hepadnaviridae missed. Class VII
Describe Baltimore system about viral genome classification
Scheme for classifying viruses based on the type of genome and its replication strategy
How are viral genomes structurally different?
- Linear 2. Circular 3. Gapped 4. Segmented 5. Single stranded + 6. Single stranded - 7. SS Ambisense 8. Double-stranded 9. Cross-linked ends of DS DNA 10. Covalently attached proteins 11. DNA with covalently attached RNA
Virus diversity
Inter and intra species genetic recombination, and mutation, reassortment of segmented viruses.
RNA polymerase proofreading activity none, 1 misincorporation every 10^4-10^5 nucleotides polymerized
purpose- disease emergence, vaccine failure, drug resistance, virulence
Reassortment
Exchange of genetic segments between different strains of a segmented virus that have co-infected the same cell.
Information encoded in viral genome
Gene products and regulatory signals for:
1. Replication and efficient expression of viral genome
2. Modulation of host defences
3. Spread to other hosts and cells
4. Assembly and packaging of the genome
5. Regulation of the timing of the replication cycle
Information not encoded in viral genome
(No genes for)
- Membrane biosynthesis and energy production
- Complete protein synthesis machinery
- Centromere (for segregation of genomes) and telomere (for maintenance of genome)
Giant virus
90% of genes encoded are novel
Some may encode components of the protein synthesis machinery
Class I - DsDNA
Adenoviridae, polyoma, papilloma
Class II- SS DNA
Circoviridae, parvoviridae
Class III - DsRNA
Reoviridae
Class IV - (+) SsRNA
Viruses from 8 families infect mammals
Picornaviridae (Poliovirus)
Class V - (-) SSRNA
Rhabdoviridae (Rabies)
Segmented Genome: Orthomoxyviridae
Reassortment
Some are ambisense:
Arenaviridae, bunyaviridae (Also segmented)
Class VI - +SSRNA with DNA intermediate
One viral family: Retroviridae
2 Human pathogens
1. HIV
2. Human T-lymphotropic virus (HTLV)
Class VII - Gapped dsDNA
Hepadnaviridae,
Hepatitis B virus
Wild-type
Laboratory-adapted, original, from which mutants are selected
May not be identical to field isolates or clinical isolates (natural hosts)
DNA-mediated transformation
Introduction of foreign DNA into cells
Transfection
Production of infectious virus after transformation of a cell with viral DNA (first done with bacteriophage lambda)
Mutation
Changes in DNA or RNA comprising base changes and nucleotide insertion, deletion, arrangements. Includes nonsense, missense mutations.
How can you make a mutant of virus?
- Chemical treatments (Screen for desired phenotype; plaque size, drug resistance)
+ RNA polymerase error prone
Modern way:
Take viral DNA/RNA, make DNA copy, amplify it in bacterial plasmid, take that dna and put into cell
For influenza virus: It has 8 segmented genomes, we make cDNA out of all, we put it in 8 plasmids, transfect each 8 plasmids in cells, and then get infectious influenza, both RNA pol I and II will make + and - RNA and proteins.
Infectious DNA clone: Transfection
Modern validation of Hershey-chase experiment; Deletion, insertion, substitution, nonsense, missense.
Nature of viruses
One should avoid anthropomorphic analyses:
1. do not think
2. do not achieve their goals in a human centered manner
3. passive agents
Are viruses alive? Why?
No.
1. Non-cellular structures (basic unit of life)
2. Depend on host cell to reproduce (Obligate intracellular parasite)
What serves as the vehicle for the transmission of the viral genome to the next host cell or organism?
Progeny virion assembled during the infectitious cycle
Since when, prevention of virus infections in practice without knowledge of agent?
11th century
Ebolavirus classification
Filoviridae -> Ebolavirus -> Zaire ebolavirus
Raw sewage flowchart
Global locations -> Sample collection (10L) - Virion purification (1mL) : flocculation, DNAse treatment -> Pyrosequencing: NA extraction, random amplification -> Informatics: Removal of duplicates, low complexity sequences, identify by BLAST, taxonomic distribution
Deep highthroughput sequencing used in metagenomics, identification of new virus particles from environmental samples, identification of new pathogens
Examples of viruses that are inoculated in chorioallantoic membrane of embrynoated chicken eggs?
HSV, Poxvirus, Rous sarcoma
Examples of viruses that are inoculated in allantoic of embrynoated chicken eggs?
Mumps, newcastle disease, avian adenovirus, influenza
Examples of viruses that are inoculated in yolk sac of embrynoated chicken eggs?
HSV
Examples of viruses that are inoculated in amniotic of embrynoated chicken eggs?
Influenza, mumps
What is added to the cell cultures growing viruses to keep the pH neutral?
5% CO2
PCR product not the same as infectious virus
15 days after zikv infection of male mice, plaque assay of seminal fluid revealed no infectious virus.
However, ZIKV virus RNA reverse transcribed to DNA and measured by PCR, and still detected after 60 days.
When was influenza influenza virus resurrected?
1918
