Introduction to virology Flashcards
What are Viruses?
- Sub-microscopic, acellular, obligate intracellular parasites
- More than 80% of infectious diseases are caused by viruses
- Assemble, not grow
- Lack essential apparatus for generation of energy or protein synthesis
- Entirely dependent on host cell function
Describe the Production of viral +mRNA
- Double stranded DNA virus:
Needs DNA dependent RNA polymerase to use 1 strand of the DNA as a template to make mRNA
- A positive single stranded DNA virus:
First it has to make a negative complementary strand Needs DNA dependent DNA polymerase to synthesise a negative complementary strand
then it can make more positve strand and +mRNA copies
- A negative single stranded DNA virus:
DNA dependent RNA polymerase to make a complementary strand and +mRNA
- Double stranded RNA viruse:
Needs RNA dependent RNA polymerase
One of the strands used as a template for +mRNA synthesis if it is from the negative strand
- Negative single stranded RNA virus:
Needs RNA dependent RNA polymerase
strand can be used as a template to synthesise +mRNA
- Positive strandded RNA virus:
First it has to make a negative complementary strand
then it can make more positve strand and +mRNA copies
What are viruses produced from?
What do viruses lack?
- Virus particles are produced from the assembly of pre-formed components, whereas other agents such as cells ‘grow’ from an increase in the integrated sum of their components & reproduce by division. Virus particles (virions) themselves do not ‘grow’ or undergo division.
- Viruses lack the genetic information which encodes apparatus necessary for the generation of metabolic energy or for protein synthesis (ribosomes).
- No known virus has the biochemical or genetic potential to generate the energy necessary for driving all biological processes, e.g. macromolecular synthesis. They are therefore absolutely dependent on the host cell for this function.
Where did viruses come from?
- Tracing the origins of viruses is difficult
- do not form fossils
- some viruses integrate into host genomes
- infect basically any organism
- started as big bits of cellular DNA and then became independent?
2 hypothesis:
1) complex enveloped viruses = from small cells, probably prokaryotic, that parasitized larger more complex cells → retrograde evolution (become more simple).
2) cellular nucleic acids that have become partially independent.
Explain the Origins of Virology
- English doctor Edward Jenner, the pioneer of smallpox vaccination.
- In 1796, he carried out his famous experiment on eight-year-old James Phipps. Jenner took pus from a cowpox pustule and inserted it into an incision on the boy’s arm. He was testing his theory, drawn from the observation, that milkmaids who suffered the mild disease of cowpox never contracted smallpox, one of the greatest killers of the period, particularly among children. Jenner subsequently proved that having been inoculated with cowpox Phipps was immune to smallpox. He submitted a paper to the Royal Society in 1797 describing his experiment, but was told that his ideas were too revolutionary and that he needed more proof. Undaunted, Jenner experimented on several other children, including his own 11-month-old son. In 1798, the results were finally published and Jenner coined the word vaccine from the Latin ‘vacca’ for cow.
- Subsequently, Pasteur worked extensively on rabies, which he identified as being caused by a ‘virus’ (from the Latin for ‘poison‘, however, he did not discriminate between bacterial & other agents of disease). Louis Pasteur developed his rabies vaccine by growing the virus in rabbits, then drying the affected nerve tissue to weaken the virus. On July 6, 1885, the rabies vaccine was administered to Joseph Meister, a 9-year-old boy who had been attacked by a rabid dog. The boy survived and avoided contracting rabies.
- Good thing it worked: Pasteur was not a licensed physician and could have been prosecuted had the vaccine failed. The legalities were forgotten and Pasteur instead became a national hero.
Who are the Pioneering Virologists?
Dimitri Ivanovsky (1892)
Filtered diseased tobacco plant extracts
Martinus Beijerinick (1898)
Confirmed Ivanovsky data
Defined term ‘virus’
Freirich Loeffler (1898)
Discovered Foot & Mouth
Although Louis Pasteur and Edward Jenner developed the first vaccines against viral infections, they did not know that viruses existed
- On 12th February 1892, Dmitri Ivanovski, a Russian botanist, presented a paper to the St. Petersburg Academy of Science which showed that extracts from diseased tobacco plants could transmit disease to other plants after passage through ceramic filters fine enough to retain the smallest known bacteria. This is generally recognised as the beginning of Virology. Unfortunately, Ivanovski did not fully realize the significance of these results.
- A few years later, in 1898, Martinus Beijerinick confirmed & extended Ivanovski’s results on tobacco mosaic virus & was the first to develop the modern idea of the virus, which he referred to as contagium vivum fluidum (‘soluble living germ’).
- Also in 1898, Freidrich Loeffler & Paul Frosch showed that a similar agent was responsible for foot-and-mouth disease in cattle. Thus these new agents caused disease in animals as well as plants.
- In spite of these findings, there was resistance to the idea that these mysterious agents might have anything to do with human diseases. This view was finally dispelled by Landsteiner & Popper (1909), who showed that poliomyelitis was caused by a ‘filterable agent’ - the first human disease to be recognized as having a viral cause.
IDescribe Ivanovsky’S Experiment
- Extracted sap from tabacco plant with toacco mosaic disease
- Passed sap through a porecelain filter known to trap bacteria
- Rubbed filtered sap onto healthy tobacco plant
- Healthy plants become infected
Explain the discovery of Bacteriophages
- Frederick Twort (1915)
- Félix d’Herelle (1917)
- Bacteriophages = bacterial eaters
In 1915, British bacteriologist Frederick Twort discovered a small agent that infected and killed bacteria. One of his theories was that the agent must be a virus.
Twort’s research was interrupted by the onset of WWI and a shortage of funding.
Independently, French-Canadian microbiologist Felix d’Herelle, working at the Pasteur Institute in Paris discovered “an invisible, antagonistic microbe of the dysentery bacillus”. He was sure of the nature of his discovery - a virus parasitic on bacteria. D’Hérelle called the virus a bacteriophage, a bacteria-eater (from the Greek phagein meaning “to devour”). He also recorded a case of a man suffering from dysentery who was restored to good health by the bacteriophages. D’Herelle conducted much research into bacteriophages and introduced the concept of phage therapy.
Describe the relative size of viruses
- Virions come in many shapes and sizes.
- Most viruses are smaller than prokaryotic cells, ranging in size from 0.02 to 0.3 μm (20–300 nanometers, nm).
- Smallpox virus, one of the larger viruses, is about 200 nm in diameter, which is about the size of the smallest known bacterial cells.
- Poliovirus, one of the smallest viruses, is only 28 nm in diameter, which is about the size of a ribosome.
Describe the two typical virus structures
Viral nucleic acid is surrounded by a protein coat called a capsid. Each capsid is made up of capsomeres. Capsomeres bond together and give the capsid structural symmetry. Some viruses also have a lipid envelope. In enveloped viruses, the inner structure of nucleic acid plus capsid protein is called the nucleocapsid.
Envelopes are formed of host cell lipid bilayer decorated with viral glycoproteins. They are firmly embedded in the envelope bilayer and form spikes or other structures on the outside of the virion and can be used to attach to a host cell.
Viruses can carry also enzymes, sometimes on surface (e.g. influenza), or mostly inside (e.g. retroviruses) necessary for their infection/replication.
Virion, an entire virus particle, consisting of an outer protein shell called a capsid and an inner core of nucleic acid (either RNA or DNA). The core confers infectivity, and the capsid provides specificity to the virus.
Virion structure must overcome two basic problems:
1) It must be strong enough to protect the viral nucleic acid
2) It must be able to shed the protein coat upon entry into a host cell
The virion protects the viral genome when the virus is outside the host cell, and proteins on the virion surface are important in attaching it
to its host cell. Most bacterial viruses are naked, whereas many animal viruses contain an outer layer consisting of protein plus lipid called the envelope
Describe a typical virus structure
- Viruses are either icosahedral, helical, or complex
- Viruses are highly symmetric
Icosahedral shape is derived from 20 triangular faces that make up the capsid
There are two types of icosahedral viruses:
Simple
Complex
There are two shapes of helical viruses:
Rod – straight and relatively rigid
Filamentous – flexible, curved, or coiled
The nucleic acid of a virion is always surrounded by its capsid. The capsid is composed of a number of individual protein molecules called capsomeres that are arranged in a precise and highly repetitive pattern around the nucleic acid. Two kinds of symmetry are recognized in viruses, which correspond to the two primary viral shapes, rod and spherical. Rod-shaped viruses have helical symmetry while spherical viruses have icosahedral symmetry.
1) An icosahedron is a polygon with 12 vertices and 20 faces. Two types of capsomeres constitute the icosahedral capsid: pentagonal (pentons) at the vertices and hexagonal (hexons) at the faces. There are always twelve pentons, but the number of hexons varies among virus groups. In electron micrographs, capsomeres are recognized as regularly spaced rings with a central hole. Icosahedral symmetry is identical to cubic symmetry.
2) Helical- The protomers are not grouped in capsomeres, but are bound to each other so as to form a ribbon-like structure (hollow tubes with protein wall). This structure folds into a helix because the protomers are thicker at one end than at the other. The diameter of the helical capsid is determined by characteristics of its protomers, while its length is determined by the length of the nucleic acid it encloses. Arranges as a helix or spiral, produces long, rigid tube.
3) Complex- e.g., that exhibited by poxvirus, rhabdovirus, phage. This group comprises all those viruses which do not fit into either of the above two groups or is a combination of both.
When the viral particle has entered a host cell, the host cellular enzymes digest the capsid and its constituent capsomeres, thereby exposing the naked genetic material (DNA/RNA) of the virus, which subsequently enters the replication cycle.
Viral Genome
- DNA or RNA (not both)
- double stranded (ds) or single stranded (ss)
- linear or circular
- RNA + ve strand
- RNA – ve strand
- Only +ve strand mRNA can be translated into viral protein
All 4 types are found in animal viruses, most plant viruses are ssRNA, most phages are dsDNA. A very few highly unusual viruses use both
DNA and RNA as genetic material, but at different stages of their life cycle, they can be linear or circular. Viral genomes of the plus configuration
have the exact same base sequence as that of the viral mRNA that will be translated to form viral proteins. By contrast, viral genomes of the minus configuration are complementary in base sequence to viral mRNA. Viral genomes encode from a few up to about 350 genes.
Viral genomes. The genomes of viruses can be either DNA or RNA, and some use both at different stages in their replication cycle. However, only one type of genomic nucleic acid is found in the virion of any particular type of virus.
What is the 5 main steps in the life cycle of a bacteriaphage?
- Attachment (adsorbtion)
- Penetration (entry, uncoating)
- Synthesis (NA and proteins)
- Assembly (maturation, packaging)
- Release
Animal viruses are known in all genomic categories, The majority of important human viral diseases are caused by RNA viruses
Unlike a bacteriophage infection, in which one of only two outcomes—lysis or lysogeny is possible depending on the virus, other events are possible in an animal virus infection
- Attachment/Adsorbtion
Binding of attachment sites of the virus with receptor sites of the host cytoplasmic membrane
- Some viruses require a co-receptor to attach. Without the co-receptor, there is no infection
- Many different host cell molecules can be used as viral receptors
- Some viruses use more than one type
- Some receptors are shared by many viruses
- Receptors can determine host range of virus
- Virus-receptor binding is highly specific
Penetration and Uncoating: Naked viruses
Interaction of the capsid and host cell membrane causes rearrangement of the capsid proteins to allow nucleic acid entry.
Penetration and Uncoating: Enveloped viruses
The virus enters the host cell and the viral envelope is removed. The capsid is then enzymatically removed.
What happens after fusion of viral envelope with host membrane?
- Capsid enters or the whole virus is endocytosed
- Endosomal enzymes can aid in virus uncoating and low pH often triggers uncoating, in some instances envelope fuses with endosomal membrane and nucleocapsid released into cytosol.
- Once in cytosol, viral NA may be released from the capsid upon completion of uncoating or may function while still attached to capsid components.
- Vesicle acidification causes a capsid conformational change, the altered capsid contacts vesicle membrane and either releases viral NA into the cytoplasm through a membrane pore or ruptures the membrane to release the virion.
Synthesis and Assembly
The viral genome directs the host cell replication and translation machinery to synthesize viral components
Why must DNS viruses enter the host nucleus?
- DNS viruses must enter the host nucleus (e.g. eukaryotic cell infecting viruses) before it is able to replicate. These viruses require host cell polymerases to replicate the viral genome and thus are dependent on the cell cycle.
- All viruses depend on host cell machinery for viral protein synthesis. Host cell DNA synthesis is inhibited by the virus, all polymerases and proteins concentrate on viral DNA synthesis.
- Capsids self-assemble.
All virions must complete a common set of assembly reactions:
- Formation of structural subunits for the capsid
- Assembly of the capsid
- Association of the viral genome within the capsid
Synthesis and Assembly: Nucleus (DNA virus) vs cytosol (RNA virus)
- RNA virus carries/encodes specific viral enzymes
- RNA-dependent RNA polymerases
- RNA-dependent DNA polymerases
Describe the release process
In naked viruses the host cell deteriorates and the virus is released.
What are the 3 different modes of release?
- lysis
- budding
- exocytosis
- Virion release differ between naked and enveloped viruses.
- naked viruses usually released by host cell lysis
- enveloped viruses bud out through cell membrane or can be exocytosed
How does Virion release differ between naked and enveloped viruses?
- naked viruses usually released by host cell lysis
- enveloped viruses bud out through cell membrane or can be exocytosed
Release: enveloped viruses
- Some viruses obtain their envelope from the host cell membrane upon release via budding.
- Others obtain their envelope from internal membranes are released by exocytosis.
There are at least four different outcomes for animal viruses:
- Virulent infection results in lysis of the host cell; this is the most common outcome.
- Latent infection, viral DNA does not replicate and the host cells are unharmed.
- Persistent infection, budding may be a slow process and the host cell may not be lysed.
- Transformation, certain animal viruses can convert a normal cell into a tumour cell.
Analysis of phages: Plaque assay
- Phage dilution and bacterial cells are added to top agar
- Mixture poured onto an agar plate
- Prepare a nutrient agar plate
- Sandwich the top agar and nutrient agar
- Incubate overnight
- There will be phage plaques and a lawn of host cells
What can be seen from a plaque assay?
- Plaque is a clear zone in the bacterial lawn
- Each plaque originates from a single phage particle
Lytic cycle: 1
Adsorption
- The bacteriophage binds to receptors on the bacterial cell wall
- specific proteins on membrnae interact with host cell proteins
Lytic cycle: stage 2
Penetration
The bacteriophage injects its genome into the cytoplasm of the bacterium
Lytic cycle: stage 3
Replication
The bacteriophage genome replicates and its components begin to be produced by the host bacterium’s metabolic machinery
Lytic cycle: Stage 4
Maturation/Assembly
The bacteriophage components assemble
Lytic cycle: Stage 5
Release
- A bacteriophage-coded enzyme breaks down the peptidoglycan in the bacterial cell wall causing osmotic lysis
- From 50 to 200 phages may be produced per infected bacterium
Give a summary of the lytic cycle
- Attachment- adsoprtion of phage virlon
- Penetration of viral nucleic acid
- Synthesis of viral nucleic acid and protein
- Assembly and packaging of new viruses
- Cell lysis and release of new virlons
Lysogenic cycle: Stage 1
Adsorbtion
The bacteriophage binds to receptors on the bacterial cell wall
Lysogenic cycle: Stage 2
Penetration
- The bacteriophage injects its genome into the cytoplasm of the bacterium
- All known bacteriophage use lytic enzymes to penetrate the cell wall
Lysogenic cycle: Stage 3
Prophage formation
The bacteriophage inserts its genome into the bacterium’s nucleoid to become a prophage or if the conditions are favourable it will make more viral particles
Lysogenic cycle: Stage 4
Maintenance
As the bacterium replicates, the prophage replicates as a part of the bacterium’s nucleoid.
Lysogenic cycle: Stage 5
Spontaneous induction
The phage genome is excised from the bacterial nucleoid and becomes lytic
Lysogenic cycle: Stage 6
Replication
The bacteriophage genome replicates and its components begin to be produced by the host bacterium’s metabolic machinery
Lysogenic cycle: Stage 7
Maturation
The bacteriophage components assemble
Lysogenic cycle: Stage 8
Release
A bacteriophage-coded enzyme breaks down the peptidoglycan in the bacterial cell wall causing osmotic lysis
Give a summary of the lysogenic cycle
- Attachment of the virus to the host cell
- Injection of viral DNA
- Viral DNA is integrated into host DNA
- Viral DNA is replicated with host DNA ar cell division
What needs to be considered to determine the effectivenes of phage treatment?
- Dose and momemnt of treatment
- Phage concentration
- Specificity
- Phage administration
- Resistance to phage
- Acessibility to tager bacteria
- Environment conditions
- Neutralisation or destruction e.g by antiodies
What is baltimore classification of viruses based on?
How they produce their mRNA
Viruses can be classified on the basis of the hosts they infect as well as by their genome structure. E.g. bacterial viruses, archaeal viruses, animal viruses, plant viruses, protozoan viruses, etc.
I: These types of viruses must enter the host nucleus (unless it is a prokaryotic virus) before it is able to replicate. These viruses require host cell polymerases to replicate the viral genome. Eukaryotic dsDNA viruses are highly dependent on the cell cycle. Proper infection and production of progeny requires that the cell be in replication, as it is during replication that the cell’s polymerases are active. The virus may induce the cell to forcefully undergo cell division, which may lead to transformation of the cell and, ultimately, cancer. Examples: Herpesviridae, Adenoviridae.
II: Most have circular genomes (exception: parvoviruses).
III: Replicates in the cytoplasm, not having to use the host replication polymerases to as much a degree as DNA viruses.
IV: The positive-sense RNA viruses can be directly accessed by host ribosomes to immediately form proteins. Reproduce in the cytoplasm.
V: The negative-sense RNA viruses cannot be directly accessed by host ribosomes to immediately form proteins. Instead, they must be transcribed by viral polymerases into a “readable” form, which is the positive-sense reciprocal.
VI: Positive-sense single-stranded RNA viruses that replicate through a DNA intermediate. A well-studied family of this class of viruses include the retroviruses. One defining feature is the use of reverse transcriptase to convert the positive-sense RNA into DNA which is then used to transcribe mRNA. DNA intermediate is integrated into the host genome using integrase. Replication can then commence with the help of the host cell’s polymerases.
VII: Double-stranded DNA viruses that replicate through a single-stranded RNA intermediate
This small group of viruses (e.g. Hepatitis B virus) virus have a double-stranded, gapped genome that is subsequently filled in to form a covalently closed circle (cccDNA) that serves as a template for production of viral mRNA and a subgenomic RNA. The pregenome RNA serves as template for the viral reverse transcriptase for production of the DNA genome.
