Microbiology 4- Viral properties

Question 1

Compare the size of viruses with bacteria

Answer 1

Viruses are smaller than bacteria and can be passed through a filter.

Question 2

Which human virus was the first to be described

Answer 2

The first human virus to be described was Yellow Fever Virus. In 1901 Colonel Walter Reed of the US Army was intensely researching this disease because it caused havoc during the building of the Panama canal. There was a clear link with mosquitoes. Reed injected filtered serum from a patient into nonimmune individuals and reproduced the disease, thus fulfilling Koch’s postulates

Question 3

Describe Koch’s postulates

Answer 3

The microorganism must be found in large numbers in all diseased animals, but not in healthy ones.
The organism must be isolated from a diseased animal and grown outside the body in a pure culture.
When the isolated microorganism is injected into other healthy animals, it must produce the same disease.
The suspected microorganism must be recovered from the experimental hosts, isolated, compared to the first microorganism, and found to be identical.

Question 4

Define a virus

Answer 4

Viruses are infectious OBLIGATE intracellular PARASITES.

Question 5

List some of the properties of viruses

Answer 5

They are energy-less, they float around until they come into contact with an appropriate cell
A virus has a genome that comprises DNA or RNA- never both.
Structural simplicity: Virus particles are structurally simple. They have highly repetitive units that are so regular they can be crystallized. TMV was crystaliized in 1932. This achievement fed the debate as to whether viruses were alive or not. Their capacity to form crystals made them seem more like an inorganic substance.
They are basic life forms composed of a protein coat, called a capsid, that surrounds genetic material. Viruses do not have organelles or ribosomes. Certain viruses are further enclosed by an external lipid bilayer membrane that surrounds the capsid and may contain glycoproteins. Some viruses also carry structural proteins and enzymes inside their capsid.
Within an appropriate cell, the viral genome is replicated and directs the synthesis, by cellular systems, of more viral components and genomes.
The components effect the transport of replicated viral genomes through the environment to new host cells.

Question 6

How do we detect viruses now

Answer 6

Unethical to detect viruses by seeing whether they reproduce disease in healthy individuals. We use molecular methods, such as electron microscopy, to detect viruses and compare with health individuals/

Question 7

Explain the symmetry of viruses

Answer 7

Viruses are symmetrical, they can multiple copies of the same protein arranged similarly.

Question 8

What is meant by tegument

Answer 8

All the viral proteins carried in a virus

Question 9

What are the 4 levels of viral morphology

Answer 9

Nucleic acid:
DNA or RNA
Ds vs ss
Single or segmented pieces of nucleic acid
Positive or negative stranded RNA
Complexity of genome

Capsid:
Icosahedral
Helical

Envelope:
Naked
Enveloped

Size:
Diameter of the helical capsid viruses
The number of capsomers in icosahedral capsids.

Question 10

Describe icosahedral symmetry capsids

Answer 10

Take 1 or more polypeptide chains and organise them into a globular protein subunit. This will be the building block of our structure and is called a capsomere. Place the capsomers into an equilateral triangle and place 20 triangles together to form an icosahedron. Package the genome inside the icosahedral capsid.

Question 11

Describe helical symmetry capsids

Answer 11

In helical symmetry the protein capsomers are bound to RNA (always RNA because only RNA viruses have helical symmetry) and coiled into a helical nucleoprotein capsid. Most of these assume a helical shape except for the rhabdoviruses (rabies) which assume a bullet-shaped capsid.

Question 12

Describe how a virus obtains an envelope

Answer 12

Viruses acquire this membrane by budding through the host cell nuclear or cytoplasmic membrane and tearing off a piece of the membrane as they leave.

Question 13

Explain the difference between naked and enveloped viruses

Answer 13

Viruses that do not have membranes are referred to as naked viruses. Those with membranes are referred to as enveloped.

Question 14

List some naked viruses

Answer 14

Symmetrical protein capsid
Adenovirus
Picornavirus
Calicivirus
Non-enveloped

Question 15

List some enveloped viruses

Answer 15

Enveloped
Lipid envelope derived from host membrane
Pleiomorphic: measles virus
Typical shape: Ebola virus

Combination of capsid
and envelope
Herpes virus

Question 16

Describe how viruses have been named previously

Answer 16

Virus names are random. Viruses have been historically named after:
associated disease, e.g. poliovirus, rabies,
type of disease e.g. murine leukemia,
place in the body where isolated, e.g. rhinovirus
geographical location where first found e.g. Sendai, Coxsackie,
person who discovered it e.g. Epstein Barr,
way imagined to be spread e.g. dengue means evil spirit, influenza means influence of bad air

Question 17

Describe the Baltimore classification system

Answer 17

Commonly used nowadays is the Baltimore Classification system which places viruses into groups depending on the pathway they use to make their genomes into mRNAs.

Question 18

What is the central dogma of the genome

Answer 18

DNA — RNA — Protein

Question 19

Describe the differences in RNA viruses

Answer 19

Positive RNA- just like mRNA. On entering a host, its RNA can immediately be translated by host ribosomes into protein.
When negative stranded RNA viruses enter a cell, they are not able to begin translation immediately. They must first be transcribed into a positive strand. To do this, negative stranded RNA viruses must carry, in their capsid, an enzyme called RNA-dependent RNA polymerase, which will carry out transcription of the negative strand into the positive. Humans or mammals do not have RNA-dependent RNA polymerase.

Question 20

List the exceptions to these RNA viruses

Answer 20

Retroviruses, of which HIV is a member, are unique because of their ability to incorporate into the human genome, using reverse transcriptase to convert ssRNA into dsDNA.
Reoviridae, including rotavirus, is unique as they are the only viruses with a dsRNA genome.
dsRNA is converted into mRNA.

Question 21

Describe the differences in DNA viruses

Answer 21

Most DNA viruses have both a negative strand and positive strand. The negative strand is read, while the positive strand is ignored. Parvo-viruses are the exception and have a single-stranded positive DNA.

Question 22

Give an example of a ssDNA virus

Answer 22

Parvovirus B19; fifth disease/Slap face

Question 23

Give examples of dsDNA viruses

Answer 23

HSV1; cold sores, smallpox,

Papillomavirus; warts

Question 24

Give examples of dsDNA viruses that use RT

Answer 24

Hepatitis B; hepatocellular carcinoma

Question 25

Give examples of + ssRNA viruses that use RT

Answer 25

HIV; AIDS

Question 26

Give examples of +RNA viruses

Answer 26

SARS, norovirus; winter vomitting disease, poliovirus; poliomyelitis, dengue virus; DHF,

Question 27

Give examples of dsRNA

Answer 27

Rotavirus; diarrhoea

Question 28

Give examples of - ssRNA viruses

Answer 28

Ebola, rabies, Lassa fever, Influenza, Measles, Mumps

Question 29

Describe the consequences of viral genome type

Answer 29

RNA viruses and retroviruses use their own polymerase to replicate.
These lack proof reading capacity leading to high mutation rate.

RNA viral genomes are limited in size due to inherent instability to RNA vs DNA. The largest RNA viruses are coronaviruses genome size around 30kb. RNA viruses often use complex coding strategies to make more proteins than expected from a small RNA genome.

DNA viruses have genomes up to 100s kb. There is plenty of room for accessory genes that can modify the host immune response. These genes are often lost in passage in culture.

Segmented genomes allow an additional easy form of recombination known as reassortment, but also impose more difficult packaging strategies. Influenza has 8 different RNA segments, rotavirus has 11!

Question 30

Why do viruses have a high mutation rate

Answer 30

Viruses have no proof-reading mechanisms- high mutation rate.
Beneficial when strong selection pressure is applied.

Question 31

Does the disease that the virus causes have any association with the way it replicates

Answer 31

Disease virus causes has little association with the way it replicates, different viruses cause similar symptoms despite having different nucleic acids.

Question 32

Describe the role of accessory genes

Answer 32

Contribute to pathogenicity- encode immune antagonists- allow viruses to evade the immune system.

Question 33

Describe the 4 basic steps in viral reproduction

Answer 33

Adsorption and penetration
Uncoating of the virus- to allow nucleic acids and proteins to interfere with host cell
Synthesis and assembly of viral products (as well as inhibition of the host cell’s own DNA, RNA and protein synthesis).
Release of virions from host cell either by lysis or budding.

Question 34

Describe how the virus enters the host cell

Answer 34

Attachment to the host cell by specific interaction between the virus attachment protein and a host cell receptor (a molecule on the surface of a host cell that has a completely different role but that the virus has evolved to use as its key for entry to the interior of the cell). Viral encoded protein is usually on the capsid or glycoprotein. Unlike the bacteriophage which injects its DNA, these viruses are completely internalised, capsid and nucleic acid. This internalisation occurs by endocytosis or by fusion of the virion envelope with the host cell membrane.

Question 35

Describe the other steps in viral replication

Answer 35

Entry of the virus genome to the host cell and synthesis of viral mRNA (primary transcription).
Translation of viral mRNAs into viral proteins by host cell ribosomes in the cytoplasm.
Replication of the viral genome, usually by making a small number of complementary copies that are then themselves copied at high numbers into new genomes (asymmetric replication).
Assembly of the virus proteins into new virions, association and encapsidation of the new genomes.
Exit from the infected cells and dissemination to new hosts.

Question 36

Describe the difference between late and early proteins

Answer 36

Early proteins are regulatory- required for DNA replication and for further transcription of late mRNA
Late proteins are structural- transcribed after viral DNA replication has begun and is transcribed from progeny DNA. The capsid structural proteins are synthesised from the late mRNA genome.

Question 37

How do naked virions exit the cell

Answer 37

Cell lysis or exocytosis

Question 38

How do enveloped virions exit the cell

Answer 38

Acquires its new clothing by budding through the Golgi apparatus, nuclear membrane, or cytoplasmic membrane. tearing off a piece of host bilayer as it exits.

Question 39

What do +ssRNA need to be converted into before replication

Answer 39

-ssRNA to use as template strand, RNA dependent RNA polymerase is translated to do this.

Question 40

What do -ssRNA need to be converted into before replication

Answer 40

+ssRNA using a pre-made viral RNA dependent RNA polymerase that is carried by the virion.

Question 41

How are viruses visualised

Answer 41

Viruses are very small (10-500 nm diameter) and are visualized with the electron microscope.

Question 42

Describe another method of detecting viruses

Answer 42

Cytopathic effect is usually a result of the virus lysing the cell.
This could be due to shut down of host protein synthesis or accumulation of viral proteins.
Lysed cells are stained black to show infected cells- plaques- hole in cell when cell dies- viruses form plaques in monolayers

Question 43

Describe the plaque assay

Answer 43

Dilutions of virus can be assayed to find the end point at which they no longer produce such effects. This allows their quantification.
A specific example is the plaque assay in which foci of infection can be counted.

Question 44

Describe syncytia

Answer 44

Viruses with surface proteins that can fuse at neutral pH often fuse cells together

Question 45

How can we detect viruses that produce little effect on cells

Answer 45

Some viruses produce little visible change on the cells but can be detected using antibodies to their proteins that will be expressed during replication. Immune fluorescence or immunohistochemistry allow the antibody binding to be seen. Antibodies generated in the lab to unique virus proteins indicate which cells are infected
or where in the cell the virus proteins are located.

Question 46

Describe some techniques for seeing viruses that do not use cell cultures

Answer 46

Techniques for ‘seeing’ viruses that do not use cell cultures include detection of the particles or the viral proteins, or the virally encoded nucleic acid.
Viruses that attach to red blood cells can be visualized using the haemagglutination assay.
Cells infected by viruses can be lysed and antibodies to viral antigens used in Western Blot or enzyme-linked immunosorbent assay (ELISA).
Nucleic acid detection is usually by the polymerase chain reaction (PCR)- process genome in different ways depending on how it replicates
Viruses can be manipulated in the laboratory in order to study them.
Detecting virus particles EM- expensive, not specific, HA
Detecting virus cytopathic effect in cultured cells (Virus isolation)

Detecting antibodies to virus (serology)- difficult in acute infections- response is too quick.

Question 47

Describe how we can propagate viruses

Answer 47

We can passage viruses in the laboratory by providing permissive cells, often continuous lines of transformed cell cultures.
Viruses may accumulate mutations that adapt them to the ‘new’ host.
This can lead to attenuation and was the basis for generation of vaccines in the past.

For some viruses we have no permissive cell lines: norovirus ‘Winter Vomitting Disease’. And Hepatitis C virus remain difficult to study in the laboratory.

Question 48

What is meant by continuous cell lines

Answer 48

Continuous cell lines are monocultures propagated indefinitely due to their transformation. They may grow as monolayers on a plastic support or in suspension.

Question 49

What is meant by a primary cell culture

Answer 49

Primary cell cultures may include several different cell types and may retain aspects of the original tissue

Question 50

Describe how we can manipulate viral genomes

Answer 50

Virus genomes are so small they can be synthesized.
When introduced into permissive cells they direct synthesis of all their component are new viruses are made de novo.
This allows reverse genetics, the creation of viruses at will with engineered mutations in their genomes.
New generation of vaccines- synthesis viral genome- remove genes that contribute to pathogenicity- but keep genes that allow their recognition and ability to replicate-basis behind live attenuated vaccines

Question 51

Describe classical genetics of manipulating viral genomes

Answer 51

Classical genetic techniques involve growing the virus under different conditions, for example temperature or in the presence of a drug, that may induce changes in the genome and studying their effects.