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Virologie > Virus > Flashcards

Virus Flashcards

1
Q

What is a virus?

A
  • Very small, infectious, obligate intracellular parasites
  • Viral genome consists of DNA or RNA
  • In a suitable host cell the viral genome is replicated and determines the synthesis through virus-derived or cellular components
  • New viruses are generated with newly synthesized components within the host cell „de novo“
    -The viruses, which are synthesized in this replicative cycle are the vehicles for the transmission of the viral genome into the new host cell or organism, in which the uncoating of the virion and the next replicative cycle starts
  • 20 % -> number two cause of death
  • most prominent ones: pneumonia, tuberculosis, diarrhoeal diseases, malaria, measles, Sars Cov-2 and HIV/AIDS
  • Loeffler and Frosch are acknowledged to be the founders of virology
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2
Q

First vaccines against viral diseases

A
  • Human pox (Variola) -> Virus of cowpox
  • Rabies -> attenuated rabies virus
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3
Q

Koch Henleschen Postulate for the identification of pathogens

A
  1. Pathogen must be detectable in all diseased animals, not in healthy ones
  2. Cultivating pathogen in pure culture
  3. Pathogen can reproduce disease in healty individuals
  4. Pathogen can be reisolated from the newly diseased animals

Viruses were pathogens, whose cultivation was not possible with the methodes of Koch!!

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4
Q

The first described viruses

A
  • Tobacco-mosaic-virus (TMV)
  • Foot-and-mouth disease virus
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5
Q

Yellow fever virus (Flavivirus)

A
  • Wide-spread in tropical regions
    e.g. 1853 epidemic in New Orleans (28% mortality) -> Until today about 200.000 cases and 30.000 death per year
  • Observation: No transmission from patient to patient!
    -> 1880 Finlay (physician on Cuba): Mosquitoes as vector (at this time only a hypothesis)
    -> 1899 Walter Reed (US Army): Tests with soldiers, Blood filtrates infectious for humans, Passing on via mosquitos proven (Arthropode borne = Arbo virus)
    -> 1901: Identification of yellow fever virus
    Mosquito as the vector
    -> 1930: Max Theiler, attenuation in chicken embrio cell culture, Vaccine(17D)(Nobel price 1951)
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6
Q

Isoliation of pathogenic bacteria was significantly more efficient than of pathogenic viruses
Why?

A

Characteristics of viruses:
- Viruses are submicroscopical genetic parasites; they need the cellular system of the host for their replication
S.E. Luria
- obligate intracellular!
Need for identification of a permissive (animal) host
Effective cell culture systems required for pure cultures!

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7
Q

Influenza A Virus

A

Pandemic: 1918/1919, ca. 20-40 millionen death
- Identification of Influenza Virus not before 1933
- Replication of virus in lung-tissue of ferrets
(normal mice allow only very inefficient virus replication)
Later: fertilized chicken eggs (Allantois fluid/membrane)
- highly efficient
- statements on virulence/ attenuation possible
Still a general problem in virus isolation and propagation: Identification of a permissive host (cell)
e.g. human Hepatitis B Virus Proliferation only in primates
New: primary liver cells from
Tupaia belangeri
- human Hepatitis C Virus: primates

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8
Q

Virus proliferation in the laboratory

A

Problem of animals as model organisms for viral diseases
- ethics
- costs
- hazards
- space requirements
- reproducibility

Technical solutions: Working with cells instead of whole organisms

Easiest variant: Bacteria and phages
- E.coli phages, T(ype)-phages (1-7)
- Replicative cycle <1h
- Basic principles of molecular biology

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9
Q

Eukaryotic cell culture

A
  • Proliferation of animal and human viruses without an organism (e.g. animal) as a host
    Making a cell culture:
  • extracting tissue/organ
  • dissipation (scalpel)
  • digestion by trypsin
  • filtration/centrifugation
  • seeding on plates
  • monolayer/contact inhibition

Primary cell culture: cells directly from an animal
- cell culture passage: secondary, tertiary ect. cultures
Problems:
- dedifferentiation, loss of host factors required for viral amplification
- dying after only a few passages (apoptosis or necrosis)
Permanent cell culture
- Only a small number of cells survives spontaneously, hence > use of tumor cells from patients
- Artificial immortalisation of cells via
> Tumor viruses
> Chemical / physical noxa
> Genetechnical manipulation

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10
Q

Efficient cell culture techniques (since ca 1955)

A

Requirements
- sterile working
- antibiotics (1929, Penizillin by Fleming)
- culture medium
- growth factors
> serum
> plasma
> lymph
> extracts of embryos
> factors produced by molecular biology
- immortalisation
Advantage of established cell culture?
Reproducibility when working with viruses

  • discovery of new viruses, which had not been discovered because of the lack of a suited, experimental host
  • adenovirus
  • measles virus
  • rubella virus
  • attenuation of viruses in cell culture: Example poliovirus
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11
Q

HeLa-cells

A

Isolated from tumor patient: Henrietta Lacks
- mother of 5 children, Baltimore, USA
- developed uterus cancer (died in 1951)
- George Gey received tissue sample (John-Hopkins-Hospital)
HeLa-cells are the first human cell line which divides continously in the lab (still today!) – cell division each 48 h
- HeLa-cells can be found in most cell culture labs around the world:
- Molecular principles of cancer development
- Approaches on therapy / drug development

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12
Q

WI-38 cells

A
  • derived 1962 from the lungs of an embryo after a legal abortion in Sweden
  • Leonard Hayflick at Wistar Institute in Philadelphia prepared cell strain which
    stops dividing at around passage 50: normal cells (not cancer cells like HeLa)
  • models for cellular aging (express a beta-galactosidase, stainable at pH 6)
  • cells were used for producing vaccines against rubella, polio, measles, chickenpox (people were afraid to use cancer cells for vaccine production)
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13
Q

Poliovirus

A
  • Pathogen of infantile paralysis (polio)
  • Massive epidemics at the beginning of the 20th century e.g. 1916 in New York State, more than 13.000 cases
  • Poliovirus is transmittable by injection of spinal fluid of a child, which died from the disease, onto an ape
  • News: Transmission between human host and animal host is possible
  • Currently: World wide poliovirus eradication program (Countries that are still problematic are Afghanistan, Pakistan)

Poliovirus in cell culture
- 1949 John Enders cultivates poliovirus
- 1953 Jonas Salk, dead vaccine
- 1961Albert B. Sabin, poliovirus live attenuated vaccine in HeLa-cells

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14
Q

Further fundamental principles of molecular biology, clarified with phages

A
  • DNA replication
  • principle of „self-assembly“ for proteins and nucleic acids
  • genetic code
  • temporal regulation of gene expression
  • principle of lysogenesis – Operon model, integration of foreign DNA
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14
Q

Further fundamental principles of molecular biology, clarified with phages

A
  • DNA replication
  • principle of „self-assembly“ for proteins and nucleic acids
  • genetic code
  • temporal regulation of gene expression
  • principle of lysogenesis – Operon model, integration of foreign DNA
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15
Q

Role of viruses in the analysis of eukaryotic gene regulation

A

SV40: Transcriptional enhancer element, Transacritption factors, poly(A)signal
Adenoviruses: RNA pol III promoter recognition, RNA splicing, RNA transport
Poxviruses: mRNA polyadenylation
Reoviruses: Cap and methylation of 5’ end of mRNA
Poliovirus: Translational regulation
Numerous viruses: Trafficking, post translational processing: proteases, CHO and fatty acid additions, phosphorylation

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16
Q

Use of viruses and their gene products in gene technology

A
  • Retroviral reverse transcriptase: for the generation of complementary (c)DNA
  • SV40 DNA as first ecpression vector in mammalian cells
  • Retroviruses, adenovirus, Aden-asscociated virus etc. as gene transfer vectors
  • Promoters (cytomegalovirus, CMV)
  • Translation elements (internal ribosomal entry site (IRES); SV40 poly(A) signal)
  • T Phages: RNA polymerase, RNA/DNA Ligase, Polynukleotide Kinase
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17
Q

Comparison: virus/flash memory stick

A
  • Stores information, but cannot translate it
    -> Only in the computer (host cell) this information can be read and
    translated
  • The information has always one of two possible forms, RNA or DNA.
    -> Compare: MS-DOS and Macintosh format; very different, but convertable
  • RNA- or DNA- information carrier has protective cover made of proteins compare: Plastic body of stick

Viruses are obligate intracellular parasites no own protein translation apparatus and no own energy generation

18
Q

Structure of viruses

A
  1. nucleic acid
  2. Capsid (always!)
  3. Envelope (facultative)
  4. Lipid membrane (facultative)
19
Q

Helical viruses

A
  • the simplest way to arrange multiple, identical protein subunits is to use rotational symmetry
    -> helix -> de- fined by amplitude and pitch -> number of subunits per turn µ, and axial rise per subunit p
  • pitch of the helix: P = µ x p
    -> for TMV: µ = 16.3 -> 16.3 coat protein molecules per helical turn
    -> p = 0.14 nm
    -> pitch of the TMV helix is 16.3 x 0.14 = 2.28 nm.
20
Q

Criteria for classification of viruses

A

Type of nucleic acid for the genome
– DNA, RNA
– Size
– Single-stranded(ss),double-stranded(ds)
– Linear, circular, segmented, a.o.
* Existence of a (lipid)-envelope
* Symmetry of the capsid (helical, icosahedral)
* Arrangement of the genes
* Replication strategy (Baltimore-scheme)
* No criteria are:
– Host range
– Pathogenicity and kind of disease

-> Viruses with ambisense genomes: ssRNA, genome and antigenome are coding

21
Q

Classification of viruses

A
  • classes I - VII (Baltimore Schema)
  • ICTV nomenclature
    order: virales
    family: viridae
    genus: virus
    species: name
22
Q

New ICTV Rules for Taxonomy of Viruses

A

4 Realms:
Riboviria (RNA genome), “Monodnaviria (Parvo-, Circo-, Polyomaviridae),” “Varidnaviria (Mimi,- Adenoviridae),” and “Duplodnaviria (e.g. Herpesviridae)”

23
Q

Genomes of different human DNA virus families

A

For comparison:
- Human genome (L-ds DNA segment): 3.3 x 10^9 bp
- E.coli (C-ds DNA): 5.5 x 10^6 bp
- L-ds DNA (Pandoraviridae -> 2475 kb, Poxviridae -> 130 - 375 kb, Herpesviridae -> 125 - 240 kb, Adenoviridae -> 26 - 45 kb)
- C-ds DNA (Papillomaviridae -> 6.8 - 8.4, Polyomaviridae -> 4.7 - 5.2 kb)
- L-ss DNA (Parvoviridae -> 4 - 6 kb)
- C-ss DNA (Circoviridae -> 1.7 - 3.3 kb)
- L-ds RNA segmented (Reoviridae (Rotaviruses, Enteritis) = 20 - 30 kb)
- L-RNA (Order Mononegavirales -> 9 - 19 kb (Paramyxoviridae (mumps), Rhabdoviridae (rabies), Filkoviridae (Ebola, Marburg), Bornaviridae)
- L-RNA segmented (Orthomyxoviridae (Influenza) -> 10 - 14.6 kb, Bunyaviridae (Hanta) -> 8 -12 kb)
- L +/- RNA segmented/ambisense (Arenaviridae (Lassa) -> 11 kb)
- C-RNA (Deltavirus (Hepatitis D) virusoid -> 1.7 kb)

L = linear, ds = double-stranded, ss = single-stranded, C = circular

24
Q

Genomes of different plus-strand RNA viruses

A
  • Picornaviridae (non enveloped) (Poliovirus, Rhinovirus, Hepatitis A V.) -> 7 - 8.5 kb
  • Flaviviridae (enveloped) (Yellow fever V., Dengue V., Hepatitis C) -> 9.6 - 12.3 kb
  • Caliciviridae (subgenomic RNA, non enveloped) (Gastroenteritis-Viruses) -> 7.4 - 8.3 kb
  • Togaviridae (subgenomic RNA, enveloped) (Arboviruses, Rubella virus) -> 9 - 11.8 kb
  • Coronaviridae (multiple subgenomic RNA enveloped) (Gastroenteritis Viruses, SARS9 -> 27.6 - 31 kb)
25
Q

Viruses which replicate via reverse transcription

A
  • L+RNA (Retroviridae -> HIV, HTLV, Rous-Sarkom Virus) -> 7 - 11 kb
  • C-ss/ds DNA (Hepadnaviridae, Hepatitits-B-Virus) -> 3.2 kb
26
Q

Approximate number of genes in different taxa

A
  • 30.000 - 40.000 human
  • 500 - 10.000 bacteria
  • 4 - 300 (1.120) viruses
  • 1 virusoid
  • 0 viroid
27
Q

Viruses and sub viral infectious agents

A
  • Viruses
    – Nucleic acid and proteins
    – Glycans and lipids facultative
  • Virusoids (e.g. Hepatitis Delta Virus)
    – Helper-dependent virus; encode no capsid proteins (but still encodes a protein)
  • Viroids
  • infectious RNA, encodes no proteins
  • Prions
  • infectious proteins, no nucleic acid
28
Q

Virusoids

A
  • 1.7 kB RNA (neg. polarity)
  • needs Hepatitis B Virus capsid proteins for packaging
  • codes for hepatitis delta antigen (HD Ag; L and S form)
  • S-HD Ag important for genome replication, L for virion assembly
  • cell. RNA Pol II (DdRp) is involved in genome replication
29
Q

Satellite virus

A
  • genome slightly larger than in viroids
  • Coding viral proteins but no capsid proteins
  • The virusoid-nucleic-acid is packaged by the capsid
    -> proteins of the helper-virus
30
Q

Viroids

A
  • small (200-400 nt), circular RNA
  • self-complementary, rod-like secondary structure
  • encode no protein
  • no capsid or envelope
  • replicate in nucleus or chloroplast
  • cause severe diseases of plants
  • 20 species, 2 families
  • replication involves DNA dep. RNA Pol II of the host
  • „Vaccination“ possible by avirulent form
    -> Still unknown: How do viroids cause disease? 4 nucleotides may make the difference!
31
Q

Prions

A

Novel concept: Proteins with “infectious nature”
* Prion-diseases:
– Classical Creutzfeldt-Jakob disease
– Scrapie (sheep)
– BSE (bovine spongiform encephalopathy)
– New variant of Creutzfeldt- Jakob disease (-> eating BSE-diseased beef did cause vCJD)
– Chronic wasting disease of mule deer, elk (wapiti), and moose
Pathogenic form of PrP enters the nervous system in the gut and is transported via neurons into the CNS -> No neural tissue in food chain

32
Q

Fundamental techniques -> Cell culture

A

Primary cell culture: Culture of cells obtained from original tissue cultivated in vitro for the first time. They may be sub-cultured or grown to „strains“ but are not immortal.

Cell line: Immortal cell cultures are called cell lines. Immortalization can occur spontaneously during passage of a cell strain, or it can be induced.

33
Q

Detection of virus infection in cell culture

A
  • Cytopathic effect (CPE)
  • Development of ‚inclusion bodies‘
  • Hemadsorption
  • MOI (multiplicity of infection) - average number of viruses added per cell
    in an infection
34
Q

Virus Proliferation in the lab -> Viral replicative cycle

A
  • Adsorption
  • Penetration
  • Replication
  • Generation of new particles
  • Release by cell lysis, „virus burst“ (often 8 -10 h p.i.)
    Lytic virus proliferation „virus burst“,
    as contrast to the cell division in bacteria
    For the infection of a cell, one „infectious unit“ is sufficient, but: During lysis of a single infected cell a huge number of infectious units“ is released
35
Q

How to count viruses?

A

Bacteriology: Plating of a dilution series on agars, Counting of colonies
Virology: Addition of dilution series on tissue culture cells, Counting of, „plaques“ in the monolayer -> blue: remaining stained cells

-> cytolytic viruses: plaques in the monolayer unit: plaque forming units (PFU)
-> non cytolytic viruses (e.g. classical swine fever virus): Detection via Ab/dye reaction
unit: focus forming units (FFU)

Isolation of a biologically cloned virus:
- covering the cell culture with agar
- local cell lysis
- viral progeny is locally fixed/stained
- isolate plaque material with pipette 140

36
Q

Quantification of viruses -> Plaque assay (e.g. Vaccinia virus)

A

Ability of a single infectious viral particle to form a macroscopical, cytopathic effect in a monolayer of cultivated cells

Isatin beta- Thiosemicarbazone (IBT): Vaccinia virus inhibitor

Plaque: equivalent of one infectious virus
-> Quantifies ONLY infectious particles = PFU (plaque forming units)

37
Q

Quantification of viruses -> Focus assay

A

Ability of a single infectious viral particle to form a macroscopical, cytopathic effect in a monolayer of cultivated cells
(here: Transformation of cells by a retrovirus).

38
Q

Quantification of viruses

A
  • Plaque assay
  • Focus assay
  • Direct quantification by EM
  • Pock assay
  • Hemaglutinin (HA) assay

-> Number of viruses always depends on the method used for determination!
-> Infectious particles (in a spec. system!) vs total particles vs subunits

39
Q

Quantification of viruses -> Hemaglutinin (HA) assay

A

Certain virions (e.g. Influenza) can bind directly (via their Hemagglutinin protein) to N- acetylneuraminic acid on the surface of erythrocytes. Due to the fact, that both, viruses and erythrocytes, have multiple binding sites, viruses can lead to the formation of cell latices (not visible). Without a virus erythrocytes settle out of suspension forming a „red dot“. By starting with a defined number of erythrocytes viruses can be counted as „HA Units“.
HA-units correlate with amount of
viral hemagglutinin in a virus preparation

Important value for vaccine production!

Variation thereof: HA inhibition assay:
By the addition of antibodies against
the viral hemagglutinin the effect of the virus
on eythrocytes can be inhibited; thus the amount of antibodies or virus can be calculated

40
Q

Detection of viral antigens (proteins) via specific antibodies

A

Polyclonal serum
- wide reactivity
- low specificity

Monoclonal Ab
- low range of reactivity
- high specificity

41
Q

Detection of viral antigens

A

“in situ” (at its natural site)
- cells or tissue (cell-containing samples)
- Virus propagation in cell culture

“in vitro”
- isolated antigens

Additional informations:
- Localization of Ag in tissue, cell population, sub cellular compartment

42
Q

Detection of viral antigens “in situ” (at their natural site)

A

Steps of procedure
- Sample preparation smear on slide
tissue: sliced preparation
- Fixation / precipitation or crosslinking - Permeabilisation
- Immunolabeling
antigen specific primary antibody with label
- Detection via
-> Immunocytochemestry (chem. dye reaction, light microscopy)
-> Immunofluorescence (Fluorescence microscopy, flow cytometry)

43
Q

Detection of viral antigens via immunoblot (Western blot)

A
  • Mincing sample, removal off insoluble components
  • Denaturing proteins by boiling in SDS-buffer
  • Amino acid chains unfolded
  • Due to SDS negatively charged
  • Separation of proteins by their size via SDS-polyacrylamid- gel-electrophoresis (SDS PAGE)
  • Electric transfer of the proteins in the gel onto the membrane (blot)

Chromogenic substrat: Staining reaction
Chemiluminescence-substrat: Light emission detection e.g. x-ray film; detection unit

Virologie (11 decks)

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