What is a virus?
- 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
First vaccines against viral diseases
- Human pox (Variola) -> Virus of cowpox
- Rabies -> attenuated rabies virus
Koch Henleschen Postulate for the identification of pathogens
- Pathogen must be detectable in all diseased animals, not in healthy ones
- Cultivating pathogen in pure culture
- Pathogen can reproduce disease in healty individuals
- Pathogen can be reisolated from the newly diseased animals
Viruses were pathogens, whose cultivation was not possible with the methodes of Koch!!
The first described viruses
- Tobacco-mosaic-virus (TMV)
- Foot-and-mouth disease virus
Yellow fever virus (Flavivirus)
- 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)
Isoliation of pathogenic bacteria was significantly more efficient than of pathogenic viruses
Why?
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!
Influenza A Virus
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
Virus proliferation in the laboratory
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
Eukaryotic cell culture
- 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
Efficient cell culture techniques (since ca 1955)
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
HeLa-cells
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
WI-38 cells
- 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)
Poliovirus
- 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
Further fundamental principles of molecular biology, clarified with phages
- 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
Further fundamental principles of molecular biology, clarified with phages
- 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
Role of viruses in the analysis of eukaryotic gene regulation
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
Use of viruses and their gene products in gene technology
- 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
Comparison: virus/flash memory stick
- 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
Structure of viruses
- nucleic acid
- Capsid (always!)
- Envelope (facultative)
- Lipid membrane (facultative)
Helical viruses
- 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.
Criteria for classification of viruses
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
Classification of viruses
- classes I - VII (Baltimore Schema)
- ICTV nomenclature
order: virales
family: viridae
genus: virus
species: name
New ICTV Rules for Taxonomy of Viruses
4 Realms:
Riboviria (RNA genome), “Monodnaviria (Parvo-, Circo-, Polyomaviridae),” “Varidnaviria (Mimi,- Adenoviridae),” and “Duplodnaviria (e.g. Herpesviridae)”
Genomes of different human DNA virus families
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
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Virus44
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Virus genomes and viral replication strategies54
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RNA Viruses - Principles of replication and mRNA-synthesis of non-retroviral RNA viruses31
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Replication and RNA synthesis of (-)-strand RNA viruses38
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Nidovirales25
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Gene expression of viral genomes: Translation strategies36
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Genetic economy: one primary transcript- multiple translation products21
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Replication of ds RNA Viruses26
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Hepatitis C Virus85
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(Viruses in the ocean)11
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Altklausuren133
