Mim - Lecture 25

Front 1

25.1

methods for virus cultivation

Back 1

1. susceptible animals
2. egg inoculations
3. cell cultures

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25.2

primary cell culture

Back 2

1 animal organ (monkey kidney) or embryo (chicken or mouse)
2 organ is minced and treated with trypsin to dissolve tissue into individual cells
3. the cells are dispersed into sterile tubes or dishes and covered with a buffered grwoth medium containing serum
4. cells adhere to surface of the container, grow, and become confluent forming a monolayer )contact inhibition)
5 cells can be infected with the virus
6 unlike bacterial cultures that can be transferred indefinitely, primary cell cultures can be subcultured to initate a limited number of secondary cultures (5-10 cell divisions)
- diploid mixed cells
- 1-2 passages
- influenza, parainfluenza, enteroviruses

Front 3

25.3

diploid cell strains

Back 3

1. some primary or secondary cultures usually from fetal tissue undergo changes which permet them to muliply for a number of generations (50-70 subcultures). These cells become morphologically altered even though maintaining original x-some number
2. one of the most widely used is WI-38 strain derived from human embryonic lung.
- diploid, fibroblasts, limited passages (50-70)
- human diploid, fibroblasts (WI38, MRC5 or HEL)
- herpes simplex, cytomegalovirus, varicella zoster, rhinovirus

Front 4

25.4

continuous or established cell lines

Back 4

1. Cells rom cancer tissue
2. Examples: Hep-2 ( epidermoid carcinoma of the larynx) and HeLa (human carcinoma of the cervix).
3. continuous cell lines -> indefinite propagation in vitro.
4. These cell lines grow faster, form multi-layers of cells, and are aneuploid (ie: altered numbers of chromosomes)
5. Many continuous cell lines derived from transformed primary cultures produce tumors when inoculated into susceptible animals.
- adenovirus, respiratory syncytial virus

Front 5

25.5

cytopathic effect (CPE)

Back 5

- necrosis of cells is produced by many viruses
- cells balloon up and demonstrate clumping

Front 6

25.6

plaques

Back 6

- areas of CPE containing dead cells seen in an agar overly. Each plaque represents infection of a cell by one infectious virus particle, referred to as plaque forming unit (PFU)

Front 7

25.7

pock

Back 7

- plaques produced in chorio-allantoic membrane (CAM) of embryonated chicken egg (herpes, influenza virus, pox viruses)

Front 8

25.8

hemadsorption

Back 8

- cells infected with orthomyxoviruses or paramyxoviruses which bud from cytoplasmic membranes have the ability to absorb RBCs. This is due to the incorporation into the plasma membrane of a newly synthesized viral protein (hemagglutinin_ that can adsrob to RBCs.

Front 9

25.9

viral inclusion-body formation

Back 9

- intranuclear or intracytoplasmic structure that appear in virus infected cells and can be diagnostic important
- ex: rotovirus in stool filtrate
- take a stool sample, dilute, centrifuge and see if you find virus - usually there is so much in stool supernant that it appears cloudy

Front 10

25.10

syncytia

Back 10

- formation of multinucleate giant cells. These are formed when viral-induced alterations in the cell membrane result in fusion of contiguous cells

Front 11

25.11

viral-cell interactions

Back 11

- cell transformation (non-permissive)
- virus replication: cell survives or cell is killed, lysed (permissive)
- abortive infection: viral infetion is started up, but stoppped
- asymptomatic infection - latent infection: instead of developing lytic herpes (legions), HSV1 migrated into your ganglia

Front 12

25.12

steps in viral replication

step 1

Back 12

1. recognition of target cell
a. match up with cell receptor to recognize cell (lipoprotein receptors in primate cells recognize poliovirus; mucoprotein receptors of respiratory epithelial recognize influenza virus)
b. electrostatic forces b/w the cells and the virus have a predominant role, a cationic environment is important (virus and mammalian cells are negatively charged)
c. enter by phagocytosis or pinocytosis
d. also possible to enter through a pore

Front 13

25.13

steps in viral replication

step 2-4

Back 13

2. attachment
3. penetration
4. uncoating:
a. in order to replicate, the virus must lose its capsid. If it does not lose capsid, viral nucleic acid is not free to replicate
b. period of uncoating is reflected in the growth curve as the eclipse period

Front 14

25.14

steps in viral replication

5-8

Back 14

5. macromolecular aynthesis: early mRNA and nonstructural protein synthesis, genes for enzymes and nucleic acid proteins
6. viral assembly
a. subunits of capside come together, replicated nucleic acid is incorporated
b. together, viral assemble and macromolecular synthesis are the latent period of viral replication
7. budding of enveloped viruses
8. release of virus - viral burst
a. for picornaviruses, rhabdoviruses and togaviruses, the burst size can be > 100,000 particles per cell. Orthomyxo and paramyxoviruses bust size = 5-6000 particles per cell. retroviruses (1000 particles per cell)

Front 15

25.15

one-step growth curve

Back 15

1. cells infected simultaneously by using a high m.o.i. (multiplicity of infection).
2. ncrease in infectious virus over time -> sequential samples and titration.
3. after adsorption, infectious virus can not be demonstrated.
4. The eclipse period = the time from adsorption until new intracellular virus appears. 3-12 hours.
5. latent period = time from adsorption until the release of extracellular virus into the medium
6. The growth curve and burst size can vary.

Front 16

25.16

penetration:

viropexis
fusion or metling
receptor-mediated endocytosis
direct penetration

Back 16

1. Viropexis - engulfment of naked viral particles by cells by a process of endocytosis.
2. Fusion or melting - the viral envelope fuses with the plasma membrane resulting in release of the nucleocapsid into the cytoplasm (e.g. herpesvirus).
3. Receptor-mediated endocytosis - interaction of the virion with receptor sites on cell surface -> viral capsid alteration and nucleic acid released directly into the cytoplasm.
4. Direct penetration- naked virion nucleocapsids are not thought to directly enter into the cytoplasm.

Front 17

25.17

uncoating

Back 17

poliovirus (non-enveloped)
adenovirus (non-enveloped)
poxvirus (enveloped)

Front 18

25.18

poliovirus (non-enveloped)

Back 18

interaction of cell receptors with virions at the cell surface leads to disruption of the viral capsid which leads to direct release of RNA into the cytoplasm.

Front 19

25.19

adenovirus (non-enveloped)

Back 19

since penetration is via viropexis, the virus is exposed to cellular lysosomal enzymes leading to virion disruption.

Front 20

29.20

poxvirus (enveloped)

Back 20

2 stage uncoating mechanism.
a. After engulfment by phagocytosis, vaccinia’s outer envelope is removed by lysosomal enzymes, and the resulting viral cores are released into the cytoplasm.
b. DNA in viral cores initiates mRNA synthesis via viral DNA dependent RNA polymerase. This mRNA is translated to an uncoating enzyme which removes the core membrane thus releasing free vaccinia DNA in the cytoplasm where it can then start replicating.

Front 21

29.21

replication of RNA virus

Back 21

- replication of viral nucleic acid can occur either in the cytoplasm or the nucleus of the infected cell (most RNA viruses replicate in cytoplasm)
- orthomycoviruses as well as retroviruses utilize the cell nucleus in their replicative cycels
- the cells do not have the machinery to duplicate the viral RNA - there is no RNA to RNA polymerase available in mammalian cells: virus must bring this along to survive
- unless the virus is +RNA which is actually mRNA, the cell can work with this and translate it like cellular mRNA
- RNA viruses are prone to mutation

Front 22

29.22

picornavirus, togavirus, flavivirus, calicvirus, coronavirus

Back 22

- RNA virus type

- ssRNA that is non-segmented, + RNA used to make polymerase which uses - strand to duplicate

Front 23

29.23

orthomyxovirus, paramyxovirus, rhabdovirus, filovirus, bunyavirus

Back 23

-RNA genome = ssRNA that is segmented, makes series of complementary RNAs smaller than genome

Front 24

29.24

reovirus

Back 24

dsRNA, makes subviral particles which have viral genome, RNA dependent RNA polymerase, and capside proteins, only - strand is transcribed

Front 25

29.25

retrovirus

Back 25

- has diploid RNA genome, + strand used to make DNA, then DNA -> RNA -> viral protein

Front 26

29.26

replication of DNA virusews

Back 26

- DNA viruses have different structures (ss or dsDNA; linear or circular; covalently closed circles)
- mode of replication varies depending upon the virus family
- viral genes must use the hosts' system for replication, exception for translation (pox virus codes for its down DNA dependent DNA polymerase, DNA dependent RNA polymerase, splicing proteins and enzymes -> denucleated cell can be infected with Pox virus
- larger viruses have more ocntrol over the cell: more room it has in its capsid for protein codes that the host might not have. more genes to control the cell's machinery

Front 27

29.27

papovavirus

Back 27

- circular dsDNA, needs host cell enzymes to replicate, both strands replicate at the same time like in mammals

Front 28

29.28

adenovirus

Back 28

- linear dsDNA, also replicated from both ends but one does not see Okasaki fragments

Front 29

29.29

pox virus

Back 29

- can replicate in cytoplasm with own DNA synthesis, replication/transcription machinery codes

Front 30

29.30

herpesvirus

Back 30

- can stimulate cell growth (hence can cause caner), has own polymerase and deoxyribonucleotides, replicates in rolling circle

Front 31

29.31

hepadnavirus

Back 31

+ ssRNA used as intermediate to make DNA while using its own viral reverse transcriptase

Front 32

29.32

parvovirus

Back 32

ssDNA with innate hairpin structures at both ends which act as signal for hosts' DNA polymerase, also needs cells undergoing DNA synthesis to replicate