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Virology, Luis Materon, Ph.D. - UTPA

9/3/12 12:38 PM

Virology, Luis Materon, Ph.D. - UTPA

Virology, Luis Materon, Ph.D. - UTPA 9/3/12 12:38 PM Virology, Luis Materon, Ph.D. - UTPA <afaculty.utpa.edu | Home | Notes | Messages | Class Syllabus | Lab Syllabus | Lab Projects | Reading | Images | Journals | Links | Contact | Size Range Measure in nm = nanometers (1 nm = 1 thousandth of a micrometer or 1 millionth of a mm). ​ ​ Smallest have the size of ribosome (25-30 nm). ​ ​ More than 2,000 bacterial viruses could fit into an average bacterial cell. Bacteriophages average 65 x 200 nm. ​ ​ More than 50 million polioviruses could be accommodated by an average human cell. ​ ​ Animal viruses range in size from the small parvoviruses (20 nm = 0.02 µm in diameter). Poxviruses can be as large as bacteria (up to 450 nm in length). Vaccinia (210 nm). Some cylindrical viruses (TMV) are relatively long (800 nm = 0.8 µm) but narrow in diameter (15 nm = 0.015 µm). http://www.instapaper.com/text?u=http%3A%2F%2Ffaculty.utpa.edu%2Flmateron%2F4404%2Fviro-5.html Page 1 of 8 " id="pdf-obj-0-10" src="pdf-obj-0-10.jpg">

Size Range

  • Measure in nm = nanometers (1 nm = 1 thousandth of a micrometer or 1 millionth of a mm).

​ ​ Smallest have the size of ribosome (25-30 nm). ​ ​ More than 2,000 bacterial
Smallest have the size of ribosome (25-30 nm).
More than 2,000 bacterial viruses could fit into an average bacterial cell.
Bacteriophages average 65 x 200 nm.
  • More than 50 million polioviruses could be accommodated by an average human cell.

  • Animal viruses range in size from the small parvoviruses (20 nm = 0.02 µm in diameter).

 
  • Poxviruses can be as large as bacteria (up to 450 nm in length). Vaccinia (210 nm).

  • Some cylindrical viruses (TMV) are relatively long (800 nm = 0.8 µm) but

 

narrow in diameter (15 nm = 0.015 µm).

 

Virology, Luis Materon, Ph.D. - UTPA

9/3/12 12:38 PM

  • Host cells are often 1,000 times the volume of a virus infecting it. 1/10 to 1/3 of bacterial size.

Cultivation of Viruses

Initially it was believed that viruses, as obligatory intracellular parasites, could not replicate in any cell-free medium, no matter how complex.

This dictum was nullified in 1991 by the demonstration that infectious poliovirus could be synthesized in a cell-free extract of human cells incubated with viral RNA.

Despite this finding, most work on viruses is done in:

  • Cultured cells

  • Embryonated eggs

  • Laboratory animals

Background on Virus Cultivation

Human and other animals cells were first cultured in the early 1900’s, microbial contamination make routine work on such systems extremely difficult.

Most viruses were grown in laboratory animals.

Poliovirus multiplied in cultured cells not of neuronal origin (1949). This Nobel Prize discovery led the way to:

The propagation of many other viruses in cultured cells.The discovery of new viruses. The development of viral vaccines (poliomyelitis, measles, and rubella). Studies on biochemistry and molecular biology. Resolution of 3D structural composition of viral particles.

Virology, Luis Materon, Ph.D. - UTPA

Preparation of Cell Cultures

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Cell culture is the most common method for the propagation of viruses.

  • Tissues are dissociated into a single-cell suspension by mechanical disruption followed by treatment with proteolytic enzymes.

 
  • Cells are suspended in culture medium and placed in plastic flasks or plates.

  • As cells divide, they cover the plastic surface.

  • Epithelioid and fibroblastic cells attach to the plastic and form a monolayer, whereas blood cells settle but do not adhere.

 
  • Cells are grown in a chemically-defined medium of an isotonic solution of salts,

 

glucose,vitamins, coenzymes, and amino acids buffered to a pH between 7.2 - 7.4 and often supplemented with antibiotics to inhibit microbial growth.

  • Serum is usually added to the cell culture medium to provide a source of growth factors required by the cells.

  • Cell lines double in 24 to 48 hr in such media.

Types of Cell Cultures Primary Cell Cultures

  • Are prepared from animal tissues.

Virology, Luis Materon, Ph.D. - UTPA

9/3/12 12:38 PM

  • Consist of several cell types and have a limited cell span, no more than 5 or 10 cell divisions.

​ ​ ​ Mostly derived from monkey kidney, human embryonic amnion, kidney, or foreskin; and chicken
Mostly derived from monkey kidney, human embryonic amnion, kidney, or
foreskin; and chicken or mouse embryos.
  • Used in vaccine production ( e.g. attenuated poliovirus vaccine strains are propagated in primary monkey kidney cells.

Diploid Cell Strains

  • Homogenous population of a single type and can divide up to 100 times before dying.

 
  • Despite numerous divisions, they retain the diploid chromosome number.

  • The most used are those cells established from human embryos, such as the WI- 38 strain derived from human embryonic lung.

Continuous Cell Lines

  • Consist of a single cell type that can be propagated indefinitely in culture.

  • These immortal lines are usually derived from tumor tissue or by treating a primary cell culture or a diploid strain with a mutagenic chemical, or a tumor virus.

 
  • Such cell lines do not resemble the cell of origin; they are less differentiated

 

(having lost the morphology and biochemical features that they possess in vivo).

 

Virology, Luis Materon, Ph.D. - UTPA

9/3/12 12:38 PM

  • Often are abnormal in chromosome morphology and number (aneuploid), and can be tumorigenic ( e.g. in mice).

  • Can be propagated indefinitely and, like diploid cell strains, they retain viability after being frozen at -70 to -196°C.

  • Can also be maintained in suspension cultures, in which cells are continuously stirred by a spinning magnet.

Examples of Cell Lines

Hep-2 and HeLa cells derived from human carcinomas. Vero cells from African green monkey kidney. L and 3T3 cells derived from mice. BHK-21 cells derived from hamster kidneys.

Evidence of Viral Growth in Cultured Cells

Some viruses kill the cells in which they replicate, and the infected cells may eventually detach from the plastic cell culture plate.

As more cells are infected, the changes become visible and are called cytopathic effects.

Changes include:

Rounding up and detachment of cells from the plastic culture dish Cell lysis.Swelling of nuclei.Formation of fused cells (syncytia).Fragmentation of organelles.

Virology, Luis Materon, Ph.D. - UTPA

Formation of viral crystals.Duplication of membranes.Clumps of ribosomes in virions.

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Enteroviruses and herpes viruses can cause cytopathic effects in 1 to 2 days and destroy the cell monolayer in 3 days.

Cytomegalovirus, rubella and some adenoviruses may not produce cytopathic effects for several weeks.

Many viruses multiply in cells without causing an obvious cytopathic effect (members of Arenaviridae, Paramyxoviridae and Reoviridae).

Embryonated Eggs

Before the advent of cell culture, many viruses were propagated in embryonated chicken eggs.

At 5 to 14 days after fertilization, a hole is drilled in the shell and virus is injected into the site appropriate for its replication.

This method is used for the influenza virus, mumps virus, Newcastle virus and avian adenovirus.

Robust yields have led to their widespread use in research labs and for vaccine production.

Laboratory Animals

In the early 1900’s it was necessary to maintain virus stocks by continuous passage of virus from animal to animal.

It led to the selection of viral mutants. Cell culture has large supplanted the use of animals for propagating viruses, but some

Virology, Luis Materon, Ph.D. - UTPA

viruses cannot be grown in cell culture. Hepatitis C virus replicates only in chimpanzees.

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Many viruses that cause human gastroenteritis, such as Norwalk virus, can not be grown in cell culture.

Understanding how the immune system or any complex organ system reacts to a virus can not be achieved without research on living animals.

The development of viral vaccines, antiviral drugs, and diagnostic tests has also benefitted from research on viral diseases in experimental animals.

Examples

Polio virus in monkeys led to an understanding of the basis of the disease and the development of the polio vaccine.

The development of the hepatitis B vaccine was due to experimental studies with chimpanzees.

Animals are increasingly being discarded for the following reasons:

1.

Breeding and maintenance of animals infected with pathogenic viruses are

expensive.

2.

Whole animals are complex systems, in which it is sometimes difficult to discern

events.

3.

Results obtained are not always reproducible, due to host vaccination.

4.

They are rapidly being overtaken by ‘modern science’ — cell culture and

molecular biology.

Virology, Luis Materon, Ph.D. - UTPA

9/3/12 12:38 PM

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