Virology 1.4-Virus Cultivation and Assays How do you grow viruses and then determine how many there are in a sample? (Physical, Chemical, Biological approaches)

Slides:



Advertisements
Similar presentations
Essentials of Virology
Advertisements

Bacteriophage Prokaryotes as host Subcellular structure without metabolic machinery Double stranded DNA, single stranded DNA, RNA Virulent phage vs. template.
Viruses. General Characteristics of viruses 1.Depending on one’s viewpoint, viruses may be regarded as exceptionally complex aggregations of nonliving.
VIROLOGY.
 Non-living entities  Can infect organisms of every domain  Commonly referred to by organism they infect  Viruses that infect bacteria: Bacteriophage.
Viruses, Viroids, and Prions
Section 18.1: Viruses Vocabulary: virus, host cell, bacteriophage, capsid, lytic cycle, lysogenic cycle, provirus, retrovirus, prions, viroids.
 Classification of animal viruses › Taxonomic criteria based on  Genomic structure  DNA or RNA  Single-stranded or double-stranded  Virus particle.
VIROLOGY – The Study of Viruses Are NOT Small Bacteria Are NOT Living Cells Lack ALL (nearly) enzymes necessary for metabolism and biosynthesis ARE Protein.
Virus Quantification & Neutralization
Virology Lab 6.
CELL CULTURE AND DIAGNOSTIC VIROLOGY. Since the discovery by Enders (1949) that polioviruses could be cultured tissue, cell culture has become a very.
Detecting Mutagens and Carcinogens. introduction - Increased number of chemicals used and present as environmental contaminats, testes for the mutagenicity.
Viruses Part II. It was not until the 1950s that scientists were able to isolate and identify how viruses multiplied in a host It was not until the 1950s.
ISOLATION, QUANTIFICATION AND identification OF VIRUSES
Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Lecture Slide Presentation prepared by Christine L. Case Microbiology.
Viruses, Prions and Viroids Infectious Agents of Animals and Plants
Chapter 19 Page 478. The phylogeny of the virus No fossil evidence Only speculation by scientists about how viruses came to be --- Some think that their.
Virus Isolation in tissue culture & Virus Quantitation Lab 5.
13-a Viruses pp H1N1. 2 Viruses Size, Structure, Morphology Taxonomy Growth Identification.
Lecture 9 Viruses, Viroids, Prions
Introduction to Viruses By Christine Herrmann, PhD Courtesy of B.V.V. Prasad BioEd Online.
Viruses Viral Structure Growing (Cultivating) Prokaryote Types Eukaryote Types Obligate Intracellular Parasites: Require living host cell in order to replicate.
Viruses, Viroids, and Prions
Viruses Viral Structure Growing (Cultivating) Bacteriophage Life Cycles Obligate Intracellular Parasites: Require living host cell in order to replicate.
Branches of Microbiology Bacteriology Virology Mycology Parasitology Immunology Recombinant DNA technology.
VIRUS REPLICATION PETER H. RUSSELL, BVSc, PhD, FRCPath, MRCVS Department of Pathology and Infectious Diseases, The Royal Veterinary College, Royal College.
Viruses, viroids, and prions For Dr. Wright’s Bio 27 Class
BTY328: Viruses Dr William Stafford
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Lecture Slide Presentation prepared by Christine L. Case M I C R.
LAB. DIAGNOSIS OF VIRUSES 5 methods are used for diagnosis in the virology laboratory: 1.Direct microscopy 2.Cultivation of viruses 3.Serology 4. Detection.
Viruses Living or Not ???????. Viruses Virus – Latin for “_Poison__” Virus – Latin for “_Poison__” Russian Biologist Dmitri Ivanovski (1892) – Tobacco.
Characterizing and Classifying Viruses and Prions
Chapter 13 Viruses, Viroids, and Prions Part 1. General Characteristics of Viruses Very small in size –Need an electron microscope to visualize and determine.
professor in microbiology
Viruses Chapter Viruses  Discovery – 1892, Dimitri Iwanowski, A Russian Scientist- Disease agent is filterable.  1935, Wendell Stanley, an American.
ISOLATION OF BACTERIOPHAGE CLERIGO, GEHAN ALYANNA V. DIMAANO, PETER BOB Z. DECIO, JOHN LAWRENCE GOCO, AMELIA BERNADETTE O.
Viruses The Interface Between Living and Nonliving.
VIRUSES - Small non-cellular particles - Seen only with an electron microscope - Classified according to their shape - Classified according to their molecular.
Chapter 6 - Viruses Obligate Intracellular Parasites – only demonstrate characteristics of life while “inside” a host cell: Bacteria, animal, plant.
METHODS IN VIROLOGY.
 Virus: A biological particle composed of nucleic acid and protein  Intracellular Parasites: organism that must “live” inside a host.
Methods in Virology.
Laboratory virology. From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Table Specimens for viral diagnosis.
An Introduction to the Viruses Non-Living Etiologies
Characterizing and Classifying Viruses, Viroids, and Prions
Cell Culture and Diagnostic Virology
Cell Culture and Diagnostic Virology
Virology Lab 6.
TISSUE Culture and Virology
Haemagglutination assay
What is a virus? Tiny organisms that may lead to mild to severe illnesses in humans, animals, and plants.
Introduction to Virology
LAB. DIAGNOSIS OF VIRUSES
Plaque Forming Unit (PFU)
VIRUSES Text p
ISOLATION, QUANTIFICATION AND identification OF VIRUSES
Introduction to Virology
MBI 140 panel (semester 1; H)
VIRUSES Text p
Viral Diseases How To Diagnose By: Dr. Amr. Viral Diseases How To Diagnose By: Dr. Amr.
Cell Culture and Diagnostic Virology
Viruses, Viroids, and Prions
Lecture-16 Viruses.
Introduction to Virology
Incorporation of the B18R Gene of Vaccinia Virus Into an Oncolytic Herpes Simplex Virus Improves Antitumor Activity  Xinping Fu, Armando Rivera, Lihua.
Virus Notes.
Introduction to Virology
Introduction to Virology
This work is licensed under a Creative Commons Attribution 4
Presentation transcript:

Virology 1.4-Virus Cultivation and Assays How do you grow viruses and then determine how many there are in a sample? (Physical, Chemical, Biological approaches)

Initial Detection Usually symptoms in infected host But rarely detection of unusual appearance of cultured cells Body fluids or culture medium tested on uninfected host

Cultivation Maintained in laboratory by serial passage through intact organisms or cell cultures (or in the freezer) Laboratory stocks expanded for bulk harvesting Virus maintained in the lab will not have the same properties as the virus from nature

Direct particle counts-a physical method  Limited usefulness  Internal standard  Electron microscopy or or cytometry cytometry

Example Mix virus stock with known concentration of virus-sized plastic spheres. Count # virions & spheres per EM field. From the ratio of spheres to virus, calculate virus “concentration”. Are the virus particles “alive”, “dead”, “pseudovirions” or just “empties” (e.g. bromoviridae)?

Example of particle counts Direct Counts of Viruses in Natural Waters and Laboratory Cultures by Epifluorescence Microscopy, by Kilian P. Hennes and Curtis A. Suttle © 1995 American Society of Limnology and Oceanography. American Society of Limnology and OceanographyAmerican Society of Limnology and Oceanography

Chemical Measurement By nucleic acid (genome) concentration: The MW of the genome must be known to use this method. Determine DNA or RNA amount Divide by “molecular weight” of one genome Virus particle count can be approximated.

Example A solution contains 1 ug of pure DNA. Each virus genome contains 1 fg of DNA. 1 ug pure DNA/ 1 fg DNA per copy = 1,000,000,000 copies

qPCR is another way to measure the number of genomes

Hemagglutination assay Based on Hemagglutinin (HA) protein Causes RBCs to agglutinate agglutinate Coat well bottom evenly evenly HA titer Frequently used with influenza A

Biological Methods: Infectivity assays Measurement of key biological activity Ability to cause an infection detects “live” viruses (signs, symptoms, death, etc.)

Better methods to measure infections How many infections are caused by a How many infections are caused by a defined amount of my sample? defined amount of my sample? Two types of assays used Two types of assays used Dilution (quantal) Dilution (quantal) Titration (preferred method-quantitative) Titration (preferred method-quantitative)

Dilution or quantal assays Dilution End Point (DEP) Infectious Dose 50% (ID 50 ) n/10 individuals infected

Dilution end point Highest (or greatest) dilution of sample that still shows infectivity Cartoon illustrates methodology with cultured cells on a plate (not all the methodology with cultured cells on a plate (not all the data) data)

More data from the experiment SampleDilutionLogDilution # Infected Wells Total # OfWells%Infected None / / / /

Graphic data from a similar experiment ID 50 or TCID 50

Titration  Most common quantitative assay  “Titer” used as noun or verb  More accurate count  All cells in sample must get the same treatment  Involves same geometry

Titration invented for lytic bacteriophage  Allow adsorption of phage to occur in a small volume  Dilute mixture  Plate samples in top agar  Incubate 8-16 hrs Holes in the lawn will form = plaques

 Plaque forming unit (PFU)  Provides a count of infectious units in sample  Phage titer = dilution x average plaque count/volume plated

Phage plaques  101 plaques per plate  0.1 ml plated  1 x dilution  Titer = ?  PFU = plaque forming units Additional examples of phage plaques

Animal virus titration  Technically difficult (use of live animals)  No comparable system to bacteria at first  Type of data depends on host-virus interaction  Some viruses could be roughly titrated in eggs

Use of eggs to quantitate virus

Animal virus titration  Dependent upon ability to culture host cells in vitro (1948-)  Dulbecco adapts plaque assay to animal viruses 1952 At Caltech I continued to work with phages for a few years. One day I was told by Delbrück that a rich citizen had given Caltech a fund for work in the animal virus field. He asked me whether I was interested. My medical background and the experience gained in Levi's laboratory came back to me and I accepted. After visiting the major centers of animal virus work in the US I set out to discover the way to assay animal viruses by a plaque technique, similar to that used for phages, using cell cultures. Within less than a year, I worked out such a method, which opened up animal virology to quantitative work. I used the technique for studying the biological properties of poliovirus. These successes brought me an appointment first to associate professor, then to full professor at Caltech.

Primary cell culture

Terminology Organ culture, Explant culture, cell culture Cell culture Primary cell cultures cell strains cell lines Adherent Contact inhibition Passage Secondary culture

TerminologySelection Senescence or crisis Transformation (aka cell transformation) ImmortalTumorigenic Continuous cell lines Euploid/aneuploid Growth factor dependent/independent

Formation of a cell line “Crisis” “Immortal” Continuous cell line

HeLa Cells Henrietta Lacks-1951 Cervical tumor First highly prolific human cell line

Animal virus plaques HSV on Vero cells Polio on Hela cells HSV on Vero cells Polio on Hela cells Vital stain-taken up by living cells

Antiviral resistance measured by plaque assay Wild type AndMutantInfluenzaVirus Effect of Ribavirin ©2003 by National Academy of Sciences

Transforming viruses  Viruses that cause transformation in host cells  “ viral transformation”  Transformed cells on a plate form a clump = “focus”  Focus assay, expressed in Focus Forming Units or FFU

Focus Assays Loss of contact inhibition Immortalized Able to move more freely Cytoskeleton and other organelles change appearance Biochemical changes

Cytopathic Effect or CPE The viruses are cytopathic-not lytic infected cells are really sick Misshapen Abnormal Fused (Syncytium) Inclusion bodies

Limitations  Titration methods error-prone  100% error?  Measure only those particles that can cause a detectable infection in the system used  Does not count particles that do not cause infection

Efficiency of Plating or EOP  Absolute EOP- titer/number of virions  Relative EOP- ratio of virus titer on two hosts  Particle/Pfu ratio- inverse of absolute EOP Variable but always more than 1.0!!! Variable but always more than 1.0!!! Some particles in the preparation Some particles in the preparation cannot start an infection. cannot start an infection.

Multiplicity of infection (m.o.i.)  Experimental parameter  Ratio of infectious units to cells in an experiment  Necessary to ensure that each cell has a chance to collide with a virus  Poisson distribution The more virus you use in the experiment (high moi)-the more likely it is that each cell will encounter a virus. Common sense!!!!

Titration Statistics-Poisson Distribution  Allows calculation of probability that any given cell in a sample will come in contact with n virus particles.  Moi must be known  Equation P(k) = e -m m k /k! P(k) probability that any cell will be infected with k particles m= moi k! = factorial of k

Poisson Distribution P(k) values moi k

Plant virus assays “One sick plant” TMV in tobacco-systemic infection Signs: Mottling with borders-mosaic ChlorosisRugosity

Different host-different reaction TMV on pinto bean leaves Localized infection Local-lesion host Very tricky assay, dependent on host plant status

Local lesions and assay Local lesion assay Alfalfa mosaic virus on P. vulgaris Half-leaf only is shown Right: TMV on Samsun tobacco NN Left: mock-inoculated

Utility of plaque assays  D’Herelle discovers bacteriophage plaques by 1921; viruses must be particulate  Delbruck decides to use phage as the atom of biology in the 1930’s  Ellis and Delbruck demonstrate the one step growth curve in 1939  Later, Luria and Delbruck use plaque assays to demonstrate that mutations are spontaneous

One Step Growth Total Phage Extracellular Phage Latent or eclipse phase Intracellular accumulation phase Time after Infection Number of Infectious Particles Lysis

Interpretation  Phage are fundamentally different from their hosts because they have a different growth curve.  “Eclipse” or “latent” phase  “burst” as phage are released from infected cells

Plaque Assay Dose-Response Animal viruses and phage display single hit kinetics with few exceptions.

Some additional reading and some advertisements