Methods in Virology Flashcards
Introduction to methods in virology
- Significance of virus necessitated their study.
- Means of studying viruses was derived.
- Techniques in viral research involves:
- Propagation or growth of virus
- Concentration and purification•Immunological methods
- Molecular methods
- Research methods are been updated from time to time.
Virus purification, and Virus isolation
- Virus cultivation or culture: techniques involving the use of specific characteristics of a virus for multiplication.
- Virus isolation: use for obtaining identical strains of a virus.
- Virus purification: E.g. centrifugation
Virus identification techniques
- Microscopy: the use of highly sensitive microscopes for the analysis of virus architecture for identification.
- Electrophoresis: separation of viral DNA and proteins by differential movement of charged particles through a membrane for identification.
- Gene analysis techniques: PCR, RT-PCR, Real-time PCR, Microarrays.
Cultivation of viruses
•This refers to the propagation or growing of a virus for research purposes.
2 major techniques:
- Cell free culture (absence of living cells): Made from extraction of internal components of different organismsfor the cultivation of viruses.
- Cell culture: Majority of viruses requires supply of appropriate cells to enable growth and multiplication
Virus cell culture
•Cells for the culture of viruses are provided based on the host type
- Phages: virus-infecting bacteria are supplied with bacteria cultures.
- Plant viruses: may be supplied with specially grown plants or with cultures of chloroplast.
- Animal viruses: requires either:
- whole organism, such as transgenic mice,
- eggs containing chick embryos,
- insect larvae or
- cultured animal cells
Requirements for animal cell culture
continuous cell lines
- Continuous cell lines: This involves the propagation of viruses using cells from animals or humans that have been immortalized either in the body of an animal or in the laboratory.
- The cells can be sub-cultured for many years or indefinitely for research purpose
Requirements for animal cell culture
media
- Media: this provides nutrients for a virus to grow. Most media are supplemented with animal serum which contains nutrients needed for the growth of many cell lines.Also, Media ensures the maintenance of optimum osmotic pressure and pH
Requirements for animal cell culture
growth vessel
3.Growth vessel: A virus can be cultivated in immortalized cells in glass or plastic flask or plates where the cells bathed or suspended in the growth medium.
Cells forming monolayer: cells growing in a single layer on a growth vessel.
Cells in suspension: Need to be stirred to keep them in suspension
Requirements for animal cell culture
antibiotic
- Antibiotic: To prevent cell contamination with unwanted microorganisms (fungi and bacteria
Requirements for animal cell culture
sterile cabinet
- Sterile cabinet: provides a sterile work environment which help prevent contamination of cells, self, others or the environmen
Requirements for animal cell culture
incubator
- Incubator: Used to provide the optimum concentration of carbon dioxide for the culture of viruses
summary of virus isolation techniques
- Majority of viruses can be isolated due to their ability to form discrete visible zones called plaquesin layers of host cells.
- Plaques are formed as confluent areas on infected cell on the culture plate or dish which shows signs of cell alteration or cell death.
- Plaques continues to spread as the virus infect more cells.
- Plagues could be formed as monolayers (single patches) if overlaid in agarosegel or lawns by phages of bacterial growth (figure 5).
- Extraction and re-culturing of individual plagues result in a purified plague
Virus isolation techniques
clone
f a plague is assumed to be from a single virus, it is referred to as a clone (genetically identical)
Virus isolation techniques
isolate
arises from a clone that is genetically identical
Virus isolation techniques
strain
an isolate different form the parent isolate is regarded as a strain
Virus isolation techniques
purified plaque
are genetically pure virus strain derived from the re-culturing of a plaque derived from monolayers of the 2 or more virions
Virus isolation techniques
passaging
a term used for each virus sub-culture process
Virus isolation techniques
virus efficiency
viruses replicate more efficiently after repeated subculture
Virus isolation techniques
laboratory strain
occurs when an isolated virus strain has undergone numerous replication cycles in the lab and is now quite different genetically from the wild type of virus
Centrifugation
•Centrifugation employs rotational gravitational force to separate particles in a solution by density.•The process separates virus strain from host cell debris and other contaminants to obtain a pure concentrate for various experimental purpose
Differential centrifugation
involves alternating cycles of low and high speed resulting in partial purification of the virus
density gradient centrifugation
involves the centrifugation of viral particles in a solution of increasing concentration which separates each particle by density
this results in a more purified form of the virus
•Sucrose is commonly used as a solute due to its high solubility.•
Two types of density gradient centrifugation are:
i. Rate zonal Density gradient centrifugation
ii. equilibrium density gradient centrifugation
.
Techniques used in structural investigation of viruses
light microscopy
more are not efficient in virus identification due to their low resolution power compared to the minute size of a virus
Techniques used in structural investigation of viruses
confocal microscope
is a more sensitive type of light microscope used in virus investigation to study cytopathic effects
Generally, it employs the use of a laser to scan the virus particle, producing excellent images of thick specimens and fluorescing specimens.It can be used to investigate live viral cells or transfer of proteins from a virus or host cell which are tagged with appropriate fluorescence labels. E.g. green fluorescence protein from a jelly fish protein.Images of specimens arising from confocal microscopy can be represented in 3-D
Techniques used in structural investigation of viruses
electron microscopy
Highly sensitive and commonly used in virus investigation to obtain fine details of the suspected organism.Most procedure requires staining of suspected virus particles or ultrathin section of a virus infected cell in form of a micrograph.Two methods are used to reveal fine details of the virus or ultra-thin section of a virus-infected cell
Techniques used in structural investigation of viruses
negative staining
: generate contrast using heavy metal-containing compounds. E.g. potassium phosphotungstateand ammonium molybdate.In an electron micrograph of virus, the stains appears as dark areas around the virions, allowing the overall visualization of virionshape and size.Examples are scanning electron microscope and transmission electron microscope
Techniques used in structural investigation of viruses
ultra low temperature cooling
Cryo-electron microscopy: wet samples are frozen to below -1600C, freezing the water in the glass-like material. Images are recorded while the specimen is frozen and processed by a computer. Thereafter, data from multiple images of a specimen are constructed into a three-dimensional images of the virus particl
Techniques used in structural investigation of viruses
x-ray crystallography
- It reveals very fine details of the three dimensional structures of a virionincluding DNA, proteins and DNA-protein complexes.
- It involves producing crystals of the virus particle which are placed in a beam of X-ray, where they are diffracted based on repeating arrangements of the molecules/atoms in the crystal.
- Analysis of diffraction patterns of molecules or atoms will help determine their position in the cell/tissue
Detection of virus DNA and proteins
electrophoretic techniques
- Electrophoretic techniques: Mixtures of DNA or proteins can be separated in gel composed of agaroseor polyacrylamide.
- Each DNA or protein in a mixture is visualized as a band (horizontal line) in the gel matrix after staining in appropriate reagent.
- Each DNA molecule or protein in a complex mixture is allowed to migrate through a gel matrix according to its molecular weight or size and charge which are monitored my reference markers.
- There 3 major form of electrophoresi
Detection of virus DNA and proteins
agarose gel electrophoresis
Used for detecting the size of a virus specific DNA using already-known DNA templates as marker
Detection of virus DNA and proteins
1D SDS page
One-dimensional Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE): Used for estimating molecular weight of a virus protein using known weights of proteins as reference
Detection of virus DNA and proteins
2D SDS page
Two-dimensional SDS-PAGE: Proteins are separated in two dimensions based on their isoelectric point and molecular weig
Detection of virus and virus components
- Detection of virus components are important for identification and diagnosis of viral diseases.
- The techniques can be categorized into four:
i. Detection of virions
ii. Detection of virus infectivity
iii. Detection of virus antigens
iv. Detection of virus nucleic acid
Detection of virions
- Its involves negative staining of virionsfor examination using confocal or electron microscopy.
- Negative staining: generate contrast using heavy metal-containing compounds. E.g. potassium phosphotungstateand ammonium molybdate.
- In an electron micrograph of virus, the stains appears as dark areas around the virions, allowing the overall visualization of virionshape and size.
- Examples are scanning electron microscope and transmission electron microscope.
- The technique can be applied in detecting rotavirusesin faecalsample from patient suffering gastroenteritis.
- Disadvantageof the technique are; high cost of equipment, limited sensitivity requiring a minimum detectable concentration of 106virion
Detection of infectivity using cell cultures
- Virus infectivity: refers to the ability of a virus to replicate itself in a host.
- Virus inoculation: Suspected viruses in infected host cellscan be detected by inoculation of suspected specimen in a culture of cells, or an host organism.
- Microscopy: After incubation in desired temperature, cultures are observed under the light microscope for virus-induced pathology (cytopathiceffect).
Detection of virus antigens
- Virus proteins/antigens can be detected using various immunological techniques involving antigen-antibody interactions.
- It can be done using specific antisera or monoclonal antibodies raised against the virus protein.
- Direct testing: Probing of virus antigen with anti-virus antibody could be direct if antibody was probed on the antigen directly.
- Indirect testing: Asecondary antibody (anti-IgG) was used after reacting the virus antigen and anti-virus antibody to detect the immunoreaction.
- Positive samples are detected with the appearance of a immunoreaction or label due to binding of antigen-antibody
Figure 11: Detection of virus antigens using anti-virus antibody alone (left) and reaction with anti-virus antibody and a secondary anti-IgGantibodies (right).Note:
- Anti-virus antibody was induced by injecting virus antigen into an animal. The anti-virus antibody contains a label.
- A secondary anti-IgGwas made by injecting IgGfrom one animal into a second animal of the same species. The second antibody contains the la
Detection of virus nucleic acid
1.hybridization:
•Several techniques can be used in detecting virus nucleic acid:
1.hybridization: Employs sequence specific-DNA probes carrying appropriate labels for the detection of specific viral DNA or messenger RNAs (mRNAs).
•It may be done on the surface of a membrane after Southern blotting(DNA probing) or northern blotting(RNA probing)
.•In situhybridization: Thin sections of a virus-infected tissue are probed for the presence of virus specific nucleic acid.
•DNA microarrays: Made up of a substrate containing hundreds to millions of tiny spots of specific DNA probes where specific DNA or RNA molecules in a sample can be detected by hybridization with specific spots
Figure 12: Hybridization technique detecting virus specific nucleic acid using a labeled DNA probe Note that virus nucleic acid could be DNA or RNA.For RNA, Thymine (T) is replaced by Uracil (U).
Detection of virus nucleic acid
- PCR
- Polymerase chain reaction (PCR): Enables the multiplication of DNA fragment into millions of copy. Important for detecting small amount of viral DNA in a sample.PCR technique requires specific oligonucleotide primers specific to viral sequences. Agarosegel electrophoresis can be used to separate the amplified DNA, which is detected by probing in specific label
Detection of virus nucleic acid
- Revers Transcriptase (RT) PCR
3.Reverse Transcriptase (RT) PCR: The RNA of viruses can be copied to DNA using Reverse Transcriptasesand amplified using Reverse Transcriptase PCR
Detection of virus nucleic acid
- Real time PCR
- Real-time PCR: Is a quantitative technique for detecting the number of copies of a specific nucleic acid in a sample. Increase in DNA is monitored using fluorescing labels whose glow increases as number of DNA is multiplied
Infectivity Assay
- Measures the titer or concentration of infective virus in a specimen or preparation.
- Test virus samples are inoculated into suitable hosts: bacteria culture, plantor animal and responses are observed for infective virus.Two categories:
- Quantitative assays: Host response can be assigned values. E.g. number of plagues on a culture plate.
•A plague assay estimates the concentration of an infective virus in a culture dish and results are calculated as plague-forming unit per milliliter solution (PFU/ml). - QuantalAssay: It detects if a host has responded to the presence of a virus or not depending on the desired variable. •In cell culture, it determines the virus dose that can infect 50% of inoculated tissue culture (TCID50 : Tissue culture Infective dose)
•In animal assay, it determines either the amount of virus that can infect 50% of test animal (ID50)or cause the death of 50% of test animals (LD50).
Viral genetics
genome sequencing
- Genome sequencing: developed by Fred Sanger and colleague at Cambridge, UK.
- It is used to determine the sequence of bases in a DNA molecule usually after PCR.
- Derived DNA sequences are search against the genome database (BLAST) to find a match which in turn provide information on the identity, role, function or characteristics of the gene.
Viral genetics
gene manipulation
Viral genetics•Genemanipulation: Enable the manipulation of virus nuclei acid for research and health purposes.
•It involves the isolation of specific fragments of a genome using restriction endonucleases, the cloning of the fragments in bacterial plasmids and the introduction of specific-site mutations into the virus genome.
Viral genetics
gene recombination
•Gene recombination: The natural processes of recombination and re-assortment that produce new combinations of virus genes can be harnessed to produce new viral genotype in the laborator
Investigating gene function and expression
The technique assesses the role of a virus gene by blocking its expression using specific enzymes resulting in a mutant gene. Hence, the mutant gene is unable to replicate or perform the same role as the wild type and becomes a conditional lethal mutants.
Techniques in studying gene function and expression are:
- Gene mutation
- Reverse genetics
- RNAi
- Microarray
Gene mutation
: Alteration of the normal conditions (temperature, chemical)for a gene (DNA) to generate a mutant whose functions are compared with the wild type
Reverse genetics
: The gene of an RNA virus can be Reverse Transcribed into DNA where mutations are induced before transcription back to mRNA
RNA interference (RNAi) or RNA silencing
: Short sequences of specific dsRNAmolecules can be used to inhibit the expression of virus genes and thereafter investigate the function of the gene
Microarray tech
Allows researchers to monitor the expression of hundreds and thousands of genes.
Virus RNA are detected by copying RNA from infected cells into DNA using a reverse transcriptase, amplify the DNA via PCR, label with fluorescing dye and added to the microarray. The probes that bind DNA from the sample are detected by scanning with a laser at a wave length that excites the fluorescent dye
