Lecture 7 and 8!
How do we study viruses?
Through isolation and cultivation:
a) Animals/Eggs
-First method used for virus cultivation
-Inconvenient
-Safety in handling animals.
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGEKDXCimKF86A8JrUWtkEf3ezpXIhfS-9yk5ItoS4nPRwYCxCYPNOaF95hCqZ0Td1902HMtwMCtoWrgz501qqoF_YlZhPFPFYn6aHokshHUYo-Uviy898tsQfQCMQQ6CpoJt44lfJULQ/s320/images.jpg)
Egg embryo
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcwd-H6VnYnn4Sc6B_gswAMtrDeJ5Z8ltznT4sdo69r8venvjm2brOLebhaOr-CKisn-M-Om3GPNBTgIl7VdusyH4XpEqevU50Dcr1XpoWf1cIocd6kGeBOlquX8pZ3v4FOtqoZzu0TSE/s320/mouse+infection.jpg)
Mouse infection data for analysing
b) tissue culture
Cell tissue culture
-Cells grown in vitro
-primary cell culture
-continuous cell lines
Some viruses do not grow in vitro.
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiqp5DrGId9cVOQPvagi45OaAFGPYuN9wCOJStfLZgrzq-mzGtuN_Mt3HHAnL3ugfagJs7EOg269IKAAMxHoRQf4jFdOirk4E9qR6yA5lwC9FZ_1jXN5AdR34Oh_Xnl_LHtvlKEBL8FIl8/s320/G25JYWCAT06N7ICA7TN8I2CAO7LUJYCAIEODLTCA2JAKMYCAO0N0M3CAULXT0OCAGHEUQFCA0EKU0ACA6E1BZHCAIZJ7NQCAOWZ7BACAH8U4YUCA5DKSDTCA9E1FBUCATOYM9UCA73209PCAI4Z327.jpg)
Primary cell culture
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiY4x9BmfT4xe76JD2_cPO94BKzsr9kJYQaKMQgcfFW0vidAodWpukaJZzGiwQRSGx601T8HP2yiW53MjTZ22SGd_Fyyv4YgB-RDHK1paVqFgJzCSxwNd93A5dfXKlWEyZC9oFp2vrt7Ms/s320/R4S7NACAFIHFJ9CA8N14B4CAINQH69CAV7WN4OCAFNHN9FCA6Q7E2MCAX3S8QOCAFHF826CANH4Q2XCAKHLNTUCA7QJPV0CAC94EO8CACM2ATRCAMORH5ICAAG427RCAIQIFQKCAD8S4KMCAPJOEMH.jpg)
Continuous cell line cultivation
Through detection, identification and diagnosis:
1) Tissue culture methods:
(a) Cytopathic Effect (CPE)
Feature:
· Relatively slow method.
· Not all viruses will grow on cell sheet.
· Degenerative changes in cells, such changes include: altered shape, detachment from substrate, lysis, membrane fusion, altered membrane permeability, inclusion bodies, apoptosis.
· Associated with the multiplication of certain viruses.
· Spread of virus is restricted by an overlay of agar which lead to formation of plaque. · Identification of a viral infection by examining the characteristic CPE produced on different cell sheets.
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3NqrFxHY3PngpmgCRgGMvyIiZx52cg7s8flhQ5ELWu8B_jyEcmJe7pLiT-kHY7kO2DSPmZ2Yn942lTI-8qyIHcM5FS7opJcp4_xylFfAFSOKu_4HyhBubcHg_ylxTfljr90EVqssoUK0/s200/CPE.bmp)
CPE effect on vero cell after a few days(b) Plaque Assay
· Renato Dulbecco, 1952
~Based on original plaque assays for bacteriophage
· Observing cell death in infected cell culture
· One virus infects one cell and spread to surrounding cells
· Higher accuracy at lower concentration
Virus Dilution:
Virus Count- Features:
· Counts only viable virus (virus capable of multiplying)
· Useful for samples vith very low virus counts
· Requires time for incubation
Plaque Assay- Features:
· Very time-consuming
· Very simple method
· Only works for viruses that infect monolayer cells
· Only works for viruses that causes cell lysis
· Uses only the principle that one virus on the monolayer produces one plaque
Viral concentration can be determined by:
Virus concentration = (number of plaques) X (dilution factor) pfu/ml
2) Physical methods:
(a) X-ray crystallography Feature:
· Determine the arrangement of atoms within a crystal
· Beam of X-rays strikes a crystal and scatters into many different directions
· Results are very accurate![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEierIzHRTvwck5hDUp4YHMktfTmKed2sAJN9V4htowYkSfX31J1Xr35ffA8v9LzF5Akc-N_NQv7_2Pd9xUwiDU5QDN1Aqj_aeeqTZqkUZ14giF8VjPygfpUVmzzg_s8Aolfj-1hWm0CcKQ/s200/x+ray+crystallgraphy.png)
Workflow of the structure of the molecule
X-ray Diffraction Patterns
· Depends on spacing between scattering bodies and wavelengths if incident radiation
· Patterns depend on orientation of the crystal; the intensities of spots yield information on atomic positions.
· A crystal acts as a 3D diffraction grating for x-rays as the interatomic spacings in a crystal are
the same size as the wavelengths of x-rays.
· Diffraction from crystals - oscillating electrons emits x-rays in all directions. X-ray diffraction
locates electron density.
(b) Electron microscopy
Feature
· Type of microscope that uses electrons to illuminate a specimen and create an enlarged image.
· Greater resolving power than light microscope.
· Obtain much higher magnifications than light microscope (up to 2 million times).
· Use electrostatic and electromagnetic lenses in forming the image by controlling the electron beam to focus it at a specific plane relative to the specimen.
Types:
*Transmission EM· Uses a high voltage electron beam to create an image![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjweYODH045yiiFt0HZ-1VutcdezX6G0n_-7whRBkA9fKmM9L4p94yc7DvURFNQoOyDmGDY3xZ7QXp9FRiXMUvbhe7aRZUAV_KQiRJf8_STwp80QjpRqKM9t_PWao_v6NuR2QawphefczU/s200/transmission+Electron_Microscope.png)
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiO1M13QolS5My6JDX3xKZvnQk8Xha5bRUqFcaIgDWvE9qfHySAaJ-W9wMERh3S9B8tXXDdTg4VaEQiaK7j7v7VChBiQr5w_oXkNIxQcSKbi2SpZFvg2ltsl4vz10ylN7JKdwu44iPyJ0U/s200/transmission+Electron_Microscope.1.jpg)
In the laboratory
*Scanning EM
· Does not at any time carry a complete image of the specimen.
· SEM produces images by probing the specimen with a focus electron beam that is scanned across a rectangular area of the specimen.
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj-WhUSAAHZdlB6FJEtMeQYIXVfal5E-i5SzyLPydb99tssXkH_Ks4VNZJeBztykGOtWpQuPJfqpDqfdLaFwGVfcmfFpG5ACedq7Urq2X2uP2Qc5QoIxyPNv9fG2ivjluDgp2KeymVzjiE/s200/scanning+EM.jpg)
In the laboratory
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGGiuipIzt28R_hFxPVWVfjKLUI7TA36wICy5gwj7wuzuGPdJKZE_MlXGL8kzldH43Qk7fzLvqoHxkK3ZqhbCJ2elwywYy60UknlPOacc9LGpZNU-cJKFW_h21m9ZKfSZ5z2ZQTTnheJI/s200/180px-Ant_SEM.jpg)
An image of an ant from SEM
*STEM
Focus incident probe across a specimen that has been thinned to facilitate detection of electrons scattered through the specimen.
· Occur before the electrons hit the specimen in the STEM.
· STEM's use of SEM-like beam rastering simplifies annular dark-field imaging
(c) Ultracentrifugation
Feature· Centrifuge optimized for spinning a rotor at very high speeds.
· Capable of generating acceleration as high as 1,000,000 g (9,800 km/s²).· Method- by purification.![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiglWg8dDu1a-JPlNVbVA1zTIteBY_i3N-2fqxHMaNnGuwGkfedyoeRsHDdJowhAQDcNuRmHwSGPafknm3EJu07g3qv5gBFpu5tdvM5-E9-n1Nb1AytPiDir9ODqNqipt9lb6iMjhV3Oa4/s200/lab+Ultracentrifuge.jpg)
The Laboratory Ultracentrifugation
3) Serological/ Immunological methods:
Basic Immunology
· Antigens
~Any foreign particle or molecule not recognised by the body.
~Virus, bacteria, toxins, foreign proteins, etc.
Antibodies
Antibody Neutralising Virus
Methods:
(a) Haemagglutination (HA)
· Influenza and other viruses
· Two spike proteins
~Neuraminidase
~Haemagglutinin
-binds specifically to red blood cells
(b) Haemagglutination Inhibition (HI)
· HA conducted in the presence of antibody
· Neutralisation of virus inhibits agglutination
(c) Virus neutralisation
· Antibody neutralisation prevents/ lowers virus infectivity.
(d) Complement fixation
· Mediated by antibody.
· Antibody binds to antigen.
· Complement cascade of molecules in blood serum intiated, causing lysis of infected cell or pathogen.
serum without antibodies
serum with antibodies(e) Immunostaining ~ Immunoflourescnce
· Antibody tagged with fluorescent dye
· Antibody attached specifically to antigen
· View specimen under exciting light
· Fluorescence microscope
Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody.~ Immunogold Electron Microscopy
· Same principle as immunoflourscence
· Gold particles attached to antibodies (nanometer size particles)
· View under EM to localise specific proteins or antigens
Detection of antibody to bovine coronavirus by immunogold staining.
(f) Immunoprecipitation / Immunoblot
· Antigens radioactively labelled
· Reaction with antibody
· Isolate antibody-antigen complex
· Run through SDS-PAGE
· Detect through x-ray film
~Immunoblot
· Western blot analysis
· Whole protein sample run through SDS-PAGE
· Transfer to nitrocellulose membrane
· Antibody attaches specifically to one protein
· Antibody labelled with sensitive indicator (eg. Horse radish peroxidase)
· Colour reaction with streptavidin
~Western bolt
Analysis of proteins separated by SDS-PAGE gradientgel electrophoresis.
(g) ELISA
· Enzyme-linked immunosorbant assay
· Antibody to detect antigen
· Antibody labelled with indicator (eg. Horse radish peroxidase)
· Colour reaction
· System requires one molecule to be attached to solid surface
A 96-well microtiter plate being used for ELISA
4) Others and Molecular Biology:
Molecular Virology
· Study genome organisation
· Expression of viral genome
· Replication of genome and progeny virus
· Molecular basis of viral replication cycle
~Attachment and entry
~Replication and translation
~Assembly of virions
~Virus-host interactions
Molecular Biology and Others
· Analysis of viral proteins
~PAGE/ SDS PAGE
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiLWS1ITQSPxB3amgGJBgc9xutBxcmNUt_ybWatboD6TFLKCgzA5VfMSraa7lmo5VV45aCCANf3u0_jSVTV1fagY-Xqf3XKYMZTXT65soGGNvpMK1pGj5-RSQE2JXHD1tWeV7PkpgkpVtQ/s200/230px-SDS-PAGE.jpg)
~Western blot
~Protein Sequencing
Protein Sequencing Facility
~X-ray crystallography
X-ray crystallography facility
· Analysis of viral genome
~Agarose gels
DNA of different size can be separate by agarose gel electrophoresis
~Restriction analysis
restriction enzymes
~Sequencing
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEijaELILyOiN_E-PlR39AgdPpljGWrbQ56jp11jU0L9ZFVUQRzA6VMeI5_3-hHcea0DXu3psWbes5Ilaf7s_vge0nDp4_4QSP_Q1GjL0tEDfw2xuMHlDuNbmIvOUBrBLwjNYVcROXusHl8/s200/531613833_6dbd2f337a.jpg)
DNA sequencing
~Southern blot
~Northern blot
-Based on southern blot
-Analysis of RNA instead of DNA
-Named to reflect different moleules studies
~PCR/ RT-PCR
-Invented by Kary Mullis, 1993 Nobel Prize winner
-Photocopier- Xeroxing DNA
-Generates millions of copies of specific DNA in a few hours
-Quick and simple way of cloning genes in test tube
How does PCR work?
· Cycles of three steps repeated again and again
· First step: Heating à separate double stranded DNA
· Second step: Cooling à primers bind
· Third step: increase temperature à polymerase work
· Step four: repeat the cycle for about 20-30 times
PT-PCR
· RNA is single stranded, fragile, not stable
· Convert back to DNA called cDNA ( “complementary” DNA)
· First step: used enzyme reverse transcriptase (RT) to produce double0stranded form-
Complementary DNA
· Second step: normal PCR cycle
References
http://en.wikipedia.org/wiki/Cytopathic_effect
http://www.medscape.com/content/2004/00/49/59/495916/art-nm495916.fig4.jpg
http://stemcells.nih.gov/StaticResources/info/scireport/images/figurec1.jpg
http://www.ebiotrade.com/buyf/productsf/Clontech/p67_fig1.gif
http://www.microbiologybytes.com/video/virus.html