PRACTICAL VIROLOGYIntroduction

Lab. 1

Laboratory Safety

• It is important to remember that whenever one works with an infectious agent there is the possibility of infection to oneself or to others by negligence

• Furthermore, organisms that are referred to as "nonpathogenic“ are still potential pathogens • For example, certain "non-human“ viruses, such as

Newcastle disease virus (NDV), have been known to cause conjunctivitis when inadvertently introduced into the human eye

Laboratory Safety

• Cell cultures can also be potentially hazardous since they can infect the laboratory worker with an endogenous or a latent virus

• One should adopt the same precautions when working with cell culture as when working with a virus

General Laboratory Safety Procedures

• There must be NO EATING, DRINKING, SMOKING, OR APPLYING OF COSMETICS IN THE LABORATORY.

• Unauthorized persons, particularly children and infants, should NOT be allowed in the laboratory.

• NO MOUTH-PIPETING should be permitted due to the Possibility of accidental ingestion of virus-contaminated materials, cell-contaminated products, or toxic chemicals. Automatic pipetors or pipeting bulbs must always be used.

• Hands should be washed when coming in the laboratory, after handling cells or virus, and before leaving the laboratory.

General Laboratory Safety Procedures

• One should NOT walk around touching door knobs and surfaces with contaminated gloves. Contaminated gloves should be discarded in the decontamination pan.

• Avoid touching the eyes, nose, mouth, or face while working in the laboratory.

• Laboratory coats should be worn when doing cell culture or virus Work. Laboratory coats should NOT be worn while eating or outside of the laboratory.

General Laboratory Safety Procedures

• Whenever working with syringes and needles, special containers (that are sealed and autoclavable) should be provided for their disposal.

• DONOT replace the cap on the needle (this is where many accidental injections occur). Drop the uncapped syringe into the disposal container immediately after use. These containers are then autoclaved prior to disposal.

• Viruses, cells, or their products, should NEVER be disposed of in the drainage system.

STERILIZATION

Autoclaving• Autoclaving is used when ever possible, especially when dealing with liquid solutions and materials that should not dry out.

• The use of Autoclave tape on all materials to be autoclaved permits the identification of those materials that have been autoclaved and prevents the accidental opening of contaminated materials.

• The standard cycle is 121°C, 15 p.s.i., for 15 minutes.

Autoclave Tape

Dry Heat Sterilization

• Effective for glassware provided there are no rubber or plastic-lined parts.

• The standard cycle is at 300°C for 2 hours.

• Proper loading of the oven is also very important so as to reduce insulating effects

Filtration

• Filtration (using a 0.22 μm membrane filter) is used for aqueous solutions and medium components that are heat labile.

ASEPTIC TECHNIQUE

• Even when sterile materials are available, proper aseptic technique must be rigorously enforced in order to avoid accidental contamination of cell cultures

• Biohazard hoods are preferable for cell culture work

• It is important to disinfect the working area with an appropriate disinfectant just prior to and after working.

• It is helpful to keep flasks and bottles open for as short period as possible.

• Remember to flame the lips of the bottles, flasks, and tubes after opening, and to use only sterile pipets tips.

• If a sterile pipet tip accidentally touches a non-sterile surface, it should be discarded and another one used in its place

VIRUSES

Viruses

• Virus is a small infectious agent that replicates only inside the living cells of other organisms

• Viruses can infect all types of life forms from animals and plants to bacteria

Viruses• They are small, nanometer “nm” is the measuring unit

Reasons for Studying Viruses

• Viruses can infect all cellular life forms

• It is undoubtedly the case that the viruses that have been discovered represent only a tiny fraction of the viruses on the Earth

(Virology Principles & Applications Book, p1)

Reasons for Studying Viruses• Viruses are important agents of many human, animal and

plant diseases• There is therefore a requirement to understand:

• the nature of viruses, • how they replicate • and how they cause disease

• This knowledge permits the development of effective means for: • Prevention, through the production of vaccines, • Diagnosis, through the production of diagnostic reagents and

techniques • and treatment of virus diseases, through the production of antiviral

drugs

Reasons for studying viruses

• Some viruses are studied because they have useful current or potential applications

• Phage typing of bacteria: • Some groups of bacteria,

such as some Salmonella species, are classified into strains on the basis of the spectrum of phages to which they are susceptible

Reasons for studying viruses

• Sources of enzymes: • A number of enzymes used in

molecular biology are virus enzymes (eg. reverse transcriptases from retroviruses and RNA polymerases from phages)

• Pesticides: • Some insect pests are controlled

with baculoviruses and myxoma virus

Reasons for studying viruses

• Gene vectors for protein production: • Viruses such as certain baculoviruses and adenoviruses

are used as vectors to take genes into animal cells growing in culture

• Gene vectors for treatment of genetic diseases:• Children with severe combined immunodeficiency have

been successfully treated using retroviruses as vectors to introduce into their stem cells a non-mutated copy of the mutated gene responsible for the disease

Reasons for studying viruses

• Anti-cancer agents: • Genetically modified strains of viruses, such as herpes

simplex virus and vaccinia virus, are being investigated for treatment of cancers

Study of Viruses

• Characteristics:• Acellular• Obligate intracellular parasites• No ribosomes or means of protein synthesis• No ATP generating system• NOT ALIVE!

METHODS USED IN VIROLOGY

Cultivation of viruses

•Three methods:• Living animals• Chicken embryos• Cell culture

Isolation of viruses

• Many viruses can be isolated as a result of their ability to form discrete visible zones, plaques (areas where cells are killed or altered by the virus infection) in the host cells

Plaques formed by a phage on Bacterial culture

Centrifugation• After a virus has been propagated it is usually necessary

to remove host cell debris and other contaminants before the virus particles can be:• used for laboratory studies, • for incorporation into a vaccine, • or for some other purpose

Differential centrifugation• Involves alternating cycles of low-speed centrifugation,

after which most of the virus is still in the supernatant, and high-speed centrifugation, after which the virus is in the pellet

Partial purification of virions by differential centrifugation

Density gradient centrifugation

• Involves centrifuging particles (such as virions) in a solution of increasing concentration, and therefore density

Purification of virions by density gradient centrifugation

Structural investigations of cells and virions

• Light microscopy• has useful applications in detecting virus-

infected cells, for example by observing cytopathic effects

• Fluorescence microscopy• by detecting a fluorescent dye linked to

antibody molecules that have bound to a virus antigen

• Electron microscopy• Many investigations of the structure of

virions or of virus-infected cells involve electron microscopy

Electron microscopy

Electrophoretic Techniques

• Mixtures of proteins or nucleic acids can be separated by electrophoresis in a gel composed of agarose or polyacrylamide

DETECTION OF VIRUSES AND VIRUS COMPONENTS

• A wide range of techniques has been developed for the detection of viruses and virus components and many of them are used in laboratories involved with diagnosis of virus diseases.

• The techniques can be arranged in four categories: • detection of virions,• detection of virus infectivity, • detection of virus antigens • and detection of virus nucleic acids.

Detection of virions

• Specimens can be negatively stained and examined in an electron microscope for the presence of virions

• Limitations to this approach are:• the high costs of the equipment • and limited sensitivity;

• the minimum detectable concentration of virions is about 106/ml.

Detection of infectivity using cellcultures• To determine whether a sample or a specimen contains infective virus it can be inoculated into a culture of cells, or a host organism

• After incubation of an inoculated cell culture at an appropriate temperature it can be examined by light microscopy for characteristic changes in the appearance of the cells resulting from virus-induced damage• A change of this type is known as a cytopathic effect

(CPE)

Detection of virus antigens

• Virus antigens can be detected using virus-specific antisera or monoclonal antibodies.

Detection of virus nucleic acids

• Hybridization • Virus genomes or virus messenger RNAs (mRNAs) may

be detected using sequence-specific DNA probes carrying appropriate labels

• Polymerase chain reaction (PCR)