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Study of Virus
Contents
• Structure of virus

• Classification of virus

• Morphological types of virus

• Cultivation of virus

– Laboratory animals

– Embryonated eggs

– Tissue culture

Intended Learning objectives

• Explain the virus structure

• Draw and explain the different morphological types of virus

• Classify virus

• Explain the methods for viral cultivation

Discovery of virus
• The concept of a virus as a distinct entity dates back only to the very late 1800s

• In 1886, the Dutch chemist Adolf Mayer showed that tobacco mosaic disease (TMD) was transmissible from a diseased
plant to a healthy plant.

• Ivanoski reported in 1892 that extracts from infected leaves were still infectious after filtration through a
Chamberland filter-candle.

• A new world of filterable pathogens wa

• Beijerinck, in 1898, was the first to call 'virus', the causative agent of the tobacco mosaic.

• He showed that the virus was able to migrate in an agar gel, therefore being an infectious soluble agent

He described the agent of mosaic disease of tobacco as a “contagium vivum fluidum”, or contagious living fluid, because
he was convinced the infectious agent had a liquid nature.

• The first human disease associated with a filterable agent was yellow fever.

• By the 1930s, scientists had begun using the word virus, the Latin word for poison, to describe these filterable agents.

• Advances in the molecular biological techniques in the 1980s and 1990s led to the recognition of several new viruses
including

– Human Immunodeficiency Virus (HIV)

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– SARS associated coronavirus.

• Wendell M. Stanley crystallized and described the molecular structure of the tobacco mosaic virus (TMV) in 1935,
showing that it has properties of both living and non-living matter.

• At about the same time, the invention of the electron microscope made it possible to see viruses

Virus – living or Dead?

Viral size

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How do we define virus?

• Contain a single type of nucleic acid, either DNA or RNA.

• Contain a protein coat that surrounds the nucleic acid.

• Sometimes enclosed by an envelope of lipids, proteins, and carbohydrates

• Multiply inside living cells by using the synthesizing machinery of the cell.

• Cause the synthesis of specialized structures that can transfer the viral nucleic acid to other cells.

Viral structure
• A ‘virion’ is a complete, fully developed, infectious viral particle

• It is composed of nucleic acid and surrounded by a protein coat

• The protein coat protects it from the environment and is a vehicle of transmission from one host cell to another.

• The viral nucleic acid can be linear or circular

Capsid
• The nucleic acid of a virus is protected by a protein coat called the capsid

• The structure of the capsid is ultimately determined by the viral nucleic acid and accounts for most of the mass of a
virus

• Capsid is composed of protein subunits call©eRdamcaiaah Upnisveorsitmy ofeAprpleiedsSciences

Envelope
• In some viruses, the capsid is covered by an envelope

• Usually consists of some combination of lipids,proteins, and carbohydrates

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Viral envelope

Enveloped virus

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Spikes
• Depending on the virus, envelopes may or may not be covered by spikes

• These are carbohydrate- protein complexes that project from the surface of the envelope

• Some viruses attach to host cells by means of spikes

• They can be used as a means of identification

• Viruses whose capsids are not covered by an envelope are known as non-enveloped viruses

Viral Structure

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Viral Morphology
 Classified into several different morphological types on the basis of their capsid architecture.

 The structure of these capsids has been revealed by electron microscopy and X-ray crystallography.

 Helical Viruses

 olyhedral Viruses

 Enveloped Viruses

 Complex Viruses

Helical virus

Polyhedral virus
• Icosahedron, a regular polyhedron with 20 triangular faces and 12 corners

• The capsomeres of each face form an equilateral triangle

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Enveloped virus

• Enveloped viruses are roughly spherical

• When helical polyhedral viruses are enclosed by envelopes they are called enveloped helical or enveloped polyhedral
viruses.

Complex virus

Viral taxonomy
Virus are classified based on

 Nucleic acid type

 Envelope

 Morphology

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Classification of virus

Cultivation of virus
 Viruses cannot multiply outside a living host cell

 Viruses must be provided with living cells instead of a fairly simple chemical medium.

 Bacteriophages – Bacterial cell cultures

 Animal virus – Laboratory animals, embryonated eggs, tissue cultures

• Viruses not only need living cells to grow in but also they are specific about the type of cell they infect and grow in

• There is no universal cell that will support all viruses

• Therefore in the laboratory the suspected virus must be grown in a culture method known to support its growth

• Viruses tend to be host specific; therefore:

– Human viruses grow best in cells of human origin,

– Bovine viruses in bovine cells,

– Canine viruses in canine cells,

– While some viruses will not grow in vitro at all

The primary purposes of viral cultivation are:

• To isolate and identify viruses in clinical specimens

• To prepare viruses for vaccines

• And to do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells

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Growing Bacteriophages in the Laboratory

• Bacterial cultures on solid media – laque method is used for detection

• The concentrations of viral suspensions measured by the number of plaques -plaque- forming units (FU)

Animal inoculation

• Animals are used for studying viruses which do not grow in cell cultures or eggs, and for testing vaccines

• Animal inoculation may be used as a diagnostic procedure for identifying and isolating a virus from a clinical specimen

Inoculation of virus in animals

• Laboratory animals play an essential role in studies of viral pathogenesis

• Live animals such as monkeys, mice, rabbits, guinea pigs, ferrets are widely used for cultivating virus

• Mice are the most widely employed animals in virology

• The different routes of inoculation in mice are:

– intracerebral

– subcutaneous

– intraperitoneal

– or intranasal

• After the animal is inoculated with the virus suspension, the animal is:

– observed for signs of disease

– visible lesions

– or is killed so that infected tissues can be examined for virus

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Inoculation of Virus in Embryonated Eggs

• Eggs provide a suitable means for:

– The primary isolation and identification of viruses

• The cells and extraembryonic membranes of the chicken embryo provide varied substrates that allow the growth of
many viruses.

• Due to the ability of virus to alter their tropism and to adapt to a new host species, many viruses become capable of
growing in chicken embryo tissues and may even attain a higher concentration than in the tissues of the natural host.

• The stage of incubation at which a particular infectious agent is inoculated is determined by the time at which the
target tissues are most prominent.

• The chicken embryo develops from a single cell to a hatchling chick in 21 days of incubation in a humid 38⁰C
environment.

• The egg itself has a blunt end where there is an air space or air sac. Underlining the shell is the fibrous eggshell
membrane.

• The shell membrane does not contain live cells; consequently, it will not support virus replication.

• After 4 to 5 days, readily detected by candling.

Detection of suitable embryo by candling

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Routes of inoculation

• Routes of inoculation include the chorioallantoic sac, chorioallantoic membrane, yolk sac, amniotic sac, intracerebrally,
and intravascularly

• Requires removal of a small piece of shell over a major vein

Inoculation of influenza virus

Detection of viral growth

– Death of the embryo

– Defects in embryonic development

– Localized areas of damage in the membranes, resulting in discrete opaque spots called pocks

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• If a virus does not produce obvious changes in the developing embryonic tissue, virologists have other methods of
detection

• Embryonic fluids and tissues can be prepared for direct examination with an electron microscope

• Certain viruses can also be detected by:

– Their ability to agglutinate red blood cells

– Or by their reaction with an antibody of known specificity

Advantages
• An embryo is an early developmental stage of animals marked by rapid differentiation of cells

• Birds undergo their embryonic period within the closed protective case of an egg, which makes an incubating bird egg
a nearly perfect system for viral propagation

• It is an intact and self-supporting unit, complete with its own sterile environment and nourishment

• It furnishes several embryonic tissues that readily support viral multiplication

• Defense mechanisms are not involved in embryonated eggs

• Cost- much less, Maintenance-easier, less labor and readily available

Cell Culture
• rior to the advent of cell culture, animal viruses could be propagated only on whole animals or embryonated chicken
eggs

• Cell cultures have replaced embryonated eggs as the preferred type of growth medium for many viruses

• Cell culture consists of cells grown in culture media in the laboratory

• These cultures can be propagated and handled like bacterial cultures; they are more convenient to work with than
whole animals or embryonated eggs

Growing Animal Viruses in the laboratory

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Advantages of cell culture techniques

Summary
• Virus – obligate intracellular parasite

• They do not belong to prokaryotic or eukaryotic cell type

• Basically made up of nucleic acid (DNA or RNA) covered by capsid

• Envelope may or may not be present

• Morphologically virus are classified as helical, polyhedral and complex virus

• Virus can be cultivated in lab animals, embryonated eggs or in tissue culture

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