Cultivating Viruses: Techniques and Requirements

Cultivation of Viruses

• Viruses are not unicellular microorganisms
• They are obligate intracellular parasites
  • They lack the machinery for protein and nucleic acid synthesis
• Viruses rely on the host's machinery for growth and survival
• Viral multiplication involves complex processes
• Viruses are inactive outside host cells but show living characteristics inside them

Cultivation of Viruses

• Viruses cannot grow on inanimate culture mediums
  • As obligate intracellular parasites, they need living cells
• Viruses can be cultivated within suitable hosts
  • Such as a living cell
• The primary objectives of viral cultivation include
  • Isolating and identifying viruses in clinical specimens
  • Preparing viruses for vaccines
  • Conducting detailed research

Viral Growth Specificity

• Viruses require living cells to grow and are specific about the cell type
• There is no universal cell that supports all viruses
• Viruses are host-specific
  • Best growth for human viruses is in human cells
  • Bovine viruses in bovine cells
  • Canine viruses in canine cells
  • Some viruses will not grow in vitro at all
• In the lab viruses are grown in a culture that supports its growth

Virus Cultivation Methods

• There are three methods that can be employed
  • Inoculation of virus into animals
  • Inoculation of virus into embryonated eggs
  • Tissue culture
• Animals are used to study viruses
  • Specifically ones that do not grow in cell cultures or eggs
  • Additionally used for testing vaccines
• Eggs can support a range of viruses
  • Of importance for diagnostic service
• Cell cultures use different cell lines that will support different virus types

Inoculation of Virus in Animals

• Lab animals serve an essential role in viral pathogenesis studies
• Live animals used for cultivation include
  • Monkeys
  • Mice
  • Rabbits
  • Guinea pigs
  • Ferrets
• Mice are the most widely used animals in virology

Mouse Inoculation Routes

• The different routes of inoculation in mice include
  • Intracerebral
  • Subcutaneous
  • Intraperitoneal
  • Intranasal
• After inoculation, the animal is
  • Observed for signs of disease
  • Examined for visible lesions
  • Killed to asses the infected tissues for the virus

Cell Cultures

• Before cell cultures, animal viruses could only propagate on whole animals or embryonated eggs
• Cell cultures have replaced embryonated eggs for growth for many viruses
• Cell culture consists of cells grown in culture media in the laboratory
• They can be propagated and handled like bacteria
  • More convenient to work with than whole animals or embryonated eggs

Viral Inoculation in Embryonated Eggs

• Goodpasture and Burnet were the first to use embryonated hen's eggs in 1931
• The cultivation process depends on the type of egg being used
• Eggs provide a means for
  • Viral identification/isolation
  • Stock culture maintenance
  • Vaccine production

Embryonated Egg Terms

• Terms to describe eggs
  • Embryonated is defined as having an embryo
  • Unembryonated is defined as not having an embryo
  • De-embryonated is defined as having lost an embryo
• Embryonated eggs are eggs in an advanced stage of development

Embryonated Eggs are Advantageous

• An embryo is an early developmental stage of animals
  • Marked by rapid cell differentiation
• Birds undergo their embryonic period in a closed protective egg
  • Nearly perfect system for viral propagation
• It is an intact and self-supporting structure
  • Complete with its own sterile environment and nourishment
• Furnishes embryonic tissues that readily support viral multiplication
• Defense mechanisms are not involved in embryonated eggs
• Cost-effective and require less maintenance and labor

About Viral Inoculation

• Chicken, duck, and turkey eggs are the common choices for inoculation
• The eggs for cultivation must be sterile
  • The shell should be intact and healthy
• Rigorous sterile techniques must be used to prevent contamination by bacteria and fungi

Viral Inoculation Process

• The egg is typically injected through a drilled hole in the shell
• In some cases small window can be made in the shell
• The viral suspension or suspected virus-containing fluid is injected into the egg fluid
• The type of tissue to inoculate into is dependent on
  • Type of virus being propagated
  • Goals of the experiment

Detecting Viral Growth

• Viruses multiplying in embryos may or may not have effects visible to the naked eye
• Signs of viral growth include
  • Death of the embryo
  • Defects in embryonic development
  • Localized areas of damage in the membranes, resulting in discrete opaque spots called pocks

Viral Growth

• Additional detection methods are used if viruses do not produce obvious changes in embryonic tissue
• Embryonic fluids and tissues can be prepared for direct examination with an electron microscope
• Viruses can be detected by
  • Their ability to agglutinate red blood cells
  • Their reaction with an antibody of known specificity

Embryonated Egg Parts

• The air sac is relevant to the development of the embryo
  • Respiration and pressure adjustments
• The shell and shell membrane function both as a barrier and an exchanging system
  • Responsible for gases and liquid molecules
• The chorioallantoic sac (allantoic fluid) functions to remove waste products of the developing embryo
  • This membrane increases in size as the embryo grows
• The yolk sac provides nourishment for the developing embryo
  • As the embryo grows, the yolk sac shrinks as it is absorbed into the embryo digestive system
• The amnion is a thin membrane that encloses the embryo
  • Protects it from physical damage
  • Serves as an exchange system

Viral Routes of Inoculation

• An embryonated egg offers various sites for the cultivation of viruses
  • Chorioallantoic membrane (CAM)
  • Amniotic Cavity
  • Allantoic Cavity
  • Yolk sac
• The chosen route of inoculation and the age of the embryo are determined by the virus
  • Infectious bronchitis virus is propagated in the yolk sac of a 5-6 day old embryo
  • Rous-sarcoma is inoculated on the chorioallantoic membrane of a 9-11 day old embryo

Candling Eggs

• Candling uses a strong light above or below the egg
  • Meant to observe the embryo
• A candling lamp consists of a strong bulb covered by a container
  • Typically made of plastic or aluminum that has a handle and aperture

CAM - Chorioallantoic Membrane

• Used in veterinary virology
• Growth of viruses or adaptation to growth
• Viruses produce visible foci or 'pocks', inclusion bodies, oedema or other abnormalities
• Each virus particle forms one pock
• Viruses grown include
  • Herpes viruses
  • Poxviruses

Amniotic Cavity Inoculation

• The virus is introduced directly into the amniotic fluid that bathes the developing embryo
• The volume of fluid used is small
• Recommended for the primary isolation of human viruses
  • mumps virus
  • influenza A, B and C viruses
• Limited application in veterinary virology
• Newly isolated influenza viruses require passages to adapt to growth
  • Can be done through other routes, such as allantoic

Allantoic Cavity Inoculation

• Viruses can grow
  • Such as Newcastle disease virus
• Other viruses require amniotic passages to adapt
  • Influenza
  • before becoming adapted to the egg and then grown in the allantoic cavity
• Allantoic inoculation is fast and easy
  • Yields large amounts of virus-infected egg fluids (8–15 ml)

Yolk Sac

• Simplest for the growth and multiplication of a virus
• Mostly for isolation for mammalian viruses
• Immune interference mechanism can be detected in most avian viruses
• Also used for the cultivation of some bacteria
  • Chlamydiae and Rickettsiae