General Microbiology 200 Level First Semester Lecture Notes | MCB 211 UNIUYO

General Microbiology 200 Level. Learn about viral pathogenesis, tissue cultures, viral replication and infection mechanisms.

General Microbiology 200 Level Lecture: First Semester

VIRAL REPLICATION

Viruses bind to host cells via specific receptors.

This specificity identifies in part the tropism of a virus for a particular host or cell. Following entry, the virus uncoats, nucleic acid is released, and transcription occurs followed by the production of viral proteins.The viral genome is replicated and new ‘progeny’ virus particles (virions) are assembled and released to infect neighboring cells and tissues. The details of this process depend on the particular virus and on the metabolic state of the host cell.

For example, picornaviruses (small RNA viruses) take around 8 hours to produce new virions, whereas human cytomegalovirus (a DNA virus) may take up to 48 hours.Viruses are extremely diverse in their ability to infect, persist and initiate disease in a host.

Entry is commonly at mucosal surfaces; puncturing skin (e.g. by insect bites or needles) is another very efficient means of introducing virus directly into the blood stream.Replication usually occurs at epithelial surfaces, followed in some cases by viraemia (blood-borne spread) to infection other tissues.

Recovery from the infection can involve the elimination of the virus from the host. Some viruses however, (e.g. herpes virus) persist in a latent (non-infectious) form after the acute infection is resolved, and can reactivate to produce new infectious virions.

Other viruses can persist in an infectious form despite the presence of the immune response (e.g. hepatitis B virus and lymphocytic chorio-meningitis virus). In scrapie and CID there is no acute stage; these agents persist as a slow infection, producing disease after many years.

Unlike viruses, prions do not provoke an immune response nor is interferon produced following infection.

However, dendritic cells can be infected by these agents and constitute an important step in the pathogenesis of infection, by transporting the agents from tissue sites (e.g. gut, skin) to the lymphoid system, where an amplification of prions takes place.

Therefore, through subsequent lymphocyte trafficking, the lymphoid system serves to aid in the transmission of the agent to the nervous system.

Read also: UNIUYO BCH 200 Level Biochemistry Past Questions & Answers

Outcome of Virus-Cell Interaction

Virus infection causes a wide variety of potentiality victorious changes in the many different kinds of cells that occur in the animal host.

✓ Disruption of cellular function ✓ Induction of cell death ✓ Transformation ✓ Activation of an inappropriate immune response

Types of Virus-Cell Interactions

✓ Cytolytic infection – Cell die ✓ Non cytolytic infection – Cell remain intact ✓ Productive Infections – Virus replicate and viral progeny produce and they are fully infective ✓ Nonproductive Infections/ abortive infections – Virus replicate inside the cell but defective virus or incomplete progeny will produce

➢ Permissive cells – They support the complete replication of viruses ➢ Non permissive cells – Not allow the replication of virus. viral replication may be blocked at any point from viral attachment through to the final stages of virion assembly and release.

CULTIVATION OF VIRUSES

In the early years of virus research, the use of animals was mandatory for the recognition of viruses, and rapid, quantitative results were often difficult. For example, poliomyphitis research was limited as long as the presence of the virus could be detected only by monkey inoculation.

At present, many viruses can be grown in cell cultures or infertile eggs under strictly controlled conditions. Growth of virus in animals is still used for the primary isolation of certain viruses and for the study of pathogenesis of viruses.

I. CHICK EMBRYO

Virus growth in an embryonated egg may result in the death of the embryo (e.g. encephalitis virus), the production of plaques on the chorioallantoic membrane (e.g. herpes, smallpox, vaccinia), the development of hemagglutinins in embryonic fluids or tissues (e.g. influenza) or the development of infective virus (e.g. poliovirus type 2). Inoculation may be into the allantoic or amniotic cavities into the yolk sac or on to the chorioallantoic membrane, the precise route used depending on the particular virus being cultivated.

The amniotic route is the method of choice for the isolation of the influenza and mumps viruses. The alantoic route, which is technically the simplest, is used mainly for the passage of influenza viruses that have already been established in embryo.

Yolk sac inoculation is of particular value for the propagation of rickettsince. Inoculation on to the chorioallantoic and characterization of the poxviruses and herpes simplex.

Tissue Cultures

Most of the humans’ pathogenic viruses may be propagated in tissue cultures derived from a variety of animal species. Since, however, a number of common viruses e.g. the enteroviruses and the adenoviruses will grow only in the cells of primate tissues, the tissue cultures used in diagnostic virology are generally derived from monkey or human sources.

Tissue culture techniques may be divided broadly into three groups: (1) Fragment cultures, (2) cell cultures and (3) organ cultures.

1. Fragment Cultures

The simplest form of fragment culture is Maitland type of culture which consists of fragments of tissue suspended in a fluid medium. The cell remains viable for several days-sufficiently long to permit virus growth but do not multiply.

In plasma clot cultures, the tissue fragments are fixed by a plasma clot to the sides of tubes on rattler; new cells, mostly fibroblast, then grow out from the tissue fragments. Fragment cultures have no application in routine diagnostic virology.

2. Cell Cultures

These are prepared from cell suspensions obtained from intact tissue or from a prior tissue culture. Dispersal of cells is usually achieved by treatment of the tissue or tissue culture with a proteolytic enzyme usually trypsin or with the chelating agent versene (EDTA, ethylenediamine tetra-acetic acid, sequenstrene).

This results in the release of single cells and small aggregates of cells capable of initiating growth. Cell culture may be prepared as suspended cultures or as monolayer cultures.

(a) Suspended cell cultures resemble Maitland type culture in that the cells are simply suspended in nutrient medium. They are used for metabolic inhibition tests which may, in diagnostic work, be applied to the estimation of antiviral antibody. Uninfected cells metabolize and actively produce acid. This causes a colour change in an appropriate pH indicator incorporated in the nutrient medium. If the cells are infected by virus, their metabolism is interfered with and the indicator change does not occur.

If however the virus is neutralized by antibody the indicator change occurs as in a normal uninfected culture.

(b) In monolayer cell cultures, the cells are allowed to settle on the sides of a tube or flat bottle and are covered with nutrient medium. Antibiotics are incorporated in order to control, bacterial and fungal contamination.

After two to seven days incubation at 37oC the growing cells will have formed into a continuous sheet or monolayer, no called because it is one cell thick, adherent to the glass of the tube or bottle.

The cell growth medium is then removed and replaced with a maintenance median which is nutritionally less rich than the initial growth medium but adequate to maintain the viability of the tissue cells. Then the virus inoculation is introduced and culture again incubated. During incubation tubes are maintained in a slightly sloped and bottles in a horizontal position.

Many viruses when propagated in a monolayer culture produce degenerative changes in the tissue cells readily visible under the lower power objective or if the area involved is sufficiently large, to the naked eye. This is known as a cytopathogenic effect (cytopathic effect, CPE). The type of cellular change produced differs with different viruses.

Cell cultures may be divided into three types according to the damage history of cells used for their preparation:

i) Primary and secondary cell cultures – primary cell cultures are prepared from cells obtained directly from the tissues. A secondary cell culture is the first subculture of a primary cell culture, the cells of which are dispersed by treatment with typsin or versene for the preparation of the secondary culture.

This procedure is particularly convenient for the preparation of monkey Kichey cell cultures.

ii) Continuous cell lines – As a rule when tissue cultures are prepared directly from an animal tissue, the cultures cannot be serially propagated, the cells dying out after a few subcultures.

A number of lines of mammalian cells are, however, available (mainly derived from fetal and malignant tissues) which can serially be propagated more or less indefinitely. These established cells lines have the advantage of the reccorsity for procuring fresh animal tissue for each set of cultures.

They have the disadvantage, on the other hand, that on prolonged subculture the tissue is liable to undergo spontaneous changes in its susceptibility to infection. The established cell lines most frequently employed are Hela cell culture which was derived originally from a human cervical caocinoma, and the HEp-2 cell derived from a carcinoma of the larynx.

iii) Diphoid cell cultures – The cells of certain tissues, notably human embryo cells can be serially propagated for about 50 subcultures without transformation. Genetically, they differ from continuous cell lines and resemble normal cells. Diphoid cell cultures are highly susceptible to infection by viruses such as the H rhinoviruses and the cytomegalovirus which may be difficult to propagate in other systems.

3. Organ Culture

Organ culture has been recommended for the isolation of cold viruses which cannot be propagated by other procedures. This is done by use of tissue from appropriate organ for the isolation of viruses.

III. Animal Inoculation

Of the ordinary laboratory animals the mouse is the most generally useful and depending on the virus, mouse can be infected by nasal instillation or by intraperitoneal (stomach) or intracerebral inoculation.

The presence of virus in the inoculated material is shown by the development of appropriate symptoms, diagnostic pathological changes or specific antibody.

Viral Tropism

Viral tropism is the ability of a given virus to productively infect a particular cell (cellular tropism), tissue (tissue tropism) or host species (host tropism). Virus tropism refers to the virus’ preferential site of replication in discrete cell types within an organ.

In most cases, tropism is determined by the ability of the viral surface proteins to fuse or bind to surface receptors of specific target cells to establish infection. Thus, the binding specificity of viral surface proteins dictates tropism as well as the destruction of particular cell populations, and is therefore a major determinant of virus pathogenesis.

However, co-receptors are sometimes required in addition to the binding of cellular receptors on host cells to viral proteins in order to establish infection. For instance, HIV-1 requires target cells to express co-receptors CCR5 or CXCR4, on top of the CD4 receptor for productive viral attachment.

Interestingly, HIV-1 can undergo a tropism switch, where the virus glycoprotein gp120 initially uses CCR5 (mainly on macrophages) as the primary co-receptor for entering the host cell. Subsequently, HIV-1 switches to bind to CXCR4 (mainly on T cells) as the infection progresses, in doing so transitions the viral pathogenicity to a different stage.

Apart from cellular receptors, viral tropism can also be governed by other intracellular factors, such as tissue-specific transcription factors.

An example would be the JC (John Cunningham) polyomavirus, in which its tropism is limited to glial cells since its enhancer is only active in glial cells, and JC viral gene expression requires host transcription factors expressed exclusively in glial cells. The accessibility of host tissues and organs to the virus also regulates tropism.

Accessibility is affected by physical barriers, such as in enteroviruses, which replicate in the intestine since they are able to withstand bile, digestive enzymes and acidic environments.

Frequently Asked Questions (FAQs)

1. What is viral replication and how long does it take?

Viral replication is the process by which viruses bind to host cells, uncoat their nucleic acid, produce viral proteins, replicate their genome, and assemble new virions. The duration varies by virus type: picornaviruses (small RNA viruses) take approximately 8 hours to produce new virions, while human cytomegalovirus (a DNA virus) may require up to 48 hours for complete replication.

2. What are the different types of virus-cell interactions?

There are four main types of virus-cell interactions: cytolytic infection (where cells die), non-cytolytic infection (where cells remain intact), productive infections (where fully infective viral progeny are produced), and nonproductive/abortive infections (where defective or incomplete viral progeny are produced).

3. What methods are used for cultivating viruses in the laboratory?

Viruses can be cultivated using three primary methods: chick embryo (embryonated eggs), tissue cultures (including fragment cultures, cell cultures, and organ cultures), and animal inoculation.

4. What is viral tropism andwhat determines it?

Viral tropism is the ability of a viruss to productively infect a particular cell, tissue, or host species. It is primarily determined by the binding specificity of viral surface proteins to receptors on target cells. However, co-receptors, tissue-specific transcription factors, and the accessibility of host tissues (affected by physical barriers) also regulate tropism. For example, HIV-1 requires both CD4 receptors and co-receptors (CCR5 or CXCR4) for productive infection.

5. How do viruses persist in the host after infection?

Viruses can persist in hosts through different mechanisms. Some viruses like herpes persist in a latent (non-infectious) form after acute infection and can reactivate later. Others, such as hepatitis B virus, persist in an infectious form despite immune responses. Prions cause slow infections that produce disease after many years without provoking immune responses, and they amplify in the lymphoid system before spreading to the nervous system through lymphocyte trafficking.