Fundamentals of microbiology: virus

Microorganisms are ubiquitous in nature and are vital components in the cycle of life.

The majority are free-living organisms growing on dead or decaying matter whose prime function is the turnover of
organic materials in the environment.

Pharmaceutical microbiology, however, is concerned with the relatively small group of biological agents that cause human disease, spoil prepared medicines or can be used to produce compounds of medical interest.

In order to understand microorganisms more fully, living organisms of similar characteristics have been grouped together into taxonomic units.

The most fundamental division is between prokaryotic and eukaryotic cells, which differ in a number of respects (Table 13.1) but particularly in the arrangement of their nuclear material.

Eukaryotic cells contain chromosomes, which are separate from the cytoplasm
and contained within a limiting nuclear membrane, i.e. they possess a true nucleus.

Prokaryotic cells do not possess a true nucleus, and their nuclear material is free within the cytoplasm, although it may be aggregated into discrete areas called nuclear bodies.

Prokaryotic organisms make up the lower forms of life and include Eubacteria and Archaeobacteria.

One characteristic shared by all microorganisms is the fact that they are small; however, it is a philosophical argument whether all infectious agents can be regarded as living.

Some are little more than simple
chemical entities incapable of any free-living existence.

 Viroids, for example, are small circular, single-stranded RNA molecules not complexed with protein.

One particularly well-studied viroid has only 359 nucleotides (1/10 the size of the smallest known virus) and yet causes a disease in potatoes.

Prions are small, self-replicating proteins devoid of any nucleic acid.

The prion associated with Creutzfeldt–Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle has only 250 amino acids and is highly resistant to inactivation by normal sterilization procedures.

Viruses are more complex than viroids or prions, possessing both protein and nucleic acid.

Despite being among the most dangerous infectious agents known, they are still not regarded as living.

Table 13.2 shows the major groups of viruses infecting humans.

Fundamentals of microbiology: virus

Viruses

Viruses are obligate intracellular parasites with no intrinsic metabolic activity, being devoid of ribosomes and energy-producing enzyme systems.

They are thus incapable of leading an independent existence and cannot be cultivated on cell-free media, no matter
how nutritious.

The size of human viruses ranges from the largest poxviruses, measuring approximately 300 nm, to the picornaviruses, such as poliovirus,
which is approximately 20 nm.

When one considers that a bacterial coccus measures 1000 nm in diameter,
it can be appreciated that only the very largest virus particles may be seen under the light microscope, and electron microscopy is required for visualizing
the majority.

It will also be apparent that few of
these viruses are large enough to be retained on the 200 nm (0.2 µm) membrane filters used to sterilize thermolabile liquids.

Viruses consist of a core of nucleic acid (either DNA as in vaccinia virus or RNA as in poliovirus) surrounded by a protein shell, or capsid.

Most DNA viruses have linear, double-stranded DNA but in the case of the parvoviruses it is single stranded.

The majority of RNA-containing viruses contain one molecule of single-stranded RNA, although in reoviruses it is double stranded.

The protein capsid constitutes 50% to 90% of the weight of the virus and, as nucleic acid can only synthesize approximately 10% its own weight of protein, the capsid must be made up of a number of identical protein molecules.

These individual protein units are called
capsomeres and are not in themselves symmetrical but are arranged around the nucleic acid core in characteristic symmetrical patterns.

Additionally, many of the larger viruses possess a lipoprotein envelope surrounding the capsid arising from the
membranes within the host cell.

In many instances the membranes are virus modified to produce projections
outwards from the envelope, such as haemagglutinins or neuraminidase as found in influenza virus.

The enveloped viruses are often called ether sensitive, as ether and other organic solvents may dissolve the
membrane.

The arrangement of the capsomeres can be of a number of types.

  • Helical. The classic example is tobacco mosaic virus (TMV), which resembles a hollow tube with capsomeres arranged in a helix around the central nucleic acid core
  • Icosahedral. Such viruses often resemble spheres on cursory examination but when studied more closely, they are seen to be made up of icosahedra that have 20 triangular faces, each containing an identical number of  capsomeres. Examples include the poliovirus
    and adenovirus.
  • Complex. The poxviruses and bacterial viruses (bacteriophages) make up a group whose members have a geometry that is individual and complex.

Reproduction of viruses

Because viruses have no intrinsic metabolic capability, they require the functioning of the host cell machinery
in order to manufacture and assemble new virus particles.

It is this intimate association between the
virus and its host that makes the treatment of viral infections so complex.

Any chemotherapeutic approach which damages the virus will almost inevitably cause injury to the host cells and hence lead to side effects.

An understanding of the life cycle of the virus is, therefore, vital in determining suitable target sites for antiviral chemotherapy.

The replication of viruses within host cells can be broken down into a number of stages.

Adsorption to the host cell

The first step in the infection process involves virus adsorption onto the host cell.

This usually occurs via an interaction between protein or glycoprotein moieties on the virus surface with specific receptors on the host cell outer membrane.

Different cells possess receptors for different viruses. For example, the human immunodeficiency virus (HIV) possesses two proteins involved in adsorption to T lymphocytes; these are known as gp41 and gp120.

There are receptors on the lymphocyte surface to which HIV will bind.

The main receptor is CD4, to which the protein gp120 attaches. Other receptors are CXCR4 and CCR5, to which the protein gp41 binds.

Both attachments are necessary for infection and lead to conformational changes in the HIV envelope proteins, resulting in membrane fusion.

Penetration

Enveloped viruses fuse the viral membrane with the host cell membrane and release the nucleocapsid directly into the cytoplasm.

Naked virions generally penetrate the cell by phagocytosis.

Bacteriophages are viruses which specifically attack bacteria, and they inject their DNA into the host cell, while the rest of the virus remains on the outside.

Uncoating

In this stage the capsid is removed as a result of attack by cellular proteases, and this releases the nucleic acid into the cytoplasm.

These first three stages are similar for both DNA viruses and RNA viruses.

Nucleic acid and protein synthesis

The detailed mechanisms by which DNA- and RNAcontaining viruses replicate inside the cell are outside the scope of this article.

After nucleic acid replication, early viral proteins are produced, the function of which is to switch off host cell metabolic activity and direct the activities of the cell towards the synthesis of proteins necessary for the assembly of new virus particles.

Assembly of new virions

Again, there are differences in the detail of how the viruses are assembled within the host cell, but construction of new virions occurs at this stage, and up to 100 new virus particles may be produced
per cell.

Release of virus progeny

The newly formed virus particles may be liberated from the cell as a burst, in which case the host cell ruptures and dies. Infection with influenza virus results in a lytic response.

Alternatively, the virions may be
released gradually from the cell by budding of the host cell plasma membrane.

These are often called ‘persistent’ infections, an example being hepatitis B.

Latent infections

In some instances, a virus may enter a cell but not go through the replicative cycle outlined in the previous sections and the host cell may be unharmed.

The genome of the virus is conserved and may become integrated into the host cell genome, where it may be replicated along with the host DNA during cell
division.

At some later stage the latent virus may
become reactivated and progress through a lytic phase, causing cell damage/death and the release of new
virions.

Examples of this type of infection are those which occur with the herpes simplex viruses associated with cold sores, genital herpes and also chickenpox, where the dormant virus may reactivate to give shingles later in life.

Oncogenic viruses

Oncogenic viruses have the capacity to transform the host cell into a cancer cell.

In some cases, this may lead to relatively harmless, benign growths, such
as warts caused by papovavirus, but in other cases more severe, malignant tumours may arise.

Cellular transformation may result from viral activation or mutation of normal host genes, called protooncogenes,
or the insertion of viral oncogenes.

Bacteriophages

Bacteriophages (phages) are viruses that attack bacteria but not animal cells. It is generally accepted that the interaction between a phage and a bacterium
is highly specific, and there is probably at least one phage for each species of bacterium.

In many cases the infection of a bacterial cell by a phage results in lysis of the bacterium; such phages are termed virulent. Some phages, however, can infect a bacterium without causing lysis.

In this case the phage DNA becomes incorporated within the bacterial genome.

The phage DNA can then be replicated along with the bacterial cell DNA; this is then termed a prophage.

Bacterial cells carrying a prophage are called lysogenic, and phages capable of inducing lysogeny are called temperate.

Occasionally some of the prophage genes
may be expressed, and this will confer on the bacterial cell the ability to produce new proteins.

The ability to produce additional proteins as a result of prophage DNA is termed lysogenic conversion.

The discovery of bacteriophages in the early 20th century is attributed to two workers, Frederick Twort and Felix d’Herelle.

In 1896 Ernest Hankin had made
an observation that the waters of the Ganges River possessed antibacterial properties which may have led to a reduction in cases of dysentery and cholera in the areas surrounding the river.

Twort and d’Herelle independently came to the conclusion that this effect
must be due to a virus.

Twort did not continue with his research, but d’Herelle quickly established the potential of bacteriophages in antibacterial therapy 10 years before the advent of antibiotics.

It was the discovery of penicillin by Alexander Fleming in 1928 that led to the demise of bacteriophage therapy, but
interest is now increasing again due to the emergence of antibiotic-resistant strains of bacteria.

Archaea

Archaea are a fascinating group of prokaryotic microorganisms that are frequently found living in hostile environments.

They differ in a number of respects from Eubacteria, particularly in the composition of their cell walls. They comprise methane producers, sulfate reducers, halophiles and extreme
thermophiles.

However, at present they have not
been found to be of any value from a pharmaceutical or clinical standpoint and so will not be considered further.

Eubacteria

Eubacteria constitute the major group of prokaryotic cells that have pharmaceutical and clinical significance.

They include a diverse range of microorganisms, from the primitive parasitic rickettsias that share some of
the characteristics of viruses, through the more typical free-living bacteria to the branching, filamentous actinomycetes, which at first sight resemble fungi rather than bacteria.

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