Immunology and Virology - prima parte

Virus: Structure and classification

Viruses are not just purveyors of bad news, because they are everywhere around us! Viroma = the human viroma is the set of all viruses in the human body. Viruses are the most numerous infectious agents on the planet. Humans are constantly exposed to many genetically different viruses with new genotypes, species, and strains that evolve rapidly (just think that the number of viruses in our body is bigger than the number of our bacteria and of our eukaryotic cells).

• Viruses 67.7%
• Bacteria 9.5%
• Eukaryote (includes human) 3.6%
• Other sequences 4.4%
• Unknown 14.7%

Every person has a unique viroma, which is not static but rapidly changing (lifestyle, geographic location, age, and even the season). The susceptibility to viral disease depends on the individual's genetic and immune response. Our viroma is like our fingerprint, as it totally represents our person: everybody has their own viroma!

Composition of the viroma

The viroma is composed of different viruses and retroviruses. We have different viruses for different kinds of diseases in specific organs in our body: most of them have “target organs”, so they can infect only a certain region and organize in different places. The same virus can infect more than one organ! Most of them cause persistent infections: they remain always in the same place in our body for the entire life (for example, herpes, chickenpox, …). However, sometimes viruses can also be transient, as they pass, stay for a little while, and then go away.

Nature of viruses

Viruses (or virions) are not cells (have no genome) and don’t have autonomy; they have been described for the first time as filterable agents, defined as organized combinations of macromolecules, biological organizations, or biological entities with a subcellular (but very simple) structure. They are obligate intracellular parasites that depend entirely on the host biochemical machinery for replication. Dimensions: the unit of measurement for viruses is the nanometer (it means they are very small!).

Types of viruses

• Animal viruses: Include viruses of insects and vertebrates.
• Bacterial viruses: Are genus-specific, and called “bacteriophages” or “phages”.
• Plant viruses: Are the second cause of infections in plants.

Virion structure

The virion may contain essential enzymes for initial replication; the complexity of the viral genome reflects the degree of independence from the cell. Inside every virus, there’s nucleic acid, representing the genome:

• DNA
• RNA

We can’t observe them at the same time in a virus! Around the genome, there’s the capsid, composed of capsomers: while naked viruses only have the “capsid”, some others called “enveloped viruses” present a lipidic structure outside the capsid, known as the “envelope”.

Sometimes, together with the genome, there are some specific proteins like DNA/RNA polymerases, used by viruses to correct possible transcription mistakes. In fact, viral proteins can be divided into three classes:

• Structural proteins that form capsid and are part of the envelope.
• Functional proteins suitable for the replication of nucleic acids (DNA polymerase, RNA polymerase, proteases, etc.); some of those specific functional proteins can change and chemically transform normal cells into cancer cells.

Genome

The genome contained inside the capsid can be DNA or RNA. Most animal viruses have an RNA genome, which represents at least 70% of the entire structure.

• Ribovirus (RNA): From 7 to 30 kb (kilobytes), it can be:
  • Double-stranded (+/-): Is an mRNA ready to be translated by ribosomes.
  • Single-stranded positive sense (+): Is complementary to mRNA but unable to produce proteins (needs special polymerases).
  • Ambisense: Containing positive (+) and negative (-) regions of RNA attached end to end (it depends on the situation).

RNA can be segmented, so that viruses can have from 2 up to 12 segments of RNA (“Segmented Viruses”). The genome is haploid, except in retroviruses.

• Deossiribovirus (DNA): From 5 to 370 kb (kilobytes), it is:
  • Haploid
  • Double-stranded:
    • Linear (Parvovirus, Adenovirus, Herpes virus, Poxvirus)
    • Circular (Papillomavirus, Hepadnaviruses, Polyomavirus)

With the exception of:

• Parvovirus, that has a single-strand genome.
• Hepadnaviruses, that have a partially double-strand genome.

DNA viruses show less variability and have their own polymerase: DNA polymerase is precise and equipped with a proofreader, so the DNA doesn’t change for a long time. However, some of them are not proofreaders, so it means that the DNA virus can change frequently.

Capsid

The capsid is a rigid protein structure that consists of proteins able to self-assemble; its main role is to contain and protect the genome. Thanks to those different proteins (produced by cells), the capsid is able to resist unfavorable environmental conditions. Naked capsid viruses are also resistant to drying, acids, and detergents (naked viruses are resistant in the gastrointestinal tract).

For example, Hepatitis A is a naked virus that can be contracted just by eating food, and its target organ is the liver. The influenza virus cannot survive in the stomach or the intestine because it has an envelope that can be immediately destroyed by the enzymes of the intestinal tract.

The protein which forms the capsid is held together by non-covalent bonds, as the structure has to be broken. The capsomer is composed of 5 protomers, that together form a pentamer (or penton); 12 pentamers create a “procapsid”, which represents the structure of the mature virion.

Nucleocapsid structure

The capsid can have different structures:

• Structure with cubic-icosahedral symmetry: The capsid is composed of viral protein subunits organized in capsomers, forming together a solid made of 20 equilateral triangular identical faces and 12 vertices.
• Helical symmetry structure (helical rods): The capsid is made from identical protein subunits (protomers) that interact with the other and with nucleic acids (DNA or RNA) to form tubule-filamentary structures; the helix length is determined by the length of the nucleic acid. The structure can also be rigid (plant viruses) or flexible (Orthomyxovirus).
• Structure with complex and/or unknown symmetry: Examples are the Poxvirus and the Bacteriophages DNA.

Pericapsid or envelope

The pericapsid is a phospholipid bilayer composed of lipids, proteins (peplomers), and virus-specific glycoproteins. The envelope is derived from cell membranes and is acquired during the budding process; it shows both cellular proteins and viral proteins (those taken from an infected organism) useful to bind receptors expressed on the host cell.

The Viral Attachment Protein (VAP) is a special structure that allows the virus to enter only specific cells because it recognizes particular signals on those cells; it can be:

• Outside the envelope, in enveloped viruses.
• On the capsomer, in naked viruses.

One virus (with the VAP) can infect only the “Sensitive Cells”, namely the cells that express on their membrane the VAP receptor for specific viruses. This means that they can be infected only by certain viruses. Virus with envelopes does not necessarily kill the cell.

The structure is sensitive to drying, acids, detergents, and solvents. Viruses provided with envelopes survive in aqueous environments and are transmitted through fluids such as blood, secretions, and aerosols, inside small droplets, that can reach long destinations from one person to another (in which the virus is able to survive for a period of time); however, they do not survive in the gastrointestinal tract!

Just under the envelope, attached on its inner face, there’s a protein layer called the “Matrix” or the “Tegument”, which contains enzymes and other kinds of proteins.

Classification of viruses

There are two ways to classify viruses:

• Classification ICTV (International Committee on Taxonomy of Viruses), divides viruses into:
  • Orders (-es) 6
  • Families (-viridae) 87
  • Subfamilies (-virinae) 19
  • Genres (virus) 384
  • Species 2288
• Baltimore Classification: Divides viruses into 7 groups according to the characteristics of the genome and the replication mechanism (so it can say many things about a virus!).

Viruses culture

Viruses are intracellular parasites; they must:

• Enter in contact with a host cell (adhesion).
• Penetrate into a host cell where they can replicate.

The cells viruses enter into are called “sensitive” and “permissive” cells. Isolation of the virus allows subsequent analysis and archiving of samples, but may put individuals at risk of infection. A virus can be grown in tissue culture, embryonated eggs, and experimental animals. Although embryonated eggs are still used for the growth of the virus for some vaccines (e.g., influenza), they have been replaced by cell cultures for routine virus isolation in clinical laboratories. Experimental animals are rarely used in clinical laboratories for the purpose of isolating viruses.

Inoculation in animals laboratory

Some viruses cannot be cultivated in the laboratory, but they can be grown in experimental animals (rabbits, guinea pigs, dogs, cats, mice, chickens). This was the first method used to isolate viruses. In animals, we can observe the ability of the virus to kill or produce injury and clinical symptoms. The inoculation is done into embryonated chicken eggs, because they are sterile and do not have an immune system; usually, eggs of 10-12 days of life are used. So we use very young animals, in which the immunity system is barely functional.

Viruses like the Influenza virus, Mumps virus, Herpes virus, etc., are cultivated in different parts of the eggs, such as in the allantoic cavity, in the yolk, or in the amniotic cavity.

Organ culture

The organ culture gives the possibility to use organs to cultivate viruses: is a development from tissue culture methods of research, able to accurately model functions of an organ in various states and conditions by the use of the actual in vitro organ itself. The main objective is to maintain the architecture of the tissue and direct it towards normal development. In this technique, it is essential that the tissue is never disrupted or damaged. It thus requires careful handling. The media used for growing organ culture are generally the same as those used for tissue culture. The techniques for organ culture can be classified into:

• Those employing a solid medium.
• Those employing liquid medium.

For example, a slice of skin is usually infected with a certain virus that is able to produce light: when the skin is infected, a special camera is used to see viruses that are reproducing and growing. This is a new way to study viruses - VZV ORF7 deletion virus in skin organ culture. The left panel is an image of ex vivo skin organ culture (SOC) maintained in Netwell inserts. The right panel is an image from the IVIS assay of the same SOC tissue infected with parental and ORF7 deletion (ORF7D) VZV LUC viruses. Ex vivo growth curve analysis of ORF7D VZV LUC in human fetal skin organ cultures (SOC). Skin tissues were inoculated with 5×10³ PFU of either WT VZV LUC, ORF7D VZV LUC, or ORF7 rescue (ORF7R) VZV LUC viruses, in parallel. VZV replication was monitored daily by IVIS for one week as bioluminescence emitting from each skin culture was measured. Each line represents an average of the data from 3 different skin tissue samples, all infected with the same virus.

Tissue culture

Tissue culture is a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to grow, survive, and function. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an organ. Cells in culture may multiply; change size, form, or function; exhibit specialized activity (muscle cells, for example, may contract); or interact with other cells.

Cells may be grown in a culture medium of biological origin such as blood serum or tissue extract, in a chemically defined synthetic medium, or in a mixture of the two. A medium must contain proper proportions of the necessary nutrients for the cells to be studied and must be appropriately acid or alkaline. Cultures are usually grown either as single layers of cells on a glass or plastic surface or as a suspension in a liquid or semisolid medium.

To initiate a culture, a tiny sample of the tissue is dispersed on or in the medium, and the flask, tube, or plate containing the culture is then incubated, usually at a temperature close to that of the tissue’s normal environment. Sterile conditions are maintained to prevent contamination with microorganisms. Cultures are sometimes started from single cells, resulting in the production of uniform biological populations called clones. Single cells typically give rise to colonies within 10 to 14 days of being placed under culture conditions.

Types of tissue culture

Specific types of tissue culture cells are used to grow viruses.

• Primary: Normal cells are cultured without any change in their division rate.
• Continuous:
  • Finite: Single cell type roughly thirty times of division, enhanced by growth factors.
  • Indefinite: It is nearly the same as finite, but the cells here can divide indefinitely by transformation into tumor cells; they are called “cell line”.

Primary cell culture

Primary cultures consist of normal cells, tissues, or organs (such as kidney, lungs, or liver) that are excised directly from tissue collected by biopsy from a living or dead organism (animals). Primary cell cultures are obtained by dissociating specific animal organs with trypsin or collagenase, through mechanical fragmentation, chemical or enzymatic treatment, and placed in culture dishes. The cells obtained by this method are then grown as monolayers (fibroblast or epithelial) or in suspension (lymphocyte) in artificial media supplemented with bovine serum or another source of growth factors. Primary cells can be dissociated with trypsin, diluted, and allowed to grow into new monolayers (passed) to become secondary cell cultures.

Primary cultures are advantageous in that they essentially model the natural function of the cell, tissue, or organ under study. However, the longer the samples are maintained in culture, the more mutations they accumulate, which can lead to changes in chromosome structure and cell function. In addition, primary cultures generally are mortal. Cells undergo an aging process whereby they multiply for only 50 to 100 generations, after which the rate decreases markedly. The point at which cells in primary cultures stop growing, or undergo replicative senescence, marks the so-called Hayflick limit (named for its discoverer, American microbiologist Leonard Hayflick).

The only limit is lifespan, as their survival time is generally one month. After a dead animal has been dissected, cells that derive from a specific organ are cut and added to an enzyme and put in a medium. Mediums are usually cultivated in specific flasks: cells attach on its bottom and grow till they reach a monolayer; at this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Advantages:

• Similar chromosome number as parent tissue.
• Perform specialized biochemical properties as parent tissue (growth factors and hormone secretion).
• Contact inhibition

The genome of these cells is usually diploid!

Secondary cell culture

Diploid cell lines are cultures of a single cell type, derived from primary cultures, that are capable of being passed a large but finite number of times (from 10 up to 100 steps) before they senesce or undergo a significant change in their characteristics (for example, lymphocytes from the blood can be cultivated many times). Their survival time is about 2-3 months, and their genome is also a diploid one!

Continuous cell culture

In the last century, the possibility to use more cell lines was discovered: cancer cells have the possibility to be propagated indefinitely. Generally, they have this ability because they have been transformed into:

• Tumor cells
• Viral oncogenes
• Chemical treatments

So, tumor cell lines and immortalized cell lines, usually initiated from human or animal tumors or by treatment of primary cells with oncogenic viruses or chemicals, consist of single cell types that can be passed continuously without senescing. This has the disadvantage of having retained very little of the original in vivo characteristics.

HeLa cell line originates from a deadly cervical tumor taken from a patient named Henrietta Lacks, who died in 1951. HeLa cells were the first human cells able to grow and be used in the laboratory; after that, many cell lines have been discovered: we have to know the origin of the cell line (tumor, cancer, etc.) because their characteristics are different.

Examination questions

• Structure and composition of viruses
• Classification of viruses
• Cultivation of viruses in the laboratory

Viral life cycle

Every kind of virus has a different type of replication, which also depends on the type of genome it has: a virus is an ultramicroscopic, metabolically inert, infectious agent that can replicate only within the cells of living hosts (animals or plants). The host range is the set of different cell types and tissues, as well as the different species, which can be infected by a specific virus (different viruses have different hosts and different types of cells). The binding of viral attachment proteins (VAP) to cellular receptors is crucial for infection.