Morphology and structure of viruses. Cultivation of viruses. Indication of viruses.

Serological test in virology.

Viruses are the smallest infectious agents (20-300 nm in diameter), containing one kind of nucleic acid (RNA or DNA) as their genome, usually as single molecule.

The nucleic acid is encased in a protein shell, and the entire infectious unit is termed a virion. Viruses replicate only in living cells. The viral nucleic acid contains information necessary for programming the infected host cell to synthesize a number of virus-specific macromolecules required for the production of virus progeny. During the replicative cycle, numerous copies of viral nucleic acid and coat proteins are produced. The coat protein assemble together to form the capsid, which encases and stabilizes the viral nucleic acid against the extracellular environment and facilitates the attachment and perhaps penetration of the virus upon contact with new susceptible cells.

The nucleic acid once isolated from the virion, can be hydrolyzed by either ribo- or deoxyribonuclease, whereas the nucleic acid within the intact virus is not affected by such treatment. In contrast, viral antiserum will neutralize the virion because it reacts with the antigens of protein coat. However, the same antiserum has no effect on the free infectious nucleic acid isolated from the virion.

The host range for a given virus may be extremely limited, but viruses are known to infect unicellular organisms such as mycoplasmas, bacteria, and algae and all higher plants and animals.

Much information on virus-host relationships has been obtained from studies on bacteriophages, the viruses that attack bacteria.

Some Useful Definitions in Virology

Capsid: The symmetric protein shell that en closes the nucleic acid genome. Often, empty capsids are by-products of the viral replicative cycle.

Nucleocapsid: The capsid together with the enclosed nucleic acid.

Structural units: The basic protein building blocks of the capsid.

Capsomeres: Morphologic units seen in the electron microscope on the surface of virus particles. Capsomeres represent clusters of polypeptides, which when completely assembled form the capsid.

Virion: The complete infective virus particle, which in some instances (adenoviruses, papovaviruses, picornaviruses) may be identical with the nucleocapsid. In more complex virions (herpesviruses, myxoviruses), this includes the nucleocapsid plus a surrounding envelope.

Detective virus: A virus particle that is functionally deficient in some aspect of replication. Defective virus may interfere with the replication of normal virus.

Pseudovirus: During viral replication the capsid sometimes encloses host nucleic acid rather than viral nucleic acid. Such particles look like ordinary virus, particles when observed by electron microscopy, but they do not replicate. Pseudovirions contain the “wrong” nucleic acid.

Primary, secondary, and tertiary nucleic acid structure: Primary structure refers to the sequence of bases in the nucleic acid chain. Secondary structure refers to the spatial arrangement of the complete nucleic acid chain, i.e., whether it is single- or double-stranded, circular or linear in conformation. Tertiary structure refers to other elements of fine spatial detail in the: helix, eg, presence of supercoiling. breakage points, regions of strand separation.

Transcription: The mechanism by which specific information encoded in a nucleic acid chain is transferred to messenger RNA.

Translation: The mechanism by which a particular base sequence in messenger RNA results in production of a specific amino acid sequence in a protein.

Evolutionary origin of viruses. The origin of viruses is not known. Three hypotheses have been proposed:

(1) Viruses became parasites of primitive cells, and the 2 evolved together. Many viruses today cause no host cell damage and remain latent in the host.

(2) Viruses evolved from parasitic bacteria. While this possibility exists for other obligatory intracellular organisms, eg, chlamydiae, there is no evidence that viruses evolved from bacteria.

(3) Viruses may be components of host cells that become autonomous. They resemble genes that escape the regulatory control of the host cell. There is evidence that some tumor viruses exist in host cells as unexpress genes. The likehood is great that some small viruses evolved in this fashion. On the other hand, large viruses of the pox or herpes groups show very limited resemblance to host cell DNA.

CLASSIFICATION OF VIRUSES. Basis of Classification. The following properties, listed in the order of preference or importance, have been used as a basis for the classification of viruses. The amount of information available in each category is not uniform for all viruses. For some agents, knowledge is at hand about only a few of the properties listed.

(1) Nucleic acid type: RNA or DNA; single-stranded or double-stranded; strategy of replication.

(2) Size and morphology, including type of symmetry, number of capsomeres, and presence of membranes.

(3) Presence of specific enzymes, particularly RNA and DNA polymerases concerned with genome, and neuraminidase necessary for release of certain virus particles (influenza) from the cells in which they were formed.

(4) Susceptibility to physical and chemical agents, especially ether.

(5) Immunologic properties.

(6) Natural methods of transmission.

(7) Host, tissue, and cell tropisms.

(8) Pathology; inclusion body formation.

(9) Symptomatology.

Classification by Symptomatology. The oldest classification of viruses is based on the diseases they produce, and this system offers certain conveniences for the clinician. However, it is not satisfactory for the biologist because the same virus may appear in several groups, since it causes more than one disease depending upon the organ attacked.

A. Generalized Diseases: Diseases in which virus is spread throughout the body via the bloodstream and in which multiple organs are affected. Skin rashes may occur. These include smallpox, vaccinia, measles, rubella, chickenpox, yellow fever, dengue, enteroviruses, and many others.

B. Diseases Primarily Affecting Specific Organs: The virus may spread to the organ through the bloodstream, along the peripheral nerves, or by other routes.

1. Diseases of the nervous system – Poliomyelitis, aseptic meningitis (polio-, coxsackie-, and echoviruses), rabies, arthropod-borne encephalitides, lymphocytic choriomeningitis, herpes simplex, meningoencephalitis of mumps, measles, vaccinia, and "slow" virus infections.

2. Diseases of the respiratory tract – Influenza, parainfluenza, respiratory syncytial virus pneumonia and bronchiolitis, adenovirus pharyngitis, common cold (caused by many viruses).

3. Localized diseases of the skin or mucous membranes – Herpes simplex type 1 (usually oral) and type 2 (usually genital), molluscum contagiosum, warts, herpangina, herpes zoster, and others.

4. Diseases of the eye – Adenovirus conjunctivitis, Newcastle virus conjunctivitis, herpes keratoconjunctivitis, and epidemic hemorrhagic conjunctivitis (enterovirus-70).

5. Diseases of the liver-Hepatitis type A (infectious hepatitis) and type B (serum hepatitis), yellow fever, and, in the neonate, enteroviruses, herpesviruses, and rubella virus.

6. Diseases of the salivary glands – Mumps and cytomegalovirus.

7. Diseases of the gastrointestinal tract – Rotavirus, Norwalk type virus.

8. Sexually transmitted diseases – Until recently, only bacteria (Neisseria gonorrhoeae, Treponema pallidum, and Chlamydia trachomatis) were included in this category of disease. It is now recognized that herpes simplex virus, hepatitis B virus, papilloma virus, molluscum contagiosum virus, and probably cytomegalovirus are all venereal pathogens.

Classification by Biologic, Chemical, and Physical Properties.

Viruses can be clearly separated into families on the basis of the nucleic acid genome and the size, shape, substructure, and mode of replication of the virus particle. Table 1 shows one scheme used for classification. However, there is not complete agreement among virologists on the relative importance of the criteria used to classify viruses.

Within each family, genera are usually based on antigenicity. Properties of the major families of animal viruses are summarized in Table 1, are discussed briefly below.

DNA-Containing Viruses

A. Parvoviruses: Very small viruses with a particle size of about 20 nm. They contain single-stranded DNA and have cubic symmetry, with 32 capsomeres 2-4 nm in diameter. They have no envelope. Replication and capsid assembly take place in the nucleus of the infected cell. Parvoviruses of rodents and swine replicate autonomously. The adenoassociated satellite viruses are defective, i.e., they require the presence of an adenovirus or a herpesvirus as a ''helper''. Some satellite viruses occur in humans.

B. Papovaviruses: Small (45-55 nm), ether-resistant viruses containing double-stranded circular DNA and exhibiting cubic symmetry, with 72 capsomeres. Known human papovaviruses are the papilloma (wart) virus and agents isolated from brain tissue of patients with progressive multifocal leukoencephalopathy (JC virus) or from the urine of immunosuppressed renal transplant recipients (BK virus). In animals, there are papilloma, polyoma, and vacuolating viruses. These agents have a slow growth cycle and replicate within the nucleus. Papovaviruses produce latent and chronic infections in their natural hosts, and all can induce tumors in some animal species.

C. Adenoviruses: Medium-sized (70-90 nm) viruses containing double-stranded DNA and exhibiting cubic symmetry, with 252 capsomeres. They have no envelope. At least 37 types infect humans, especially in mucous membranes, and they can persist in lymphoid tissue. Some adenoviruses cause acute respiratory diseases, pharyngitis, and conjunctivitis. Some human adenoviruses can induce tumors in newborn hamsters. There are many serotypes that infect animals.

D. Herpesviruses: Medium-sized viruses containing double-stranded DNA. The nucleocapsid is 100 nm in diameter, with cubic symmetry and 162 capsomeres. It is surrounded by a lipid-containing envelope (150-200 nm in diameter). Latent infections may last for the life span of the host.

Human herpesviruses include herpes simplex types 1 and 2 (oral and genital lesions); varicella-zoster virus (shingles and chickenpox); cytomegalovirus; and EB virus (infectious mononucleosis and association with human neoplasms). Other herpesviruses occur in many animals.

E. Poxviruses: Large brick-shaped or ovoid (230 x 400 nm) viruses containing double-stranded DNA, with a lipid-containing envelope. All poxviruses share a common nucleoprotein antigen and contain several enzymes in the virion, including a DNA-dependent RNA polymerase. Poxviruses replicate entirely within cell cytoplasm. All poxviruses tend to produce skin lesions. Some are pathogenic for humans (smallpox, vaccinia, molluscum contagiosum), others for animals. (Some of the latter can infect humans, eg, cow-pox, monkeypox).

RNA-Containing Viruses

A. Picornaviruses: Small (20-30 nm), ether-resistant viruses containing single-stranded RNA and exhibiting cubic symmetry. The groups infecting humans are rhinoviruses (more than 100 serotypes causing common colds) and enteroviruses (polio-, coxsackie-, and echoviruses). Rhinoviruses are acid-labile and have a high density; enteroviruses are acid-stable and have a lower density. Picornaviruses infecting animals include foot-and-mouth disease of cattle and encephalomyocarditis of rodents.

Table 1. Classification of viruses into families based on chemical and physical properties

Nucleic Acid Core	Capsid Symmetry	Virion: Enveloped or Naked	Ether Sensitivity	No. of Capsomeres	Virus Particle Size (nm)*	Physical Type of Nucleic Acid	No. of Genes (Approx.)	Virus Family
DNA	Icosahedral	Naked	Resistant	32 72 252	18-26 45-55 70-90	SS DS circular DS	3-4 5-8 30	Parvoviridae Papovaviridae Adenoviridae
		Enveloped	Sensitive	162	100**	DS	160	Herpesviridae
	Complex	Complex coats	Resistant***		230 X 400	DS	300	Poxviridae
RNA	Icosahedral	Naked	Resistant	32****	20-30 60-80	SS DS segmented	4-6 10-12	Picornaviridae Reoviridae
		Enveloped	Sensitive	32?	30-90	SS	10	Togaviridae
	Unknown or complex	Enveloped	Sensitive		50-300 80-130 ~100	SS segmented SS SS segmented	10 30 >4	Arenaviridae Coronaviridae Retroviridae
	Helical	Enveloped	Sensitive		90-100 80-120 150-300 70 X 175	SS segmented SS segmented SS SS	>3 >8 >10 >5	Arenaviridae Coronaviridae Retroviridae

"Diameter, or diameter X length.

**The naked virus, i.e., the nucleocapsid, is 100 nm in diameter; however, the enveloped virion varies up to 200 nm.

***The genus Orthopoxvirus, which includes the better studied poxviruses, eg, vaccinia, variola, cowpox, ectromelia, rabbitpox,

monkeypox, is ether-resistant. Some of the poxviruses belonging to other genera are ether-sensitive.

****Reoviruses contain an outer and an inner capsid. The inner capsid appears to contain 32 capsomeres, but the number on the outer capsid has not been definitely established. A total of 92 capsomeres has been suggested.

B. Reoviruses: Medium-sized (60-80 nm), ether-resistant viruses containing a segmented double-stranded RNA and having cubic symmetry. Reoviruses of humans include rotaviruses, which cause infantile gastroenteritis and have a distinctive wheel-shaped appearance. Antigenically similar reoviruses infect many animals. Orbiviruses constitute a distinct subgroup that includes Colorado tick fever virus of humans and other agents that infect plants, insects, and animals (blue tongue of cattle and sheep).

C. Arboviruses: An ecologic grouping of viruses with diverse physical and chemical properties. All of these viruses (more than 350) have a complex cycle involving vertebrate hosts and arthropods as vectors transmitting the viruses by their bite. Arboviruses infect humans, mammals, birds, and snakes, and mosquitoes and ticks as vectors. Human pathogens include dengue, yellow fever, encephalitis viruses, and others. Arboviruses belong to several groups, including toga-, bunya-, rhabdo-, arena-, and reoviruses, described here.

D. Togaviruses: Most arboviruses of antigenic groups A and B and rubella virus belong here. They have a lipid-containing envelope, are ether-sensitive, and their genome is single-stranded RNA. The enveloped virion measures 40—70 nm. The virus particles mature by budding from the host cell membrane. Some togaviruses, eg, Sindbis virus, possess a 35-nm nucleocapsid and within it a spherical core 12-16 nm in diameter. Sindbis virus may have 32 capsomeres in an icosahedral surface lattice.

E. Arenaviruses: RNA-containing, enveloped viruses ranging in size from 50 to 300 nm. They share morphologic, biologic, and antigenic properties of arboviruses of the Tacaribe complex, Lassa fever, and lymphocyticchoriomeningitis. Some produce "slow" virus infections.

F. Coronaviruses: Enveloped, 80- to 130-nm particles containing an unsegmented genome of single-stranded RNA; the nucleocapsid is probably helical, 7-9 nm in diameter. They resemble orthomyxoviruses, but coronaviruses have petal-shaped surface projections arranged in a fringe like a solar corona. Coronavirus nucleocapsids develop in the cytoplasm and mature by budding into cytoplasmic vesicles. Human coronaviruses have been isolated from acute upper respiratory tract illnesses— "colds". Coronaviruses of animals include avian infectious bronchitis virus among many others.

G. Retroviruses: Enveloped viruses whose genome contains duplicate copies of high-molecular-weight single-stranded RNA of the same polarity as viral messenger RNA. The virion contains various enzymes including reverse transcriptase (RNA – DNA). Leukemia and sarcoma viruses of animals, foamy viruses of primates, and some "slow" viruses (visna, maedi of sheep) are included.

H. Bunyaviruses: Spherical, 90- to 100-nm particles that replicate in the cytoplasm and acquire an envelope by budding through the cell membrane. The genome is made up of a triple-segmented, single-stranded RNA. About 70 are antigenically related to Bunyamwera virus; 50 others are not but are mor-phologically similar.

Orthomyxoviruses: Medium-sized, 80- to 120-nm enveloped viruses containing a segmented single-stranded RNA genome and exhibiting helical symmetry. Particles are either round or filamentous. Most Orthomyxoviruses have surface projections as part of their outer wall (hemagglutinin, neuraminidase). The internal nucleoprotein helix measures 6-9 nm, and the RNA is made up of 8 segments. During replication, the nucleocapsid is formed in the nucleus, whereas the hemagglutinin and neuraminidase are formed in the cytoplasm. The virus matures by budding at the cell membrane. Orthomyxoviruses are sensitive to dactinomycin. All Orthomyxoviruses are influenza viruses that infect humans or animals.

J. Paramyxoviruses: Similar to but larger (150-300 nm) than Orthomyxoviruses. The internal nucleocapsid measures 18 nm, and the molecular weight of the single-stranded nonsegmented RNA is 4 times greater than that of Orthomyxoviruses. Both the nucleocapsid and the hemagglutinin are formed in the cytoplasm. Paramyxoviruses are resistant to dactinomycin. Those infecting humans include mumps, measles, parainfluenza virus, and respiratory syncytial virus. Others infect animals.

K. Rhabdoviruses: Enveloped virions resembling a bullet, flat at one end and round at the other (Fig 27-35), measuring about 70 x 175 nm. The envelope has 10-nm spikes. The genome is single-stranded RNA. Particles are formed by budding from the cell membrane. Rabies virus is a member of this group along with many other viruses of animals and plants.

L. Other Viruses: Insufficient information to permit classification. This applies to hepatitis viruses, to agents responsible for some immune complex diseases and for sortie "slow" virus diseases, and to some viruses of gastroenteritis

M. Viroids: Small infectious agents causing diseases of plants and possibly animals and humans. They are nucleic acid molecules (MW 70,000-120,000) without a protein coat. Plant viroids are single-stranded, covalently closed circular RNA molecules consisting of about 360 nucleotides and comprising a highly base-paired rodlike structure with unique properties. They are arranged in 26 double-stranded segments separated by 25 regions of unpaired bases embodied in single-stranded internal loops; there is a loop at each end of the rodlike molecule. These features provide the viroid RNA molecule with structural, thermodynamic, and kinetic properties very similar to those of a double-stranded DNA molecule of the same molecular weight and G + C content. Viroids replicate by an entirely novel mechanism in which infecting viroid RNA molecules are copied by the host enzyme normally responsible for synthesis of nuclear precursors to messenger RNA. Thus, DNA-dependent RNA polymerase purified from healthy plant tissue is capable of synthesizing linear (—) viroid RNA copies of full length from (+) viroid RNA templates in vitro.

The infectious agents of degenerative neurologic disorders such as kuru or Creutzfeldt-Jakob disease, or scrapie of sheep, may fit into this category. (The agent of the latter may be a DNA molecule similar in size to plant viroid RNA).

CULTIVATION; QUANTIFICATION; INCLUSION BODIES; CHROMOSOME DAMAGE

Cultivation of Viruses

At present, many viruses can be grown in cell cultures or in fertile 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 and of viral on-cogenesis.

A. Chick Embryos: Virus growth in an embryonated chick egg may result in the death of the embryo (eg, encephalitis virus), the production of pocks or plaques on the chorioallantoic membrane (eg, herpes, smallpox, vaccinia), the development of hemagglutinins in the embryonic fluids or tissues (eg, influenza), or the development of infective virus (eg, polio virus type 2).

B. Tissue Cultures: The availability of cells grown in vitro has facilitated the identification and cultivation of newly isolated and previously known viruses. There are 3 basic types of cell culture. Primary cultures are made by dispersing cells (usually with trypsin) from host tissues. In general, they are unable to grow for more than a few passages in culture, as secondary cultures. Diploid cell strains are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages) but which retain their normal chromosome pattern. Continuous cell lines are cultures capable of more prolonged (perhaps indefinite) culture which have been derived from cell strains or from malignant tissues They invariably have altered and irregular numbers of chromosomes.

The type of cell culture used for virus cultivation depends on the sensitivity of the cells to that particular virus In the clinical laboratory, multiplication of the virus can be followed by determining the following.

1 The cytopathic effect, or necrosis of cells in the tissue culture (polio-, herpes-, measles-, adenovirus, cytomegalovirus, etc).

2 The inhibition of cellular metabolism, or failure of virus-infected cells to produce acid (eg, enteroviruses).

3 The appearance of a hemagglutinin (eg, mumps, influenza) or complement-fixing antigen (eg, poliomyelitis, varicella, measles).

4 The adsorption of erythrocytes to infected cells, called hemadsorption (parainfluenza, influenza). This reaction becomes positive before cytopathic changes are visible, and in some cases it is the only means of detecting the presence of the virus 5 Interference by a noncytopathogenic virus (eg, rubella) with replication and cytopathic effect of a second, indicator virus (eg, echovirus).

6 Morphologic transformation by an oncogenic virus (eg, SV40, Rous sarcoma virus), usually accompanied by the loss of contact inhibition and the piling up of cells into discrete foci Such alterations are a heritable property of the transformed cells.

Quantification of Virus

A. Physical Methods: Virus particles can be counted directly m the electron microscope by comparison with a standard suspension of latex particles of similar small size However, a relatively concentrated preparation of virus is necessary for this procedure, and infectious virus particles cannot be distinguished from noninfectious ones.

Hemagglutination. The red blood cells of humans and some animals can be agglutinated by different viruses Both infective and noninfective particles give this reaction, thus, hemagglutination measures the total quantity of virus present The orthomyxoviruses contain a hemagglutinin that is an integral part of the viral envelope Once these viruses have agglutinated with the cells, spontaneous dissociation of the virus from the cells can occur The dissociated cells can no longer be agglutinated by the same virus species, but the recovered virus is able to agglutinate fresh cells. This is due to the destruction of specific mucopolysaccharide receptor sites on the surface of the erythrocyte by the enzyme neuraminidase of the virus particles.

Paramyxoviruses growing in cell culture can be detected by hemadsorption Erythrocytes adsorb to each infected cell.

Poxviruses have an agglutinin for red cells (a phosphohpid-protein complex) that can be separated from the infective virus particle.

Arboviruses and others have hemagglutinins that appear to be identical with the virus particle The union between hernagglutinin and red blood cells is irreversible.

B. Biologic Methods: Quintal assays depend on the measurement of animal death, animal infection or cytopathic effects in tissue culture upon end point dilution of the virus being tested The titter is exprcssei.1 as the 50% infectious dose (ID5o), which is the reciprocal of the dilution of virus that produces the effect 111 50% of the cells or animals inoculated Precise assays require the use of a large number of test subjects.

The most widely used assay for infectious virus is the plaque assay. Monolayers of host cells are inoculated with suitable dilutions of virus and after adsorption are overlaid with medium containing agar or carboxymethylcellulose to prevent virus spreading Alter several days, the cells initially infected have produced virus that spreads only to surrounding cells, producing a small area of infection, or plaque Under controlled conditions a single plaque can arise from a single infectious virus particle, termed a plaque-forming unit (PFU) The cytopathic effect of infected cells within the plaque can be distinguished from uninfected cells of the monolayer, with or without suitable staining. and plaques can usually be counted macroscopically. The ratio of infectious to physical particles vanes widely, from near unity to less than 1 per 1000.

Certain viruses such as herpes or vaccinia form pocks when inoculated onto the chorionallantoic membrane of the embryonated egg. Such viruses can be quantitated by relating the number of pocks counted to the virus dilution.

Inclusion Body Formation. In the course of virus multiplication within cells, virus-specific structures called inclusion bodies maybe produced. They become far larger than the individual virus particle and often have an affinity for acid dyes (eg, eosin) They may be situated in the nucleus (herpesvirus), in the cytoplasm (pox virus), or in both (measles virus) In many viral infections, the inclusion bodies are the site of development of the virions (the virus factories) In some infections (molluscum contagiosum), the inclusion body consists of masses of virus particles that can be seen in the electron microscope to ripen to maturity within the inclusion body. In others (as in the intranuclear inclusion body of herpes), the virus appears to have multiplied within the nucleus early in the infection, and the inclusion body appears to be a remnant of virus multiplication. Variations in the appearance of inclusion material depend largely upon the fixative used

The presence of inclusion bodies may be of considerable diagnostic aid The intracytoplasmic inclusion in nerve cells, the Negri body, is pathognomonic for rabies.

Negri body

Chromosome Damage. One of the consequences of infection of cells by viruses is derangement of the karyotype. Most of the changes observed are random. Frequently, breakage, fragmentation, rearrangement of the chromosomes, abnormal chromosomes, and changes in chromosome number occur Herpes zoster virus interrupts the mitotic cycle of human cells in culture, resulting in formation of micronuclei and fragmentation of some chromosomes. Chromosome breaks have also been observed in leukocytes from cases of chickenpox or measles These viruses, as well as rubella virus, cause similar aberrations when inoculated into cultured cells. Cells infected with or transformed to malignancy by SV40, polyoma, or adenovirus type 12 also exhibit random chromosomal abnormalities.

The Chinese hamster cell has a stable karyotype composed of 22 chromosomes Inoculation of these hamster cells with herpes simplex virus results in chromosome aberrations that arc not random in distribution Most of the breaks occur in region 7 of chromosome No. 1 and in region 3 of the X chromosome The Y chromosome is unaffected Replication of the virus is necessary for induction of the chromosome aberrations. To date, no pathognomonic chromosome alterations have been identified in virusinfected cells in humans.

STRUCTURE AND SIZE OF VIRUSES

Virus Particles

Advances in x-ray diffraction techniques and electron microscopy have made it possible to resolve fine differences in the basic morphology of viruses. The study of virus symmetry in the electron microscope requires the use of heavy metal stains (eg, potassium phosphotungstate) to emphasize surface structure The heavy metal permeates the virus panicle as a cloud and brings out the surface structure of viruses by virtue of "negative staining "

Virus architecture can be grouped into 3 types based on the arrangement of morphologic subunits. (1) those with helical symmetry, eg, paramyxo- and orthomyxovimses, (2) those with cubic symmetry, eg, adenoviruses, and (3) those with complex structures, eg, poxviruses All cubic symmetry observed with animal viruses to date is of the icosahedral pattern. The icosahedron has 20 faces (each an equilateral triangle), 12 vertices, and 5-fold, 3-fold, and 2-fold axes of rotational symmetry. Capsomeres can be arranged to comply with icosahedral symmetry in a limited number of ways, expressed by the formula N = 10(n-I)² + 2, where N is the total number of capsomeres and n the number of capsomeres on one side of each equilateral triangle shows the number of capsomeres where n varies from 2 to 6, in several virus groups.

Icosahedral structures can be built from one simple, asymmetric building unit, arranged as 12 pentamer units and x number of hexamer units. The smallest and most basic capsid is that of the phage fX-174, which simply consists of 12 pentamer units Viruses exhibiting icosahedral symmetry can also be grouped according to their tnangulation number, T, which is the number of small triangles formed on the single face of the icosahedron when all its adjacent morphologic subunits are connected by lines. One class has T values of 1,4,9,16, and 25; a second class, values of 3 and 12, and a third class, values of 7, 13, 19, and 21 The number of morphologic units (capsomeres) is expressed by the formula M = 10T + 2. Table 2 shows the tnangulation number for several virus groups This formula for tnangulation number originated in the idea that those viruses would be formed from small subunits so as to give a surface lattice representing the minimum-energy design for closed shells arranged from identical units.

An example of icosahedral symmetry is seen in Fig 1. The adenovirus (n = 6) model illustrated shows the 6 capsomeres along one edge (Fig 1[a]). Degradation of this virus with sodium lauryl sulfate releases the capsomeres in groups of 9 (Fig 1 [b], [c]) and possibly groups of 6 The groups of 9 lie on the faces and include one capsomere from each of the 3 edges of the face, and the groups of 6 would be from the vertices The groups of 9 form the faces of the 20 triangular facets, making the adenovirus icosahedron account for 180 subunits, and the groups of 6 which form the 12 vertices account for 72 capsomeres, thus totaling 252.

Figure 1. (a) Representation of the capsomere arrangement of an adenovirus particle, as viewed through the 2 fold axis of symmetry. (b) Arrangement of capsomere group of 9, obtained by treatment of an adenovirus with sodium lauryl sulfate. (c) Orientation of the capsomere group of 9 on the adenovirus particle If the model were marked to show the maximum number of small triangles formed on one face of the icosahedron by drawing a line between each adjacent morphologic subunit, it would yield the tnangulation number for the adenovirus particle, which in this case turns out to be 25.

Measuring the Size of Viruses. Small size and ability to pass through filters that hold back bacteria are classic attributes of viruses. However, because some bacteria may be smaller than the largest viruses, filtrability is no longer regarded as a unique feature of viruses.

The following methods are used for determining the sizes of viruses and their components.

A. Filtration Through Collodion Membranes of Graded Porosity: These membranes are available with pores of different sizes. If the virus preparation is passed through a series of membranes of known pore size, the approximate size of any virus can be measured by determining which membranes allow the infective unit to pass and which hold it back. The size of the limiting APD (average pore diameter) multiplied by 0.64 yields the diameter of the virus particle. The passage of a virus through a filter will also depend on the physical structure of the virus; thus, only a very approximate estimate of size is obtained.

B. Sedimentation in the Ultracentrifuge: If particles are suspended in a liquid, they will settle to the bottom at a rate that is proportionate to their size. In an ultracentrifuge, forces of more than 100,000 times gravity may be used to drive the particles to the bottom of the tube. The relationship between the size and shape of a particle and its rate of sedimentation permits determination of particle size. Once again, the physical structure of the virus will affect the size estimate obtained.

C. Direct Observation in the Electron Microscope: As compared with the light microscope, the electron microscope uses electrons rather than light waves and electromagnetic lenses rather than glass lenses. The electron beam obtained has a much shorter wavelength than that of light, so that objects much smaller than the wavelength of visible or ultraviolet light can be visualized. Viruses can be visualized in preparations from tissue extracts and in ultrathin sections of infected cells. Electron microscopy is the most widely used method for estimating particle size.

D. Ionizing Radiation: When a beam of charged particles such as high-energy electrons, alpha particles, or deuterons passes through a virus, it causes an energy loss in the form of primary ionization. The release of ionization within the virus particle proportionately inactivates certain biologic properties of the virus particle such as infectivity, antigenicity, and hemagglutination. Thus, the size of the biologic unit responsible for a given function in a virus particle can be estimated.

E. Comparative Measurements: (See Table 1.) For purposes of reference, it should be recalled that: (1) Staphylococcus has a diameter of about 1000 nm. (2) Bacterial viruses (bacteriophages) vary in size (10-100 nm). Some are spherical or hexagonal and have short or long tails. (3) Representative protein molecules range in diameter from serum albumin (5 nm) and globulin (7 nm) to certain hemocyanins (23 nm).

The relative size and morphology of various virus families see Lecture 4. Particles with a 2-fold difference in diameter have an 8-fold difference in volume. Thus, the mass of a pox virus is about 1000 times greater than that of the poliovirus particle, and the mass of a small bacterium is 50,000 times greater.

CHEMICAL COMPOSITION OF VIRUSES

Viral Protein. The structural proteins of viruses have several important functions. They serve to protect the viral genome against inactivation by nucleases, participate in the attachment of the virus particle to a susceptible cell, and are responsible for the structural symmetry of the virus particle. Also, the proteins determine the antigenic characteristics of the virus.

Virus structural proteins may be very specialized molecules designed to perform a specific task: (1) vaccinia virus carries many enzymes within its particle to perform certain functions early in the infectious cycle; (2) some viruses have specific proteins for attachment to cells, eg, influenza virus hemagglutinin;

and (3) RNA tumor viruses contain an enzyme, reverse transcriptase, that makes a DNA copy of the virus RNA, which is an important step in transformation by these viruses.

Viral Nucleic Acid. Viruses contain a single kind of nucleic acid, either DNA or RNA, that encodes the genetic information necessary for the replication of the virus. The RNA or DNA genome may be single-stranded or double-stranded, and the strandedness, the type of nucleic acid, and the molecular weight are major characteristics used for classifying viruses into families (Table 1).

The molecular weight of the viral DNA genome ranges from 1.5 X 10⁶ (parvoviruses) to 160 X 10⁶ (poxviruses). The molecular weight of the viral RNA genome ranges from 1 x 10⁶ (for bromegrass mosaic virus) to 15 x 10⁶ (for reoviruses).

The sequence and composition of nucleotides of each viral nucleic acid are distinctive. One of the properties useful for characterising a viral nucleic acid is its guanine + cytosine (G + C) content.

Most viral genomes are quite fragile once they are removed from their protective protein capsid, but some nucleic acid molecules have been examined in the electron microscope without disruption, and their lengths have been measured. If linear densities of approximately 2 X 10⁶ per mcm for double-stranded nucleic acid and 1 x 10⁶ per mcm for single-stranded forms are used, molecular weights of viral genomes can be calculated from direct measurements (Table 1).

All major DNA virus groups in Table 1 have genomes that are single molecules of DNA and have a linear or a circular configuration. This circle is often supercoiled in the virion.

Viral RNAs exist in several forms. The RNA may be a single linear molecule (eg, picomavirus). For other viruses (eg, orthomyxovirus), the genome consists of several segments of RNA that may be loosely linked together within the virion. The isolated RNA of picomaviruses and toga viruses is infectious, and the entire molecule functions as a messenger RNA within the infected cell. The isolated RNA of other RNA viruses is not infectious. For these virus families, the virions carry an RNA polymerase which in the cell transcribes the genome RNA molecules into several complementary RNA molecules, each of which may serve as a messenger RNA.

Molecular hybridization techniques (DNA to DNA, DNA to RNA, or RNA to RNA) permit the study of transcription of the viral genome within the infected cell as well as the relatedness of different viruses.

The number of genes in a virus can be approximated if one makes certain assumptions about (1) triplet code, (2) the molecular weight of the genome, and (3) the average size of a protein (Table 1).

Viral Lipids. A number of different viruses contain lipids as part of their structure (eg, Sindbis virus [Fig 2]). Such lipid-containing viruses are sensitive to treatment with ether and other organic solvents (Table 1), indicating that disruption or loss of lipid results in loss of infectivity. Non-lipid-containing viruses are generally resistant to ether.

Figure 2. Proposed structure of Sindbis virus

The specific phospholipid composition of a virion envelope may be determined by the ' 'budding " of the virus through specific types of cell membranes in the course of maturation. For example, herpes viruses bud through the nuclear membrane of the host cell, and the phospholipid composition of the purified virus reflects the lipids of the nuclear membrane. The different ways in which various animal viruses acquire an envelope are suggested in Fig 3. Budding of virions occurs only at sites where virus-specific proteins have been inserted into the host cell membrane.

Figure 3. Diagrammatic relationship between lipid-containing viruses and host cell membranes

Glycosphingolipids occur in the surface membrane of animal cells. When cultured cells are transformed by some oncogenic viruses, there are alterations in the various sphingolipids. These may be related to the loss of contact inhibition and to changes in surface antigens that result from viral transformation.

Viral Carbohydrates. Virus envelopes contain glycoproteins. The sugars added to vims glycoproteins often reflect the host cell in which the vims is grown. The glycoproteins are important vims antigens. As a result of their position at the outer surface of the virion, they are frequently involved in the interaction of the vims with neutralizing antibody.

PURIFICATION AND IDENTIFICATION OF VIRUSES

Purification of Virus Particles. For purification studies, the starting material is usually large volumes of tissue culture medium, body fluids, or infected cells. The first step involves concentration of the virus particles by precipitation with ammonium sulfate, ethanol, or polyethylene glycol or by ultrafiltration. Hemagglutination and elution can be used to concentrate myxoviruses. Once concentrated, virus can be separated from host materials by differential centrifugation, density gradient centrifugation, column chromatography, and Rhabdovirus electrophoresis.

The minimal criteria for purity are a homogeneous appearance in electron micrographs and the failure of additional purification procedures to remove “contaminants” without reducing infectivity.

Rate-Zonal Centrifugation. A sample of concentrated virus is layered onto a preformed linear density gradient of sucrose or glycerol, and during centrifugation the virus sediments as a band at a rate determined primarily by the size and weight of the virus particle. Samples are collected by piercing a hole in the bottom of the centrifuge tube. The band of purified virus may be detected by optical methods, by radiolabeling the virus, or by assaying for infectivity.

Equilibrium Density Gradient Centrifugation. Viruses can also be purified by high-speed centrifugation in density gradients of cesium chloride (CsCI), potassium tartrate, potassium citrate, or sucrose. The gradient material of choice is the one that is least toxic to the virus. Virus particles migrate to an equilibrium position where the density of the solution is equal to their buoyant density and form a visible band. Virus bands are harvested by puncture through the bottom of the plastic centrifuge tube and assayed for infectivity.

Additional methods for purification are based on the chemical properties of the virus surface.

As shown by column chromatography, virus is bound to a substance such as DEAE or phosphocellulose, then eluted by changes in pH or salt concentration. Zone electrophoresis permits the separation of virus particles from contaminants on the basis of charge.

Identification of a Particle as a Virus. When a characteristic physical particle has been obtained, it should fulfill the following criteria before it is identified as a virus particle.

(1) The particle can be obtained only from infected cells or tissues.

(2) Particles obtained from various sources are identical, regardless of the cellular species in which the virus is grown.

(3) The degree of infective activity of the virus varies directly with the number of particles present.

(4) The degree of destruction of the physical particle by chemical or physical means is associated with a corresponding loss of virus activity.

(5) Certain properties of the particles and infectivity must be shown to be identical, such as their sedimentation behaviour in the ultracentrifuge and their pH stability curves.

(6) The absorption spectrum of the purified physical particle in the ultraviolet range should coincide with the ultraviolet inactivation spectrum of the virus.

(7) Antisera prepared against the infective virus should react with the characteristic particle, and vice versa. Direct observation of an unknown virus can be accomplished by electron microscopic examination of aggregate formation in a mixture of antisera and crude virus suspension.

(8) The particles should be able to induce the characteristic disease in vivo (if such experiments are feasible).

(9) Passage of the particles in tissue culture should result in the production of progeny with biologic and serologic properties of the virus.

REACTION TO PHYSICAL & CHEMICAL AGENTS

Heat and Cold. Virus infectivity is generally destroyed by heating at 50-60 °C for 30 minutes, although there are some notable exceptions (eg, hepatitis virus, adenoassociated satellite virus, scrapie virus).

Viruses can be preserved by storage at subfreezing temperatures, and some may withstand lyophilization and can thus be preserved in the dry state at 4 °C or even at room temperature. Viruses that withstand lyophilization are more heat-resistant when heated in the dry state. Enveloped viruses tend to lose infectivity after prolonged storage even at —90 °C and are particularly sensitive to repeated freezing and thawing; however, in the presence of dimethyl sulfoxide (DMSO) at concentrations of more than 5%, these viruses are stabilized.

Stabilization of Viruses by Salts. Many viruses can be stabilized by molar concentrations of salts, i.e., they are not inactivated even by heating at 50 °C for 1 hour. The mechanism by which the salts stabilize virus preparations is not known. Viruses are preferentially stabilized by certain salts. Molar MgCl₂ stabilizes picorna- and reoviruses, molar MgSO₄ stabilizes orthomyxo- and paramyxo-viruses, and molar NazSO₄ stabilizes herpes-viruses.

The stability of viruses is important in the preparation of vaccines. The ordinary nonstabilized polio-vaccine must be stored at freezing temperatures to preserve its potency. However, with the addition of salts for stabilization of the virus, potency can be maintained for weeks at ambient temperatures, even in the high temperatures of the tropics.

Heating of some virus preparations in the presence of high salt concentrations can be used to remove adventitious agents. For example, heating poliovirus suspensions in molar MgCI₂ will inactivate such simian contaminants as SV40, foamy virus, and herpes B virus but has no deleterious effect on the infectivity and potency of poliovirus.

PH. Viruses are usually stable between pH values of 5.0 and 9.0. In hemagglutination reactions, variations of less than one pH unit may influence the result.

Radiation. Ultraviolet, x-ray, and high-energy particles inactivate viruses. The dose varies for different viruses.

Vital Dyes. Viruses are penetrable to a varying degree by vital dyes such as toluidine blue, neutral red, and proflavine. These dyes bind to the viral nucleic acid, and the virus then becomes susceptible to inactivation by visible light. Impenetrable viruses like poliovirus, when grown in the dark in the presence of vital dyes, incorporate the dye into their nucleic acid and are then susceptible to photodynamic inactivation. The coat antigen is unaffected by the process.

Ether Susceptibility. Ether susceptibility can distinguish viruses that possess a lipid-rich envelope from those that do not. The following viruses are inactivated by ether: herpes-, orthomyxo-, paramyxo-, rhabdo-, corona-, retro-, arena-, toga-, and bunyaviruses. The following viruses are resistant to ether: parvo-, papova-, adeno-, picorna-, and reoviruses. Poxviruses vary in sensitivity to ether.

Antibiotics. Antibacterial antibiotics and sulfonamides have no effect on viruses. However, rifampin can inhibit pox virus replication.

Antibacterial Agents. Quaternary ammonium compounds are not effective except for a few viruses. Organic iodine compounds are also ineffective. Larger concentrations of chlorine are required to destroy viruses than to kill bacteria, especially in the presence of extraneous proteins. For example, the chlorine treatment of stools adequate for typhoid bacilli is inadequate to destroy poliomyelitis virus present in feces. Formalin destroys resistant poliomyelitis and coxsackieviruses. Alcohols such as isopropanol and ethanol are relatively ineffective against certain viruses, especially picornaviruses.

REPLICATION OF VIRUSES

Viruses multiply only in living cells. The host cell must provide the energy and synthetic machinery and also the low-molecular-weight precursors for the synthesis of viral proteins and nucleic acids. The viral nucleic acid carries the genetic specificity to code for all the virus-specific macromolecules in a highly organised fashion. In some cases, as soon as the viral nucleic acid enters the host cell, the cellular metabolism is redirected exclusively toward the synthesis of new virus particles. In other cases the metabolic processes of the host cell are not altered significantly, although the cell synthesises viral proteins and nucleic acids.

During the replicative cycle, viruses transfer genetic information in several ways from one generation to another. The essential theme, however, is that specific mRNAs must be transcribed from the viral nucleic acid for successful expression and duplication of genetic information. Once this is accomplished, viruses use cell components to translate the mRNA. Various classes of viruses use different pathways to synthesize the mRNAs depending upon the structure of the viral nucleic acid. Some viruses (eg, rhabdo-viruses, myxoviruses) carry RNA polymerases to synthesise mRNAs. RNA viruses of this type are called negative-strand viruses, since their single-strand RNA genome is complementary to messenger RNA, which is conventionally designated positive-strand. Table 3 summarises the various pathways of transcription (but not necessarily those of replication) of the nucleic acids of different classes of viruses.

Most of the viral mRNAs possess a sequence of polyadenylic acid [Poly (A)] at their 3'-end and an unusual blocked, methylated structure at the 5'-end called a cap. The precise function of these features is yet to be elucidated, but the capped structure appears to enhance initiation of translation. Viral mRNA is not always an exact copy of the genome template, since some mRNAs are processed or spliced to delete certain sequences.

Table 3. Pathways of nucleic acid transcription for various virus classes.

Type of Viral Nucleic Acid	Intermediates	Type of mRNA	Example	Comments
± DS DNA	None	+mRNA	Most DNA viruses (eg, herpesvirus, T4 bacteriophage)
+ SS DNA	± DS DNA	+mRNA	fX bacteriophage	See Bacteriophage
± DS RNA	None	+mRNA	Reovirus	Virion contains RNA polymerase that transcribes each segment to mRNA.
+ SS RNA	± DS RNA	+mRNA	Picornaviruses, togaviruses	Viral nucleic acid is infectious and serves as mRNA. For togaviruses, smaller + mRNA is also formed for certain proteins.
-SS RNA	None	+mRNA	Rhabdoviruses, paramyxoviruses, orthomyxoviruses	Viral nucleic acid is not infectious; virion contains RNA polymerase which forms + mRNAs smaller than the genome. For orthomyxoviruses, + mRNAs are transcribed from each segment.
+ SS RNA	- DNA,±DNA	+mRNA	Retroviruses	Virion contains reverse transcriptase; viral RNA is not infectious but complementary DNA from transformed cell is.

DS = double-stranded; SS = single-stranded; “–” indicates negative strand; “+” indicates positive strand;

“+” indicates a helix containing a positive and a negative strand

Virus multiplication was first studied successfully in bacteriophages. For animal viruses, some of the steps of the interaction between the infecting virus and susceptible cells have now been elucidated.

The following sections describe the replication of an RNA and a DNA virus.

RNA Virus Replication (Fig 4). Poliovirus contains a single-stranded RNA as its genome. All of the steps are independent of host DNA and occur in the cell cytoplasm. Polioviruses adsorb to cells at specific cell receptor sites (step 1), losing in the process one virus polypeptide (VP4), which may, therefore, be important in adsorption. The sites are specific for virus coat-cell interactions. Whereas intact poliovirus infects only primate cells in culture, the isolated RNA also infects nonprimate cells (rabbit, guinea pig, chick) and completes one cycle of multiplication. Multiple cycles of infection are not observed in nonprimate cells because the resulting progeny possess protein coats and will again infect only primate cells. After attachment, the virus particles are taken into the cell by viropexis (similar to pinocytosis) (step 2), and the viral RNA is uncoated (step 3). The single-stranded RNA then serves as its own messenger RNA. This messenger RNA is translated (step 4), resulting in the formation of an RNA polymerase that catalyzes the production of a replicative intermediate (RI), a partially double-stranded molecule consisting of a complete RNA strand and numerous partially completed strands (step 5). At the same time, inhibitors of cellular RNA and protein synthesis are produced. Synthesis (+) and (—) strands of RNA probably occurs by similar mechanisms; this is completely elucidated only (+) strands. Here the RI consists of one complete ( strand and many small pieces of newly synthesized ( strand RNA (step 6). The replicative form (RF) consists of 2 complete RNA strands, one (+) and one (-).

The single (+) strand RNA is made in large amounts and may perform any one of 3 functions: serve as messenger RNA for synthesis of structural proteins, (b) serve as template for continued RI replication, or (c) become encapsidated, resulting mature progeny virions. The synthesis of viral cap proteins (step 7) is initiated at about the same time RNA synthesis.

The entire poliovirus genome acts as its own mRNA, forming a polysome of ~350S, and is translated to form a single large polypeptide that is processed during and after translation to form the various viral polypeptides. Thus, the poliovirus genome serves as a polycistronic messenger molecule. The giant polypeptide is cleaved to form a capsid precursor protein and 2 noncoat proteins one of which undergoes further processing. The capsid precursor protein cleaved into coat proteins VPO, VP 1, and VP3. During encapsidation, VPO is cleaved into coat proteins VP2 and VP4.

Completion of encapsidation (step 8) produces mature virus particles that are then released when cell undergoes lysis (step 9).

DNA Virus Replication (Fig 5). In pox virus replication, synthesis of virus components and assembly of virus particles occur wit the cytoplasm of the infected cell. The replication of other DNA viruses (including the adeno-, herpes-, and papovavirus families) differs in that viral DNA is replicated in the nucleus, where viral proteins are synthesized in the cytoplasm, 1 lowed by their migration to and assembly within nucleus. Fig.5 shows the steps in the replication adeno virus, a double-stranded DNA virus. Adsorption (step 1) and penetration (step 2) of the virus into cell are similar to steps described for poliovirus. addition to viropexis, enveloped viruses penetrate fusion of the virus envelope with the plasma membrane, releasing the nucleocapsid into the cytoplasm.

After the virus enters the cell, the protein coat removed (step 3), presumably by cellular enzymes and the viral DNA is released into the nucleus. One both DNA strands are transcribed (step 4) into spec mRNA, which in turn is translated (step 5) to synthesize virus-specific proteins, such as a tumor anti, and enzymes necessary for synthesis of virus DNA. This period encompasses the early virus functions. Host cell DNA synthesis is temporarily elevated an then suppressed as the cell shifts over to the manufacture of viral DNA (step 6). As the viral DNA continues to be transcribed, Iate virus functions become apparent. Messenger RNA transcribed during the later phase of infection <step 6) migrates to the cytoplasm and is translated (step 7). Proteins for virus capsids are synthesized and are transported to the nucleus to be: incorporated into the complete virion (step 8). The migration of some structural proteins of certain viruses from the cytoplasm to the nucleus can be inhibited when arginine is absent from. the growth medium. Assembly of the: protein subunits around the viral DNA results in the formation of complete virions (step 9), which are released after cell lysis.

Summary of Viral RepIication. The molecular events that have been discussed above are summarized in Fig. 6. Virus genomes containing double-stranded (ds) nucleic acid proceed along most of the: steps shown in this figure.

Viruses with single-stranded (ss) nucleic acid only some of the steps. For the orthomyxovirus, the RNA template is utilized for the synthesis of complementary RNA strand that produces the replicative form of the nucleic acid. This in turn serves template for the synthesis of the progeny viral RNA. For the retroviruses, the ssRNA acts as a template for the RNA-dependent DNA polymerase (revertase transcriptase) to synthesize dsDNA. The dsDNA molecules are then used as templates for the transcription and synthesis of ssRNA molecules that serve either as viral mRNA molecules or as viral genomes for encapsidation by the viral structural proteins.

Figure 4. Replication of poliovirus, which containing an RNA genome

Figure 5. Steps in the replication of adenovirus, which contains DNA in its genome

Control Mechanisms of Virus Replication. In the course of virus replication, all the virus-specified macromolecules are synthesized in a highly organized sequence, although virus components are usually made in excess. In some virus infections, early viral proteins are synthesized soon after infection and late proteins are made only late in infection, after viral DNA synthesis. Early genes may or may not be shut off when late products are made. In addition to these temporal controls, quantitative controls also exist, since not all virus proteins are made in the same amounts. Virus-specific proteins may regulate the extent of transcription of genome or the translation of viral messenger RNA. .Although the exact mechanism of these controls is unknown, we do know something about the mechanism of mRNA synthesis. Small animal viruses and bacteriophages are good models for study of gene expression. Their small size has enabled the total nucleotide sequence of a few small DNA phages and SV40 to be elucidated. This led to the discovery that some pieces of DNA are expressed twice by mRNA, being read off either in 2 reading frames or by 2 mRNA molecules with different starting points being read in the same frame.

One surprising discovery has been the observation that animal "virus mRNA molecules (at least for adeno virus and SV40) are not direct copies of their DNA genomes. In these viruses, the mRNA sequences coding for a given protein are preceded in the mRNA molecule by short sequences from farther "upstream” on the DNA template, with intervening sequences spliced out. This suggests the possibility that modification and control of virus gene expression could occur at the level of mRNA construction or "splicing”.

Laboratory diagnosis of viral infections is based on either detecting a causative agent or demonstrating specific antibodies in the blood.

Virus isolation from a patient is performed, using cell cultures, chicken embryos, or experimental animals. Isolated viruses are identified with such serological tests as virus neutralization, haemagglutination inhibition, and others.

Specific antiviral antibodies in patients' blood are studied over time, using paired sera. An increase in the serum antibody titre becomes diagnostically significant only when it is of at least a fourfold magnitude. Rapid diagnostic techniques (immunoelectron microscopy, immunofluorescence test, radioimmunoassay, enzyme-linked immunosorbent assay, etc.) are increasingly gaining in importance.

VIRUS DETECTION AND IDENTIFICATION IN CELL CULTURES

There are primary, diploid, and continuous cell cultures. Primary cultures are obtained directly from animal or human tissue by breaking the intercellular substance with proteolytic enzymes (trypsin, collagenase, pronase). Dissociated (dispersed) cells placed in a culture medium are capable of adhering to the surface of a culture vessel and of proliferating there. Since cells of most primary cultures remain viable for several generations, they may be repeatedly subcultured (passaged). Several passages may produce a diploid culture, i.e., a population of fibroblast-like cells which can be rapidly reproduced and endure 30 to 60 passages still retaining their initial sets of chromosomes. Human diploid cells are highly sensitive to numerous viruses and are extensively used in virology. Both human (WI-38, MRC-5, MRC-9, IMR-90, etc.) and animal (cow, swine, sheep, and lamb) diploid cell cultures have been obtained.

Continuous cell cultures can be subcultured endlessly. They are derived from the primary cultures of cells due to their genetic variability during the growing process, rapidly become dominant in the cell population, and have chromosomal sets typical of all continuous cell lines. Continuous (stable) cell cultures have been obtained from various normal and neoplastic human tissues: amnion (A-0, A-1, FL), kidneys (Rh), cervical carcinoma (HeLa), laryngeal carcinoma (Hep-2), bone marrow from patients with lung cancer (Detroit-6), human embryo rhabdomyosarcoma (RD), etc.

Continuous cell lines are stored in liquid nitrogen and thawed before use.

Cells are cultivated in glass or plastic vessels of various size and shape, preferably disposable, with sterility strictly observed at all stages of cultivation. Nutrient media for cell cultures contain the whole range of amino acids, vitamins, and growth factors. Commercially available are fluid (medium 199, Eagle's medium, lactic albumin hydrolysate) and dry media or concentrates which are dissolved before use.

There are growth and maintenance culture media. Cell cultures are grown, using growth media enriched with human or animal sera, e.g., bovine or foetal (embryonic) cow serum. The serum makes up 2 to 30 per cent of the medium, depending on the properties of the cell culture and composition of the medium.

The maintenance media are used to preserve the established cell monolayers during virus inoculation. These media contain a smaller amount of serum, or they are added to the culture without it. Before the medium is used, antibiotics are added to it in order to prevent the growth of possible extraneous microorganisms. Culture media are sterilized; if they contain unstable constituents, filtration is carried out. Using buffer systems (commonly, bicarbonate buffer), the pH of the medium is maintained at 7.2-7.6. An indicator is added to the media, e.g., phenol red which becomes orange-yellow in acid medium or crimson in alkaline medium.

Obtaining Cell Culture

Primary cell cultures are obtained from any animal or human embryonic tissue, since embryonic cells have a high ability for growth and proliferation. Cultures are commonly prepared of a mixture of several tissues, such as skin, bone, and muscle. Commonly used are human embryonic fibroblasts, chicken embryonic fibroblast, and human kidney cells. Human embryonic tissues from aborted pregnancies and 8-12-day-old chicken embryos are used for cell cultures.

Preparing cell suspension. The tissue is washed in Hanks' solution or antibiotic-containing phosphate buffer to remove blood, fat, cell detritus or other admixtures, chopped with scissors, and washed once again until the solution is clear. Then, it is immersed in trypsin (200-300 ml per 100 g of tissue) and dispersed with a magnetic mixer or pipette. The supernatant containing trypsin-treated cells is decanted and stored in a refrigerator at 4 °C.

Trypsinization is repeated several times. The cell suspension is centrifuged at 600 X g for 5-10 min, resuspended in the medium, and stained with fuchsine, methylene blue, or other dyes. Cell concentration is determined in a Goryaev counting chamber. The suspension is diluted with nutrient medium to a concentration of 400 000-800 000 cells per ml, dispensed into culture flasks, tightly stoppered with rubber plugs, and incubated at 35-37 °C for 48-96 hours (flasks are tilted at a 5 degree angle); thereafter, cultures with well-formed monolayers are harvested.

Passage of continuous (stable] cell cultures. After pipetting off the nutrient medium, pour solution of 0.25 per cent trypsin or 0.02 per cent versene warmed to 37 °C into cell-containing flasks and place them in an incubator for 3-5 min. Then, remove trypsin or versene, add a small amount of the nutrient medium into the vessel, and make the cells suspend in the medium by vigorous shaking. Count the cells, adjust the cell concentration to the required level, and dispense the continuous culture into new flasks.

Suspension cultures. Most continuous cell cultures can proliferate in the medium in a suspended state, which is achieved by automatic rotation in a special drum or cultivation in fermenters.

For virus isolation, the medium is decanted from test tubes with established monolayers, and cells are washed several times with Hanks' solution to remove serum antibodies and inhibitors. A 0.1-0.2-ml portion of the tested material, examined for sterility and appropriately pretreated for virological examination, is placed in every test tube (see p. 189). At least two tubes are used for one test. Some 30-60 min after inoculation, 1 ml of maintenance medium is added to each tube which is then placed in a 37 °C incubator.

If the examined material (e.g., faeces) has a toxic effect on the cell monolayer, it is initially diluted with 1 ml of nutrient medium and, after a 30-60-min exposure to the cell culture, is removed and replaced with the maintenance medium.

Virus Detection

Detection of viruses in the cell culture is based on their cytopathic effect, electron microscopic identification of intracellular inclusions, immunofluorescence, and haemadsorption and also on interference and the "plaque" formation phenomena. A positive haemagglutination test indicates the presence of a virus in the culture fluid.

A cytopathic effect (CPE) represents degenerative cell alterations resulting from intracellular virus reproduction. It is manifested within the first days after cell culture inoculation with some viruses '(variola, polio, etc.) and much later (in 1-2 weeks) when others (adenoviruses, parainfluenza viruses, etc.) are used. The nature of CPE primarily depends on a virus species.

Monolayer cell degeneration is subdivided into total and partial. Total degeneration due to such viruses as polio, Coxsackie and ECHO significantly affects monolayer cells, with great numbers of them sloughing off the slide. The remaining separate cells are shrunken (nuclear and cytoplasmic pyknosis) and characterized by double refraction, i.e., strong fluorescence on microscopy.

Partial degeneration of cultured cells falls into several types:

(1) Racemose formation: rounding, enlargement, and partial confluence of cells producing characteristic racemose aggregates. Degeneration of this type is caused by adenoviruses.

(2) Focal degeneration: local cell injuries (microplaques) appearing against the background of a largely intact monolayer. This type of degeneration is induced by certain strains of variola, variola vaccines, and influenza viruses.

(3) Symplast formation: virus-induced cell aggregation resulting in the formation of giant multinuclear cells (symplasts or syncytia). Symplast formation is caused by measles, mumps, parainfluenza, respiratory-syncytial, and herpes viruses.

Certain oncogenic viruses cause malignant transformation of cells provoking their intense proliferation, in other words changes of a proliferative type.

If the CPE in infected cell cultures is absent or mild, "blind passages" are performed, i.e., new cell cultures are inoculated with the culture fluid.

Intracellular inclusions occur when certain viruses are reproduced in cell nuclei and cytoplasm (variola, rabies, influenza, herpes viruses, etc.). They are detected by light microscopy after staining a monolayer-carrying slide with the Romanowsky-Giemsa solution or with other dyes, or by the luminescent microscopy, using acridine orange (1:20000).

Depending on a virus type, solitary virions or their crystalloid clusters can. be visualized in cell nuclei and cytoplasm with the electron microscope.

A specific virus antigen can be detected in virus-infected cell cultures. using the direct immunofluorescence test.

Plaque formation. Plaques, or negative virus colonies, are sites of virus-destroyed cells in the agar-coated monolayer. Infective virus activity is quantified by counting these colonies.

To obtain the plaques, different dilutions of virus suspension are streaked onto one-layer tissue cultures in Hat vials or Petri dishes and overlaid with a layer of nutrient agar; virus reproduction and CPE are thus confined to initially infected and adjacent cells. Sites of cell degeneration, i.e., plaques, are identified by staining the culture with neutral red which is either included in the composition of the agar layer or added immediately before reading the results. Consisting of dead cells, the plaques are not stained with neutral red and, therefore, are recognized as light spots on a pink-red cell monolayer.

Other techniques of detecting virus plaques in cell cultures are also available, e.g., demonstration of plaque formation under a bentonite layer. Finely dispersed purified bentonite is added to a fluid nutrient medium, and the infected cell monolayer is immersed with this mixture. Because of adsorption of bentonite particles on cell surfaces, the monolayer becomes milk-coloured. At sites of virus reproduction (plaques), cells are not covered with bentonite, and are partially or completely stripped off the slide.

Virus plaques are identified under the bentonite nutrient layer, using mono-layer cultures of continuous human or animal cells sensitive to the tested virus; 1-2-day-old thin cell monolayers are also suitable for this purpose.

Ten-fold dilutions of the material to be tested are prepared, and at least two culture matrasses (Erienmeyer flasks or penicillin vials) are inoculated with every dilution. After virus adsorption (30-40 min) the monolayers arc washed 3-4 times with sterile sodium chloride solution and coated with a bentonite nutrient layer: bidistilled water (415ml), 5-fiper cent bentonite gel (5 ml), Earle's solution, ten-fold concentrate (50ml), native bovine serum (15 ml), 7.5 per cent sodium hydrocarbonate solution (15 ml), penicillin (200 U/ml), streptomycin or lincomycin (100 U/ml).

An infected cell monolayer in 50-ml Erienmeyer flasks is covered with 20-30 ml and that on bottoms of penicillin vials with 5-6 ml o[ bentonite.

Bentonite gel is obtained from dry mineral. Sorbent properties of bentonite are improved by impregnating it with sodium cations. Finally, it is sterilized for 40 min at 111 °C. Bentonite gel may be stored at room temperature for years without any changes in its sorbent properties.

Time of plaque formation under a bentonite layer varies with different viruses. Enterovirus plaque formation is evaluated, for example, in 36-48 hours. Culture flasks are inverted with the monolayer upward, washing degenerated cells away with the medium. Plaques formed by different enteroviruses vary in size, development, and margin patterns.

Since one infective viral particle (virion) produces one plaque, the plaque formation test accurately measures both the number of infective units in the specimen and the neutralizing activity of virus antibodies.

Haemagglutination test is based on the ability of certain viruses to clump (agglutinate) red blood cells obtained from animals of definite species. Influenza and some other viruses with supercapsid membrane contain the surface antigen haemagglutinin responsible for the erythrocyte agglutination.

The HA test is performed in test tubes, on special plexiglass plates, and in a Takata apparatus. A virus-containing specimen is double-diluted in 0.5 ml of isotonic saline. Half a millilitre of 1°n erythrocyte suspension thrice washed in isotonic saline is added into all test tubes, and 0.5 ml of erythrocyte suspension is mixed with an equal volume of virus-free isotonic sodium chloride solution, to be used as control. The mixture may be incubated at 37°, 20° or 4 °C, depending on the properties of the tested virus.

Test results are assessed at 30-60 rain after complete erythrocyte sedimentation in the control, with the findings reading as follows:

(++++), intense and rapid erythrocyte agglutination with a star-like, marginally festooned sediment ("umbrella"}; (++4-), residue of erythrocytes has clearings; (++), a less marked residue; (+), a floccular sediment surrounded with lumps of agglutinated erythrocytes, and (–), a markedly localized erythrocyte sediment ("rouleaus"), as in the control.

Using HA, one can detect the presence of an agglutinating virus in the specimen and determine its titre. The virus titre is defined as the maximum virus dilution at which erythrocyte agglutination still occurs. This dilution is accepted as containing one haemagglutinating unit of the virus.

Results of the haemagglutination test are influenced by several factors: species and individual sensitivity of erythrocytes, temperature and pH of the medium, etc. Furthermore, erythrocyte haemagglutination may be induced by certain microorganisms, such as staphylococci, Escherichia, salmonella, shigella, cholera vibrio El Tor. Therefore, in cases where viruses are detected in bacteria-contaminated material, caution should be exercised while interpreting the results obtained.

Haemadsorption test makes it possible to reveal the virus before the onset of CPE due to the appearance of the virus-specific antigen (haemagglutinin) on the surface of an infected cell. After a period of incubation appropriate for a virus, 0.2 ml of 0.5 per cent erythrocyte suspension is added to the cell culture (both control and virus-infected) so that the monolayer is covered, and the culture is stored for 15-20 min at 4°, 20° or 37 °C (depending on virus properties). Then, the test tubes are shaken in order to remove unadsorbed erythrocytes, and erythrocyte clusters are counted on single cells or throughout the monolayer by low-magnification microscopy. Uninfected cells should carry no erythrocytes.

VIRUS DETECTION AND IDENTIFICATION IN CHICKEN EMBRYOS

Virus demonstration in chicken embryos. Viruses are cultivated in 6-15-day-old chicken embryos. The material to be assayed is introduced with a syringe onto the chorio-allantoic membrane (CAM), into the yolk sac, and into the amniotic and allantoic cavity.

To infect the CAM (Fig. 7, 2), the eggshell is treated with iodine and alcohol, punctured above the air sac and a 7x2 mm opening is made laterally at the place of the greatest vascular ramification. Without destroying the shell-underlying membrane, 0.1-0.2 ml of virus-containing material is placed onto the CAM with a short thin needle. The damaged sites of the shell are coated with sterile paraffin or collodion. Then, the embryos are put into an incubator, putting the eggs horizontally.

Figure 7. Different methods of chicken embryo inoculation

For inoculation into the allantoic cavity the tested material is introduced through the lateral opening of the shell ID-15 mm deep.

When the amniotic cavity is to be inoculated, the virus-containing specimen is injected through the opening at the obtuse end of the egg; the needle should be directed toward the embryonic body so as to ensure penetration of the virus into different organs and tissues of the embryo. Puncture sites are sealed with paraffin or collodion.

The infected embryos are stored in an incubator at 35-37 °'C for 48-72 hrs, depending on the species of the assayed virus. Then, the eggs are cooled at 4 °C for 18 hrs for maximum constriction of the embryonic blood vessels and opened under sterile conditions. The amniotic and allantoic fluid is aspirated with a syringe, and the membranes and embryo are transferred into sterile Petri dishes.

Virus reproduction in chicken embryos is evidenced by characteristic changes on the CAM. Viruses in the amniotic and allantoic fluid can be recovered by means of HA.

Variola, variola vaccine, and herpes simplex viruses produce plaques on the CAM, which look like whitish convex spots 1 to 2 mm in diameter, with their number corresponding to the number of infectious particles.

Erythrocyte agglutination induced by the allantoic and amniotic fluid of infected embryos is a marker of orthomyxovirus and paramyxovirus accumulation. Quantitatively, the virus is determined by the haemagglutination litre (maximum dilution of virus-containing fluid causing erythrocyte agglutination).

Viruses can be titrated on pieces of the chorio-allantoic membrane. Pieces of the shell from an 11-12-day-old chicken embryo with the intact CAM are placed in wells on sterile plates, 0.5-1 ml of virus-containing fluid diluted ten-fold with a buffer (NaCl (8.0 g), KC1 (0.6 g), glucose (0.3 g), MgCl₂x6H₂O (0.05 g), CaCl₂-6H₂O (0.8 ml), phenol red 1:100 (10.0 g), NaOH 1M (0.2 ml), gelatine (2.0 g), antibiotics) is added, and the plates are covered with foil and incubated at 35-37 °C. The shell is removed from the wells in 24-72 hrs and 0.5 per cent chicken erythrocyte suspension is added to the remaining medium. A positive HA test indicates virus reproduction.

Viruses cultivated in chicken embryos are identified with the aid of the neutralization test which is interpreted as positive if plaque formation is inhibited on the CAM and haemagglutination is absent; other identification techniques include the HAI, PG, CF, and IF tests.

According to the nature of the material to be tested and the procedures utilized, the methods for diagnosing viral infections may be categorized into rapid, viroscopic, virological, and serological (Table).

Table

Methods of the Diagnosis of Viral Infections

Method	Purpose of examination
Rapid diagnosis	Detection and identification of the virus-specific antigen arid diagnosis viral particles in the patient's material within 2-3 hrs, which is done with the aid of such methods as EM, IEM, IF, RIHA, ELISA, RIA, PG, and HadsSM. Detection and identification of the virus-specific antigen and viral particles in the patient's material or in biological systems following the preliminary cultivation of the virus with the help of EM, IEM, IF, RIHA, ELISA, RIA, PG. HA, HAI, and CF
Virological	Isolation of the virus through its cultivating in sensitive systems, enrichment for the virus, serological identification, and investigation of the biological properties of the virus by means of such reactions as N, CF, PG, HAI, IF, RIA, ELISA, Hads, and Hadsl
Serological	Determination of the growth in the anti-virus antibodies and identification of immunoglobulins by the CF, HAI, N, RH, IF, IHA, RIA, and ELISA tests

Most of the relevant diagnostic techniques rely on the interaction between virus antigens and homologous antibodies in a fluid medium (complement-fixation (CF) test, haemagglutination inhibition (HAI) test, indirect haemagglutination (IHA) test, reversed indirect haemagglutination (RIHA) test, reversed indirect hemagglutination inhibition (RIHAI) test, radioimmunoassay (RIA), or in gel (the test of precipitation in gel (PG), radial hemolysis (RH) test, immunoelectrophoresis (IEP) test, or during fixation of any ingredient in a solid medium (enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), haemadsorption on a solid-medium (HadsSM) test, immunofluorescence (IF) test, haemadsorption (Hads) test, and haemadsorption inhibition (Hadsl) test). In order to improve test sensitivity, antigens or antibodies are adsorbed on erythrocytes (IHA, RIHA, RIHAI, HadsSM, RH) or linked to enzymes (ELISA), isotopes (RIA, PG), and fluorochromes (IF); an alternative principle is erythrocyte lysis induced by the antigen-antibody interaction in the presence of complement (CF, RH).

The appropriate test procedures are described in detail in chapters dealing with serological diagnosis and with virus detection and identification in cell cultures. This chapter is devoted to the specific features of these tests and modifications which are used in the diagnosis of viral infections.

Haemagglutination inhibition is based on blocking viral haemagglutinin by antibodies. The test is performed on plexiglass plates and interpreted as positive if erythrocytes fail to agglutinate on adding them to mixture of the virus and specific serum. In order to remove or destroy non-specific haemagglutinaton inhibitors, test sera are pretreated with potassium periodate, kaolin, bentonite, acetone, or other agents. Then, the sera are diluted two-fold in isotonic podium chloride solution, and every dilution is supplemented with an equal amount of virus-containing fluid which has four haemagglutinating units. The mixture is incubated for 30-60 min at a temperature optimal for a given virus (0°, 4°. 20°, 37 °C), and an equal volume of 0.5-1 per cent erythrocyte suspension is added. The mixture is reincubated for 30-45 min, and the results of the test are read. The serum titre is defined as the greatest serum dilution at which haemaglutination is inhibited.

Microhaemagglutination inhibition test using Takata's micro-panel and loop is also widely employed.

Haemadsorption inhibition test is used for identifying haemadsorbing viruses and determining serum antibody titres. Specific serum (0.2 ml) diluted 1:5 is placed in test tubes with a culture of virus-infected tissue and following its incubation for 30-60 min. 0.2 ml of 0.5 per cent erythrocyte suspension is added. Nonimmune serum from the same animal species and erythrocytes are instilled in the control test tubes. The tubes are incubated for 20-30 min at a temperature which is optimal for the haemadsorption of the virus to be isolated. A conclusion about a species of the virus is based oil the absence of erythrocyte adsorption in the test tubes in the presence of typical haemadsorption in the control test tubes.

Neutralization test for an infective and cytopathic effect of viruses is performed in virus-sensitive live systems. A virus-containing specimen is serially diluted, and specific serum, diluted to a titre indicated on the ampoule label, is added. The mixture is incubated for 30-60 min at 37 °C and is used to infect tissue culture, chicken embryos, or laboratory animals. A sensitive system inoculated with the virus treated in normal serum serves as control.

Neutralization test is considered positive if the cell culture displays no CPE, chicken embryos show no changes, and the animals live without exhibiting any signs of disease. The findings obtained are used to determine a neutralization index which is a ratio of the virus titre in the control (where CPE is observed) to the test titre. The test is considered negative if the neutralization index is below 10, ambiguous if it varies from 11 to 49, and positive with an index of 50 or higher (significant virus-antiserum correlation).

The most sensitive version of the N test is inhibition of virus plaque formation by virus-specific antiserum (virus plaque reduction test). For this test, a virus-containing specimen (50-100 plaque-forming units) is supplemented with antiserum (diluted to a specified titre), and, after 30-60-min incubation in a heating block, the mixture is applied onto monolayers of sensitive cell cultures. Matching of the virus to the employed antiserum is expressed in reduced plaque formation as compared with control. The N test helps to ascertain the virus species and type (variant).

Colour test (colorimetric neutralization test). Cell activity in the nutrient medium results in accumulation of acid products, which induces a corresponding change in the pH (making the medium orange-coloured). Inoculation of the cell culture with cytopathogenic viruses (enteroviruses, reoviruses, etc.) leads to inhibition of cell metabolism. As a result, the pH of the medium undergoes no change and the medium remains red.

0.25-ml portions of the working virus dilution (100-1000 CPE₅₀) and the respective serum dilution are pipetted into the test tubes. Let the mixture stand for 30-60 min at room temperature, and, after adding 0.25 ml of the cell suspension into each test tube, stopper them with rubber plugs, or pour sterile vaseline oil into them. The mixture is incubated at 37 °C for 6-8 days. The results are interpreted colorimetrically: pH equal to or above 7.4 (red-coloured medium) indicates virus reproduction, whereas pH of 7.2 or less (orange-coloured medium) suggests virus neutralization by antibodies.

Enzyme-linked immunosorbent assay (ELISA) or the immunoenzymic test relies on the capacity of the enzyme antibody label to break down the substrate with the formation of stained products. Antibodies linked to the enzyme regain their ability to conjugate with antigens. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining.

Peroxidase and alkaline phosphatase are commonly utilized as enzymes while 5-aminosalicylic acid, orthophenylendiamine, and other substances are used as the substrate for peroxidase.

Currently, a solid phase modification of ELISA is most often employed in microbiology. The essence of this variant consists in the fact that at first antigens (or antibodies) are sorbed on a solid material and only after that the remaining ingredients of the serological reaction are added. Plastic plates, beads, films or tubes made of various synthetic inert materials (polystyrene, methacrylate, etc.) are usually used as a solid phase carrier of antibodies or antigens. Being adsorbed on the surface of such materials, antibodies or antigens, even in a dry state, retain their immunological specificity and ability to participate in serological reactions for a long time.

There are numerous methodological variants of immunoenzymic detection of antigens; in most cases the antigen is caught by antibodies bound to the solid phase. Following incubation with the material, the antigen tested attaches to the antibody and thus to the solid phase. Then the "linked" antigen is demonstrated by means of enzyme-labelled antibodies against this antigen, the direct variant of ELISA- In an indirect variant anti-species (antiglobulin) enzyme-labelled sera are used. The amount of enzyme linked to the solid phase is equal to the amount of the antigen. Activity of the enzyme is determined quantitatively by the intensity of post-incubation staining with the appropriate substrate. This analysis can be made by means of an automatic device, with the results being registered by a special spectrophotometer.

ELISA is distinguished by a fairly high sensitivity and rapidity of obtaining the results (within 2 hours). Improvement in the sensitivity of the solid phase ELISA modification requires the use of antibodies with a high degree of specificity. Despite their relatively low-affinity, monoclonal antibodies appear promising in this regard. Hence, the development of methods for obtaining highly affinitive monoclonal antibodies is one of the top priorities facing modern microbiologists.

The following buffer solutions are necessary to perform ELISA.

1. Coupling buffer: 0.05 M of sodium-carbonate-bicarbonate buffer (CBB) (pH 9.5-9.7) for sorption of the antigen or antibodies on a solid carrier. The composition of the buffer is as follows: 1.18 g of Na₂CO₃, 3.47 g of NaHCO₃ and 200 mg of NaNO₃. The volume of the buffer is adjusted to 1 L with distilled water,

2. Incubation buffer: phosphate-salt solution (pH 7.3-7.5) which is used for diluting the components introduced into the reaction after sorption of the first component on the carrier. The composition of the buffer is as follows:

17.9 g of Na₂HPO₄H₂O; 0.8 g of NaH₂PO₄HaO; 42.5 g of NaCI; 2.5 ml of Twin-20. The volume of the buffer is brought to 5 L with distilled water and the buffer is stored at 20-25 °C.

3. Washing buffer: isotonic sodium chloride solution containing 0.05 per cent. of Twin-20. Phosphate-salt solution with 0.05 per cent of Twin-20 may also be utilized as a washing buffer. Orthophenylendiamine or 5-aminosalicylic acid serves as a substrate for peroxidase.

Orthophenylendiamine is prepared ex tempore in the following manner:

10 mg of orthophenylendiamine, 6.1 ml of 0.1 M of citric acid, 6.4 ml of 0.2 M of Na₂HPO₄12H₂O (for complete solution the mixture is heated on a water bath), 12.5 ml of distilled water, 0.35 ml of 3 per cent H₂O₂.

Dilute 80 mg of 5-aminosalicylic acid in 100 ml of distilled water, adjust the pH of the solution to 6.0 ex tempore with the help of 1 M NaOH. Prior to using, add 1 ml of 0.05 per cent H₂O₂ to each 9 ml of the solution.

To perform ELISA, one should have polystyrene plates with flat-bottom wells and automatic pipettes. To quantitate the results, the spectrophotometer (a registrator of extinction at a 492 nm wave length) is used.

Procedure. The first stage of ELISA is sorption of the corresponding dilution of antibodies or antigen (in concentration of 10-20u.g/ml)on carbonate-bicarbonate buffer in a 0.2-ml portion on a solid phase for 1-2 hrs at 37 °C and 10-12 hrs at 4 °C (sensitization). Then, the wells are washed (to remove the antibody or antigen which has not been sorbed on the carrier) with tap water and washing buffer containing 0.05 per cent Twin-20 for 5 min (twice) at room temperature. After that place into each well (solid phase) 0.2 ml of 1 per cent solution of bovine serum albumin in CBB and incubate for 1 hr at 37 °C to ensure covering of those sites of the well surface, which have remained free after sensitization, sorption of the first component of the reaction on the solid carrier- Wash the well to remove the unbound bovine serum albumin and introduce the material to be tested (antigen or antibodies) (in 0.2 ml aliquots) diluted with a phosphate-salt solution (pH 7,2) containing 0.05 per cent Twin-20. Each dilution of the material is pipetted into two wells and placed in a 37 °C incubator for 1-3 hrs. Wash off the antigens or antibodies which have not reacted in the immune test and introduce 0.2-ml portions of conjugated antibodies against the test antigen or antibodies in a working dilution on a phosphate-salt solution containing 0.05 per cent Twin-20. Then, incubate the mixture at 37 °C for two hours. The unbound conjugate is washed off with buffer three times for 10 min.

Put 0.1 ml of substrate (chromogen) solution into the well and allow it to stand for 30 min in the dark at mom temperature. In the process of incubation in the presence of peroxidase orthophenylendiamine is stained yellow and aminosalicylic acid, brown.

To stop the reaction of substrate splitting, add 0.1 ml of 1 N H₂SO₄ (or 1 M NaOH) into the well.

Control of the reaction: the test antigen or antibodies are replaced with a homologous component of the reaction.

Control of the conjugate: 0.2 ml of 1 per cent bovine serum albumin per CBB + 0.2 ml of conjugated antibodies in the working dilution.

The results of the reaction are read either visually or instrurnentally. In the first case, one looks for the greatest dilution of the material tested in which the staining is more intense than in the control (by bovine serum albumin). In reading the results of the test with the help of a spectrophotometer, a positive dilution is the greatest dilution of the material tested at which the level of extinction exceeds by at least two times the level of extinction of the corresponding dilution of the heterologous component of the reaction.

To obtain antibodies, conjugated with the enzyme, one needs highly active precipitating sera against the antigen or against animal or human globulins from which the gamma-globulin fraction is isolated by precipitation with polyethylene glycol, ammonium sulphate, and by means of the rivanol-alcohol technique. Immunoglobulins are conjugated by the enzyme with the help of glutaraldehyde. Non-conjugated enzyme is removed by dialysis or chromatography on Sefadex. To prevent bacterial growth, merthiolate in a volume of up to 0.01 per cent of the mixture is added to the conjugates and the latter are kept at 4 °C or in the frozen state.

Radioimniunoassay (RIA). The antigen or antibodies for RIA are labelled with radioactive isotopes, most commonly with ¹²⁵I. RIA is very sensitive and allows the detection of 1-2 ng of the substance tested, or even less. Special radiometric equipment is necessary to perform this assay.

Variable RIA modifications are available, with the solid phase variant being the one most frequently utilized in practice. As in the case of solid phase ELISA, antibodies (antigen) are sorbed on a solid phase carrier [on the surface of plates with wells, beads, and films from polystyrene or other polymer synthetic materials). Adsorbed (immobilized) antigens and antibodies preserve their capacity to participate in serological reactions for a long time.

Figure 1 presents the diagrams of conducting RIA by three methods, viz., competitive, reverse, and indirect.

In the competitive method of RIA antibodies specific in relation to the antigen tested are sorbed on the surface of polystyrene wells. Then, the antigen-containing material to he assayed is placed into the wells and after a definite period of time sufficient for the specific interaction of the antigen with immobilized antibodies to take place, the purified antigen labelled with a radioactive isotope is added. With regard to antigenic specificity, it should correspond to the antibodies immobilized on the surface of wells.

If the material to be examined contains the antigen corresponding to immobilized antibodies, some of the active centres of the latter are blocked. In this case the labelled antigen placed into the -wells will conjugate with immobilized antibodies to a lesser degree (as compared to the control), the difference being expressed in varying levels of radioactivity