Theme 7. Microbial antagonism. Basis of chemotherapy. Antibiotics. Examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

Theme 7. Microbial antagonism. Basis of chemotherapy. Antibiotics. Examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

Theme 8. Morphology and ultrastructure of viruses. Cultivation of viruses. Indication of viruses.

Various chemical substances comparatively harmless for the macroorganism but with a lethal action on pathogenic micro-organisms are widely used in medical practice for treating patients with infectious diseases and in some cases for prophylaxis.

This method was known long ago to ancient people, and was used for treating certain diseases. The Peruvian Indians discovered the therapeutic action of cinchona bark, and in the 18th century cinchona bark was brought to Europe. The inhabitants of Brazil successfully employed the root of the ipecacuanha for treating amoebiasis. Mercury has been extensively employed in the therapy of syphilis. In the middle of the 16^th century this method became known to the people of Europe.

The basis of modem chemotherapy was founded by P. Ehrlich and D. Romanowsky, who formulated the main scientific principles and the essence of chemotherapy. They showed that in the treatment of each infection a substance should be found which, during injection into the diseased body, will bring the least harm to it and cause the most destructive action to the pathogenic (causative) agent. P. Ehrlich devised the principles of synthesis of medicinal substances by chemical variations: methylene blue, derivatives of arsenic–salvarsan (‘”606″), neosalvarsan (“914”). By the further development of chemistry new medicinal preparations could be obtained.

Extensive experimental and clinical tests of chemopreparations were carried out by E  Metchnikoff.

Chemopreparations should have a specific action, a maximal therapeutic effectiveness, and a minimal toxicity for the body.

As a characteristic of the quality of a medicinal preparation, P. Ehrlich introduced the chemotherapeutic index which is the ratio of the maximal tolerated dose to the minimal curative dose:

 

maximal tolerated dose (DT—Dosis tolerata)

                —————————————————————-         > 3

minimal curative dose (DC—Dosis curativa)

 

The chemotherapeutic index should not be less than 3 Chemotherapeutic preparations include a number of compounds used in medicine

Arsenic preparations (novarsenol, myarsenol, aminarsone, osarsol, etc.) are administered in syphilis, relapsing fever, trypanosomiasis, amoebiasis, balantidiasis, anthrax, sodoku, and other diseases.

Bismuth preparations (basic bismuth nitrate, xeroform, basic bismuth salicylate, bioquinol, bismoverol, bithiurol, pentabismol, etc.) are used against enterocolitis and syphilis

Antimony compounds (tartaric antimony potassium salt, stibenil, stibozan, surmine, solusurmine, etc.) are used for treating patients with leishmaniasis and venereal lymphogranulomatosis.

Mercury preparations (mercury salicylate, mercuric iodide, mercury cyanide, calomel, unguentum hydrargyri cinereum containing metallic mercury, etc.) are prescribed for treating patients with syphilis and are used as antiseptics in pyogenic diseases.

Acridine preparations (rivanol, tripaflavine, acriflavine, acricide, flavicide, etc.) are recommended for pyogenic diseases and inflammatory processes of the pharynx and nasopharynx

Antimalarial substances include more than 30 preparations, e g , chinine hydrochloride, quinine sulphate, mepacrine (acrichine), rodochin (plasmocide), proguanyl (bigumal), pyrimethamine (chloridine), resochine, quinocide sulphones and sulphonamides, sulphadiamine, etc.

Alkaloid preparations (emetine, etc.) are used for treating patients with amoebiasis.

Sulphonamide preparations. The introduction into practice of compounds of the sulphonamide group (streptocid, ethasole, norsulphazol, sulphazine, methylsulphazine, sulphadimezin, urosulphan, phthalazole, sulgine, sulphacyi, soluble sulphacyl, disulphormin, etc.) marked a revolution in the chemotherapy of bacterial infections.

Sulphonamide preparations are used for treating pyogenic diseases, tonsillitis, scarlet fever, erysipelas, pneumonia, dysentery, anaerobic infections, gonorrhoea, cystitis, venereal  lymphogranulomatosis, psittacosis, ornithosis, trachoma, blennorrhoea in the newborn, etc.

There are several points of view concerning the mechanism of action of sulphonamides on microbes.

 

Antibiotics (Fr. anti against, bios life) are chemical substances excreted by some micro-organisms which inhibit the growth and development of other microbes (in recent years several antibiotics have been obtained semisynthetically).

 

 

 

 

 

Antibiotics are obtained by special methods employed m the medical industry For the production of antibiotics strains of fungi, actmomycetes, and bacteria are used, which are seeded in a nutrient substrate After a definite growth period the antibiotic is extracted, purified and concentrated, checked for inocuousness and potency of action In composition a number of antibiotics (penicillin, streptomycin, gramicidin, etc ) have optically distorted molecules The antibacterial properties of some antibiotics are associated with optical inversion of their molecules which have the same physicochemical properties as normal molecules and can easily be bound to the enzyme Since they lack the ability to participate in biochemical reactions, this binding is accompanied by a blockade of enzymes, and consequently, a growth inhibition followed by death of the microorganism.

According to the character of action, antibiotics are subdivided into bacteriostatic (tetracyclines, chloramphenicol, and others) and bactericidal (penicillines, ristomycin, and others). Each antibiotic is characterized by a specific antimicrobial spectrum of action. Some antibiotics are inactivated in the presence of animal and plant proteins. Only a few antibiotics have a powerful antibacterial action, which does not decrease in the presence of protein matter of animal tissues and at the same time is not toxic (in certain concentrations) for the human being.

The mechanism of action of antibiotics varies. Penicillin inhibits the synthesis of polymers of the bacterial cell wall (it hinders the use of muramic acid by bacteria), which leads to an increase of cells  incapable of multiplication. Sometimes the action of penicillin leads to the formation of L-forms in the shape of pleomorphic protoplasmic structures. Thus, penicillin has a lethal effect not on the given population, but on its off-spring. The selective action of penicillin on microbes hinders the penetration of glutamic and other amino acids through the cytoplasmic membrane of pathogenic cocci unable to synthesize amino acids which are vitally important for the existence of these bacteria. Penicillin inhibits the ability of the bacterial cell to absorb protein components — aminoacids, and it inhibits the synthesis of the enzyme system and also of adaptive enzymes.

 

 

 

 

Streptomycin inhibits the incorporation of some amino acids in protein synthesis and attacks the bacterial enzyme with the participation of  which the introduction of pyruvic acid into the tricarbonic acid cycle by its union with oxalacetic acid takes place. This antibiotic inhibits the activity of biotin-containing enzymes catalysing the union of carbon dioxide with carbonic acids; it disturbs reading of the genetic code and synthesizes leucine instead of alanine.

Of special interest is the mechanism of action of streptomycin on tubercle bacilli. This preparation does not have a sterilizing action, but inhibits the respiration of tubercle bacilli, which leads to the inhibition of cell reproduction and toxin formation. At the same time stimulation of tissue respiration occurs in the patient as well as an increase in the ability of the macro-organism to destroy tubercle bacilli and their toxins.

The selective action of streptomycin on the tubercle bacillus is due to the fact that the permeability of cell membranes in the bacilli and in the tissue cells  of animals and man differs due to the dissimilar chemical structure of the cytoplasm of these organisms.

There are data showing that streptomycin inhibits the capacity of bacterial cells of the colibacillus to oxidize fumaric and glutamic acids. This leads to an inhibition of adaptive enzyme production.

Chloramphenicol is a specific inhibitor of the biosynthesis of bacterial protein. It comes into action with the peptidyl transferase area of 50S ribosome. Competing with the aminoacyl end of the aminoacyl tRNA, chloramphenicol blocks the formation of the peptide bond.

Tetracyclines, lincomycin, erythromycin, kanamycin, neomycin, spectinomycin, sparsomycin, fucidine and others belong to the group of antibiotics which inhibit protein biosynthesis in bacteria at the ribosome level. The antibiotic rifampicin suppresses protein biosynthesis by inhibiting the activity of RNA polymerase.

Antifungal antibiotics impair the intactness of the cytoplasmic membrane in fungi; antineoplastic antibiotics suppress the synthesis of nucleic acids in bacterial and animal cells and bind with DNA which serves as the matrix for RNA synthesis; bruneomycin leads to sharp inhibition of the synthesis of DNA or to its destruction.

 

There are various hypotheses and theories which have not entirely revealed the mechanism of action of antibiotics, and this question has not been completely solved.

 

The activity of antibiotics is expressed in international units (IU). Thus, for example, 1 IU of penicillin (Oxford unit) is the smallest amount of preparation inhibiting the growth of a standard Staphylococcus aureus strain. Recently the method of determining the activity of antibiotics according to the weight of the preparation has received wide application.

One unit of activity (AU) corresponds to the activity of 0.6 micrograms (ug) of. the chemically pure crystalline sodium salt of benzylpenicillin. Consequently, in 1 mg of sodium salt of benzylpenicillin there may  be 1667 AU, and in 1 mg of potassium salt — 1600 AU. For practical purposes both preparations are manufactured with an activity not less than 1550 AU.

The concentration of dry preparations as well as of solutions is expressed as the number of micrograms of active substance in 1 g of preparation or in 1 mg of solution.

 

Antibiotics are classified according to the chemical structure of the dmg, the molecular mechanism, and the spectrum of activity exerted on the cells.

 

According to origin, antibiotics are subdivided into the following groups.

 

Antibiotics produced by fungi. Penicillin is produced by the fungi Penicillium notatum, Penicillium chrysogenum, etc. Penicillin is produced as sodium and potassium salts. It dissolves readily in water, but its solutions are not stable. It is a dipeptide consisting of dimethylcysteine and acetylserine.

Penicillin is used in staphylococcal, streptococcal, and meningococcal infections, anaerobic infections, gonorrhoea, syphilis, leptospirosis, anthrax and other diseases.

Penicillin preparations include ecmonovocillin which is a form of long-acting penicillin, maintaining the necessary therapeutic concentration of penicillin in the blood. It is used only for intramuscular injections. The indications are the same as for the application of penicillin.

Semisynthetic penicillins (methicillin, oxacillin) are used in infection with penicillin-resistant staphylococci; ampicillin is prescribed in mixed infections. Resistance, however, develops faster to semisynthetic preparations than to natural ones. Novobiocin and ristomycin cause a favourable therapeutic effect.

Antibiotics produced by actinomycetes. 1. Streptomycin is obtained from Streptomyces griseus. Chemically it consists of two components: the nitrous base of streptidin and streptobiosamine. Streptomycin is a base and forms salts with acids, which readily dissolve in water and are insoluble in organic solvents. It has a bacteriostatic property in relation to Gram-negative as well as to Gram-positive pathogenic microbes.

Streptomycin has a good therapeutic action on tuberculosis, tuberculous meningitis, plague, brucellosis, tularaemia, whooping cough, etc.

2. Chloramphenicol is obtained from the cultural fluid of a strain of Streptomyces venezuelae, isolated from the soil in tropical South America. It has a good therapeutic effect during dysentery, enteric fever, typhus fever and other rickettsioses.

3. Chlortetracycline (biomycin, aureomycin) is produced by Streptomyces aureofaciens. It is employed during staphylococcal infections, pneumonia, subacute septic endocarditis, rickettsioses, amoebiasis, dysentery, whooping cough, gonorrhoea, brucellosis, tularaemia, trachoma, psittacosis, peritonitis, surgical sepsis and other diseases.

4. Tetracycline is a derivative of chlortetracycline. It is obtained by reductive dechlorination of chlortetracycline. Tetracycline has a wide spectrum of action, it inhibits many species of Gram-positive, Gram-negative and acid-fast microbes. It also inhibits the development of many rickettsiae and some protozoa. It is used in treating patients with cholera, pneumonia, subacute septic endocarditis, amoebiasis, dysentery, whooping cough, gonorrhoea, in diseases of the urogenital tract, typhus fever and other rickettsioses, and for the prevention of suppurative processes in surgery. Tetracycline hydrochloride is manufactured in the form of pills with pure tetracycline or in combination with nystatin.

5. Oxytetracycline (terramycin) is obtained from Streptomyces rimosus. In spectrum and mode of action it is close to chlortetracycline. Rondomycin (6-methyl-5-hydroxytetracycline) is a homologue of oxytetracycline. It is absorbed rapidly. Rondomycin possesses a broad spectrum of action (suppresses Gram-positive and Gram-negative bacteria, i. e. cocci, Salmonella organisms, Shigella organisms, pathogenic E. coli serotypes) and is administered per os.

6. Erythromycin is obtained from Streptomyces erythraeus. It is administered in streptococcal diseases. In experiments on animals it has proved to be effective in diseases caused by Gram-positive and Gram-negative bacteria, rickettsiae, chlamydias, intestinal amoebae and trichomonads. Diphtheria bacilli are quite sensitive to erythromycin

7. Neomycin has been isolated from Streptomyces fradiae. It has a bacteriostatic action against Gram-negative and Gram-positive bacteria. The preparation is slightly toxic. It is prescribed mainly for the local treatment of suppurative processes, caused by staphylococci which are resistant to penicillin and to other antibiotics, and also during colienteritis, the causative agents of which are the pathogenic serotypes of E. coli.

8. Nystatin has been extracted from the cultural fluid of Streptomyces noursei. It inhibits many pathogenic fungi and some pathogenic protozoa. It is non-toxic when used per os. It has received wide application in treatment of candidiasis.

9. Kanamycin is an antibiotic produced by Streptomyces kanamycetius. In mode of action it resembles streptomycin and neomycin. It inhibits the growth of Gram-positive and Gram-negative bacteria. Kanamycin is used for treating patients with tuberculosis in whom the causative agent became resistant to antituberculous chemopreparations and antibiotics. It is prescribed for treating anthrax, gonorrhoea and for acute and chronic forms of infections of the urinary tract, and diseases caused by resistant strains of staphylococci.

Cycloserine obtained from Streptomyces lavendulae and other actinomycetes belongs to this group of antibiotics. It produces a beneficial therapeutic effect in tuberculosis; it disturbs the synthesis of the cell wall of mycobacteria and other Gram-positive micro-organisms. Oleandomycin obtained from Streptomyces antibioticus culture fluid inhibits the vital activity of Gram-positive bacteria, Mycobactenum tuberculosis, rickettsia, and Chlamidobacteriales organisms. Levorin produced by Actinomyces levoris is employed for treating superficial and deep candidiases.

Amphotericin (A and B) are antimycotic antibiotics obtained from Streptomyces nodosum. They are effective against yeast-like fungi, pathogens of deep and systemic mycosis, particularly, histoplasmosis, chromomycosis, sporotrichosis.

                Chemical structure  of differens antibiotics

 

Antibiotics produced by bacteria. 1. Gramicidin isolated from a culture of B. brevis has a bacteriostatic and bactericidal action on some pyogenic cocci.

2. Polymyxins  are produced by Bac. polymyxa. They are prescribed in diseases caused by Gram-negative bacteria.

 

Semisynthetic antibiotics. This group includes some penicillins obtained on the basis of6-aminopenicillmic acid, the nucleus of penicillin (methicillin, oxacillin, dioxacillm, ampicillin, etc.).

 

Semisynthetic penicillins

 

Semisynthetic penicillins

 

 

 

           Semisynthetic cephalosporins

 

 The antibiotic levomycetin (an analogue of natural chloramphemcol) is obtained  by synthesis. Combined preparations have also been produced on a mass scale, e. g. vitacycline (tetracycline with vitamins C, b(and B, and some others). New medicinal forms of tetracyclines having weaker side effects have been devised.

 

Resistance of microbes to antibiotics. With the extensive use of antibiotics in medical practice, many species of pathogenic micro-organisms became resistant to them.

Resistance may develop to one or simultaneously to more antibiotics (multiple resistance).

The molecular mechanism of the production of resistance to antibiotics is determined by genes localized in the bacterial nucleoids or in the plasmids, the cytoplasmic transmissible genetic structures.

Resistance to antibiotics occurs as the result of disturbed translation of genetic information and altered synthesis of the polypeptide chain, diminished permeability of the cytoplasmic membrane and cell wall, and the formation, due to the effect of R-plasmids, of enzymes inactivating antibiotics (ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, etc.).

Mutations according to the nucleoid genes, leading to antibiotic resistance, form with a frequency of 10^–6 to 10^–12. Owing to this, the occurrence of simultaneous mutations to two and more antibiotics is excluded; they may develop, however, as the result of independent mutation in a strain primarily resistant to one of the antibiotics.

Resistance to penicillin is linked with penicillinase (B-lactamase) synthesis controlled by one of the genes of the R-factor. Penicillinases are synthesized under the effect of not only the R-factor genes but also the nucleoid genes. Resistance to chloramphenicol is determined by the action of the enzyme — chloramphenicol acetyl-phenicolacetyl transferase coded by the gene of the R-factor. Five enzymes are responsible for the resistance to antibiotics of the aminoglycoside group. Inactivation of antibiotics in the R+ strains with multiple resistance is accomplished by three types of reactions, phosphorylation, acetylation, and adenylation. It has been established that a bacterial cell may be resistant to more than one antibiotic by one gene.(Fig. 8, 9).

 

 

 

Resistance plasmids (R-factors)

 

 

                                R plasmid and genes of  resistance

Due account is given in medical practice to cross-resistance to antibacterial agents which have the same chemical structure. It has been found to exist between preparations of the tetracycline series and new semisynthetic antibiotics (morphocycline, glycocycline, dibiomycin, and ditetracycline), preparations of penicillin (benzylpenicillin, phenoxymethylpenicillin, ephycillin), compounds of the nitrofuran group, and between sulphanilamides.

 

Origin of resistant forms

 

Methods of examination of antibiotic susceptibility

Method of serial dilutions in a liquid medium. Hottinger’s broth (or another medium suitable for the growth of the given micro-organism) is poured by 2-ml portions into test tubes mounted in a tube rack by ten in each row. Prepare antibiotic solution containing 100 U per ml and add 2 ml of this solution into the first test tube. Following thorough mixing, transfer with a new sterile measuring pipette 2 ml of the culture from this tube into the next one, and so on until the ninth tube is reached, from which 2 ml is poured off. The tenth tube containing no antibiotic serves as a control of cul­ture growth.

Wash the 24-hour agar culture of the studied microorganism with isotonic sodium chloride solution, determine the density of the sus­pension by the turbidity standard, and dilute to a concentration of 10000 microorganisms per ml. A sample of  0,2 ml of the obtained suspension is inoculated into all tubes of the row beginning from the control one. Thus, all tubes contain 1000 microorganisms per 1 ml. The results of the experiment are read following incubation of the tube at 37 °C for 18-20 hrs. The minimal concentration of the anti­biotic suppressing the growth of the given microorganism is deter­mined by the last test tube with a transparent broth in the presence of an intensive growth in the control one.

One may also prepare antibiotic solution in molteutrient agar to subsequently streak the tested culture onto the surface of this medium.

Another approach to antimicrobial susceptibility testing is the determination of the minimum inhibitory concentration (MIC) that will prevent microbial growth (fig. 3). The MIC is the lowest concentration of antimicrobic that prevents the growth of a microorganism in vitro.

The minimum inhibitory concentration indicates the minimal concentration of the antibiotic that must be achieved at the site of infection to inhibit the growth of the microorganism being tested. By knowing the MIC and the theoretical levels of the antibiotic that may be achieved in body fluids, such as blood and urine, the

physician can select the appropriate antibiotic, the dosage schedule, and the route of administration. Generally, a margin of safety of 10 times the MIC is desirable to ensure successful treatment of the disease.

MIC is not designed to determine whether the antibiotic is microbicidal. It is, however, also possible to determine the minimal bactericidal concentration (MBC). The MBC is also known as the minimal lethal concentration (MLC) (fig. 3). The minimal bactericidal concentration is the lowest concentration of an antibiotic that will kill a defined proportion of viable organisms in a bacterial suspension during a specified period of exposure. Generally, a 99.9% kill of bacteria at an initial concentration of 10⁵-10⁶ cells/mL during a 17- to 24-hour exposure period is used to define the MBC.

To determine the minimal bactericidal concentration, it is necessary to plate the tube suspensions showing no growth in tube dilution (MIC) tests onto an agar growth medium. This is done to determine whether the bacteria are indeed killed or whether they survive exposure to the antibiotic at the concentration being tested.

Determination of the MIC is adequate for establishing the appropriate concentration of an antibiotic that should be administered for controlling the infection in patients with normal immune response levels. Determination of the MBC is essential for patients with endocarditis (inflammation of the endocardium or lining to the heart) because the patient’s immune response cannot be relied on to remove the infecting microorganisms. It is particularly useful in determining the appropriate concentration of an antibiotic for use in treating patients with low erred immune defense responses, such as may occur in patients receiving chemotherapy

treatment in cancer.

Figure 3. The minimum bactericidal concentration (MBC) of an antibiotic requires the demonstration that microorganisms have lost the ability to reproduce. In this example, although cell growth is inhibited at concentrations of 100 and 6,25 (MIC – 6,25 mg/vL), viable cells remain, which is shown by the formation of colonies (growth) on an agar plate lacking the antimicrobic No vable cells are detected (no growth on agar plates) at 25 mg/mL, which therefore is the MBC.

 

Disk diffusion technique. Into sterile Petri dishes placed on a horizontal sur­face, pour 15 ml of solid nutrient medium (most often 2 per cent agar on Hottinger’s broth containing 0.11-0.13 per cent of amine nitrogen). On the surface of solidified and slightly dried agar, pour 1 ml of suspension of 24-hour culture of the causative agent or, if no pure culture has been isolated, of the pathological material (pus, exudate) obtained for the study and diluted with isotonic saline. Spread uniformly over the agar surface the bacterial suspension, removing its remainder with a Pasteur pipette. Disks with antibio­tics (5-6 disks per plate) are placed onto the surface of the inoculated plate at a distance of 25 mm from its centre. The plates are incubated at 37 °C for 16-18 hrs, after which the results of the test are read by measuring the zones of growth retardation of microorganisms around the disks, including the diameter of the disk itself (fig.4). The size of the zones depends on the degree of sensitivity of the causative agent to a given anti­biotic (tabl. 1). Yet, this method cannot be considered strictly quantitative.

 

Figure. Testing sensitivity of bacteria to antibiotics by the “disk method”

Table 1. Zone Size Interpretive

 

 

 

 

 

Result of medical treatment of angina by antibiotics

 

Due to the wide distribution of staphylococci resistant to antibiotics a search for new preparations became necessary. At present a semisynthetic staphylococcal penicillin has been obtained which has a distinct bacteriostatic action on resistant strains of pathogenic staphylococci With the isolation of the penicilliucleus, 6-aminopenicillinic acid (6APA), it became possible to obtain various derivatives of penicillin.

Dimethylchlorotetracycline from the group of tetracyclines is used for the treatment of many infectious diseases and in doses half as strong as tetracycline. A good result has been obtained in treatment of inflammatory processes of the urinary tract.

With the discovery of the antibiotic griseofulvin dermatology was enriched with an effective preparation with the help of which diseases of the skin, hair and nails caused by fungi imperfecti could be treated.

Some antibiotics have a poisonous effect on rats, insects and mites. They are used for exterminating rodents and arthropods, the vectors of infectious diseases.

Antibiotics (kormogrisin, chlortetracycline, etc.) stimulate the growth of animals and fowl, and are therefore widely used in agriculture.

Of interest is the very difficult problem of chemotherapy of viral diseases. At present there are no effective drugs against viral infections. This is due to the biological peculiarities of viruses as obligatory intracellular parasites, which must be acted upon by other means than those used in microbial diseases. In recent years many new antibiotics have been obtained which have a good effect in the treatment of murine leucoses. Some of them are employed successfully in agriculture for treating fowl leucoses. Antitumour antibiotics include actinomycins C, D, K, F, etc., carcinophilin, mytomycin, actinoxanthin, chrysomalin, aurantin, sarcomycin.

 

 

Side effects of antibiotics. It has been established that large doses of penicillin and streptomycin have a neurotoxic action, tetracyclines affect the liver, chloromycetin has a toxic effect on the haematopoietic organs, and chlortetracycline and oxytetracycline upon intravenous injection may lead to collapse with a lethal outcome.

 

 

Effect of tetracyclin on decolorization dental enamel

 

 Upon injection of penicillin and streptomycin a rash, contact dermatitis, angioneurotic oedema, anaphylactic reactions or allergic asthma may occur. Quite frequently allergic reactions arise during local application of antibiotics. Of the most practical importance is their indirect action which is mainly due to the development of resistant strains of micro-organisms, sometimes causing furuncles or severe generalized diseases which develop vigorously, in some cases with a lethal outcome. In case of the application of antibiotics with a wide spectrum of action infections may develop which are caused by resistant strains of Proteus and fungi. Staphylococcal colitis proceeds very severely, and is characterized by profuse diarrhoea, dehydration of the body, toxic phenomena, shock and collapse.

 

 

 

Candidosis

 

Of great hazard is the formation of resistant staphylococci which cause various postoperative complications — persistent furunculosis and staphylococcal septicaemias.

A severe complication is anaphylactic shock from the use of penicillin in which a rapid drop in blood pressure, cyanosis, superficial breathing, loss of consciousness, and convulsions are observed, and in some cases death occurs. Complications caused by penicillin are characterized by allergic reactions and proceed according to the serum sickness type.

In prolonged use of penicillin or levomycetin (in syphilis and enteric fever) collapse is one of the severe side effects.

Contact dermatitis is an allergic reaction of a medicinal origin. This disease is caused by the action of streptomycin in medical personnel and patients using this preparation over long periods. Quite often allergic manifestations are recorded in the mucous membranes such as hyperaemia and oedema of the pharynx and tongue. In children antibiotics with a wide spectrum of action cause perianal skin hyperaemia, and hyperaemia of the rectal mucosa.

 

 

 

 

Side effect after the reception of rifampin

 

In some countries the number of cases of infection with staphylococcal pneumonia in children has increased. It has been suggested that this can be explained partly by the origin of penicillin-resistant strains of staphylococci. The disease has the tendency of becoming complicated with abscesses, empyema, pneumothorax and the formation of cysts.

Antimicrobial agents cause the formation of numerous variants of microbes with weak pathogenicity (atypical strains, filterable forms, L-forms) which lead to the formation of latent forms of infections marked by recurrences and exacerbations.

Antibacterial agents may induce disorders of the genetic apparatus of the macro-organism’s cells and cause chromosomal aberrations; some of them possess a teratogenic effect leading to the development of foetal monstrosities if they are taken in the first days of pregnancy.

 

Due to the wide distribution of antibiotic-resistant pathogenic bacteria, combined treatment is recommended with the use of new antibiotics to which the causative agents of infectious diseases have yet not developed resistance. To prevent the development of resistant forms of microbes, combined preparations are prescribed: penicillin and streptomycin, erythromycin and oxytetracycline, etc.

 

 

Combined action of antibiotics

 

Tetracycline with nystatine are applied for the prevention of candidiasis.

The use of preparations which block selectively R-plasmid replication and those which promote the elimination of antibiotic modifying enzymes is believed to be promising in the control of multiple antibiotic-resistance.

 

 

MORPHOLOGY AND STRUCTURE OF VIRUSES. CULTIVATION OF VIRUSES. INDICATION OF VIRUSES.

 

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 informatioecessary 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 iewborn 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 humaeoplasms). 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 commoucleoprotein 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.

 

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 cao 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 inclu-
sion ierve 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 tnangulatioumber, 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 tnangulatioumber for several virus groups This formula for tnangulatioumber 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.

 

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 ionprimate 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 7×2 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 thieedle. 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.