General Virology Definition

 Chapter -7 

General Virology Definition

Definition

• Viruses are complexes consisting of protein and an RNA or DNA genome. They lack both cellular structure and independent metabolic processes. They replicate solely by exploiting living cells based on the information in the viral genome.

• Viruses are autonomous infectious particles that differ widely from other microorganisms in a number of characteristics: they have no cellular structure, consisting only of proteins and nucleic acid (DNA or RNA). They have no metabolic systems of their own, but rather depend on the synthetic mechanism of a living host cell, whereby the viruses exploit normal cellular metabolism by delivering their own genetic information, i.e., nucleic acid, into the host cell. The host cell accepts the nucleic acid and proceeds to produce the components of new viruses in accordance with the genetic information it contains. One thus might call viruses “vagabond genes.”    

Morphology and Structure

• Genome. The viral genome is either DNA or RNA, and viruses are hence categorized as DNA or RNA viruses (see also p. 380). The nucleic acid of DNA viruses is usually double-stranded (ds) and linear or circular depending on the family; the nucleic acid of RNA viruses is usually single-stranded (ss), with the exception of the reoviruses, and is also segmented in a number of virus families. Viruses with ssRNA are divided into two groups: if the RNA of the genome has the same polarity as the viral mRNA and can thus function directly as messenger RNA it is called a plus-strand (or positive-strand) or “sense” RNA strand and these viruses are sense or plus-strand viruses. If the genome RNA has the polarity opposite to that of the mRNA, and therefore cannot be translated into proteins until it has first been transcribed into a complementary strand, it is called a minus-strand (or negative-strand) or “antisense” RNA strand and the viruses are antisense or minus-strand viruses.

• Envelope. The envelope, which surrounds the capsid in several virus families, is always dependent on cellular membranes (nuclear or cell membrane, less frequently endoplasmic reticulum). Both cell-coded and viral proteins are integrated in the membrane when these elements are transformed into the envelope, frequently in the form of “spikes” (or peplomers. Enveloped viruses do not adsorb to the host cell with the capsid, but rather with their envelope. Removing it with organic solvents or detergents reduces the infectivity of the viruses (“ether sensitivity”).

Classification

• The taxonomic system used for viruses is artificial (i.e., it does not reflect virus evolution) and is based on the following morphological and biochemcal criteria: 

• Genome: DNA or RNA genome (important basic differentiation of virus types!) as well as configuration of nucleic acid structure: single-stranded (ss) or double-stranded (ds); RNA viruses are further subclassified according to plus and minus polarity (p. 383f.).

• Capsid symmetry: cubic, helical, or complex symmetry

• Presence or absence of an envelope.

• Diameter of the virion, or of the nucleocapsid with helical symmetry.

Replication

• The steps in viral replication are as follows:

• Adsorption of the virus to specific receptors on the cell surface

• Penetration by the virus and intracellular release of nucleic acid.

• Proliferation of the viral components: virus-coded synthesis of capsid and noncapsid proteins, replication of nucleic acid by viral and cellular enzymes

• — Assembly of replicated nucleic acid and new capsid protein.

• Release of virus progeny from the cell.

• As shown on viruses replicate only in living host cells. The detailed steps involved in their replication are shown below. The reactions of the infected cell (cytopathology, tumor transformation, etc.) are described.

• As shown on viruses replicate only in living host cells. The detailed steps involved in their replication are shown below. The reactions of the infected cell (cytopathology, tumor transformation, etc.) are described.

Penetration and uncoating. Viruses adsorbed to the cell surface receptors then penetrate into the cell by means of pinocytosis (a process also known as viropexis). In enveloped viruses, the envelope may also fuse with the cell membrane, releasing the virus into the cytoplasm. Adsorption of such an enveloped virus to two cells at the same time may result in cell fusion. The next step, known as uncoating, involves the release of the nucleic acid from the capsid and is apparently (except in the smallpox virus) activated by cellular enzymes, possibly with a contribution from cell membranes as well. The exact mechanism, which would have to include preservation of the nucleic acid in toto, is not known for all viruses.

Replication of the nucleic acid. Different processes are observed corresponding to the types and configurations of the viral genome

DNA viruses: the replication of viral DNA takes place in the cell nucleus (exception: poxviruses). Some viruses (e.g., herpesviruses) possess replicases of their own. The smaller DNA viruses (e.g., polyomaviruses), which do not carry information for their own DNA polymerase, code for polypeptides that modify the cellular polymerases in such a way that mainly viral DNA sequences are replicated.

Hantaviruses: the genome consists of an ssDNA antisense strand and a short sense strand. The infected cell transcribes an RNA sense strand (“template strand”) from the antisense strand. This template strand is integrated in virus capsids together with an RT DNA polymerase. The polymerase synthesizes a complementary antisense DNA and, to “seal off” the ends of the genome, a short sense DNA from the template strand

RNA viruses: since eukaryotic cells possess no enzymes for RNA replication, the virus must supply the RNA-dependent RNA polymerase(s) (“replicase”). These enzymes are thus in any case virus-coded proteins, and in some cases are actually components of the virus particle.

Single-stranded RNA: in sense-strand viruses, the RNA functions as mRNA “as is,” meaning the information can be read off, and the replicase synthesized immediately. Antisense-strand viruses must first transcribe their genome into a complementary strand that can then act as mRNA. In this case, the polymerase for the first transcription is contained in the mature virion and delivered into the cell. In ssRNA viruses, whether sense or antisense strands, complementary strands of the genome are produced first, then transcribed into daughter strands. They therefore once again show the same polarity as the viral genome and are used in assembly of the new viral progeny

Double-stranded RNA: a translatable sense-strand RNA is produced from the genome, which consists of several dsRNA segments (segmented genome). This strand functions, at first, as mRNA and later as a matrix for synthesis of antisense-strand RNA. Here as well, an RNA-dependent RNA polymerase is part of the virus particle.

Retroviruses also possess a sense-oriented RNA genome, although its replication differs from that of other RNA viruses. The genome consists of two single-stranded RNA segments with sense polarity and is transcribed by an enzyme in the virion (reverse transcriptase [RT]) into complementary DNA. The DNA is complemented to make dsDNA and integrated in the cell genome. Transcription into sense-strand RNA is the basis for both viral mRNA and the genomic RNA in the viral progeny    

Production of viral mRNA. In a DNA virus infection, cellular polymerases transcribe mRNA in the nucleus of the host cell from one or both DNA strands, whereby the RNA is processed (splicing, polyadenylation, etc.) as with cellular mRNA. An exception to this procedure is the poxviruses, which use their own enzymes to replicate in the cytoplasm.

The actual protein synthesis procedure is implemented, coded by the viral mRNA, with the help of cellular components such as tRNA, ribosomes, initiation factors, etc. Two functionally different protein types occur in viruses:

The “noncapsid viral proteins” (NCVP) that do not contribute to capsid assembly. These proteins frequently possess enzymatic properties (polymerases, proteases) and must therefore be produced early on in the replication cycle.

The capsid proteins, also known as viral proteins (VP) or structural proteins, appear later in the replication process.

Viral maturation (morphogenesis). In this step, the viral capsid proteins and genomes (present in multiple copies after the replication process) are assembled into new, infectious virus particles. In some viral species these particles are also covered by an envelope (p. 378f.

Release. The release of viral progeny in some cases correlates closely with viral maturation, whereby envelopes or components of them are acquired when the particles “bud off” of the cytoplasmic membrane and are expelled from the cell. In nonenveloped viruses, release of viral progeny is realized either by means of lysis of the infected cell or more or less continuous exocytosis of the viral particles.

Genetics

• Just as in higher life forms, viral genetic material is subject to change by mutation. Lack of a corrective replication “proofreading” mechanism results in a very high incidence of spontaneous mutations in RNA viruses, in turn greatly increasing the genotypic variability within each species (“viral quasispecies”). Furthermore, a potential for recombination of genetic material is also inherent in the replication process, not only material from different viruses but also from host cell and virus. This factor plays a major role in viral tumor induction and genetic engineering. Functional modifications arising from interactions between different viral species in mixed infections—e.g., phenotype mixing, interference, and complementation—have nothing to do with genetic changes.

• Mutation. Mutations are changes in the base sequence of a nucleic acid, resulting in a more or less radical alteration of the resulting protein. So-called “silent mutations” (in the second or third nucleotide of a codon) do not influence the amino acid sequence of the protei.

• Recombination. The viral replication process includes production of a large number of copies of the viral nucleic acid. In cases where two different viral strains are replicating in the same cell, there is a chance that strand breakage and reunion will lead to new combinations of nucleic acid segments or exchanges of genome segments (influenza), so that the genetic material is redistributed among the viral strains (recombination). New genetic properties will therefore be conferred upon some of the resulting viral progeny, some of which will also show stable heritability. Genetic material can also be exchanged between virus and host cell by the same mechanism or by insertion of all, or part, of the viral genome into the cell genome.

• “Quasispecies.” When viral RNA replicates, there is no “proofreading” mechanism to check for copying errors as in DNA replication. The result is that the rate of mutations in RNA viruses is about 104, i.e., every copy of a viral RNA comprising 10 000 nucleotides will include on average one mutation. The consequence of this is that, given the high rate of viral replication, all of the possible viable mutants of a viral species will occur and exist together in an inhomogeneous population known as quasispecies. The selective pressure (e.g., host immune system efficiency) will act to select the “fittest” viruses at any given time. This explains the high level of variability seen in HIV as well as the phenomenon that a single passage of the attenuated polio vaccine virus through a human vaccine recipient produces neurovirulent revertants

• Occurrence of “new” viral species. It appears to be the exception rather than the rule that a harmless or solely zoopathic virus mutates to become an aggressive human pathogen. In far more cases, changed environmental conditions are responsible for new forms of a disease, since most “new” viruses are actually “old” viruses that had reached an ecological balance with their hosts and then entered new transmission cycles as a result of urbanization, migra tion, travel, and human incursion into isolated biotopes (examples include the Ebola, Rift Valley fever, West Nile, pulmonary Hanta, and bat rabies viruses).

Host-Cell Reactions

Possible consequences of viral infection for the host cell: 

• Cytocidal infection (necrosis): viral replication results directly in cell destruction (cytopathology, so-called “cytopathic effect” in cell cultures).

• Apoptosis: the virus initiates a cascade of cellular events leading to cell death (“suicide”), in most cases interrupting the viral replication cycle.

• Noncytocidal infection: viral replication per se does not destroy the host cell, although it may be destroyed by secondary immunological reactions.

• Latent infection: the viral genome is inside the cell, resulting in neither viral replication nor cell destruction.

• Tumor transformation: the viral infection transforms the host cell into a cancer cell, whereby viral replication may or may not take place depending on the virus and/or cell type involved.

Cell Destruction (Cytocidal Infectio n, Necrosis)

• Cell death occurs eventually after initial infection with many viral species. This cytopathological cell destruction usually involves production of viral progeny. Virus production coupled with cell destruction is termed the “lytic viral life cycle.” Cell destruction, whether necrotic or apoptotic (see below) is the reason (along with immunological phenomena) for the disease manifested in the microorganism (see Pathogenesis.

• Structural changes leading to necrosis: morphological changes characteristic of a given infecting virus can often be observed in the infected cell. The effects seen in virally infected cell cultures are well-known and are designated by the term “cytopathic effect” (CPE). These effects can also be exploited for diagnostic purposes. They include rounding off and detachment of cells from adjacent cells or the substrate, formation of multinuclear giant cells, cytoplasmic vacuoles, and inclusion bodies. The latter are structures made up of viral and/or cellular material that form during the viral replication cycle, e.g., viral crystals in the nucleus (adenoviruses) or collections of virions and viral material in the cytoplasm (smallpox viruses). Although these structural changes in the host cell do contribute necrotic cytopathy, their primary purpose is to support specific steps in viral synthesis. For example, RNA synthesis and viral assembly in picornavirus infections requires specific, new, virus-induced membrane structures and vesicles that subsequently manifest their secondary effect by causing a CPE and eventual cell death.

• Apoptosis. Cells possess natural mechanisms that initiate their self-destruction (apoptosis) by means of predetermined cytoplasmic and nuclear changes. Infections with some viruses may lead to apoptosis. In rapidly replicating viruses, the viral replication process must be decelerated to allow the slow, energy-dependent process of apoptosis to run its course before the cell is destroyed by virus-induced necrosis. The body rapidly eliminates apoptotic cells before an inflammatory reaction can develop, which is apparently why virus-induced apoptosis used to be overlooked so often. Apoptosis can thus be considered a defense mechanism, although certain viruses are able to inhibit it.

Virus Replication without Cell Destruction (Noncytocidal Infection)

• This outcome of infection is observed with certain viruses that do not cause any extensive restructuring of the host cell and are generally released by “budding” at the cell surface. This mode of replication is seen, for example, in the oncornaviruses and mycoviruses' and in the chronic form of hepatitis B virus infection. However, cell destruction can follow as a secondary result of infection, however, if the immune system recognizes viral antigens on the cell surface, classifies it as “foreign” and destroys it.

Latent Infection

• In this infection type, the virus (or its genome) is integrated in a cell, but no viral progenies are produced. The cell is accordingly not damaged and the macroorganism does not manifest disease. This form of infection is found, for instance, with the adenovirus group and in particular the herpesviruses, which can remain latent for long periods in the human body. Latency protects these viruses from immune system activity and thus is part of their survival strategy. However, a variety of initiating events can initiate a lytic cycle leading to manifest disease and dissemination of the virus. Repeated activation of a latent virus is termed recidivation herpes labialis).

Tumor Transformation

• Infections by a number of viruses do not result in eventual host cell death, but rather cause tumor transformation of the cell. This means the cell is altered in many ways, e.g., in its growth properties, morphology, and metabolism. Following an infection with DNA tumor viruses, the type of host cell infected determines whether the cell reaction will be a tumor transformation, viral replication or lytic cycle. The transformation that takes place after infection with an RNA tumor virus either involves no viral replication (nonpermissive infection) or the cell produces new viruses but remains vital (permissive infection).

Carcinogenic Retroviruses (“Oncoviruses”)

• Genome structure and replication of the oncoviruses. The genomes of all oncoviruses possess gag (group-specific antigen), pol (enzymatic activities: polymerase complex with reverse transcriptase, integrase, and protease), and env (envelope glycoproteins) genes. These coding regions are flanked by two control sequences important for regulatory functions called LTR (= long terminal repeats), These sequences have a promoter/enhancer function and are responsible for both reverse transcription and insertion of the viral genome into the cell DNA. Certain oncoviruses possess a so-called “once gene” instead of the pol region (one gene = oncogene, refers to a cellular gene segment acquired by recombination, see below). These viruses also often have incomplete gag and/or env regions. Such viruses are defective and require a helper virus to replicate (complementation. An exception to this principle is the Rous sarcoma virus, which possesses both an onc gene and a complete set of viral genes and can therefore replicate itself.

• Tumor induction by oncoviruses. Both types of carcinogenic retroviruses, i.e., those with no oncogene and intact replication genes (gag, pol, env, flanked by the LTR regions) and those that have become defective by taking on an oncogene, can initiate a tumor transformation. On the whole, oncoviruses play only a subordinate role in human tumor induction.

• Retroviruses without an oncogene: LTR are highly effective promoters. Since the retrovirus genome is integrated in the cell genome at a random position, the LTR can also induce heightened expressivity in cellular protooncogenes (“promoter insertion hypothesis” or “insertion mutagenicity”), which can lead to the formation of tumors. This is a slow process (e.g., chronic leukemias) in which cocarcinogens can play an important role. The transformed cells produce new viruses.

• Retroviruses with an oncogene: a viral oncogene always represents a changed state compared with the original cellular proto-oncogene (deletion, mutation). It is integrated in the cell genome together with the residual viral genome (parts) after reverse transcription, and then expressed under the influence of the LTR, in most cases overexpressed. This leads to rapid development of acute malignancies that produce no new viruses

DNA Tumor Viruses

• Genes have also been found in DNA tumor viruses that induce a malignant transformation of the host cell. In contrast to the oncogenes in oncoviruses, these are genuine viral genes that have presumably developed independently of one another over a much longer evolutionary period. They code for viral regulator proteins, which are among the so-called early proteins. They are produced early in the viral replication cycle and assume essential functions in viral DNA replication. Their oncogenic potential derives among other things from the fact that they bind to the products of tumor suppressor genes such as p53, Rb (antioncogenes, “antitransformation proteins” see above) and can thus inhibit their functions. DNA viruses are more important inducers of human tumors than oncoviruses (example: HHV8, papovaviruses, hepatitis B viruses, Epstein-Barr viruses).

Pathogenesis

• The term “pathogenesis” covers the factors that contribute to the origins and development of a disease. In the case of viruses, the infection is by a parenteral or mucosal route. The viruses either replicate at the portal of entry only (local infection) or reach their target organ hematogenous, monogamously or by neurogenic spread (generalized infection). In both cases, viral replication induces degenerative damage. Its extent is determined by the extent of virus-induced cell destruction and sets the level of disease manifestation. Immunological responses can contribute to elimination of the viruses by destroying the infected cells, but the same response may also exacerbate the course of the disease

• Transmission. Viruses can be transmitted horizontally (within a group of individuals or vertically (from mother to offspring). Vertical infection is either transovarial or by infection of the virus in utero (ascending or diaplacental). Connatal infection is the term used when offspring are born infected.

• Portal of entry. The most important portals of entry for viruses are the mucosa of the respiratory and gastrointestinal tracts. Intact epidermis presents a barrier to viruses, which can, however, be overcome through micro traumata (nearly always present) or mechanical inoculation (e.g., bloodsucking arthropods)

• Viral dissemination in the organism. There are two forms of infection:

• Local infection. In this form of infection, the viruses spread only from cell to cell. The infection and manifest disease are thus restricted to the tissues in the immediate vicinity of the portal of entry. Example: rhinoviruses that reproduce only in the cells of the upper respiratory tract.

• Generalized infection. In this type, the viruses usually replicate to some extent at the portal of entry and are then disseminated via the lymph ducts or bloodstream and reach their target organ either directly or after infecting a further organ. When the target organ is reached, viral replication and the resulting cell destruction become so widespread that clinical symptoms develop. Examples of such infection courses are seen with enteroviruses that replicate mainly in the intestinal epithelium but cause no symptoms there.

• Clinical symptoms in these infections first arise in the target organs such as the CNS (polioviruses, echoviruses) or musculature (coxsackie viruses).

• Course of infection. The organ damage caused by viruses is mainly of a degenerative nature. Inflammatory reactions are secondary processes. The severity of the clinical symptoms depends primarily on the extent of virus-induced (or immunological, see below) cell damage. This means most of the viral progeny are produced prior to the occurrence of clinical symptoms, with consequences for epidemiology and antiviral therapies (p. 404). It also means that infections can go unnoticed if cell destruction is insignificant or lacking entirely. In such cases, the terms inapparent, silent, or subclinical infection are used, in contrast to apparent viral infections with clinical symptoms. Virus replication and release do take place in inapparent infections, as opposed to latent infections (p. 394), in which no viral particles are produced.

• Virus excretion. Excretion of newly produced viruses depends on the localization of viral replication. For example, viruses that infect the respiratory tract are excreted in expired air (droplet infection). It must be remembered that in generalized infections not only the target organ is involved in excretion, but that primary viral replication at the portal of entry also contributes to virus excretion (for example enteroviruses, which replicate primarily in the intestinal wall and are excreted in feces). Once again, since the symptoms of a viral disease result from cell destruction, production, and excretion of new virus progeny precede the onset of illness. As a rule, patients are therefore contagious before they really become ill.

Defense Mechanisms 

• The mechanisms available to the human organism for defense against viral infection can be classified in two groups. The nonspecific immune defenses, in which interferons play a very important part, come first. Besides their effects on cell growth, immune response, and immunoregulation, these substances can build up a temporary resistance to a viral infection. Interferons do not affect viruses directly, but rather induce cellular resistance mechanisms (synthesis of “antiviral proteins”) that interfere with specific steps in viral replication. The specific immune defenses include the humoral immune system, consisting mainly of antibodies, and the cellular immune system, represented mainly by the T lymphocytes. In most cases, cellular immunity is more important than humoral immunity. The cellular system is capable of recognizing and destroying virus-infected cells on the surfaces of which viral antigens are expressed. The humoral system can eliminate only extracellular viruses.

Nonspecific Immune Defenses

• The nonspecific immune defense mechanisms are activated immediately when pathogens penetrate the body’s outer barriers. One of the most important processes in these basic defenses is phagocytosis, i.e., ingestion and destruction of pathogens. Granulocytes and natural killer cells bear most of the responsibility in these mechanisms. Changes in pH and ion balance as well as fever also play a role, for example, certain temperature-sensitive replication steps can be blocked. The most important humoral factor is the complement system. Interferons, which are described below, are also potent tools for fighting off viral infections. The other mechanisms of nonspecific immune defense are described in Chapter 2, (Principles of Immunology.

• Interferons (IFN) are cell-coded proteins with a molecular weight of about 20 kDa. Three types are differentiated (leukocyte interferon = IFNa, fibroblast interferon = IFNb, and immune interferon = IFNc) of which the amino acid sequences are known and which, thanks to genetic engineering, can now be produced in practically unlimited amounts. Whereas the principal biological effects of interferons on both normal and malignant cells are antiviral and antimitotic, these substances also show immunomodulatory effects. Their clinical applications are designed accordingly. In keeping with the scope of this section, the following description of their antiviral activity will be restricted to the salient virological aspects.

• Mx protein. The observation that certain mice are resistant to influenza viruses led to the discovery of the interferon-induced, 75–80 kDa Mx proteins coded for by dominant hereditary Mx genes. Mx proteins accumulate in mouse cell nuclei and inhibit the mRNA synthesis of influenza viruses. Mx- mice are killed by influenza. In humans, Mx proteins accumulate in the cytoplasm, but their mechanism of action is unknown.
  

Specific Immune Defenses

• The specific, adaptive immune defenses include both the humoral system (antibody-producing B cells) and the cellular system (T helper cells and cytotoxic T lymphocytes). In general, viruses the antigens of which are expressed on the surface of the infected cells tend to induce a cellular response and viruses that do not change the antigenicity of their host cells tend to activate the humoral system.

• Humoral immunity. Antibodies can only attack viruses outside of their host cells, which means that once an infection is established within an organ it can hardly be further influenced by antibodies, since the viruses spread directly from cell to cell. In principle, the humoral immune system is thus only capable of preventing a generalized infection, but only if the antibodies are present at an early stage (e.g., induced by a vaccination). Class IgG and IgM antibodies are active in the bloodstream and class IgA is active on the mucosal surface. The effect of the antibodies on the viral particles (“neutralization”) is based on steric hindrance of virus adsorption to the host cells by the antibodies attached to their surfaces. The neutralizing effect of antibodies is strongest when they react with the receptor-binding sites on the capsids so as to block them, rendering the virus incapable of combining with the cellular receptors.

• Cellular immunity. This type of immune defense is far more important when it comes to fighting viral infections. T lymphocytes (killer cells) recognize virus-infected cells by the viral antigens on their surfaces and destroy them. The observation that patients with defective humoral immunity generally fare better with virus infections than those with a defective cellular response underlines the fact that the cellular immune defense system is the more important of the two.

Prevention

• The most important prophylactic measures in the face of potential viral infections are active vaccines. Vaccines containing inactivated viruses generally provide shorter-lived and weaker protection than live vaccines. Passive immunization with human immunoglobulin is only used in a small number of cases, usually as postexposure prophylaxis.

• Value of the different methods. In general, vaccination, i.e., induction of immunity (immune prophylaxis) is the most important factor in prevention of viral infection. Exposure prophylaxis is only relevant to hygienic measures necessitated by an epidemic and is designed to prevent the spread of pathogens in specific situations. Chemoprophylaxis, i.e., administration of chemotherapeutic agents when an infection is expected instead of after it has been diagnosed to block viral metabolism, is now justified in selected cases, e.g., in immunosuppressed patients (see Chemotherapy, p. 404).

• Active immunization. In this method, the antigen (virus) is introduced into the body, either in an inactivated form, or with attenuated pathogenicity but still capable of replication, to enable the body to build up its own immunity 

• & Inactivated vaccines. The immunity that develops after so-called “dead vaccines” are administered is merely humoral and generally does not last long. For this reason, booster vaccinations must be given repeatedly. The most important dead vaccines still in use today are influenza, rabies, some flavivirus, and hepatitis A and B vaccines. Some inactivated vaccines contain the most important immunogenic proteins of the virus. These so-called split vaccines induce more efficient protection and, above all, are better tolerated. Some of them are now produced by genetic engineering methods.

• Live attenuated vaccines. These vaccines confer effective and long-lasting protection after only a single dose, because the viruses contained in them are capable of replication in the body, inducing not only humoral, but sometimes cellular immunity as well, not to mention local immunity (portal of entry!). Such live vaccines are preferable when available. There are, however, also drawbacks and risks, among them stability, the increased potential for contamination with other viruses, resulting in more stringent testing and the possibility that a back-mutation could produce a pathogenic strain (see Variability.

• Vaccines with recombinant viruses. Since only a small number of (surface) viral proteins are required to induce protective immunization, viral vectors are used in attempts to express them in vaccine recipients. Suitable vectors include the least virulent virus strains among the picornaviruses, alphaviruses, and poxviruses. There must be no generalized immunity to the vector in the population so that it can replicate in vaccine recipients and the desired protein will at the same time be expressed. Such recombinant vaccines have not yet been approved for use in humans. A rabies vaccine containing the recombinant vaccinia virus for use in animals is the only practical application of this type so far (p. 390).

• Naked DNA vaccine. Since pure DNA can be inserted into eukaryotic cells (transfection) and the information it carries can be expressed, DNA that codes for the desired (viral) proteins can be used as vaccine material. The advantages of such vaccines, now still in the trial phase, include ease of production and high stability.

• Passive immunization. This type of vaccine involves the injection of antibodies using only human immunoglobulins. The protection conferred is of short duration and only effective against viruses that cause viremia. Passive immunization is usually administered as a postexposure prophylactic measure, i.e.,after an infection or in situations involving a high risk of infection, e.g., to protect against hepatitis B and rabies (locally, bite woundn list the most important vaccines.

Chemotherapy

• Inhibitors of certain steps in viral replication can be used as chemotherapeutic agents to treat viral infections. In practical terms, it is much more important to inhibit the synthesis of viral nucleic acid than of viral proteins. The main obstacles involved are the low level of specificity of the agents in some cases (toxic effects because cellular metabolism is also affected) and the necessity of commencing therapy very early in the infection cycle

• Problems of chemotherapy. As described on, viral replication is completely integrated in cell metabolism. The virus supplies only the genetic    

• formation for proteins to be synthesized by the cell. This close association between viruses and their host cells is a source of some essential difficulties encountered when developing virus-specific chemotherapeutics, since any interference with viral synthesis is likely to affect physiological cellular synthetic functions as well. Specific intervention is only possible with viruses that code for their own enzymes (e.g., polymerases or proteases), which enzymes also react with viral substrates. Another problematic aspect is the necessity of administering chemotherapeutic early, preferably before clinical symptoms manifest, since the peak of viral replication is then usually already past.

• Development of resistance to chemotherapeutics. Acyclovir-resistant strains of herpesviruses, in particular herpes simplex viruses, are occasionally isolated. Less frequently, cytomegaly viruses resistant to ganciclovir are also found. These viruses possess a thymidine kinase or DNA polymerase altered by mutation. Infections caused by resistant herpesviruses are also observed in immunodeficient patients; the pathogens no longer respond to therapy after long-term treatment of dermal or mucosal efflorescences. There are, as yet, no standardized resistance tests for chemotherapy-resistant viruses, so that the usefulness of such test results is of questionable value in confirmed cases. Also, the results obtained in vitro unfortunately do not correlate well with the cases of resistant viruses observed in clinical settings.     

Laboratory Diagnosis 

• The following methods can be used to obtain a virological laboratory diagnosis: 

• Virus isolation by growing the pathogen in a compatible host; usually done in cell cultures, rarely in experimental animals or hen embryos.

• Direct virus detection. The methods of serology, molecular biology, and electron microscopy are used to identify viruses or virus components directly, i.e., without preculturing, in diagnostic specimens

• Serodiagnostics involving assay of antiviral antibodies of the IgG or IgM classes in patient serum

• Indication and methods. Laboratory diagnostic procedures for virus infections are costly, time-consuming, and require considerable staff time. It is therefore important to consider carefully whether such tests are indicated in a confirmed case. The physician in charge of treatment must make this decision based on detailed considerations. In general, it can be said that laboratory diagnostics are justified if further treatment of the patient would be influenced by an etiological diagnosis or if accurate diagnostic information is required in the context of an epidemic or scientific research and studies.

Virus Isolation by Culturing

• In this approach, the virus is identified based on its infectivity and pathogenicity by inoculating a host susceptible for the suspected virus—in most cases cell cultures—with the specimen material. Certain changes observed in the culture (cytopathic effect indicate the presence of a virus.

• Sampling and transport of diagnostic specimens. Selection of suitable material depends on the disease and suspected viral species (see Chapter 8). Sampling should generally be done as early as possible in the infection cycle since, as was mentioned on p. 399, viral replication precedes the clinical symptoms. Sufficiently large specimens must be taken under conditions that are as sterile as possible, since virus counts in the diagnostic material are almost always quite low. Transport must be arranged quickly and under cold box conditions. The half-life of viruses outside the body is often very short and must be extended by putting the material on ice. A number of virus transport mediums are commercially available. A particular transport medium should be selected after consulting the laboratory to make sure the medium is compatible with the laboratory methods employed. Such mediums are particularly important if the diagnostic material might otherwise dry out.

• Information provided to the laboratory. The laboratory must be provided with sufficient information concerning the course and stage of the disease, etc. This is very important if the diagnostic procedure is to be efficient and the results accurate. Clinical data and tentative diagnoses must be provided so the relevant viruses can be looked for in the laboratory. Searching for every single virus potentially present in the diagnostic material is simply not feasible for reasons of cost and efficiency.

• Laboratory processing of the material. Before the host is inoculated with the specimen material for culturing, contaminant bacteria must be eliminated with antibiotics, centrifugation, and sometimes filtering. All of these manipulations of course entail the risk of virus loss and reduction of test sensitivity, so the importance of sterile sampling cannot be overemphasized. In a few cases, virus enrichment is indicated, e.g., by means of ultracentrifugation.

• Selection of a host system. The host system to be used is chosen based on the suspected (and relevant) virus infectors. Observation and incubation times, and thus how long a laboratory diagnosis will take, also depend on the viral species under investigation.

• .Identification of the viruses is based first on the observed cell changes, then determined serologically using known antibodies and appropriate methods such as immunoelectron microscopy, EIA, or the neutralization test (see p. 402 for the neutralization mechanism). Methods that detect the viral genome by means of in-situ or filter hybridization are now seeing increasing use.

• Significance of results. The importance of virus isolation depends on the virus type. In most cases, isolation will be indicative of the etiology of the patient’s disease. In some cases, (in particular the herpesvirus and adenovirus group, see Chapter 8), latent viruses may have been activated by a completely different disease. In such cases, they may of course be isolated, but have no causal connection with the observed illness.

• Amplification culture. In this method, the virus is grown for a brief period in a cell culture. Before the CPE is observed, the culture is tested using the antigen and genomic methods described. This is also known as a “shell vial assay” because the cells are grown on coverslips in shell vials (test tubes with screw caps). Using this arrangement, method sensitivity can be increased by centrifuging the diagnostic material onto the cell monolayer. The greatest amount of time is saved by detecting the virus-specific proteins produced early in the infection cycle, which is why the search concentrates on such so-called “early antigens” (see p. 388). Using this method, the time required to confirm a cytomegaly virus, for instance, can be shortened from four to six weeks to only two to five days with practically no loss of sensitivity compared to classic isolation methods.

Direct Virus Detection

• In this diagnostic approach, the viruses are not identified as infectious units per se, but rather as viral particles or parts of them. The idea is to find the viruses directly in the patient material without prior culturing or replication. Viruses in serous fluids such as the contents of herpes simplex or varicellazoster blisters can be viewed under the electron microscope (EM). It must be remembered, however, that the EM is less sensitive than virus isolation in cultures by a factor of 105. Viral antigens can be detected in secretions using enzyme immunoassay (EIA), passive agglutination, or in smears with immunofluorescence performed with known antibodies, for instance monoclonal antibodies. Analogously, the viral genome can be identified by means of filter hybridization, or in smears or tissue sections with in-situ hybridization using DNA or RNA complementary to the viral genome as a probe. 

• Sampling and transport of diagnostic specimens. Transport of patient material for these methods is less critical than for virus isolation. Cold box transport is usually not required since the virus need not remain infectious.

• Electron microscopy. For negative contrast EM, the specimen is transported to the laboratory without any additives (dilutio

Virus Detection Following Biochemical Amplification

• Polymerase chain reaction. This method provides a highly sensitive test for viral genomes. First, nucleic acid is extracted from the patient material to be analyzed. Any RNA virus genome present in the material is transcribed into DNA by reverse transcriptase. This DNA, as well as the DNA of the DNA viruses, is then replicated in vitro with a DNA polymerase as follows: after the DNA double strand has been separated by applying heat, two synthetic oligonucleotides are added that are complementary to the two ends of the viral genome segment being looked for and can hybridize to it accordingly. The adjacent DNA (toward each 50 end) is thencopied with an added polymerase, whereby the oligonucleotides act as primers. The new and old strands are once again separated by heat and the reaction is started over again. Running several such cycles amplifies the original viral DNA by a factor of many thousands. Beginning with the second generation, the newly synthesized DNA strands show a uniform, defined length and are therefore detectable by means of gel electrophoresis. The specificity of the reaction is verified by checking the sequences of these DNA strands by means of hybridization or sequencing. The amplification and detection systems in use today for many viruses are increasingly commercially available, and in some cases are also designed to provide quantitative data on the “viral load.”

Serodiagnosis

• If a viral infection induces humoral immunity (see p. 48f. and 401), the resulting antibodies can be used in a serodiagnosis. When interpreting the serological data, one is confronted by the problem of deciding whether the observed reactions indicate a fresh, current infection or earlier contact with the virus in question. Two criteria can help with this decision:
• Detection of IgM (without IgG) proves the presence of a fresh primary infection. IgM is now usually detected by specific serum against human IgM in the so-called capture test, an EIA (p. 128).
• A fourfold increase in the IgG titer within 10–14 days early on in the course of the infection or a drop of the same dimensions later in the course would also be confirmation.