Viruses were established as agents that cause human disease at the beginning of the 20th century. Their small size (approximately 2- to 60-fold smaller than a standard Gram-positive staphylococcus or streptococcus bacterium) gave viruses the distinct property of being able to pass through the conventional filters of the day, allowing their identification as "filterable agents." Today viruses have been identified that affect every kingdom—animals, plants, and bacteria. These ubiquitous agents therefore have the potential to influence the entire biota of the planet. While this chapter will focus on human viruses, the general principles discussed can be applied to other virus families.

Viruses are molecular pathogens. They possess no metabolism of their own and can be thought of as molecular parasites. A conventional virus is made up of two or three major components. A nucleic acid genome, which can be DNA or RNA (single- or double-stranded, contiguous or segmented) contains all of the genetic information and is responsible for encoding all of the

Microbial Forensics

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virus-specific macromolecules of the pathogen. Due to the aggressive application of molecular biological techniques, the sequences of many viruses are known and fully annotated. This nucleic acid genome is packaged in a protein shell called a capsid. The capsid proteins generally self-assemble to form the shell, which takes on an icosahedral, helical, or complex symmetry depending on the virus family. The functions of this capsid include packaging of the genome, protecting the genome from environmental insults, and effectively delivering the nucleic acid to the inside of living cells. Most RNA viruses and some DNA viruses are surrounded by a lipid envelope that is derived from the host cell. The membranes of enveloped viruses all contain viral-specific gly-coproteins that aid in viral tropism.

It is interesting to note that the conventional virus described above is not the simplest molecular pathogen. RNA-only agents called viroids exist that can kill plants without ever making a protein. In addition, protein-only pathogens called prions "replicate" in the absence of a nucleic acid genome by inducing a conformational change in other normal prp proteins. Prions cause degenerative neurological diseases called spongiform encephalopathies in humans. The bottom line is that it is truly a molecular jungle out there.

There are eight basic steps in a viral infection of a eukaryotic cell. The first is adsorption of the virion to the cell surface through an interaction of viral proteins with specific cellular receptors and, in some cases, coreceptor molecules. Viruses can specifically and tightly interact with proteins (e.g., HIV and CD4), carbohydrates (e.g., influenza virus and sialic acid), and lipids (e.g., B19 parvovirus and globoside). This virus-receptor interaction in large part determines the tropism of many viruses. Preventing this interaction is the goal of neutralizing antibodies that are generated by the immune system. The second step is viral penetration of the cell surface. This can be done by direct membrane fusion or endocytosis. The third step is uncoating of the viral genome inside the cell. In many cases, uncoating is due to pH changes that occur inside endocytotic vesicles that result in structural rearrangements of viral surface proteins. The fourth step is primary transcription/gene expression. Many viruses are designed to actively express their genomes immediately upon uncoating. This is accomplished via several strategies, including the packaging of viral polymerases, packaging or production of strong transcriptional transactivation proteins, and the simple fact that some viral genomes can serve directly as mRNAs. Step five is replication of the viral genome. Since viral-encoded proteins play a key role in this step for many viruses, this step has been a prime target for the development of antivirals. The next step is called "secondary transcription," or gene expression that occurs off of progeny genomes. This is an important step that viruses use to amplify their gene expression to obtain maximal yields of viral progeny. It should also be noted that many viruses make different sets of proteins at early and late times postinfection. Early proteins generally include replication and transcription factors as well as proteins that allow the virus to usurp cellular metabolism. Late protein production focuses on virion structural components. The seventh step is the packaging of progeny viral genomes. Viral capsids in most cases self-assemble, and the genomic nucleic acid must be properly inserted. The final step is the release of progeny virions from the cell. For enveloped viruses, this involves budding and the acquisition of a lipid bilayer membrane.

There are several ways to determine whether a cell is infected with a virus. First, many viral infections elicit cytopathic effects such as the rounding of cells, shrinkage, aggregation, lysis, or cell-cell fusion. These effects are due in large part to the expression of specific viral proteins or the shut-down of host cell macromolecular synthesis that occurs during the infection. Second, focal points of viral replication and assembly can sometimes be observed. These are referred to as inclusion bodies; a classic example is the detection of Negri bodies (cytoplasmic acidophilic inclusion bodies) in rabies virus-infected samples. Third, viral proteins expressed on the surface of an infected cell can sometimes bind many red blood cells, a phenomenon called "hemadsorption" that can be very striking when observed in a light microscope. This technique can be very useful if a viral infection shows very little cytopathic effect. The fourth way to directly detect viral-specific gene products is through the use of antibodies in western blots, immunofluorescence, or enzyme-linked immunosorbent assays (ELISAs). Finally, polymerase chain reaction (PCR) with virus-specific primers can be used to directly identify viral-infected tissue. The use of PCR-based assays as a powerful molecular epidemiologic tool is discussed further below.

The quantitation of virus in a sample can be approached using either physical- or biological-based assays. Physical methods such as particle counts using an electron microscope, hemaglutination assays, and ELISAs/(RIAs) can provide an approximation of the total number of viral particles in a sample, but do not address the fact that for many viruses, the particle-to-biologically active particle ratio is rather low. Therefore biological assays that include serial dilutions in conjunction with plaque assays, focus-forming assays, or determination of the infectious dose needed to kill 50% of a target laboratory animal test group are often considered the gold standard.

There are several key definitions and unique aspects of viral genetics that should be stressed. First, a field or street isolate of a virus is obtained directly from the natural host. Viruses that are passaged in cell culture often adapt to growth under in vitro conditions and may lose or gain some properties relative to the field isolate. Second, co-infection with two or more viruses that contain a segmented genome can result in a reassortment of segments in the progeny virions. This can result in dramatic changes in the biologic and/or antigenic properties of the new viruses. Third, while DNA viruses have a mutation rate of 10-8 to 10-11 per nucleotide incorporated, RNA viruses have a million-fold greater mutation rate (10-3 to 10-4). This is due to the presence of error-prone polymerases that lack the ability to proofread. Fourth, pheno-typic mixing or pseudotype formation can occur in an infection by two different viruses when progeny viruses are produced that contain the capsid of virus "A" surrounding the nucleic acid genome of virus "B." In other words, phenotypic mixing is the mixing of nonnucleic acid components between two viruses. Finally, many viruses generate defective progeny viruses as a normal part of their life cycle. While this is one reason for the low particle-to-plaque-forming-unit ratios that are observed in viral preparations, the production of defective particles can have biological consequences as well. It is well established for several RNA viruses that defective particles are naturally produced that interfere with the replication of the wild-type virus. These defective interfering particles moderate the course of an infection and can support the establishment of persistent infections.

One of the best ways to control viral infections in a susceptible population is to prevent them from occurring through the judicious use of vaccines. Vaccines have reduced the incidence of measles, mumps, and rubella by >99.7% in the U.S., and have completely eliminated natural transmission of smallpox and poliovirus. The three main types of vaccines currently in use are killed (i.e., formalin-fixed viruses), live attenuated vaccine strains, and subunit vaccines made from recombinant proteins. Additional types of vaccines, including DNA-based vaccines and use of single vectors for broad vaccination, may well be on the horizon.

Historically, the availability of effective antiviral drugs has lagged behind that of other antimicrobials. However due to advances in molecular virology and rational drug design, the arsenal of antiviral compounds is growing quickly. The prime targets of available antivirals include uncoating (e.g., amantadine prevents intraviral pH changes in influenza virions), viral polymerases to prevent replication (e.g., AZT, ribavirin, and acyclovir), viral proteases to prevent maturation of virions, neuraminidases to prevent viral release from cells, and cellular enzymes involved in protein synthesis (interferons). The application of antibodies for the treatment or prevention of viral diseases is also becoming more common. In summary, the antiviral arena is definitely an evolving field.

The International Committee on the Taxonomy of Viruses has classified the major human viral pathogens into approximately 21 families. Since more viruses remain to be discovered and the relationships of individual viruses to human disease continue to expand, this number is likely an underestimate. Viral classifications are based on a variety of factors. First is morphology—the size of the particle, its shape, and the presence or absence of membranes. Other factors include the properties of the viral genome, viral genomic organization and expression patterns, antigenic consideration, and biological relationships such as host range, etc.

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