Papovaviridae Classification Essay

1. Introduction

The electron microscope has been a powerful tool in the characterization of viruses. First constructed by Ernst Ruska and Max Knoll in the early 1930s, the electron microscope was soon used in the visualization of an orthopoxvirus, mouse ectromelia virus [1]. The potential for using this newly developed instrument for the understanding of the ultrastructure of viruses and other pathogens was quickly recognized by Helmut Ruska, a physician and the brother of Ernst [2,3]. In the ensuing years, many viruses were recognized by electron microscopy (EM), and were characterized by features such as size, shape, the appearance of the capsid, presence or absence of an envelope, surface projections, and method and site of morphogenesis. These traits can identify the virus to a family level, since, in general, the morphologic features within a given family are the same. Interestingly, the first report of the International Committee on Taxonomy of Viruses (ICTV) released in 1971 recognized only two families of viruses, Papovaviridae and Picornaviridae. All other recognized viruses were listed as “Unassigned” and mostly categorized as genera [4]. This was at a time when examining virus ultrastructure by EM permitted the grouping of viruses on a morphological basis [5,6], and in the future EM played a critical role in the taxonomic classification of viruses [7].

Electron microscopists typically use negative stain and thin section EM for diagnostic virology. Negative stain EM entails adsorbing a biological fluid (e.g., cell culture supernatant, urine, cerebral spinal fluid, etc.) onto an EM grid coated with a plastic film. The viruses that adhere to the grid are stained with a heavy metal which pools around the viruses, giving them an appearance as seen in a photographic negative, i.e., a light specimen against a dark background. Proteins on the surface of the nucleocapsid or envelope become apparent, and which allows for a morphologic differentiation among the different virus families [8]. In thin section EM, tissues or tissue culture cells are embedded in an epoxy resin and cut into ultra-thin sections (e.g., 70–90 nm). This allows the electron microscopist to examine cells and viruses in a cross-sectional view. The virus family can be determined by evaluating the morphogenesis of the virus by looking at the site of assembly, the location of envelope acquisition, and other clues that may be offered by replication complexes [9].

There are a few examples where either thin section or negative stain EM, or both, can go beyond the family level classification. Currently, there are 103 recognized virus families, of which 22 infect humans. This review will discuss six examples where morphologic features allow for diagnosis of a virus not just to the family level, but to the genus level.

2. Virus Families

2.1. Poxviridae

Poxviruses are the largest and most complex of the viruses causing human disease. The most infamous would be variola virus, the causative agent of smallpox, which was eradicated by a concerted global effort overseen by the World Health Organization; the last naturally occurring case was in 1977. Infections with a poxvirus will produce pock(s), or pustule(s), on the skin and also internally on visceral organs with some species. The genera of poxviruses that can cause human disease include Orthopoxvirus, Parapoxvirus, Molluscipoxvirus, and Yatapoxvirus.

Although poxviruses contain DNA, the DNA replication and virus assembly do not take place in the nucleus but rather in the cytoplasm. Virus factories, or virosomes, are created and nascent crescents are formed and engulf the unit genome. Immature particles are spherical, but condense down to a dumbbell-shaped intracellular mature virus (IMV). The IMV is engulfed by Golgi vesicles, migrates to the cell surface or into microvilli and fuses with the cell membrane, releasing a particle wrapped in a single membrane known as an enveloped extracellular virus (EEV).

By thin section EM, in addition to the viral factories, some poxviruses have other cytoplasmic structures know as acidophilic-type inclusions (A-type inclusions) consisting of a matrix containing the A-type inclusion protein and other proteins, with occluded intracellular mature particles [10,11] (Figure 1A). Virus species with these inclusions include cowpox, ectromelia, raccoonpox, skunkpox, volepox, and fowlpox viruses.

The viruses in the genera Orthopoxvirus and Parapoxvirus can be distinguished by negative stain EM (Figure 1B,C). Orthopoxviruses are rectangular, approximately 225 × 300 nm in size and have a surface pattern of short, whorled filaments. On the other hand, parapoxviruses are oval, average only about 150 × 200 nm in size, and have a crisscross filamentous surface pattern. Unfortunately, these two genera cannot be definitively differentiated by thin section EM.

Figure 1. (A) Thin section image of raccoonpox, showing viral factories (*) and A-type inclusions (arrows). Bar, 500 nm; (B) Negative stain image of a clinical sample of monkeypox virus (genus Orthopoxvirus). Bar, 100 nm; (C) Negative stain image of a clinical sample of orf virus (genus Parapoxvirus). Bar, 100 nm.

Figure 1. (A) Thin section image of raccoonpox, showing viral factories (*) and A-type inclusions (arrows). Bar, 500 nm; (B) Negative stain image of a clinical sample of monkeypox virus (genus Orthopoxvirus). Bar, 100 nm; (C) Negative stain image of a clinical sample of orf virus (genus Parapoxvirus). Bar, 100 nm.

2.2. Reoviridae

Reoviruses derive their name from Respiratory Enteric Orphan viruses. This paper will describe viruses that are members of the genera Orthoreovirus and Rotavirus. Orthoreoviruses are in the subfamily Spinoreovirinae and contain large spikes or turrets at the 12 icosahedral vertices of the core particle, while rotaviruses are in the subfamily Sedoreovirinae and do not have large surface projections on the core particles [12].

Orthoreoviruses are spread by the respiratory or fecal-oral routes. The genome consists of 10 segments of linear double-stranded RNA. By negative stain EM, virions are approximately 85 nm in diameter, are roughly spherical, and possess a double-layered protein capsid (Figure 2A).

Rotaviruses are the cause of severe diarrheal disease in infants and young children, and were first recognized by EM in 1973 [13]. The genome is composed of 11 segments of linear double-stranded RNA. By negative stain EM, virus particles are 70 nm in diameter and are constructed of three concentric protein layers. Virions have a wheel-like appearance (rota is Latin for “wheel”) with a sharp definition of the outer margin (Figure 2B).

Figure 2. (A) Negative stain EM image of orthoreovirus particles, with a stain-penetrated particle (arrow) showing the double capsid layers; (B) Negative stain EM image of rotavirus particles. Bars, 100 nm. (Figure B, courtesy of Charles D. Humphrey, Centers for Disease Control and Prevention, Atlanta, GA, USA.)

Figure 2. (A) Negative stain EM image of orthoreovirus particles, with a stain-penetrated particle (arrow) showing the double capsid layers; (B) Negative stain EM image of rotavirus particles. Bars, 100 nm. (Figure B, courtesy of Charles D. Humphrey, Centers for Disease Control and Prevention, Atlanta, GA, USA.)

2.3. Retroviridae

The retroviruses are divided into two subfamilies. The genera in the subfamily Orthoretrovirinae that can infect humans are Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Gammaretrovirus, and Lentivirus. Spumavirus is the only genus in the subfamily Spumaretrovirinae. The viral genome for all but the spumaviruses consists of a dimer of positive-sense, single-stranded RNA held together by hydrogen bonds. Spumaviruses contain double-stranded DNA. All retroviruses use the enzyme reverse transcriptase to transcribe an RNA template into complementary DNA.

Retroviridae is, morphologically, a multi-faceted family of viruses. As illustrated in a drawing that appeared in a publication by Gelderblom and Boller [14] (Figure 3, top), in thin section EM, members of the family can be assigned to a particular genus based on the morphogenesis of the virus and on the appearance of mature virus particles [15,16] (Figure 3, bottom).

The genera Alpharetrovirus and Gammaretrovirus, such as avian leukosis virus (ALV) and murine leukemia virus (MLV), respectively, were previously known as C-type particles; the cores form concomitantly with budding, and are centered in the middle of mature particles. Viruses previously known as B-type (such as mouse mammary tumor virus (MMTV)) and D-type (such as Mason-Pfizer monkey virus (M-PMV)) are now part of the genus Betaretrovirus. Both types are formed by envelopment of pre-formed cores, which are known as A-type particles, and mature into eccentrically located cores surrounded by the viral envelope. The viruses in the genus Deltaretrovirus, such as bovine leukemia virus (BLV), have a crescent-shaped budding profile which is composed of an electron-dense nucleoid and the nascent viral envelope. The cores of the mature virions are somewhat pleomorphic and fairly homogeneous, and there is often an electron-lucent space between the core and the envelope. Human immunodeficiency virus (HIV) is an example of the genus Lentivirus, and these viruses also have a crescent-shaped budding profile, which can be released from the cell to form a doughnut-shaped particle. The nucleoid then condenses into an electron-dense core that is cone-shaped, but can appear as a rod. The viruses in the genus Spumavirus, such as chimpanzee foamy virus (CFV), are seen as contaminants in some cell cultures derived from animal organs. Pre-formed cores are enveloped at cellular membranes or the plasma membrane, and the cores do not condense.

Figure 3. (Top) schematic diagram of the morphogenesis of the members of the family Retroviridae; (Bottom) budding profiles and mature virions. (A) Avian leukosis virus (genus Alpharetrovirus); (B) Mouse mammary tumor virus (genus Betaretrovirus); (C) Murine leukemia virus (genus Gammaretrovirus); (D) Bovine leukemia virus (Deltaretrovirus); (E) Human immunodeficiency virus 1 (genus Lentivirus); (F) Simian foamy virus (genus Spumavirus). (Top, reproduced with permission from Reference [14]. Copyright 2002 Kluwer Academic/Plenum Publishers. Bottom, reproduced with permission from Reference [17]. Copyright 1997 Cold Spring Harbor Laboratory.

Figure 3. (Top) schematic diagram of the morphogenesis of the members of the family Retroviridae; (Bottom) budding profiles and mature virions. (A) Avian leukosis virus (genus Alpharetrovirus); (B) Mouse mammary tumor virus (genus Betaretrovirus); (C) Murine leukemia virus (genus Gammaretrovirus); (D) Bovine leukemia virus (Deltaretrovirus); (E) Human immunodeficiency virus 1 (genus Lentivirus); (F) Simian foamy virus (genus Spumavirus). (Top, reproduced with permission from Reference [14]. Copyright 2002 Kluwer Academic/Plenum Publishers. Bottom, reproduced with permission from Reference [17]. Copyright 1997 Cold Spring Harbor Laboratory.

2.4. Herpesviridae

The genus Cytomegalovirus (CMV) is within the subfamily Betaherpesvirinae, and infection with these viruses typically results in an increase in cell volume (cytomeglia). CMV-infected cells have nuclear inclusions, characteristic of herpesviruses, but also have cytoplasmic inclusions. The genomes of herpesviruses in general are composed of linear, double-stranded DNA. Replication takes place in the nucleus where nucleocapsids are formed, are surrounded by an “inner” tegument, and travel to the cytoplasm by budding upon the inner nuclear membrane, passing through the perinuclear space, and fusing with the outer nuclear membrane to egress to the cytoplasm. Additional tegument proteins attach to the capsid, either within the cytosol and/or at the future envelopment site on the membranes of the Golgi complex [18] (Figure 4A).

Unlike the other members of the family Herpesviridae, the cytoplasm in cells infected by CMV contains numerous aggregations of enveloped tegument proteins that lack capsids (Figure 4B). These are known as dense bodies [19], which are highly immunogenic and have been proposed as CMV vaccine candidates since they induce both humoral and cellular immune responses [20].

Figure 4. (A) Thin section EM image of the cytoplasm of a cell infected with human herpesvirus 7. Note that although there are nucleocapsids surrounded by tegument (arrowhead), there are no dense bodies; (B) Cell infected with simian CMV, with dense bodies (arrows) and virus particles (arrowhead) in the cytoplasm. Bars, 500 nm. (Figure B, courtesy of Sara E. Miller, Duke University Medical Center, Durham, NC, USA.)

Figure 4. (A) Thin section EM image of the cytoplasm of a cell infected with human herpesvirus 7. Note that although there are nucleocapsids surrounded by tegument (arrowhead), there are no dense bodies; (B) Cell infected with simian CMV, with dense bodies (arrows) and virus particles (arrowhead) in the cytoplasm. Bars, 500 nm. (Figure B, courtesy of Sara E. Miller, Duke University Medical Center, Durham, NC, USA.)

2.5. Filoviridae

This family includes Ebolavirus and Marburgvirus genera and the recently recognized Cuevavirus genus, which is not known to cause human disease. Filoviruses are nonsegmented, negative-sense, single-stranded RNA viruses. Virions are pleomorphic, appearing as long filamentous particles, but also as branched, 6-shaped, U-shaped, or circular particles.

The viruses in the genera Ebolavirus and Marburgvirus are well-known as being some of the deadliest known viruses, with case fatality rates for Ebola virus reported at 50% to 90%, and rates for Marburg virus at 24% to 88%. Early symptoms for both diseases include sudden onset of fever, headaches, weakness, muscle pains, and a sore throat. As the diseases progress, additional symptoms such as vomiting, diarrhea, impaired kidney and liver function, and sometimes a rash and internal and external bleeding may develop.

There have been differences reported in the morphologic features of Ebola and Marburg viruses. First, the lengths of the virus particles of the two genera vary, although there have been different lengths reported. For instance, there have been reports of 665 nm, 790 nm, and 860 nm lengths for Marburg virus, and 805 nm, 970 nm, and 1200 nm for Ebola virus, but clearly Ebola viruses are longer [21,22,23] (Figure 5A,B). A second difference is found in the ultrastructure of the intermediate inclusion of the viruses. Ebola virus inclusions have distinct preformed nucleocapsids mixed with lighter-staining matrix material and, at times, naked nucleocapsids are present (Figure 5C). The inclusions in Marburg virus infections begin with light-staining nascent viral material, which increases in electron density as the infection progresses, and has 45–60 nm spheres of inclusion material surrounding the inclusion. Later, the intermediate inclusions show a dispersal of material and a loss of the spheres [23,24] (Figure 5D).

Figure 5. Negative stain images of Marburg virus (A) and Ebola virus (B), illustrating that Ebola virus has a longer length than Marburg virus. Bars, 100 nm; (C) Thin section image of a large inclusion in an Ebola virus-infected cell. Bar, 500 nm; (D) Intermediate inclusion in the cytoplasm of a Marburg virus-infected cell. Bar, 580 nm. (Figure A, courtesy of Russell Regnery, Centers for Disease Control and Prevention; Figure D, courtesy of Thomas Geisbert, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA.)

Figure 5. Negative stain images of Marburg virus (A) and Ebola virus (B), illustrating that Ebola virus has a longer length than Marburg virus. Bars, 100 nm; (C) Thin section image of a large inclusion in an Ebola virus-infected cell. Bar, 500 nm; (D) Intermediate inclusion in the cytoplasm of a Marburg virus-infected cell. Bar, 580 nm. (Figure A, courtesy of Russell Regnery, Centers for Disease Control and Prevention; Figure D, courtesy of Thomas Geisbert, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA.)

2.6. Bunyaviridae

The family Bunyaviridae contains four genera that can infect humans—Orthobunyavirus, Nairovirus, Phlebovirus, and Hantavirus. Viruses contain single-stranded RNA with three RNA segments that are negative sense, with the exception of phleboviruses which have one ambisense segment. These are zoonotic viruses, where each virus is associated with a specific vector or natural reservoir, including mosquitoes, ticks, sand flies, and rodents. Patients will usually have a hemorrhagic syndrome which is characterized by fever, increased capillary permeability, leukopenia and thrombocytopenia.

Papovaviruses undergo two types of interactions with host cells (Fig. 66-3). Permissive cells support viral replication, which results in the synthesis of progeny virus and cell death (lysis). Nonpermissive cells do not support viral replication but can be transformed. Permissive cells can, on rare occasions, be transformed if viral replication is incomplete, such as when a defective virus infects a cell. When cells are transformed, the cells survive, the cellular phenotype is altered, and no progeny virus is produced. Most of our knowledge of viral replication and cell transformation is based on studies with SV40 and mouse polyoma virus. The expression of SV40-specific events in lytic and in transforming infections is compared in Table 66-2.

Polyomaviruses

The polyomaviruses have a narrow host range. Permissive cells are derived from the natural host of each isolate (monkey cells for SV40, mouse cells for polyoma virus, and human cells for BK and JC viruses). Not all cell types from the susceptible species will support viral replication.

The infecting virion first attaches to specific receptors on permissive cells, then penetrates the plasma membrane and is transported to the nucleus, where the viral DNA is uncoated and released. During the early phase of the lytic cycle, the virus drives the cell into the S phase, thereby providing cellular enzymes associated with DNA metabolism, such as thymidine kinase and DNA polymerase. The virus uses the cellular enzymes for its own replication, as the polyomavirus genetic content is too limited to encode all of the necessary replicative functions. The induction of host cell synthetic processes depends on the expression of the early portion of the viral genome and the synthesis of large T antigen. The large T antigen binds cellular tumor suppressor proteins p53 and Rb and disrupts their normal cell cycle regulatory functions.

The early proteins (tumor antigens) are synthesized soon after infection and reach detectable levels at about 12 to 15 hours after infection. Viral DNA synthesis begins shortly after that time. The large T antigen is a prerequisite for viral DNA replication. It binds to viral DNA at the site of initiation of DNA synthesis and is essential for viral replication in permissive cells. DNA replication proceeds bidirectionally from the unique origin site. The expression of late viral genes occurs after DNA synthesis begins. Early RNA is transcribed from half of one strand of viral DNA (E strand), whereas late viral RNA is transcribed from the other half of the genome, using the opposite strand of DNA (L strand) as a template (Fig. 66-1). T antigen binding initiates transcription of late viral RNA in addition to initiating viral DNA replication.

The structural viral proteins VP1, VP2, and VP3 are synthesized from late viral mRNA and are transported into the nucleus. Progeny virions are assembled and accumulate in the nucleus, becoming detectable by 24 hours after infection. Eventually the host cells are killed. As a group, the papovaviruses have the longest (slowest) growth cycle of the DNA viruses. Cell lysis usually does not occur until 40 to 48 hours after infection. Progeny viral particles are frequently not efficiently released from cell debris.

An important biologic property of the polyomaviruses is their ability to transform cells (i.e., to convert normal cells into tumor cells). Because transformation requires cell survival and multiplication, it is not compatible with lytic (productive) infections. Transforming infections are basically abortive and may result either from viral infection of nonpermissive cells or from the infection of permissive cells with defective viral genomes (Fig. 66-3). Permanent transformation by a polyomavirus is very rare (see Ch. 47).

The virus-induced early events that are expressed in permissive cells also occur in nonpermissive cells (Table 66-2). Tumor antigens are synthesized, cell regulatory proteins are bound, and cellular DNA synthesis is stimulated. However, no free viral DNA synthesis occurs, and late viral genes that encode capsid proteins are not expressed. The viral genome becomes integrated in the cellular chromosome. Integration of viral sequences into host cell DNA is random and can occur at many different sites. In general, only one or a very few viral DNA copies are present in an individual transformed cell. The entire viral genome need not be retained in transformed cells, but an intact early region is required because the transforming protein (the large T antigen) must be synthesized continuously for a cell to remain transformed.

Viral transformation and tumor induction involve two or more separate viral functions. One event is responsible for cell immortalization (unlimited cell proliferation), whereas another event mediates structural and behavioral changes characteristic of the transformed phenotype. The large T antigen is the critical gene product in the SV40 system. The ability of large T antigen to bind cellular p53 and Rb family proteins is required for SV40 transforming activity. In transformed cells, the large T antigen localizes predominantly in the nucleus, although a small fraction (no more than 5 percent) is associated with the plasma membrane, where it is involved in virus-specific transplantation antigen reactions. In the mouse polyoma virus system, two early proteins have a role in carrying out transforming functions. Immortalization of primary cells is mediated by the large T antigen, which is localized in the nucleus. However, those cells remain phenotypically normal. In contrast, the polyoma virus middle T antigen (which associates with the plasma membrane) transforms immortalized cells, but is not able to alter primary cells. Middle T antigen binds cellular proteins, including c-src, and alters cellular growth signal transduction events.

Transformation is a stable, inherited change in cell properties. The most prominent phenotypic modifications associated with SV40-transformed cells include altered morphology (more rounded); altered growth patterns (increased growth rate, decreased requirement for serum growth factors, loss of contact inhibition, and enhanced ability to grow in semisolid medium [anchorage independence]); biochemical changes (increased metabolic rate, increased glycolysis, changes in properties of the cell membrane, synthesis of new antigens in the cell); and tumorigenicity (production of tumors when transformed cells are injected into appropriate test animals).

Papillomaviruses

Papillomaviruses have a high tropism for epithelial cells of the skin and mucous membranes. Replication of the viruses depends strongly on the differentiated state of the cell. When present, progeny virions can be detected only in nuclei of cells in the upper layers of the infected epidermis (Fig. 66-4). Viral nucleic acid is maintained in basal cells at low copy numbers, where it replicates in synchrony with the cell cycle. Vegetative viral DNA synthesis occurs predominantly in the stratum spinosum and the stratum granulosum, and capsid protein expression is restricted to the uppermost layer of terminally differentiated epidermal cells. Viral particles can be detected easily in some types of warts (e.g., hand and plantar warts), but may not be found in other types of lesions (e.g., those of the larynx, external genitalia, and cervix). Certain events in the viral life cycle presumably depend on cellular factors present in specific differentiated states of epithelial cells. This dependence of viral replication on cell differentiation is responsible for the failure of researchers to obtain a reproducible tissue culture system that is permissive for papillomavirus replication or transformation.

Figure 66-4

Schematic representation of a skin wart (papilloma). The papillomavirus life cycle is tied to epithelial cell differentiation. The terminal differentiation pathway of epidermal cells is shown on the left. Events in the virus life cycle are noted on the (more...)

Regulation of gene expression in papillomaviruses is much more complex than in polyomaviruses. Viral DNA remains episomal (free) in benign lesions, whereas it is integrated into host chromosomal DNA in malignant cells (e.g., cervical carcinoma). The E6 and E7 open reading frames are the transforming genes; both are required for cell transformation.

The papillomaviruses induce benign tumors (warts) of the epithelium in their natural hosts. A few types are associated with carcinoma development. This expression of oncogenic potential in natural hosts is in marked contrast to the actions of the polyomaviruses, which do not cause tumors in natural hosts. The papillomaviruses have a narrow host range; no interspecies transmission has been documented.

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