Chapter 4 Viruses and prions

Viruses are one of the smallest forms of microorganism and infect most other forms of life: animals, plants and bacteria. They can also cause severe acute oral and orofacial disease, produce oral signs of systemic infection, and be transmitted to patients and dental staff. The main features that characterize viruses are:

• small size (10–100 nm), averaging about one-tenth the size of a bacterium

• genome consisting of either DNA or RNA but never both; single- (ss) or double-stranded (ds); linear or circular (the encoding of the whole of the genetic information as RNA in RNA viruses is a situation unique in biology)

• metabolic inactivity outside the cells of susceptible hosts; viruses lack ribosomes – the protein-synthesizing apparatus (the corollary of this is that viruses can only multiply inside living cells, i.e. they are obligate intracellular parasites).

Structure

Viruses consist of a nucleic acid core containing the viral genome, surrounded by a protein shell called a capsid (Figs 4.1 and 4.2). The entire structure is referred to as the nucleocapsid. This may be ‘naked’, or it may be ‘enveloped’ within a lipoprotein sheath derived from the host cell membrane. In many viruses (e.g. orthomyxoviruses, paramyxoviruses), the ensheathment begins by a budding process at the plasma membrane of the host cell, while others, such as herpesviruses, ensheath at the membrane of the nucleus or endoplasmic reticulum.

Fig. 4.1 Viral structure (schematic).

Fig. 4.2 Scanning electron micrograph of a herpesvirus. Note the extensive outer lipid envelope and the icosahedral nucleocapsid.

The protein shell or capsid consists of repeating units of one or more protein molecules; these protein units may go on to form structural units, which may be visualized by electron microscopy as morphological units called capsomeres (Fig. 4.1). Genetic economy dictates that the variety of viral proteins be kept to a minimum as viral genomes lack sufficient genetic information to code for a large array of different proteins. In enveloped viruses, the protein units, which comprise the envelopes and are visualized electron microscopically, are called peplomers (loosely referred to as ‘spikes’).

Viral nucleic acid

Viral nucleic acid may be either DNA or RNA. The RNA, in turn, may be ss or ds, and the genome may consist of one or several molecules of nucleic acid. If the genome consists of a single molecule, this may be linear or have a circular configuration. The DNA viruses all have genomes composed of a single molecule of nucleic acid, whereas the genomes of many RNA viruses consist of several different molecules or segments, which are probably loosely linked together in the virion.

Viral protein

In terms of volume, the major bulk of the virion is protein, which offers a protective sheath for the nucleic acid. The viral protein is made up of two or three different polypeptide chains, although in some only one kind of polypeptide chain may be present. Virion surface proteins may have a special affinity for receptors on the surface of susceptible cells and may bear antigenic determinants.

Although most viral proteins have a structural function, some have enzymatic activity. For instance, many viruses such as the human immunodeficiency virus (HIV) contain a reverse transcriptase, whereas several enzymes (e.g. neuraminidase, lysozyme) are found in larger, more complex viruses.

Viral lipid and carbohydrate

In general, lipids and carbohydrates of viruses are only found in their envelopes and are mostly derived from the host cells. About 50–60% of the lipids are phospholipids; most of the remainder is cholesterol.

Virus symmetry

The nucleocapsids of viruses are arranged in a highly symmetrical fashion (symmetry refers to the way in which the protein units are arranged). Three kinds of symmetry are recognized (Fig. 4.3):

Fig. 4.3 Structural components and symmetry of different viruses. (A) Naked icosahedral; (B) naked helical; (C) enveloped icosahedral; (D) enveloped helical.

Page 28

• Icosahedral symmetry. The protein molecules are symmetrically arranged in the shape of an icosahedron (i.e. a 20-sided solid, each face being an equilateral triangle). Herpesviruses are an example (Figs 4.1 and 4.2).

• Helical symmetry. The capsomeres surround the viral nucleic acid in the form of a helix or spiral to form a tubular nucleocapsid. Most mammalian RNA viruses have this symmetry, where the nucleocapsid is arranged in the form of a coil and enclosed within a lipoprotein envelope.

Page 29

• Complex symmetry. This is exhibited by a few families of viruses – notably the retroviruses and poxviruses.

Taxonomy

Vertebrate viruses are classified into families, genera and species. The attributes used in classification are their symmetry, the presence or absence of an envelope, nucleic acid composition (DNA or RNA), the number of nucleic acid strands and their polarity. Classification of some of the recognized families of RNA and DNA viruses is given in Table 4.1. (Note: to memorize which viruses contain DNA, remember the acronym ‘PHAD’: P is for papova and pox, H for herpes and AD for adenoviruses. Most of the remainder are RNA viruses, including the self-evident picornaviruses.)

Table 4.1 Classification of some of the viruses causing human disease

Morphology	Virus
DNA
Enveloped, double-stranded nucleic acid	Herpesviruses
	Herpes simplex virus
	Varicella-zoster virus
	Epstein–Barr virus
	Cytomegalovirus
	Human herpesvirus 6
	Poxviruses
	Vaccinia
	Orf
Enveloped, single-stranded	Parvoviruses
Non-enveloped, double-stranded	Adenoviruses
	Papovaviruses
	Polyomaviruses
	Papillomaviruses
	Hepadnaviruses
	Hepatitis B virus
RNA
Enveloped, single-stranded	Orthomyxoviruses
	Influenzavirus
	Paramyxoviruses
	Parainfluenza
	Respiratory syncytial
	Mumps
	Measles
	Togaviruses
	Rubella
	Retroviruses
	Human immunodeficiency viruses HTLV-I, -III
	Rhabdoviruses
	Rabies
Non-enveloped, double-stranded	Reoviruses
Non-enveloped, double-stranded	Rotavirus
Non-enveloped, single-stranded	Picornaviruses
Non-enveloped, single-stranded	Rhinovirus
	Enterovirus
	Coxsackievirus
	Echovirus
	Poliovirus

HTLV-I, human T cell leukaemia virus type I.

The following is a concise description of the families of mammalian viruses.

DNA viruses

Papovaviruses

Papovaviruses are small, icosahedral DNA viruses with a capacity to produce tumours in vivo and to transform cultured cell lines. The name ‘papova’ is an acronym derived from the papillomavirus, polyomavirus and vacuolating agent simian virus 40 (SV40), which make up this family.

Papillomavirus

This genus contains human serotypes that cause benign skin tumours or warts and both oral and skin papillomas (e.g. hand and plantar warts). Although they were regarded as a cosmetic nuisance rather than a specific disease, it is now known that the papillomaviruses may be involved in genital and oral cancers.

Polyomavirus

This genus contains the polyomavirus of mice and SV40 of monkeys, which are used in experimental carcinogenesis in these animals.

Adenoviruses

Adenoviruses are icosahedral DNA viruses, commonly associated with respiratory and eye infections in humans. These viruses were so named because they were first isolated from cultured adenoid tissue eliciting cytopathic effects. Syndromes associated with adenoviruses include:

• acute febrile pharyngitis (primarily in infants and children), often indistinguishable from pharyngitis due to β-haemolytic streptococci

• acute adult respiratory disease, ranging from pharyngitis to pneumonia

• ocular infections.

Herpesviruses

Herpesviruses are the predominant viral cause of oral infections in humans; often the infections are recurrent, and latent.

Page 30

Structure

These enveloped, icosahedral viruses are 180–200 nm in diameter and contain a linear dsDNA molecule. The Herpesviridae family has over 100 members spread widely among vertebrates, and invertebrates and new species are continuously being added. Herpesviruses are unstable at room temperature and are rapidly inactivated by lipid solvents such as alcohol and other common disinfectants owing to the disruption of the outer lipid envelope.

During reproduction, maturation of the progeny begins in the nucleus of the host cell, which buds through the nuclear membrane and acquires the viral envelope. Typical and highly pathognomonic intranuclear inclusions are therefore found in cells that have undergone active virus replication. As many herpesviruses can fuse with the cells they infect, polykaryocytes or giant cells readily appear in tissue lesions. Such cells, e.g. Tzanck cells or nuclear inclusions (Lipschiitz bodies), are hallmarks of herpetic infections.

Different herpesviruses cause a variety of infectious diseases, some localized and some generalized, often with a vesicular rash. Herpesviruses establish latent infection, which can be readily reactivated by immunosuppression (Table 4.2).

Table 4.2 Latent viruses relevant to dentistry

Virus	Site of latency
Herpes simplex virus	Trigeminal ganglion
Varicella-zoster virus	Sensory ganglia
Epstein–Barr virus	Epithelial cells
	B lymphocytes
Cytomegalovirus	Salivary gland cells
Papillomaviruses	Epithelial cells
Human immunodeficiency viruses	Lymphocytes and other CD4⁺ cells (see Chapter 30)

The nomenclature of herpesviruses is contentious; there is thus a historical or a traditional (trivial) nomenclature and an official name for each virus (Table 4.3). The herpesviruses that commonly infect humans can be distinguished by their antigenic and genomic profiles, although they cannot be differentiated by electron microscopy owing to identical capsid morphology. They also have a universal ability to establish latent infection in the host in which they reside, and manifest a number of common epidemiological features. Herpes simplex virus, herpes zoster virus, Epstein–Barr virus, human cytomegalovirus and herpesviruses 6 and 8 can all cause infections in oral and perioral tissues (Fig. 4.4); see Chapter 35 for details.

Table 4.3 Official and trivial nomenclature of human herpesviruses (family Herpesviridae)

Fig. 4.4 Primary herpes simplex infection of the oral mucosa.

Poxviruses

The poxviruses are the largest viruses to infect humans or animals. Molluscum contagiosum in humans is caused by a poxvirus, as is smallpox, which is now a disease of only historical interest. Humans occasionally acquire infection by animal poxviruses, e.g. cowpox.

Parvoviruses

Parvoviruses are icosahedral viruses with ssDNA. Three serologically distinct types of autonomous parvoviruses are recognized in human disease. The first group is found in stool specimens, the second (the B19 virus) in the serum of asymptomatic blood donors, while the third has been recovered from synovial tissues of rheumatoid arthritis patients. The B19 virus is responsible for a febrile illness, particularly in children, manifesting as a maculopapular rash.

The exanthem is characterized by a fiery-red rash on the cheeks – the ‘slapped-cheek’ syndrome (also termed fifth disease).

Hepadnaviruses

Hepadnaviruses are small, spherical DNA viruses causing hepatitis, chronic liver infections and possibly liver cancer. They are of particular interest in dentistry because of their mode of transmission via blood and saliva (see Chapter 29).

RNA viruses

Picornaviruses

Picornaviruses are the smallest family of RNA viruses but incorporate a very large group of viruses, including the genus Enterovirus. Human enteroviruses have been further subdivided into three major subgroups:

• polioviruses

• echoviruses (acronym: enteric cytopathogenic, human, orphan)

• coxsackieviruses (Coxsackie, a town in the USA) types A and B.

The enteroviruses reside and multiply asymptomatically in the gut but may cause a spectrum of disease ranging from mild undifferentiated rashes, respiratory infections and pharyngitis (coxsackie A) to more serious diseases, including carditis (coxsackie B) which may be lethal in the newborn (see Chapters 21 and 35).

Orthomyxoviruses

Orthomyxoviruses are RNA viruses with a tubular nucleocapsid and a lipoprotein envelope. Influenza A viruses of birds, mammals and humans are in this category. Some of these viruses, for example, Asian influenza viruses, may cause severe and often fatal generalized infections. The nomenclature of these viruses is based on the first letter H and N of the spike glycoproteins haemagglutinin and neuraminidase, respectively. Thus, the earliest recognized virus was termed H1N1, followed by H2N2 and so on. The current bird-flu virus that causes sporadic human infections in Asia is termed H5N1. As the latter outbreaks indicate that H5N1 virus has crossed the species barrier from birds to humans, there is great concern that human-to-human transmission of this rather virulent virus may create a worldwide pandemic of avian flu. At the time of writing, there have been more than 60 human fatalities associated with avian flu transmitted directly from avian sources to humans, but no confirmed cases of human-to-human transmission (Chapter 23).

Page 31

Paramyxoviruses

Paramyxoviruses are large, pleomorphic enveloped RNA viruses. The family contains four common and important human pathogens: measles, mumps, parainfluenza and respiratory syncytial viruses. Paramyxoviruses are a common cause of croup (laryngotracheobronchitis), while respiratory syncytial viruses cause regular winter epidemics of bronchiolitis/pneumonitis in infants.

Coronaviruses

These are enveloped RNA viruses with a helical nucleocapsid. They resemble orthomyxoviruses but have petal-shaped surface projections like a solar corona, hence the name. They infect both animals and humans. Most human infections lead to mild upper respiratory tract infections including the ‘common cold syndrome’. Human coronaviruses infect the respiratory tract by the airborne route, i.e. by inhalation or aerosols by coughs and sneezes of infected individuals. Additionally, inanimate reservoirs (i.e. fomites) are a secondary factor in transmission. Rhinoviruses together with coronaviruses are the major agents of the common cold. A coronavirus that crossed the ‘species barrier’ from civet cats in China to humans is the agent of severe acute respiratory syndrome (SARS). The latter infection – considered the first emerging infection of the new millennium – spread worldwide in 2003 causing many deaths particularly among health care workers (Chapter 23). Human coronaviruses are also implicated in gastroenteritis in infants.

Retroviruses

Retroviruses are large, spherical enveloped RNA tumour viruses characterized by a unique genome, a unique enzyme and a unique mode of replication. The viral genome RNA is first transcribed into DNA by a virus-specific enzyme, reverse transcriptase. This DNA can then serve as a template for messenger RNA (mRNA) synthesis. The RNA viruses infecting humans comprise a single taxonomic group with three subfamilies:

• lentiviruses cause slowly progressive disease and include HIV types 1 and 2 (see Chapter 30)

• oneoviruses include those that cause tumours: human T cell leukaemia virus type I (HTLV-I), the agent of adult T cell leukaemia–lymphoma (ATLL), and HTLV-II, associated with hairy cell leukaemia

• spumaviruses are not recognized human pathogens.

Other RNA viruses

Other RNA viruses that are important but are not known to cause oral disease or directly relevant to dentistry include togaviruses, arenaviruses, rhabdoviruses and filoviruses.

Viroids

As a result of advances in molecular biology, two new classes of infectious agents, prions and viroids, have been discovered. These are the smallest known agents of disease. Viroids cause diseases in plants and comprise naked, covalently linked, closed circles of ssRNA, less than 300–400 nucleotides in length. Despite their minute size, they replicate using host cell enzymes. Viroids are not associated with human disease, thus far. Prions are discussed at the end of this chapter.

Page 32

Viral replication

Viral replication (Fig. 4.5) is a highly complex process and only a brief summary is given here. There are a number of general steps in the replication cycle of all viruses: adsorption, penetration, uncoating and eclipse, transcription, synthesis of viral components, assembly and release of virions. In some viruses, however, these steps may not be clearly defined and may overlap, e.g. penetration and uncoating. It is noteworthy that in some families (e.g. Herpesviridae) many of the critical events occur in the cell nucleus, while others (e.g. Picornaviridae) multiply exclusively within the cytoplasm. The period between infection and the production of the new virion (eclipse or latent period) could be as short as 3 h (e.g. Orthomyxoviridae) or as long as several months or years (e.g. HIV).

Fig. 4.5 Steps in the replication of a DNA virus.

Figure 4.5 depicts the steps in the replication of a DNA virus. However, this picture has to be somewhat modified when RNA viruses are considered, as the basic unit of information is now RNA instead of DNA. The strategies of viral replication become more complex when ds rather than ss viruses, and those with RNA of positive polarity and negative polarity are considered. The basic steps in replication are:

1. Adsorption or attachment of the virus particle to the specific receptors of the host cell plasma membrane. Firm attachment requires the presence of receptors for the virus on the plasma membrane (e.g. orthomyxoviruses and paramyxoviruses bind via an envelope protein, known as haemagglutinin, to certain glycoproteins or glycolipids on the host cell).

2. Penetration or uptake. The process by which the virus or its genome enters the host cell cytoplasm. Penetration can be achieved by three separate mechanisms:

endocytosis: most of the virions taken up by endocytosis appear to be degraded by lysosomal enzymes and therefore fail to initiate infection, but this is the normal route to successful infection by many viruses

fusion: direct fusion of the viral envelope with the plasma membrane of cells allows the nucleocapsid of some viruses to be released directly into the cytoplasm without an intervening phagocytic process

translocation: some non-enveloped viruses have the capacity to pass directly through the plasma membrane.

Page 33

3. Uncoating and eclipse. After penetration, there is a period during which no intact infectious virus can be detected. This ‘eclipse phase’ begins with uncoating of the lipid membrane and protein capsid surrounding the nucleic acid viral core. As uncoating proceeds, the viral nucleic acid becomes free to act as a template for the synthesis of virus mRNA.

4. Transcription. The virus mRNA codes for the synthesis of enzymes necessary to complete the process of uncoating itself and also to initiate early steps in viral replication. When the virus initiates the reproductive cycle within the host cell, the synthesis of host cell RNA is halted, and host ribosomes are free to receive viral mRNA and provide a focus for transcription and synthesis of viral proteins.

5. Synthesis of viral components. Viral proteins are of two types:

structural (the proteins that make up the virus particle)

non-structural (enzymes required for virus genome replication).

Structural viral proteins are synthesized on cellular polyribosomes. There is a simultaneous synthesis of progeny viral nucleic acid, using newly synthesized nucleic acid polymerases.

6. Assembly. Viral assembly is accomplished by incorporation of viral nucleic acid into putative capsomeres – procapsids. Assembly may occur in the cell nucleus, cytoplasm or (with enveloped viruses) at the plasma membrane.

7. Release may occur either through gradual budding, in the case of enveloped viruses, or by sudden rupture.

The foregoing is a brief, composite picture of processes involved in viral multiplication. It should be noted that the replication cycle of each family of viruses has unique characteristics that differ from other viruses.

Pathogenesis of viral infections

See Chapter 5.

Cellular antiviral response

The antiviral response is mostly mediated immunologically and is described in Part 2.

Prions and prion diseases

Prions (proteinaceous infectious particles) are unique elements in nature, and they are the agents of a group of chronic diseases called prion diseases or transmissible spongiform encephalopathies. They infect essentially the nerve tissues of animals and humans, and manifest with long incubation periods lasting up to decades. The relevance of this chronic disease to dentistry relates to the realization (1) that the infectious agent is extremely difficult to destroy and (2) of the probability of infection transmission (variant Creutzfeldt–Jakob disease (vCJD)) in clinical settings.

The major features of prions are given below:

• They are neither viruses nor viroids.

• Prions do not have either DNA or RNA.

• The native form of the prion protein, a normal constituent of healthy neural tissues, is designated PrP^c, while the disease-related isoform derived from the latter is designated PrP^Sc.

• The abnormal form, PrP^Sc, is derived from the native precursor by a post-translational process leading to a conformational change from an α-helical structure to an insoluble β-sheet structure.

• The abnormal (PrP^Sc) form resists destruction and accumulates in the neural tissues, causing vacuolation of cells, leading to a sponge-like appearance (hence the term ‘spongiform’).

• They have the ability to self-replicate but with a very long incubation period (up to 20 years in humans).

• The prototype prion agent caused scrapie, a central nervous system disease in sheep.

• As the organism is highly resistant to heat, chemical agents and irradiation, either special autoclaving procedures are required to sterilize contaminated instruments or disposable instruments/materials have to be used for surgical procedures on infected patients.

• The prion agent can be transmitted to cows, mink, cats and mice, for instance, when fed with infected material.

• Iatrogenic transmission of prion disease by neurosurgical instruments has been reported.

Prion-induced diseases or transmissible spongiform encephalopathies

Kuru

Kuru is the fatal neurological disease first described in societies in Papua New Guinea who consumed human brain. It is no longer prevalent owing to the cessation of this practice.

Creutzfeldt–Jakob disease

Creutzfeldt–Jakob disease (CJD) is a globally prevalent, rare, chronic encephalopathy; 10% of cases are familial and carry the mutated prion gene; the remainder are either acquired or sporadic. Onset is in middle to late life (40–60 years); the clinical course lasts for about 7–18 months.

Variant Creutzfeldt–Jakob disease

A variant form (vCJD) is localized to Europe, especially the UK; it almost always affects teenagers or young adults, with a mean age of onset of 24 years. Up to now more than 120 individuals have been affected in the UK alone. The disease is spread by consumption of prion-infected animal tissues.

Fatal familial insomnia

Characterized by progressive insomnia, disruption of circadian rhythms and motor dysfunction, fatal familial insomnia has a late onset (40–60 years) and a clinical course that lasts for about 7 months.

Page 34

Gerstmann–Straussler–Scheinker syndrome

Symptoms include profound chronic cerebellar ataxia and slow-onset dementia, early onset (20–30 years) and a clinical course of about 5 years.

Pathogenesis

Prions appear to replicate incessantly, first in lymphoid tissue, and then in brain cells where they produce intracellular vacuoles and deposition of altered host prion protein (PrP^Sc). These vacuoles give rise to the sponge-like appearance of the brain on microscopic examination. The disease is uniformly fatal.

Transmission

Kuru is transmitted in infected human brain by cannibalism.

The mode of transmission of CJD is mostly unknown. There are a few reports of iatrogenic transmission by medical and surgical procedures; hereditary acquisition occurs in familial cases; contaminated food (beef from cattle with ‘mad cow’ disease or bovine spongiform encephalopathy) is thought to cause acquired disease.

Prevention and dental implications

• There is no treatment for or vaccine against prion-induced disease.

• Hence, the only preventive measure is not consuming suspect food (especially those containing neural tissues).

• The level of infectivity in oral and dental tissues is uncertain, although in one in vitro study of the dental pulp of eight patients, no prion particles could be detected.

• A few retrospective studies indicate no evidence of dental procedures increasing transmission risk, and published iatrogenic transmission studies show no evidence of associated dental procedures.

• Due to the inconclusive data on transmission risks, there is a transatlantic divide between the infection control practices in dentistry appertaining to prion disease risk. The American guidelines are rather more rigorous than the British ones (see also Chapter 36).

North American guidelines

For patients with highly suspected or confirmed CJD or vCJD:

• Use disposable material as much as possible, or all instruments used in dental procedures must be incinerated after use.

• For patients at risk (recipients of dura mater grafts, pituitary hormone injections before the mid-1980s, those with close family members with a history of CJD):

Either use disposable instruments or clean instruments thoroughly and sterilize for 18 min at 134°C in a vacuum autoclave (i.e. repeat the standard cycle six times).

Keep instruments moist until cleaned and decontaminated (as the nerve tissue, once dried and deposited on the instruments, is difficult to clean).

Water supply should be independent using either a stand-alone suction unit or infusion.

Treat the patient at the end of the day.

British guidelines

Special precautions for patients with any form of CJD are not required, but strict adherence to standard precautions is required (Chapter 37).

Key facts

• Viruses are obligate intracellular parasites, which are metabolically inert, and can only replicate within living cells.

• The virus genome has either DNA or RNA but never both.

• The genome is protected by an outer protein coat (capsid) composed of capsomeres; the nucleocapsid is the term given to the protein and the viral genome complex.

• The nucleocapsid of viruses is arranged in one of three spatial configurations: icosahedral, helical or complex symmetry.

• When a lipoprotein surrounds the virus, it is called an envelope. Non-enveloped viruses are called naked viruses.

• Peplomers (spikes) are glycoprotein extensions from the envelope and play a role in the attachment of the virus to the target host cells.

• Viruses are classified into families, genera and species. The attributes used in classification are their symmetry, the presence or absence of envelope, nucleic acid composition (DNA or RNA), the number of nucleic acid strands and their polarity. In practice, ‘common names’ are routinely used when describing viruses.

• The stages of viral replication are adsorption, penetration, uncoating, transcription and translation of the genome, assembly of the virus particles, and release.

• Prions are unique as they are devoid of nucleic acids and are made of self-replicating, low-molecular-weight proteins (PrP); their mode of replication is unclear as yet.

• The human transmissible spongiform encephalopathies (e.g. kuru, Creutzfeldt–Jakob disease (CJD)) are caused by prions.

• In view of the difficulty of inactivating prions:

USA: special autoclaving procedures are required to sterilize contaminated instruments, or disposable instruments and materials have to be used for surgical procedures on infected or suspect patients.

UK: special precautions for patients with any form of CJD are not required, but strict adherence to standard precautions is essential.

Page 35