1452 PART 5 Infectious Diseases

which are diagnosed by serology. Serologic studies have linked C. pneumoniae to atherosclerosis; isolation and PCR detection in cardiovascular tissues have also been reported. These findings suggest an expanded

range of diseases and syndromes for C. pneumoniae. Large-scale case–

cohort studies have demonstrated some association of C. pneumoniae

with lung cancer, as evaluated by serology.

■ EPIDEMIOLOGY

Primary infection occurs mainly in school-aged children and reinfection in adults. Seroprevalence rates of 40–70% show that C. pneumoniae is widespread in both industrialized and developing countries.

Seropositivity usually is first detected at school age, and rates generally

increase by ~10% per decade. About 50% of individuals have detectable

antibody at 30 years of age, and most have detectable antibody by the

eighth decade of life. Although, as mentioned, serologic evidence suggests that C. pneumoniae may be associated with up to 10% of cases of

community-acquired pneumonia, most of this evidence is based not on

paired serum samples but rather on a single high IgG titer. Some doubt

exists about the true prevalence and etiologic role of C. pneumoniae in

atypical pneumonia, especially since reports of cross-reactivity have

raised questions about the specificity of serology when only a single

serum sample is used for diagnosis.

■ PATHOGENESIS

Little is known about the pathogenesis of C. pneumoniae infection. It

begins in the upper respiratory tract and, in many persons, persists as

a prolonged asymptomatic condition of the upper respiratory mucosal

surfaces. However, evidence of replication within vascular endothelium and synovial membranes of joints shows that, in at least some

individuals, the organism is transported to distant sites, perhaps within

macrophages. A C. pneumoniae outer-membrane protein may induce

host immune responses whose cross-reactivity with human proteins

results in an autoimmune reaction.

The role of C. pneumoniae in the etiology of atherosclerosis has been

discussed since 1988, when Finnish researchers presented serologic

evidence of an association of this organism with coronary heart disease

and acute myocardial infarction. Subsequently, the organism was identified in atherosclerotic lesions by culture, PCR, immunohistochemistry, and transmission electron microscopy; however, discrepant study

results (including those of animal studies) and failure of large-scale

treatment studies have raised doubts as to the etiologic role of C. pneumoniae in atherosclerosis. Epidemiologic studies have demonstrated an

association between serologic evidence of C. pneumoniae infection and

atherosclerotic disease of the coronary and other arteries. In addition,

C. pneumoniae has been identified in atherosclerotic plaques by electron microscopy, DNA hybridization, and immunocytochemistry. The

organism has been recovered in culture from atheromatous plaques—a

result indicating the presence of viable replicating bacteria in vessels.

Evidence from animal models supports the hypothesis that C. pneumoniae infection of the upper respiratory tract is followed by recovery of

the organism from atheromatous lesions in the aorta and that the infection accelerates the process of atherosclerosis, especially in hypercholesterolemic animals. Antimicrobial treatment of the infected animals

reverses the increased risk of atherosclerosis. In humans, two small

trials in patients with unstable angina or recent myocardial infarction

suggested that antibiotics reduce the likelihood of subsequent untoward cardiac events. However, larger-scale trials have not documented

an effect of various antichlamydial regimens on the risk of these events.

■ CLINICAL MANIFESTATIONS

C. pneumoniae was first reported as the etiologic agent of mild atypical pneumonia in military recruits and college students. The clinical

spectrum of C. pneumoniae infection includes acute pharyngitis,

sinusitis, bronchitis, and pneumonitis, primarily in young adults. The

clinical manifestations of primary infection appear to be more severe

and prolonged than those of reinfection. The pneumonitis of C. pneumoniae pneumonia resembles that of Mycoplasma pneumonia in that

leukocytosis is frequently lacking and patients often have prominent

antecedent upper respiratory tract symptoms, fever, nonproductive

cough, mild to moderate illness, minimal findings on chest auscultation, and small segmental infiltrates on chest x-ray. In elderly patients,

pneumonia due to C. pneumoniae can be especially severe and may

necessitate hospitalization and respiratory support.

Chronic infection with C. pneumoniae has been reported among

patients with chronic obstructive pulmonary disease and may play

a role in the natural history of asthma, including exacerbations. The

clinical symptoms of respiratory infections caused by C. pneumoniae

are nonspecific and do not differ from those caused by other agents of

atypical pneumonia, such as Mycoplasma pneumoniae.

■ DIAGNOSIS

Serology, PCR amplification, and culture can be used to diagnose

C. pneumoniae infection. Serology has been the traditional diagnostic

method. The gold standard serologic test is the MIF test (see section on

diagnosis of C. trachomatis genital infection, above). Any antibody titer

>1:16 is considered significant evidence of exposure to chlamydiae.

According to a CDC-sponsored expert working group, the diagnosis

of acute C. pneumoniae infection requires demonstration of a fourfold

rise in titer in paired serum samples. There are no official recommendations for diagnosis of chronic infections, although many research

studies have used high titers of IgA as an indicator. The older CF tests

and EIAs for LPS are not recommended, as they are not specific for

C. pneumoniae but identify the chlamydiae only to the genus level.

The organism is very difficult to grow in tissue culture but has been

cultivated in HeLa cells, HEp-2 cells, and HL cells. Although NAATs

are commercially available for C. trachomatis, only research-based PCR

assays are available for C. pneumoniae.

TREATMENT

C. pneumoniae Infections

Although few controlled trials of treatment have been reported,

C. pneumoniae is inhibited in vitro by erythromycin, tetracycline,

azithromycin, clarithromycin, gatifloxacin, and gemifloxacin. Recommended therapy consists of 2 g/d of either tetracycline or erythromycin for 10–14 days. Other macrolides (e.g., azithromycin)

and some fluoroquinolones (e.g., levofloxacin and gatifloxacin) also

appear to be effective.

■ FURTHER READING

Centers for Disease Control and Prevention: Sexually Transmitted Disease Surveillance, 2018. Atlanta, GA: U.S. Department of Health

and Human Services, 2019. https://www.cdc.gov/std/chlamydia/

stats.htm.

Elwell C et al: Chlamydia cell biology and pathogenesis. Nat Rev

Microbiol 14:385, 2016.

Gaydos CA, Essiq A: Chlamydiaceae, in Manual of Clinical Microbiology, 11th ed. JH Jorgensen et al (eds). Washington, DC, ASM Press,

2015, pp 1106–1121.

Goller JL et al: Population attributable fraction of pelvic inflammatory disease associated with chlamydia and gonorrhoea: A crosssectional analysis of Australian sexual health clinic data. Sex Transm

Infect 92:525, 2016.

Hammerschlag MR et al: Chlamydia pneumoniae, in Mandell,

Douglas, and Bennett’s Principles and Practice of Infectious Diseases,

9th ed. JE Bennett, R Dolin, MJ Blaser (eds). Philadelphia, Elsevier,

2020, Chapter 182.

Kuypers J et al: Principles of laboratory diagnosis of STIs, in Sexually

Transmitted Diseases, 4th ed. KK Holmes et al (eds). New York,

McGraw-Hill, 2008, pp 937–948.

Papp JR et al: Recommendations for the laboratory-based detection

of Chlamydia trachomatis and Neisseria gonorrhoeae, 2014. MMWR

63(RR-02):1, 2014.

Rowley J et al: Chlamydia, gonorrhea, trichomoniasis and syphilis:

Global prevalence and incidence estimates, 2016. Bull World Health

Organ 97:548, 2019.

1453CHAPTER 190 Principles of Medical Virology

Schachter J, Stephens RS: Biology of Chlamydia trachomatis, in Sexually Transmitted Diseases, 4th ed. KK Holmes et al (eds). New York,

McGraw-Hill, 2008, pp 555–574.

Taylor HR: Trachoma: A Blinding Scourge from the Bronze Age to the

Twenty-First Century. East Melbourne, Victoria, Australia, Centre for

Eye Research Australia/Haddington Press, 2008, 282 pp.

Workowski KA, Bolan GA: Sexually transmitted diseases treatment

guidelines, 2015. MMWR Recomm Rep 64(Rr-03):1, 2015.

Section 11 Viral Diseases: General

Considerations

190

Viruses are obligate intracellular parasites that must enter cells to replicate. Infection often injures the host cell—hence the name “virus,”

derived from the Latin word virus for poison or toxin. Viruses are one of

the simplest life forms and, at the minimum, have a nucleic acid genome

with a protein coat. They do not divide by division, as do cells; instead,

viruses are programmed to disassemble inside cells, to use their nucleic

acid genome to encode viral proteins that replicate their genomic

nucleic acid, and then to assemble the progeny genomes into viral particles. The progeny viruses are secreted or released from the host cell as

extracellular virions that infect surrounding cells. Viruses depend on the

host cell for many of the enzymes and organelles that synthesize carbohydrates, lipids, nucleic precursors and nucleic acids, and high-energy

molecules, including the host cell’s ribosomes, which are used to make

viral proteins. In the process of taking over the host cell, viruses inhibit

normal cell metabolic pathways and cause damage to the cell in a process that results in the cytopathic effect (CPE). Injury to cells and cell

death can cause tissue damage and contribute to virus-induced disease.

Viruses are distinct from other intracellular parasites such as viroids,

virusoids, prions, and intracellular bacteria. Viroids are small, circular,

single-stranded RNA infectious pathogens of plants that do not have a

protein coat, while virusoids are small, circular-RNA, infectious pathogens that depend on viruses to provide the proteins for their replication

and protein coat. Prions are misfolded proteins that spread from one cell

to another, causing the same protein molecules to misfold in the new cell.

The misfolded proteins in prions cause cellular damage (Chap. 438).

VIRUS STRUCTURE

There are many different virus structures, but nearly all are formed

from a few fundamental structural elements. The minimal virion

particle is composed of a complex of nucleic acids (the genome) and a

protein shell (the capsid) (Fig. 190-1). The combination of the genome

Principles of

Medical Virology

David M. Knipe

A

Genome

Nonenveloped

icosahedral

virion

Capsid

B

Genome

Glycoprotein

Enveloped virion

with icosahedral

capsid

Envelope

C

Genome

Enveloped virion

with helical

nucleocapsid D

Genome

Complex virion

FIGURE 190-1 Schematic diagrams of the major forms of human viruses. A. Icosahedral capsid without an envelope. B. Icosahedral capsid with a lipid envelope. C. Helical

capsid with a lipid envelope. D. Complex virion.

and the capsid is called the nucleocapsid. The genome is protected

within the capsid. The external surface of virions can consist of either

the protein capsid or a lipid envelope around the capsid (Fig. 190-1).

Viral genomes can consist of single- or double-stranded RNA or

DNA and can comprise one or more genome segments. Single-stranded

(ss) genomes are designated as positive strand (+) if they contain the

sequences encoding the open reading frames for viral proteins, while

they are designated as negative strand (–) if they contain only complementary sequences. Thus, a positive-strand RNA viral genome can

be translated into a viral protein upon entry into the host cell, while

a negative-strand genome must be copied into complementary RNA

molecules for translation. This dilemma is solved in negative-strand

viruses by the loading of transcriptases onto the viral genome prior to

encapsidation; these enzymes transcribe the genome into viral mRNA

upon entry into and uncoating within the cell.

Viral capsids are made of repeating protein subunits because their

genomes have limited coding capacity. The capsids are constructed

with a few structural units or capsomers packed into a symmetrical

arrangement. Capsids are usually organized in one of two ways: (1) an

icosahedral or spherical symmetry based on an icosahedron with two-,

three-, and fivefold axes of symmetry formed from 20 triangular faces

or (2) a helical symmetry. However, viruses occasionally have more

complex structures (e.g., the poxviruses) (Fig. 190-1).

Enveloped viruses (e.g., measles virus) are efficient in infecting

cells because the viral lipid membrane fuses easily with the plasma

membrane of the host cell or with internal membranes to deliver the

nucleocapsid to the cytoplasm of the host cell. Thus, these viruses are

highly transmissible. The lipid envelope is susceptible to disruption by

detergents or organic solvents; thus, enveloped viruses such as measles virus and influenza virus can be inactivated by soap and water or

alcohol-based hand sanitizers. In contrast, unenveloped viruses (e.g.,

norovirus or poliovirus) have a tough protein shell whose resistance

to small-intestine bile salts—a surfactant that emulsifies lipids—allows

them to infect the intestine. Unenveloped viruses, especially those that

infect the gastrointestinal tract, are not inactivated by detergents or

organic solvents and must be inactivated by peroxide or hypochlorite

or removed by washing with soap and water.

CLASSIFICATION OF VIRUSES

Viruses have been classified as a free-standing group because they are

not formally related to organisms within any of the major kingdoms.

The highest level of viral classification was originally the family, but

some families have been grouped into orders as more has been learned

about them. The major viruses of clinical interest can be conveniently

classified into a number of families (Table 190-1), each of which has

characteristic virion and genome structures (Fig. 190-2). Classification of viruses into families, genera, and species is based on multiple

criteria, including type of genomic nucleic acid (i.e., RNA or DNA;

ss positive or negative strand or double strand), capsid symmetry

(helical, icosahedral, or complex), presence or absence of an envelope,

mode of replication, and tropism (preferred cell type for replication)

or type of disease it causes. Recent sequence analysis of viral genomes

has refined and revised some of the original virus classifications.

The International Committee on Taxonomy of Viruses specifies both

formal and common names for viruses. For example, herpes simplex

virus 1 (HSV-1) is the common name for human herpesvirus 1.

1454 PART 5 Infectious Diseases

TABLE 190-1 Major Families of Human Pathogenic Viruses

FAMILY REPRESENTATIVE VIRUSES TYPE OF RNA/DNA LIPID ENVELOPE

Picornaviridae Coxsackievirus

Echovirus

Enteroviruses, including poliovirus

Rhinoviruses

Hepatitis A virus

(+) RNA No

Caliciviridae Norovirus (+) RNA No

Hepeviridae Hepatitis E virus (+) RNA No

Togaviridae Rubella virus

Eastern equine encephalitis virus

Western equine encephalitis virus

(+) RNA Yes

Flaviviridae Yellow fever virus

Dengue virus

St. Louis encephalitis virus

West Nile virus

Zika virus

Hepatitis C virus

Hepatitis G virus

(+) RNA Yes

Coronaviridae SARS-CoV-1

SARS-CoV-2

Middle East respiratory syndrome virus

(+) RNA Yes

Rhabdoviridae Rabies virus

Vesicular stomatitis virus

(–) RNA Yes

Filoviridae Marburg virus

Ebola virus

(–) RNA Yes

Paramyxoviridae Parainfluenza virus

Respiratory syncytial virus

Newcastle disease virus

Mumps virus

Rubeola (measles) virus

(–) RNA Yes

Orthomyxoviridae Influenza A, B, and C viruses (–) RNA, 8 segments Yes

Bunyaviridae Hantavirus

California encephalitis virus

Sandfly fever virus

(–) RNA, 3 segments Yes

Arenaviridae Lymphocytic choriomeningitis virus

Lassa fever virus

South American hemorrhagic fever virus

(–) RNA, 2 segments Yes

Reoviridae Rotavirus

Reovirus

Colorado tick fever virus

dsRNA, 10–12 segments No

Retroviridae Human T lymphotropic virus 1 and 2

Human immunodeficiency virus 1 and 2

(+) RNA, 2 identical segments Yes

Hepadnaviridae Hepatitis B virus dsDNA with ss portions Yes

Parvoviridae Parvovirus B19 ssDNA No

Papillomaviridae Human papillomaviruses dsDNA No

Polyomaviridae JC virus

BK virus

Merkel cell polyoma virus

… …

Adenoviridae Human adenoviruses dsDNA No

Herpesviridae Herpes simplex virus 1 and 2

Varicella-zoster virus

Epstein-Barr virus

Cytomegalovirus

Human herpesvirus 6

Human herpesvirus 7

Kaposi’s sarcoma–associated herpesvirus

dsDNA Yes

Poxviridae Variola (smallpox) virus

Orf virus

Molluscum contagiosum virus

dsDNA Yes

Abbreviations: ds, double-stranded; ss, single-stranded.

1455CHAPTER 190 Principles of Medical Virology

Positive-strand RNA viruses

Genome

size (kb)

Name

Envelope

Caspsid

symmetry

6.7–10

Picornaviridae

No

Icosahedral

Genome

size (kb)

Name

Envelope

Caspsid

symmetry

11–12

Rhabdoviridae

Yes

Helical

15–19

Filoviridae

Yes

Helical

14–22

Paramyxoviridae

Yes

Helical

7.5

Caliciviridae

No

Icosahedral

12

Togaviridae

Yes

Icosahedral

9–13

Flaviviridae

Yes

Icosahedral

25–32

Coronaviridae

Yes

Helical

Negative-strand RNA viruses

Segmented negative-strand RNA viruses Segmented double-strand

RNA viruses

Retroviruses

DNA viruses

Genome

size (kb)

Name

Envelope

Caspsid

symmetry

14

Orthomyxoviridae

Yes

Helical

12

Bunyaviridae

Yes

Helical

11

Arenaviridae

Yes

Helical

24

Reoviridae

No

Icosahedral

Genome size

Name

Envelope

Caspsid

symmetry

7–13

Retroviridae

Yes

Icosahedral

5 Kb

Parvoviridae

No

Icosahedral

5–9 kbp

Papillomaviridae

Polyomaviridae

No

Icosahedral

3 kbp

Hepadnaviridae

Yes

Icosahedral

36–38 kbp

Adenoviridae

No

Icosahedral

125–240 kbp

Herpesviridae

Yes

Icosahedral

190 kbp

Poxviridae

Yes

Complex

100 nm

FIGURE 190-2 Schematic diagrams of viruses of the major families that infect humans. The viruses are grouped by genotype, and the virions are drawn approximately to

scale. Prototype viruses of each family are listed in Table 190-1. (Source: Modified from Fig. 185-2 in Harrison’s Principles of Internal Medicine, 20th ed.)

VIRAL REPLICATION IN CELLS

Viral replication takes place in the host cell by the following steps:

binding, entry, uncoating, transport to the site of replication, transcription of mRNA, translation of viral proteins, replication of the input

genome, assembly of progeny viral particles, and egress from the cell.

All viruses must enter cells by mechanisms that allow virus binding to

the cell surface and subsequent crossing of the plasma membrane and/

or other membranes to gain entry into the cytoplasm. After entry, the

mechanisms of replication diverge for the different viruses, depending

on the nature of the viral genome.

■ VIRAL ENTRY

Viruses bind to specific receptors on the cell surface and generally

enter by one of three pathways: (1) fusion of the envelope with the

surface plasma membrane; (2) endocytosis followed by fusion with

the endosome membrane; or (3) lysis of the endosome or formation

of pores in the endosome. Viruses often bind to a charged molecule

on the surface of cells to concentrate themselves thereon. They then

bind more specifically to a protein or carbohydrate molecule, and this

binding triggers endocytosis or fusion of the viral envelope with the

cellular plasma membrane. Endocytosis can occur by any of several

mechanisms, including clathrin-mediated endocytosis, macropinocytosis, micropinocytosis, and caveolar endocytosis. After viral entry into

endocytic vesicles, acidification of the vesicles leads to conformational

changes in the viral glycoproteins, fusion of the viral envelope with

the endocytic membrane, and release of the nucleocapsid into the

cytoplasm. At the entry stage or later, the genome must be uncoated or

the capsid opened sufficiently to allow transcription, translation, and/

or replication.

■ VIRAL REPLICATION STRATEGIES

Positive-Strand RNA Viruses The RNA genomes of the picornaviruses, caliciviruses, hepeviruses, togaviruses, flaviviruses, and

coronaviruses can be translated in the cytoplasm directly after removal

of the capsid coat or uncoating. The picornaviral genomic RNA is

1456 PART 5 Infectious Diseases

translated into a polyprotein that is cleaved by viral and cellular proteases to generate (1) nonstructural proteins that replicate the genomic

RNA to complementary negative-strand molecules and then back to

positive-strand RNA molecules and (2) structural proteins that assemble capsids for progeny virions. Replication of positive-strand viral

RNA takes place in replication complexes associated with cytoplasmic

membranes, often in membrane sacs that concentrate the components,

protect them from host responses, and provide the redox environment

needed for optimal replication. Progeny virions are released when the

host cell lyses. The positive-strand genome RNA of the caliciviruses,

hepatitis E virus (a hepevirus), the togaviruses, and the flaviviruses

is translated to generate a polyprotein, which, when cleaved by viral

and cellular proteases, yields the nonstructural proteins that replicate

the viral genome to a negative-strand copy and then synthesize new

full-length positive strands and a subgenomic mRNA that encodes

the structural proteins. Progeny virions are released by cell lysis or

budding, depending on whether the virus is enveloped. The flavivirus

genome is translated into one polyprotein that is cleaved by viral and

cellular proteases to yield the nonstructural and structural proteins.

Replication of the genome to the negative strand is followed by a transition back to the positive-strand genome for translation and encapsidation. Progeny virions are released by budding.

Negative-Strand RNA Viruses The rhabdoviruses, filoviruses,

and paramyxoviruses have a single negative strand of genome RNA

that is transcribed by a virion-associated RNA polymerase (transcriptase) to yield subgenomic mRNAs that encode the replicase and

structural proteins. The replicase copies the full-length negative-strand

RNA to a full-length positive-strand RNA and then back to a fulllength negative strand, which is assembled into nucleocapsids that bud

out of the cell to form progeny virions.

The influenza viruses, bunyaviruses, and arenaviruses have segmented negative RNA genomes that are transcribed by virion-associated

transcriptases to yield mRNAs that encode nonstructural and structural

proteins. The replicase enzyme complex copies the negative-strand

RNA genomes to full-length positive-strand copies and back to fulllength negative-strand RNA molecules. The bunyaviruses and arenaviruses replicate entirely in the cytoplasm. In contrast, influenza viral

transcription takes place in the nucleus, with nascent cell transcripts

serving as primers to yield mRNAs that are transported to the cytoplasm for translation. Viral proteins are transported into the nucleus

to promote genome replication, and progeny negative-strand RNAs

are transported to the cytoplasm to bud into progeny virions. Some

of the bunyaviruses and the arenaviruses have open reading frames

on the “negative strand.” Thus, these viruses use both negative-sense

and ambisense coding of their RNA genomes. The full-length negative

strands are assembled in the correct assortment in capsid proteins and

then bud to yield infectious progeny virions.

Double-Stranded RNA Viruses The reoviruses and rotaviruses consist of multiple double-stranded (ds) RNA molecules that

are transcribed by virion-associated, RNA-dependent RNA polymerases (transcriptases) to yield mRNAs encoding nonstructural and

structural proteins. Following viral protein synthesis, replication of

positive-strand RNAs to form dsRNA molecules and assembly into

viral capsids occur in cytoplasmic viral factories. Progeny viruses are

released when infected cells lyse.

Double-Stranded DNA Viruses Most dsDNA viral genomes

are transported to the infected cell’s nucleus for transcription and

replication. The host cell recognizes foreign DNA that is not fully

loaded with histone nucleosomes with a normal pattern and tries

to epigenetically silence these molecules; DNA viruses have evolved

mechanisms to overcome these epigenetic silencing mechanisms. The

dsDNA genomes of the papovaviruses and papillomaviruses are coated

with nucleosomal chromatin in the virion and therefore are delivered

to the nucleus in a form that is not recognized as foreign. Viral early

gene expression is promoted by an enhancer adjacent to the early gene

promoter, which is transcribed by host cell RNA polymerase II to yield

the early mRNAs. The early proteins promote viral DNA replication

by host enzymes, and late genes are then transcribed. The late proteins

encode the capsid proteins to assemble progeny virions.

The dsDNA genomes of adenoviruses are delivered to the infected

cell’s nucleus coated with a viral protein that hides the viral genomes

from the host’s epigenetic silencing mechanisms. Viral DNA genomes

are transported to and released through the nuclear pores and are transcribed by host cell RNA polymerase II to yield pre-early mRNAs. The

pre-early proteins promote the transcription of early mRNAs, whose

proteins promote viral DNA replication. The late proteins encode

structural proteins of the virion.

The dsDNA genomes of the herpesviruses, which are not coated

with histones in the virion, are transported to the infected cell’s nuclear

pores and released into the nucleus. The naked DNA is rapidly loaded

with histones bearing silencing modifications by host cell mechanisms;

however, a viral enhancer and a virion protein that uses host enzymes

to drive chromatin reorganization allow immediate-early gene transcription and expression. Immediate-early proteins promote early

gene transcription. Among the E proteins, eight or nine viral proteins

including the viral DNA polymerase are essential for viral DNA synthesis. Late genes then encode proteins for virion assembly.

In contrast, the poxviruses replicate entirely in the cytoplasm—an

unusual site for replication of a dsDNA virus. As a result, they encode

many of the enzymes and factors needed for viral transcription and

genome replication. A virus-encoded, virion-associated, DNA-dependent

RNA polymerase transcribes the viral genome in the infected cell’s

cytoplasm to yield early mRNAs. The early mRNAs encode additional

transcription factors and DNA replication factors, including a viral

DNA polymerase. After DNA replication, the full set of viral proteins

needed for viral progeny assembly is generated by intermediate and

late transcription.

Single-Stranded DNA Viruses The ssDNA genomes of the

parvoviruses are delivered to the infected cell’s nucleus, and host cell

enzymes copy the ssDNA into dsDNA. The dsDNA is then transcribed

by the cell’s RNA polymerase II to yield mRNAs encoding proteins

that promote viral DNA replication and assemble progeny capsids.

How the parvoviruses deal with host epigenetic silencing mechanisms

is not known.

Retroviruses The retrovirus genome consists of two identical

positive-strand ssRNA molecules, which are not translated but instead

copied into dsDNA by the virion reverse transcriptase upon entry into

the host cell’s cytoplasm. The dsDNA is transported with the reverse

transcriptase–integrase complex into the nucleus, where the viral integrase catalyzes the integration of the viral DNA molecule into the host

cell’s chromosomes to yield the provirus. Transcription of the provirus

by host RNA polymerase II yields mRNA for translation of viral proteins and for viral full-length transcripts for assembly of progeny virions.

VIRAL EFFECTS ON THE HOST CELL

Many viruses inhibit cellular macromolecular processes, such as host

cell transcription and protein synthesis, in an attempt to optimize their

own replication by usurping the host cell’s machinery and biochemical precursors. These inhibitory events can lead to cell injury and

ultimately to cell death, or necrosis. The effects are often manifest by

progressive changes in cell structure, detachment from the substrate

and rounding up, and eventual lysis. Collectively, these changes are

referred to as the CPE. Cells may detect infection as described below

and initiate a pathway called programmed cell death, or apoptosis, in an

attempt to limit viral infection.

Some viruses induce host cell growth to optimize their own replication or to amplify the host cells. Papovaviruses, papillomaviruses, and

adenoviruses induce the cellular S phase to activate functions needed

for viral DNA replication. These viruses also target cellular proteins

that control cell growth, inactivating or degrading them to allow the

cell cycle to progress to the S phase. Studies of the mechanisms of these

viral effects on host cells have identified cellular tumor-suppressor

genes such as the p53 and retinoblastoma pRB genes. Epstein-Barr virus

induces proliferation to amplify its latent-infection host cell, a B cell.

However, the viral mechanisms sometimes induce immortalization of a

1457CHAPTER 190 Principles of Medical Virology

cell that has already undergone or later undergoes the oncogenic transformation leading to a cancer cell. Some retroviruses encode altered

versions of host genes that can induce transformation. Collectively,

these DNA viruses and retroviruses are called tumor viruses.

HOST ANTIVIRAL RESPONSES AND VIRAL

ANTAGONISTIC MECHANISMS

Host cells have evolved numerous mechanisms for resisting viral infection. They encode constitutively expressed proteins that inhibit viral

replication in a process called intrinsic resistance. One well-known host

resistance factor is the rhesus macaque Trim5α protein, which inhibits

human immunodeficiency virus (HIV) type 1 infection soon after the

viral core enters the cytoplasm.

Viruses have in turn evolved mechanisms by which to evade or

neutralize resistance factors in cells of their host species. The promyelocytic leukemia (PML) protein and its associated proteins in nuclear

domain 10 (ND-10) structures in the nucleus of human cells restrict

HSV replication, but HSV has evolved a gene product—infected cell

protein 0 (ICP0), an E3 ubiquitin ligase—that promotes the degradation of the PML protein and thwarts this antiviral mechanism.

Nevertheless, PML protein expression is increased by interferon (IFN)

signaling, and the elevated levels are sufficient to reduce wild-type

HSV infection. Thus, during HSV infection, there is a race between

IFN expression and ICP0 expression.

■ TYPES OF CELLULAR INFECTIONS

The balance of proviral and antiviral factors in a cell defines whether

it is permissive or nonpermissive for viral replication. An infection

in which progeny virus is produced is a productive infection. If a cell

becomes infected but does not die, a virus may establish a persistent

infection. A chronic infection can result if infectious virus is continually

produced. An abortive infection occurs when infection begins but is

not completed. In abortive infections, the cell may (1) die, if enough

CPEs are exerted, as described above; (2) undergo oncogenic transformation; or (3) harbor a latent infection in which no infectious virus

is found but the virus can reactivate at a later time. Examples of these

outcomes are the abortive oncogenic infection of cells by Merkel cell

polyomavirus, chronic infection of liver cells by hepatitis B virus, and

latent infection of neurons by HSV.

■ STAGES OF INFECTION OF A HOST

The stages of viral infection are (1) entry into the host, (2) primary

replication and disease at the site of entry, (3) spread through the host,

(4) secondary replication and disease at new sites, (5) persistence or

clearance by the host immune response, and (6) transmission or release

from the host. Infection of a host can be acute, chronic, or latent.

Entry Keratinized skin cells are not viable and therefore are not

good host cells for viral replication. Thus, viruses must enter the host

at a mucosal surface (e.g., at oral, respiratory, and nasal sites), through

a body opening (e.g., by inhalation or ingestion), or through a break in

the skin (e.g., the sites of mosquito or other insect bites). For example,

papillomaviruses and HSV enter at breaks in the skin, while Zika and

dengue viruses can be introduced via insect bites.

Primary Replication and Disease Viruses replicate at the site

of entry into the body (i.e., the primary site of infection), are shed

back into the environment, and may cause entry-site disease and/or

spread to cause systemic illness. For example, influenza viruses can

infect the respiratory mucosa. Noroviruses and rotaviruses can infect

epithelial cells in the gastrointestinal tract. Dengue and Zika viruses

can infect dendritic cells in the tissues after a mosquito bite. If viral

infection injures cells and tissues and causes disease at the entry site,

the incubation period between exposure and disease can be as short

as 1 or 2 days.

Viral Spread Some viral infections remain localized at the primary

site, but others spread from the primary site to secondary sites where

the viruses infect new cells and cause disease. This spread may take

place through the lymph and the bloodstream (viremia). Measles

virus, for example, replicates initially in the respiratory epithelium,

and infected dendritic cells spread through the lymph to lymph nodes

where T cells and monocytes are infected and transmit virus through

the bloodstream to organs and lymph nodes throughout the body.

Systemic disease can result from the disseminated infection, and viral

spread into the skin causes the classic measles rash. The incubation

period of 10–14 days from exposure to clinical symptoms reflects the

time involved for multiple rounds of viral replication and spread within

the body before the classic rash symptoms appear. Similarly, dendritic

cells and macrophages infected with dengue virus can travel through

the circulatory system and transmit virus to secondary sites where

infection and disease can follow.

Alternatively, viral spread may occur via neuronal pathways by

transsynaptic spread of virions. Rabies virus spreads transsynaptically

from the periphery to the central nervous system to cause encephalitis.

HSV-1 causes a primary infection at mucosal surfaces and then enters

the axon of a sensory neuron and establishes latent infection in the

neuron’s cell body. Reactivation usually leads to a recurrent infection

at the site of primary infection, but occasionally, the virus can move

along nerve tracts to the central nervous system and cause encephalitis.

Host Immune Responses Acute viral infection is blunted by the

rapid innate immune response and then controlled by the later adaptive immune response.

INNATE IMMUNITY The first arm of the host’s immune response—the

innate immune response—is rapid, with recognition of general patterns of viral molecules but not of specific antigens, whose recognition

occurs during the later adaptive response. Using pattern recognition

receptors, host cells recognize foreign molecules with patterns contained in microbes—i.e., pathogen-associated molecular patterns

(PAMPs). Recognition of the foreign molecules leads to activation of

innate signaling pathways that induce the expression of IFNs, cytokines, and other host gene products, including those attributable to

IFN-stimulated genes, which serve as antiviral effector molecules. Viral

ssRNA is recognized by Toll-like receptor 7 (TLR7) and TLR8, which

induce transcription of type I IFN genes and IFN-stimulated genes.

IFNs act on the producing cell in an autocrine manner and on surrounding cells in a paracrine manner to induce expression of antiviral

genes and to activate antiviral mechanisms. dsRNA is recognized by

TLR3, which activates expression of type I IFNs. ssRNA and dsRNA are

recognized by retinoic acid–inducible gene I (RIG-I) and melanoma

differentiation-associated antigen 5 (MDA5), which induce type I IFN

expression. Viral glycoproteins are recognized by TLR2 and TLR4.

Viral DNA is recognized by the cytoplasmic cGAS receptor, which activates type I IFN expression, and by the nuclear IFN-inducible protein

16 (IFI16) receptor, which activates IFN expression in some cell types

and epigenetic silencing of the viral DNA genome in many cell types.

IFI16 can therefore act as a constitutively expressed resistance factor or

as an IFN-stimulated gene. Innate responses also direct the induction

of the later, more specific adaptive immune responses.

ADAPTIVE IMMUNITY Viral antigens are presented as peptides to

both CD4+ and CD8+ T cells by antigen-presenting cells to induce

these T cells to develop into antigen-specific T cells. Viral antigens

are also presented to B cells, which induce differentiation of antibody-producing B cells. Antibodies can bind to virions and neutralize

their infectivity by preventing their binding to receptors, their entry,

their uncoating, or other steps in infection (Fig. 190-3). Antibodies

can also bind to viral antigens on the surface of virions and infected

cells and promote phagocytosis, antibody-dependent cytotoxicity, and

complement-mediated lysis. T cells recognize viral peptides bound to

major histocompatibility complex molecules on the surface of infected

cells and produce cytokines that exert an antiviral effect or activate

cell-killing mechanisms. Thus, the host’s adaptive immune responses

can target either virions or infected cells and can clear infection.

VIRAL EVOLUTION

Because viral RNA-dependent RNA polymerases are error prone and

do not have editing functions, sequence changes are frequently introduced into their genomes. These alterations can lead to populations

1458 PART 5 Infectious Diseases

or swarms of viruses with divergent sequences among a viral population in an individual. Upon drug selection, immune pressure, or host

restriction, preexisting variants can emerge as the new major form of a

virus. Differences in replicative ability can lead to enrichment of more

fit viruses and loss of less fit variants. This trend has been observed in

the COVID-19 pandemic as more fit variants have become the dominant forms of SARS-CoV-2 in the population.

Viruses with segmented genomes can undergo genome reassortment in cells co-infected with two viral strains, the result being a new

genetic composition for a given virus. For example, new segments can

arise in influenza virus isolates thought to be reassortants between the

extant human strains and animal or avian strains, such as those from

porcine or avian species. This type of event is the cause of the major

shifts in influenza viruses that occur periodically over a decade. These

major changes due to reassortment and acquisition of a new genome

segment are referred to as antigenic shift, as opposed to the small

changes due to sequence variation, which are designated antigenic drift.

Especially in DNA viruses but—under special circumstances—also

in RNA viruses such as coronaviruses, viral genomes can undergo

recombination between two strains of virus and generate recombinant

genomes with new combinations of genes that may be more or less fit.

Viral variants can acquire the ability to infect cells of new host

species or to jump species barriers. Zoonotic infection occurs when a

virus spreads from animals to humans, as is thought to have occurred

with both SARS-CoV-1 and SARS-CoV-2. The original viral ancestor

of these viruses—endemic in bats—is thought to have spread to other

animals sold in the markets of China, and viral variants then arose

that could efficiently infect humans. Evolution of variants that could

efficiently infect and be transmitted by humans as agents of respiratory

infection led to the COVID-19 pandemic.

MOLECULAR EPIDEMIOLOGY OF VIRUSES

Several molecular techniques allow easy genotyping of virus isolates.

Direct sequencing, analysis of polymorphisms in restriction endonuclease cleavage sites, and polymerase chain reaction (PCR) analysis

allow a search for genotypic markers in isolates, with sequencing being

the most precise definition of a viral strain. When these types of tests

are applied, some viruses (e.g., influenza virus and measles virus) are

found to have mainly one strain prevalent in the population at a given

time. Thus, only one virus strain spreads through the population. For

other viruses, such as HIV or HSV, nearly every unrelated isolate can

be differentiated by these tests, and many strains are latent and spreading within the population and are evolving in parallel. With these

molecular techniques, genotypic markers can be used to determine

whether a virus has been transmitted from one individual to another.

Genomic sequencing studies of SARS-CoV-2 have identified a number of major strains circulating at any given time. As new variants have

arisen, each has become the dominant circulating strain.

DETECTION AND QUANTIFICATION

OF VIRUSES

Viruses and viral infections need to be detected and quantified for

both clinical and scientific purposes. Diagnostic virology employs the

scientific principles described above to detect viruses and evidence of

infection in clinical samples, to define the type of virus present in a

sample, and in some cases to quantify the amount of virus or the viral

load in a patient. Scientific studies use these principles for detection

and quantification of viruses in laboratory stocks and for measurement

of viral replication.

■ DETECTION OF INFECTIOUS VIRUS

Biologic assays must be used to detect and measure infectious virus.

Infectivity can be measured as either the ability to infect animals and

cause disease or the ability to infect cultured cells and cause CPE. For

example, SARS-CoV-1 virus was first isolated by the introduction of

an oropharyngeal swab sample into Vero cell cultures and detection

of CPE.

■ DETECTION OF VIRAL PARTICLES, THEIR

COMPONENTS, AND VIRAL GENE PRODUCTS

Viral Particles Electron microscopy (EM) must be used to visualize virions directly, because viruses (other than the poxviruses) are

smaller than the resolution of the light microscope. Virions can be

visualized by EM with negative staining of the virions themselves or by

transmission EM of infected cells. As stated above, SARS viral particles

were first visualized in sections of Vero cells infected with samples

from patients. The cell culture supernatant showed coronavirus particles by negative-staining EM. The latter method has also been used

to detect viral particles in stool during outbreaks of gastroenteritis.

Antibodies specific for viral capsid proteins are often used in this assay

to concentrate the virus and enhance its detection.

Viral Nucleic Acids Viral nucleic acids are detected by amplification methods involving PCR with specific primers, which amplifies very

small numbers of viral nucleic acid molecules. These methods can use

direct amplification of DNA in clinical samples to detect and quantify

A B

Entry

Antibody

Antibody

Entry Uncoating

Synthesis

of viral

proteins

Copying

of viral

nucleic acids

Release

Uncoating

Synthesis

of viral

proteins

Copying

of viral

nucleic acids

Release

T-cell

Assembly

of progeny

virus

Assembly

of progeny

virus

FIGURE 190-3 Steps in viral infection of a host cell and effects of immune effector mechanisms. A. Steps in viral infection of a host cell. The steps include entry into the

cell, uncoating of the viral genomic nucleic acid, synthesis of viral proteins, copying of viral nucleic acids, assembly of progeny virus, egress, and release from the host

cell. B. Mechanisms of immune effector mechanisms. Antibodies can bind to the extracellular virion and neutralize infectivity by preventing binding to the cellular receptor,

preventing entry at other steps, preventing uncoating, or preventing other steps of infection. T cells recognize antigenic peptides presented on the surface of infected cells

and produce antiviral cytokines and/or activate cell killing.