Chapter 32: Adenoviruses

Adenoviruses can replicate and produce disease in the respiratory, gastrointestinal, and urinary tracts and in the eye. Many adenovirus infections are subclinical, and virus may persist in the host for months. About one-third of the 57 known human serotypes are responsible for most cases of human adenovirus disease. A few types serve as models for cancer induction in animals. Adenoviruses are especially valuable systems for molecular and biochemical studies of eukaryotic cell processes. They are also useful vectors for gene therapy approaches.

Important properties of adenoviruses are listed in Table 32-1.

Structure and Composition

Adenoviruses are 70–90 nm in diameter and display icosahedral symmetry, with capsids composed of 252 capsomeres. There is no envelope. Adenoviruses are unique among icosahedral viruses in that they have a structure called a “fiber” projecting from each of the 12 vertices, or penton bases (Figures 32-1 and 32-2). The rest of the capsid is composed of 240 hexon capsomeres. The hexons, pentons, and fibers constitute the major adenovirus antigens important in viral classification.

The DNA genome (26–45 kbp) is linear and double stranded. The GC content of the DNA is lowest (48–49%) in group A (types 12, 18, and 31) adenoviruses, the most strongly oncogenic types, and ranges as high as 61% in other types. This is one criterion used in grouping human isolates. A virus-encoded protein is covalently linked to each 5′ end of the linear genome. The DNA can be isolated in an infectious form, and the relative infectivity of that DNA is reduced at least 100-fold if the terminal protein is removed by proteolysis. The DNA is condensed in the core of the virion; a virus-encoded protein, polypeptide VII (see Figure 32-2B), is important in forming the core structure.

There are an estimated 11 virion proteins; their structural positions in the virion are shown in Figure 32-2B. Hexon and penton capsomeres are the major components on the surface of the virus particle. There are group- and type-specific epitopes on both the hexon and fiber polypeptides. All human adenoviruses display this common hexon antigenicity. Pentons occur at the 12 vertices of the capsid and have fibers protruding from them. The penton base carries a toxin-like activity that causes rapid appearance of cytopathic effects and detachment of cells from the surface on which they are growing. Another group-reactive antigen is exhibited by the penton base. The fibers contain type-specific antigens that are important in serotyping. Fibers are associated with hemagglutinating activity. Because the hemagglutinin is type specific, hemagglutination-inhibition tests are commonly used for typing isolates. It is possible, however, to recover isolates that are recombinants and give discordant reactions in antibody neutralization and hemagglutination-inhibition assays.

Classification

Adenoviruses have been recovered from a wide variety of species and grouped into five genera. All of the human adenoviruses are classified in the Mastadenovirus genus. At least 57 distinct antigenic types have been isolated from humans and many other types from various animals.

Human adenoviruses are divided into seven groups (A–G) on the basis of their genetic, physical, chemical, and biologic properties (Table 32-2). Adenoviruses of a given group have fibers of a characteristic length, display considerable DNA homology (>85%, compared with <20% of members of other groups), and exhibit similar capacities to agglutinate erythrocytes from either monkeys or rats. Members of a given adenovirus group resemble one another in the GC content of their DNA and in their potential to produce tumors in newborn rodents. Importantly, viruses within a group tend to behave similarly with respect to epidemiologic spread and disease association.

Adenovirus Replication

Adenoviruses replicate well only in cells of epithelial origin. The replicative cycle is sharply divided into early and late events. The carefully regulated expression of sequential events in the adenovirus cycle is summarized in Figure 32-3. The distinction between early and late events is not absolute in infected cells; early genes continue to be expressed throughout the cycle; a few genes begin to be expressed at “intermediate” times; and low levels of late gene transcription may occur soon after infection.

A. Virus Attachment, Penetration, and Uncoating

The virus attaches to cells via the fiber structures. The host cell receptor for some serotypes is CAR (coxsackie–­adenovirus receptor), a member of the immunoglobulin gene superfamily. The interaction of the penton base with cellular integrins after attachment promotes the internalization step. Adsorption and internalization are separate steps in the adenovirus infection process, requiring the interaction of fiber and ­penton proteins with different cellular target proteins. Adsorbed virus is internalized into endosomes; the majority of particles (~90%) move rapidly from endosomes into the cytosol (half-life ~5 minutes) by a process triggered by the acidic pH of the endosome. Microtubules are probably involved in the transport of virus particles across the cytoplasm to the nucleus. Uncoating commences in the cytoplasm and is completed in the nucleus, with release of viral DNA perhaps occurring at the nuclear membrane. Uncoating is an organized, sequential process that systematically breaks down the stabilizing interactions that were established during maturation of the virus particle.

B. Early Events

The steps that occur before the onset of viral DNA synthesis are defined as early events. The goals of the early events are to induce the host cell to enter the S phase of the cell cycle to create conditions conducive to viral replication, to express viral functions that protect the infected cell from host defense mechanisms, and to synthesize viral gene products needed for viral DNA replication.

The early (“E”) transcripts come from seven widely separated regions of the viral genome and from both viral DNA strands. More than 20 early proteins, many of which are nonstructural and are involved in viral DNA replication, are synthesized in adenovirus-infected cells. The E1A early gene is especially important; it must be expressed for the other early regions to be transcribed. Modulation of the cell cycle is accomplished by the E1A gene products. The E1B early region encodes proteins that block cell death (apoptosis) occurring as a result of E1A functions; this is necessary to prevent premature cell death that would adversely affect virus yields. The E1A and E1B regions contain the only adenovirus genes necessary for cell transformation; those gene products bind cellular proteins (eg, pRb, p300, p53) that regulate cell cycle progression. The early proteins are represented by the 75-kDa DNA-binding protein shown in Figure 32-3.

C. Replication of Viral DNA and Late Events

Viral DNA replication takes place in the nucleus. The virus-encoded, covalently linked terminal protein functions as a primer for initiation of viral DNA synthesis.

Late events begin concomitantly with the onset of viral DNA synthesis. The major late promoter controls the expression of the late (“L”) genes coding for viral structural proteins. There is a single large primary transcript (~29,000 nucleotides in length) that is processed by splicing to generate at least 18 different late messenger RNAs (mRNAs). These mRNAs are grouped (L1–L5) based on the utilization of common poly (A) addition sites. The processed transcripts are transported to the cytoplasm, where the viral proteins are synthesized.

Although host genes continue to be transcribed in the nucleus late in the course of infection, few host genetic sequences are transported to the cytoplasm. A complex involving the E1B 55-kDa polypeptide and the E4 34-kDa polypeptide inhibits the cytoplasmic accumulation of ­cellular mRNAs and facilitates accumulation of viral mRNAs, ­perhaps by relocalizing a cellular factor required for mRNA transport. Very large amounts of viral structural proteins are subsequently made.

It is noteworthy that studies with adenovirus hexon mRNA led to the profound discovery that eukaryotic mRNAs are usually not colinear with their genes but are spliced products of separated coding regions in the genomic DNA.

D. Viral Assembly and Maturation

Virion morphogenesis occurs in the nucleus. Each hexon capsomere is a trimer of identical polypeptides. The penton is composed of five penton base polypeptides and three fiber polypeptides. A late L4-encoded “scaffold protein” assists in the aggregation of hexon polypeptides but is not part of the final structure.

Capsomeres self-assemble into empty-shell capsids in the nucleus. Naked DNA then enters the preformed capsid. A cis-acting DNA element near the end of the viral chromosome serves as a packaging signal, necessary for the DNA–capsid recognition event. Another viral scaffolding protein, encoded in the L1 group, facilitates DNA encapsidation. Finally, precursor core proteins are cleaved, which allows the particle to tighten its configuration, and the pentons are added. A virus-encoded cysteine proteinase functions in some cleavages of precursor proteins. The mature particle is then stable, infectious, and resistant to nucleases. The adenovirus infectious cycle takes about 24 hours. The assembly process is inefficient; about 80% of hexon capsomeres and 90% of viral DNA are not used. ­Nevertheless, about 100,000 virus particles are produced per cell. Structural proteins associated with mature virus particles are catalogued in Figure 32-2B.

E. Virus Effects on Host Defense Mechanisms

Adenoviruses encode several gene products that counter antiviral host defense mechanisms. The small, abundant VA RNAs afford protection from the antiviral effect of interferon by preventing activation of an interferon-inducible kinase that phosphorylates and inactivates eukaryotic initiation factor 2. Adenovirus E3 region proteins, which are nonessential for viral growth in tissue culture, inhibit cytolysis of infected cells by host responses. The E3 gp19-kDa protein blocks movement of the major histocompatibility complex class I antigen to the cell surface, thereby protecting the infected cell from cytotoxic T-lymphocyte–mediated lysis. Other E3-encoded proteins block induction of cytolysis by the cytokine tumor necrosis factor α.

F. Virus Effects on Cells

Adenoviruses are cytopathic for human cell cultures, particularly primary kidney and epithelial cell lines. The cytopathic effect usually consists of marked rounding, enlargement, and aggregation of affected cells into grape-like clusters. The infected cells do not lyse even though they round up and detach from the glass surface on which they have been grown.

In cells infected with some adenovirus types, rounded intranuclear inclusions containing DNA are seen (Figure 32-4). Such nuclear inclusions may be mistaken for those of ­cytomegalovirus, but adenovirus infections do not induce syncytia or multinucleated giant cells. Although the cytologic changes are not pathognomonic for adenoviruses, they are helpful for diagnostic purposes in tissue culture and biopsy specimens.

Virus particles in the nucleus frequently exhibit crystalline arrangements. Cells infected with group B viruses also contain crystals composed of protein without nucleic acid. Virus particles remain within the cell after the cycle is complete and the cell is dead.

Species C adenoviruses establish latent infections in ­tonsils and adenoids of children, predominantly in T ­lymphocytes. Specimens from most young children ­contain viral DNA; however, it is less commonly detected in tissues from adolescents and adults. Adenovirus types 1, 2, and 5 are most commonly detected. Productive virus replication is rare in lymphocytes.

Human adenoviruses exhibit a narrow host range. When cells derived from species other than humans are infected, the human adenoviruses usually undergo an abortive replication cycle and no infectious progeny are produced.

Gene Therapy

Adenoviruses are being used as gene delivery vehicles for cancer therapy, gene therapy, and genetic immunization studies. Adenoviruses are attractive because recombinant, replication-defective viruses possess the advantages of high transduction efficiencies of many cell types and high levels of short-term expression of transduced genes; however, significant limitations include their high immunogenicity and the high prevalence of preexisting immunity in humans to subgroup C adenoviruses (types 2 and 5 are widely used as vectors). Other limitations are variable receptor (CAR) expression on different cells and the failure to integrate into chromosomal DNA to facilitate long-term transgene expression. Efforts are under way with vector design and targeting techniques to surmount these limitations.

A novel anticancer therapy uses an attenuated ­replication-competent adenovirus engineered to replicate only in targeted cancer cells. This “oncolytic therapy” is aimed at directly killing tumor cells by viral lytic replication.

Animal Susceptibility and Transformation of Cells

Most laboratory animals are not readily infected with human adenoviruses, although newborn hamsters sustain a fatal infection with type 5, and young adult animals are permissive for adenovirus 5 replication in the lung. Several serotypes, especially types 12, 18, and 31, are able to induce tumors when inoculated into newborn hamsters (see Table 32-2). Adenoviruses can morphologically transform cells in culture regardless of their oncogenic potential in vivo (see Chapter 43). Only a small part (<20%) of the adenovirus genome is present in most transformed cells.

The transforming genes of human adenoviruses are located in the early region (E1A and E1B). An exception is type 9; with it, the E4 gene is required for mammary tumorigenesis in rats. Studies of adenovirus transforming genes have revealed cellular growth control mechanisms that are altered in many types of cancer cells.

The highly oncogenic nature of adenovirus type 12 may be related to the observation that one effect of its early region is to turn off the synthesis of class I major histocompatibility antigens (H2 or HLA) in some infected and transformed cells, thereby preventing destruction by cytotoxic T lymphocytes.

Adenoviruses are not thought to be important in human cancer causation.

Pathogenesis

Adenoviruses infect and replicate in epithelial cells of the respiratory tract, eye, gastrointestinal tract, and urinary tract. They usually do not spread beyond the regional lymph nodes. Group C viruses persist as latent infections for years in adenoids and tonsils and are shed in the feces for many months after the initial infection. In fact, the name “adenovirus” reflects the recovery of the initial isolate from explants of human adenoids.

Most human adenoviruses replicate in intestinal epithelium after ingestion but usually produce subclinical infections rather than overt symptoms. The exceptions are serotypes 40 and 41, which can cause gastrointestinal disease.

Clinical Findings

About one-third of the known human serotypes are commonly associated with human illness. It should be noted that a single serotype may cause different clinical diseases and, conversely, that more than one type may cause the same clinical illness. Adenoviruses 1–7 are the most common types worldwide and account for most instances of adenovirus-associated illness.

Adenoviruses are responsible for about 5% of acute respiratory disease in young children, but they account for much less in adults. Most infections are mild and self-limited. The viruses occasionally cause disease in other organs, particularly the eye and the gastrointestinal tract.

A. Respiratory Diseases

Typical symptoms include cough, nasal congestion, fever, and sore throat. This syndrome is most commonly manifested in infants and children and usually involves group C viruses, especially types 1, 2, and 5. Infections with types 3, 4, and 7 occur more often in adolescents and adults. These cases are difficult to distinguish from other mild viral respiratory infections that may exhibit similar symptoms.

Adenoviruses—particularly types 3, 7, and 21—are thought to be responsible for about 10–20% of pneumonias in childhood. Adenoviral pneumonia has been reported to have a mortality rate up to 10% in the very young.

An outbreak of severe respiratory disease, sometimes fatal, occurred in 2007 that was caused by a new variant of adenovirus 14. Patients of all ages were affected, including healthy young adults.

Adenoviruses are the cause of an acute respiratory disease syndrome among military recruits. This syndrome is ­characterized by fever, sore throat, nasal congestion, cough, and malaise, sometimes leading to pneumonia. It occurs in epidemic form among young military recruits under conditions of fatigue, stress, and crowding soon after induction. This disease is caused by types 4 and 7 and occasionally by type 3. Because of vaccine unavailability, the United States military stopped vaccinating against adenoviruses (types 4 and 7) in the 1990s; this was followed by large epidemics affecting thousands of trainees.

B. Eye Infections

Mild ocular involvement may be part of the respiratory–pharyngeal syndromes caused by adenoviruses. Pharyngoconjunctival fever tends to occur in outbreaks, such as at children’s summer camps (“swimming pool conjunctivitis”), and is associated with types 3 and 7. The duration of conjunctivitis is 1–2 weeks, and complete recovery with no lasting sequelae is the common outcome.

A more serious disease is epidemic keratoconjunctivitis. It is caused by types 8, 19, and 37. This disease occurs mainly in adults and is highly contagious. Adenoviruses can remain viable for several weeks on sinks and hand towels, and these may be sources of transmission. The disease is characterized by acute conjunctivitis followed by keratitis that ­usually resolves in 2 weeks but may leave subepithelial opacities in the cornea for up to 2 years. Adenovirus infection of the ­cornea induces inflammation through interaction of viral capsids with host cells.

A study in Japan (1990–2001), where type 37 is the major cause of epidemic keratoconjunctivitis, showed that mutations in the viral genome occurred chronologically and that certain mutations were correlated with epidemics of disease.

C. Gastrointestinal Disease

Many adenoviruses replicate in intestinal cells and are ­present in stools, but the presence of most serotypes is not associated with gastrointestinal disease. However, two serotypes (types 40 and 41) have been etiologically associated with infantile gastroenteritis and may account for 5–15% of cases of viral gastroenteritis in young children. Adenovirus types 40 and 41 are abundantly present in diarrheal stools. These enteropathogenic adenoviruses are very difficult to cultivate.

D. Other Diseases

Immunocompromised patients may suffer from a variety of casual and severe adenovirus infections. The most common problem caused by adenovirus infection in transplant patients is respiratory disease that may progress to severe pneumonia or disseminated infection and may be fatal (usually types 1–7). Children receiving liver transplants may develop adenovirus hepatitis in the allograft. In addition, children with heart transplants who develop myocardial adenovirus infections are at increased risk of graft loss. Pediatric recipients of hematopoietic stem cell transplants may develop infections with a wide variety of adenovirus types. Patients with acquired immunodeficiency syndrome (AIDS) may experience adenovirus infections, especially in the ­gastrointestinal tract.

Types 11 and 21 may cause acute hemorrhagic cystitis in children, especially boys. Virus commonly occurs in the urine of such patients.

Immunity

Adenoviruses induce effective and long-lasting immunity against reinfection. This may reflect the fact that adenoviruses also infect the regional lymph nodes and lymphoid cells in the gastrointestinal tract. Resistance to clinical disease appears to be directly related to the presence of circulating neutralizing antibodies, which probably persist for life. Although type-specific neutralizing antibodies may protect against disease symptoms, they may not always prevent reinfection. (Infections with adenoviruses frequently occur without the production of overt illness.)

Maternal antibodies usually protect infants against severe adenovirus respiratory infections. Neutralizing antibodies against one or more types have been detected in more than 50% of infants 6–11 months old. Normal, healthy adults generally have antibodies to several types.

A group-reactive antibody response, different from the type-specific neutralizing antibody, may be measured by complement-fixation, immunofluorescence, or enzyme-linked immunosorbent assay. Group-specific antibodies are not protective, decline with time, and do not reveal the serotypes of previous viral infections.

Laboratory Diagnosis

A. Detection, Isolation, and Identification of Virus

Samples should be collected from affected sites early in the illness to optimize virus detection. Depending on the clinical disease, virus may be found in respiratory samples, conjunctiva, throat, blood, stool, or urine. Duration of adenovirus excretion varies among different illnesses: 1–7 days, respiratory samples of adults with common cold; 3–14 days, throat, stool, and eye, for pharyngoconjunctival fever; 2 weeks, eye, for keratoconjunctivitis; 3–6 weeks, respiratory samples, throat and stool of children with respiratory illnesses; 2–12 months, blood, urine, throat, and stool of immunocompromised patients.

Virus isolation in a cell culture requires human cells. Primary human embryonic kidney cells are most susceptible but usually unavailable. Established human epithelial cell lines, such as HEp-2, HeLa, and KB, are sensitive but are difficult to maintain without degeneration for the length of time (28 days) required to detect some slow-growing natural isolates. Isolates can be identified as adenoviruses by immunofluorescence tests using an antihexon antibody on infected cells. Hemagglutination-inhibition and neutralization tests measure type-specific antigens and can be used to identify specific serotypes.

Infectious adenovirus detection may be made rapidly using the shell vial technique. Viral specimens are centrifuged directly onto tissue culture cells; cultures are incubated for 1–2 days and are then tested with monoclonal antibodies directed against a group-reactive epitope on the hexon antigen. Also, nasal epithelial cells from a patient may be stained directly to detect viral antigens.

Polymerase chain reaction (PCR) assays are routinely used for diagnosis of adenovirus infections in respiratory samples, blood, tissues, or body fluids, usually by using primers from a conserved viral sequence (eg, hexon, VA I) that can detect all serotypes. PCR assays have been described that use single primer pairs that target conserved segments that bracket a hypervariable region in the hexon gene. The assays can detect all known serotypes of human adenoviruses, and sequencing of the amplicon allows serotype identification. This method is rapid compared with the weeks required for virus isolation followed by neutralization assays. Adenovirus PCR is commonly included in respiratory viral detection panels. However, the sensitivity of the PCR assay may result in detection of latent adenoviruses in some patients.

Characterization of viral DNA by hybridization or by restriction endonuclease digestion patterns can identify an isolate as an adenovirus and group it. These approaches are especially useful for types that are difficult to cultivate.

The fastidious enteropathogenic adenoviruses can be detected by direct examination of fecal extracts by electron microscopy, by enzyme-linked immunosorbent assay, or by latex agglutination test. With difficulty, they can be isolated in a line of human embryonic kidney cells transformed with a fragment of adenovirus 5 DNA (293 cells).

Because adenoviruses can persist in the gut and in lymphoid tissue for long periods and because recrudescent viral shedding can be precipitated by other infections, the significance of a viral detection must be interpreted with caution. Viral recovery from the eye, lung, or genital tract is diagnostic of current infection. Isolation of virus from nasopharyngeal or throat secretions of a patient with respiratory illness can be considered relevant to the clinical disease. Viral detection from stool of patients with gastroenteritis is inconclusive unless shown to be one of the enteropathogenic types; enzyme immunoassays are available to specifically detect these serotypes 40 and 41.

B. Serology

Infection of humans with any adenovirus type stimulates a rise in complement-fixing antibodies to adenovirus group antigens shared by all types. The complement-fixation test is an easily applied method for detecting infection by any member of the adenovirus group, although the test has low sensitivity. A fourfold or greater rise in complement-fixing antibody titer between acute-phase and convalescent-phase sera indicates recent infection with an adenovirus, but it gives no clue about the specific type involved.

If specific identification of a patient’s serologic response is required, antibody neutralization or hemagglutination-inhibition tests can be used. The neutralization test is the most sensitive. In most cases, the neutralizing antibody titer of infected persons shows a fourfold or greater rise against the adenovirus type recovered from the patient.

Epidemiology

Adenoviruses exist in all parts of the world. They are present year round and usually do not cause community outbreaks of disease. The most common serotypes in clinical samples are the low-numbered respiratory types (1, 2, 3, 5, and 7) and the gastroenteritis types (40 and 41). Adenoviruses are spread by direct contact, by the fecal–oral route, by respiratory droplets, or by contaminated fomites. Most adenovirus-related diseases are not clinically pathognomonic, and many infections are subclinical.

Infections with types 1, 2, 5, and 6 occur chiefly during the first years of life; types 3 and 7 are contracted during school years; and other types (such as 4, 8, and 19) may not be encountered until adulthood.

Although adenoviruses cause only 2–5% of all respiratory illness in the general population, respiratory disease caused by types 3, 4, and 7 is common among military recruits and young adults in group or institutional settings.

Eye infections can be transmitted in several ways, but hand-to-eye transfer is particularly important. Outbreaks of swimming pool conjunctivitis are presumably waterborne, usually occur in the summer, and are commonly caused by types 3 and 7. Epidemic keratoconjunctivitis is a highly contagious and serious disease. The disease, caused by type 8, spread in 1941 from Australia via the Hawaiian Islands to the Pacific Coast. It spread rapidly through the shipyards (hence the name “shipyard eye”) and across the United States. In the United States, the incidence of neutralizing antibody to type 8 in the general population is very low (~1%), but in Japan it is more than 30%. More recently, adenovirus types 19 and 37 have caused epidemics of typical epidemic keratoconjunctivitis. Outbreaks of conjunctivitis traced to ophthalmologists’ offices were presumably caused by contaminated ophthalmic solutions or diagnostic equipment.

The incidence of adenovirus infection in patients undergoing bone marrow transplantation has been ­estimated to be from about 5% to as high as 30%. The reported incidence is higher in pediatric patients than in adults. Patients may develop fatal disseminated infections. Types 34 and 35 are found most often in bone marrow and renal transplant recipients. The most likely source of infection in transplant patients is endogenous viral reactivation, although primary infections may be a factor in the pediatric population.

Treatment

There is no specific treatment for adenovirus infections.

Prevention and Control

Careful hand washing is the easiest way to prevent infections. Environmental surfaces can be disinfected with sodium hypochlorite. In group settings, paper towels may be advisable because dirty towels can be a source of infection in outbreaks. The risk of waterborne outbreaks of conjunctivitis can be minimized by chlorination of swimming pools and waste water. Strict asepsis during eye examinations, coupled with adequate sterilization of equipment, is essential for the control of epidemic keratoconjunctivitis.

Attempts to control adenovirus infections in the military have focused on vaccines. Live adenovirus vaccine containing types 4 and 7, encased in gelatin-coated capsules and given orally, was introduced in 1971. In this way virus bypasses the respiratory tract, where it could cause disease, and is released in the intestine, where it replicates and induces neutralizing antibody. The vaccine proved highly effective but was discontinued in 1999, and was re-approved in 2011 for U.S. military personnel only.

• Adenoviruses are nonenveloped, icosahedral viruses with a DNA genome.

• Adenoviruses exist all over the world and are present year round; community outbreaks of disease are unusual.

• Adenoviruses are excellent models for molecular studies of eukaryotic cell processes.

• Several serotypes induce tumors in laboratory animals and serve as models for studies of cancer mechanisms.

• Group C viruses establish long-term latent infections in tonsils and adenoids.

• Group C viruses cause respiratory infections in children (types 1–7) and in military recruits (types 3, 4, 7).

• Types 8, 19, and 37 cause severe eye infections (epidemic keratoconjunctivitis).

• Enteric adenoviruses, types 40 and 41, cause gastroenteritis in young children.

• Adenoviruses can cause severe disseminated disease in immunocompromised and transplant patients.

• There are no specific treatments for adenovirus infections.