Viruses in Human Disease

Viruses of medical importance to humans comprise seven families of deoxyribonucleic acid (DNA) viruses and fourteen families of ribonucleic acid (RNA) viruses. This chapter examines the specific families of viruses, including the diseases and the symptoms associated with the viral infection. Tables 66-1 and 66-2 present a quick reference to the viral families and syndromes caused by these viruses. Table 66-1 divides the virus families according to the makeup of the viral genome, either RNA or DNA. Table 66-2 lists some of the common human viral infections.

Adenoviruses

Adenoviruses (Table 66-3) are medium-sized (70 to 90 nm), icosahedral, nonenveloped, double-stranded, linear DNA viruses. This virus was first isolated from cultures of human adenoids and tonsils in the early 1950s, hence the name adenovirus. The adenoviruses belong to the family Adenoviridae and are widely distributed in nature. However, only members of the genus Mastadenovirus cause human infection. Currently, 52 serotypes of human adenoviruses have been described. Most human disease is associated with one third of the viral types. These types are then divided into seven species, A through G, with species B subdivided into two subspecies; virus serotypes are then numbered within the species classification. The viruses can cause a broad range of disease in humans. Respiratory and gastrointestinal diseases are the most common clinical manifestation associated with adenovirus infection.

Adenoviruses cause less than 5% of all acute respiratory disease in the general population, however, they account for up to 18% of respiratory infections in children. By the age of 10, most children have been exposed to and infected with at least one of the adenovirus species. In addition, adenovirus serotypes 40 and 41 cause gastroenteritis in infants and young children, and other serotypes are associated with conjunctivitis and keratitis. Although respiratory and gastrointestinal diseases are most common, disseminated disease in multiple organ systems may develop in compromised hosts.

Transmission of the virus may occur as an aerosolized droplet or maybe airborne. Respiratory disease caused by adenovirus is usually acquired through contact with contaminated respiratory secretions, stool, and fomites. The virus is very stable and can remain viable for weeks at variable temperatures on surfaces and in solution. The incubation period for respiratory disease is 2 to 14 days. Common upper respiratory tract infections caused by adenovirus include colds, tonsillitis, pharyngitis, pharyngoconjunctival fever, and sometimes croup (viral infection of the larynx). Infections of the eye and conjunctivitis often accompany respiratory infection, and in children, otitis media (ear infection) is often a complication of the respiratory disease. Lower respiratory tract infections can be quite severe in children, and adenovirus pneumonia is often fatal in infants and young children.

A unique feature of the adenoviruses is the ability to cause severe, acute respiratory disease epidemics in military recruits, often resulting in considerable morbidity and mortality. A highly effective vaccine to control the outbreaks was developed for serotypes 4 and 7 and administered to recruits from 1971 to 1996. Once the vaccination program was discontinued, the outbreaks resumed. The current adenovirus contains live serotypes 4 and 7 and is approved for military personnel between the ages of 17 and 50. In addition to the reemergence of epidemics, the emergence of a new, unusually severe lower respiratory tract infection caused by adenovirus type 14 has been identified in healthy individuals of all ages in several areas of the United States.

Adenoviruses can be detected from respiratory secretions or stool in cell culture using various epithelial cell lines, such as A-549, HEp-2, and He-La cells. Growth is usually apparent in 2 to 5 days. Adenovirus produces a characteristic grapelike cluster cytopathic effect (CPE). Viral confirmation follow-up is performed using an indirect fluorescent antibody (IFA) technique or enzyme immunoassay (EIA). Nucleic acid testing for adenovirus is becoming more popular because of the s’ detection time and the increased sensitivity over traditional cell culture. Rapid cell culture (i.e., shell vials) using centrifugation reduces detection time but is less sensitive than tube culture.

Arenaviruses

Arenaviruses, of the family Arenaviridae, include 29 spherical, enveloped RNA viruses that have T-shaped glycoprotein spikes 7 to 10 nm long surrounding the surface membrane of the virion (Table 66-4). The viruses can readily infect a variety of mammalian species, especially rodents and bats, often resulting in a deleterious effect on the reservoir rodent host. Human transmission usually occurs through inhalation of aerosols of infected rodent excrement (urine, saliva, feces, nasal secretions) or by direct contact with infected rodents. Disease in humans clinically displays a broad range of symptoms, from asymptomatic (no symptoms) to fever, prostration, headache and vomiting, to the more severe cases of meningitis and hemorrhagic fever.

The arenaviruses capable of causing disease in humans include lymphocytic choriomeningitis (LCM) virus and Lassa fever virus (first detected in Lassa, Nigeria). LCM has been identified in cases of aseptic meningitis in Europe and the Americas. Lassa has been associated with hemorrhagic fever, shock, and death in 5% to 15% of symptomatic patients (80% of cases are asymptomatic). Lassa fever virus is a significant cause of morbidity and mortality in West Africa, where economic resources are limited. Capillary leak and widespread organ involvement, accompanied by shock, respiratory distress, and/or hemorrhage, are responsible for most deaths from Lassa fever. Other, less commonly reported arenaviruses may also cause hemorrhagic fever.

Arenavirus infection is diagnosed using serologic tests or reverse transcriptase polymerase chain reaction (RT-PCR) to detect viral nucleic acid. Viral isolation using cell culture is not routinely recommended. Cell culture for viral isolation has proven to be unreliable because of inconsistent sensitivity. In addition, handling cultures and specimens puts laboratory personnel at high risk. Samples and cultures containing LCM virus require Biosafety Level (BSL) 3 facilities, and Lassa fever virus requires a BSL 4 laboratory. Serologic diagnosis is also difficult because the immunologic antibody response is delayed for several days and often weeks following symptomatic illness. An RT-PCR assay has been developed to detect arenaviruses, but it is not widely available in the acute care setting.

Bunyaviruses

Bunyaviruses, first detected in Bunyamwera, Uganda, belong to the family Bunyaviridae (Table 66-5). The virus is an RNA virus consisting of three, single-stranded RNA segments enclosed in a helical nucleocapsid that is surrounded by a lipid envelope. A unique feature of this family of viruses is their tripartite genome. The genomic structure provides a mechanism for genetic reassortment in nature, much like the orthomyxovirus family of viruses. Bunyaviruses comprise a large, diverse group of viruses (approximately 300 total members with 12 human pathogens), most of which are transmitted by mosquitoes (arboviruses).

The most important human pathogens in the United States consist of the California serogroup (CAL), which includes the California encephalitis and Lacrosse viruses (LAC). Although the name California encephalitis implies that these cases are related to the state of California, they are identified primarily in Minnesota, Wisconsin, Iowa, Illinois, Indiana, and Ohio. Disease is typically mild and self-limiting; however, severe, even fatal, encephalitis ensues in approximately 2% of patients infected. Other human disease–causing members of the family Bunyaviridae include the Cache Valley (CV), Jamestown Canyon (JC), Snowshoe hare (SSH), Tahyna, Rift Valley fever, and Inkoo viruses.

Bunyaviruses that belong to the Hantavirus genus are not arboviruses. The viruses are rodent borne and transmitted through exposure (inhalation) to aerosolized rodent excreta. Rodents develop a chronic infection that results in shedding of the virus in saliva, feces, and urine. Disruption of these animal excreta by vacuuming, sweeping, or shaking rugs aerosolizes infected particles, which are then inhaled. Evidence indicates that the chance of inhaling these particles is greater in indoor, poorly ventilated spaces than through outdoor exposure.

The disease that ensues is called hantavirus pulmonary syndrome (HPS). It was originally discovered in 1993 in the four corners area of the southwestern United States (Arizona, New Mexico, Colorado, and Utah). The discovery of this virus resulted from the outbreak of an unexplained pulmonary illness among several young, healthy people who died from acute respiratory failure. Diagnostic testing failed to identify a known cause of death. Through exhaustive analysis by the virologists at the Centers for Disease Control and Prevention (CDC) using molecular testing, the scientists were able to link the pulmonary syndrome with a previously unknown type of hantavirus. The new virus originally was called Muerto Canyon virus, but later the name was changed to sin nombre (no name) virus (SNV).

HPS begins with generalized symptoms that include headache, fever, and body aches, typically after an incubation period of 11 to 32 days. Subsequently, the symptoms become much more severe, leading to hemorrhagic fever, kidney disease, and acute respiratory failure. The deer mouse (Peromyscus maniculatus) is the primary host for the sin nombre virus. Transmission of the virus from rodent to human has been the only documented mode of human infection. No person-to-person transmission of HPS has ever been documented in the United States.

Since the discovery of SNV, several hantaviruses that cause HPS have been discovered throughout the United States. The Bayou virus, carried by the rice rat (Oryzomys palustris), was first discovered in a male from the state of Louisiana. The cotton rat (Sigmodon hispidus) is the carrier of the Black Creek Canal virus, discovered in a resident from Florida. The white-footed mouse (Peromyscus leucopus) has been implicated in a case of a hantavirus infection called the New York-1 virus. Cases of HPS stemming from related hantaviruses have been documented in Argentina, Brazil, Canada, Chile, Paraguay, and Uruguay, making HPS a panhemispheric disease.

Laboratory diagnosis relies on the identification of hantavirus-specific IgM and or IgG antibody. By the time symptoms have appeared, all patients have formed hantavirus-specific IgM antibody, and most have also developed hantavirus-specific IgG antibody. Enzyme-linked immunosorbent assay (ELISA) is usually the method of choice for diagnosis. Other available diagnostic methods include identification of viral antigen in tissue using immunohistochemistry or the presence of amplifiable viral RNA sequences in blood or tissues. Although RT-PCR assays have been developed for some hantaviruses, the variation in the viral genome reduces sensitivity, making routine identification by RT-PCR complicated for the diagnosis of hantavirus infections. Virus isolation from human sources is difficult, and to date no isolates of SNV-like viruses have been recovered from humans.

Caliciviruses

Caliciviruses are small (30 to 38 nm), rounded, nonenveloped, single-stranded, positive RNA viruses that cause acute gastroenteritis in humans. Caliciviruses (Table 66-6) have been previously recognized as major animal pathogens and have a broad host range and disease manifestation. The virus causes respiratory disease in cats, a vesicular disease in swine, and a hemorrhagic disease in rabbits. Not until the 1990s did the taxonomic status of noroviruses (formerly known as Norwalk-like viruses, named after Norwalk, Ohio) and hepatitis E virus result in classification in the family Caliciviridae. Hepatitis E virus has since been removed from the calicivirus family and included in a new family, the Hepeviridae. (Hepatitis E virus is discussed later in this chapter.)

Members of the Norovirus and Sapovirus genera are the primary cause of viral gastroenteritis in humans and are referred to as the human caliciviruses (HuCV). Previously called “Norwalk-like viruses” and “Sapporo-like viruses,” the viruses were named after their prototype strains, the Norwalk virus and the Sapporo virus, respectively. These viruses are now referred to as the “norovirus” and “sapovirus.” The HuCVs are further classified into genogroups, and within the genogroups, into genetic clusters. Human isolates in the norovirus genogroup include genogroups I, II, and IV and in the sapovirus genogroup, I, II, IV, and V.

The clinical symptoms associated with norovirus infection include nausea, abdominal cramps, vomiting, and watery diarrhea. Symptoms usually occur after a 1- to 2- day incubation period and continue for approximately 1 to 3 days. Vomiting occurs more often in children than in adults. Infection with sapovirus is similar to that with norovirus; however, sapoviruses more frequently cause disease in infants and toddlers than in school-age children, whereas norovirus infections are common to all age groups. Maximum viral shedding in the feces occurs early, at the onset of clinical symptoms, but viral shedding can occur for up to 2 to 3 weeks after cessation of the clinical symptoms. As a result, control of viral transmission is problematic, and infection does not confer long-lasting immunity.

Norovirus is the source of more than 80% of nonbacterial acute gastroenteritis cases and more than 50% of food-borne outbreaks for all ages in developed and underdeveloped countries. A major public health concern is its ability to cause large outbreaks in semiclosed environments. In recent years, noroviruses have been implicated in large outbreaks of disease on cruise ships, in nursing homes, in schools, summer camps, hospitals, and restaurants. Several factors contribute to the rapid spread of infection: fecal-oral transmission, the low infectious dose (fewer than 100 virus particles), and the virus’s high environmental stability. Noroviruses are easily transmitted in water, person to person, or in airborne droplets of vomitus. The virus persists in water despite treatment processes.

Norovirus cannot be cultivated using cell culture. The most widely used identification method is RT-PCR. Commercially available ELISA kits that use monoclonal antibodies (MoAbs) or hyperimmune sera are also available to detect norovirus but are inferior in sensitivity and specificity to RT-PCR. RT-PCR may also be used to detect the HuCVs in environmental specimens, such as drinking water or contaminated food or both.

Coronaviruses

The family Coronaviridae includes the genera Torovirus and Coronavirus (CoV) and contains many species of both human and animal origin (Table 66-7). Once considered a harmless virus capable of causing the human “cold,” the CoVs cause a wide variety of disease in animals and birds. Interest in this virus and its relationship with animals and humans was renewed after the global outbreak of the novel coronavirus severe acute respiratory syndrome (SARS) in 2002 that resulted in severe respiratory distress in the human population. (The SARS outbreak is discussed in detail later in this chapter.) Coronaviruses are pleomorphic, roughly spherical, medium-sized, enveloped RNA viruses. The prefix corona- results from the viral structure and the crownlike surface projections on the external surface of the virus that can be seen with electron microscopy. Human respiratory coronaviruses cause colds and occasionally pneumonia in adults. Together the rhinoviruses and coronaviruses cause more than 55% of the “common colds” in the human populations. Viral transmission is person to person via contaminated respiratory secretions or aerosols. The virus is present in the highest concentration in the nasal passages, where it infects the nasal epithelial cells. Coronaviruses are thought to cause diarrhea in infants based on the presence (as seen with electron microscopy) of coronavirus-like particles in the stool of symptomatic patients. Although antigen detection is available, the technique lacks sensitivity compared with nucleic acid–based testing. No practical diagnostic methods other than electron microscopy and RT-PCR are available. Many CoVs do not grow in routine cell culture. Modified cell cultures have been useful when confirmatory testing with antigen- or nucleic acid–based methods are used.

In November, 2002, SARS was identified as the cause of a worldwide outbreak. It first emerged in the Guangdong province in China. The virus is believed to have mutated and crossed into the human population from palm civets, an exotic mammal present in the live animal markets of China. More than 80% of these animals showed evidence of coronavirus infection. The proximity of humans to animals during exposure in the live animal markets probably facilitated the initial human infection. The outbreak started as a single case in a hotel in China and then snowballed, with a subsequent outbreak in a Hong Kong hospital as the virus evolved and was able to propagate through person-to-person transmission. Within months, more than 8000 patients worldwide were affected, and approximately 700 people died. The disease was characterized by a rapid onset of high fever, followed by a dry cough and dyspnea. The severe respiratory syndrome followed an incubation period of approximately 2 to 7 days after the appearance of the initial symptoms (fever, headache, myalgia, and malaise). Frequently the illness would progress to severe respiratory distress, requiring the patient to be hospitalized for supportive care and mechanical ventilation. During the hospitalizations of several patients, a secondary attack rate of more than 50% was noted among health care workers caring for the SARS patients. This secondary attack rate is a result of SARS being an unusual respiratory virus. The period of maximum infectivity and highest viral loads in the upper airways begins in the second week of illness, during the time the patients often were severely ill. The CDC soon established a case definition, and a worldwide effort in infection control began in order to stop the spread of the virus. The worldwide SARS outbreak was finally considered “contained” in July, 2003. Since the initial outbreak, the virus has not been detected in humans, but the animal reservoir and live animal markets in China are still present. This indicates that future viral strains may emerge if animal-to-human transmission occurs.

Low levels of virus in the respiratory tract during early disease provide a diagnostic challenge. Because of its sensitivity and specificity, molecular testing by RT-PCR remains the recommended method for laboratory diagnosis. The nonspecific symptoms associated with a SARS infection make laboratory testing crucial in the diagnosis and control of the virus. Although nucleic acid testing by RT-PCR is the most useful diagnostic test available, the virus is capable of growth in cell culture using the Vero-E6 cell line. The characteristic viral CPE appears as a rapid cell rounding, refractivity and detachment. BSL 3 or higher is required for propagation and manipulation of cell cultures containing this virus.

Filoviruses

The Filoviridae family of viruses (Table 66-8) is considered the most pathogenic of the hemorrhagic fever viruses. The term filo means threadlike, referring to the virus’s long, filamentous structural morphology seen with electron microscopy. The viruses are pleomorphic, enveloped, nonsegmented, single-stranded, negative sense RNA viruses. The filamentous morphology appears in many forms or configurations under the electron microscope, such as the number “6,” “U,” or circular. Marburg hemorrhagic fever virus displays the characteristic “shepherd’s hook” morphology. The term “viral hemorrhagic fever” is used to describe a severe multisystem syndrome in which multiple organ systems are affected throughout the body. The patient’s vascular system becomes damaged, and the body’s ability to regulate itself is impaired. Infection with the Marburg or Ebola virus, endemic in Africa, results in severe hemorrhages, vomiting, abdominal pain, myalgia, pharyngitis, conjunctivitis, and proteinuria. Human case fatality rates for Ebola virus infection exceed 80%; the toll for Marburg virus infection is somewhat lower, with a case fatality rate of 23% to 25%. These diseases have no cure or established drug treatment.

The first filovirus was detected in Marburg, Germany, when a group of German laboratory workers became ill and developed hemorrhagic fever after handling imported African green monkeys or monkey tissue while preparing polio vaccine. Simultaneous hemorrhagic fever outbreaks occurred in laboratories in Frankfurt, Germany, and Belgrade, Yugoslavia (now Serbia). Thirty-one individuals became symptomatic, and seven individual fatalities were recorded. Symptomatic individuals included the laboratory workers, their family members, and medical personnel. The Marburg virus was isolated from the African green monkeys and determined to be the etiologic agent of infection.

Ebola virus is the only other member of the Filovirus family. It is named after a river in Zaire (now the Democratic Republic of the Congo), where it was first identified. The genus Ebolavirus has five subspecies, based on the first location where the virus was identified: Zaire ebolavirus, Sudan ebolavirus, Cote d’Ivoire ebolavirus (formerly referred to as Ebola–Ivory Coast), Bundibugyo ebolavirus, and Reston ebolavirus. All of the Ebola subspecies cause disease in humans and nonhuman primates (i.e., chimpanzees, gorillas, and monkeys) except for Reston ebolavirus, which causes disease only in nonhuman primates. The Ebola virus was first recognized in 1976, when a total of 602 people became ill in Zaire and Sudan. Infections are acute, with no carrier state, and humans become ill after contact with an infected animal, usually a primate. Transmission of the virus is rapid. Individuals caring for the sick who come in contact with the patient’s secretions quickly develop symptoms. In fact, many of the early Ebola outbreaks were attributed to “nosocomial” infections. Personal protective equipment (e.g., gowns, masks, and gloves) often were not used by those caring for sick patients. Also, objects such as needles and syringes often were not sterilized before reuse, and many people were exposed to the virus through contaminated syringes and needles. In the initial Ebola outbreak, 431 people died, a fatality rate greater than 70%.

The natural animal reservoir for the Ebola and Marburg viruses has never been determined, although the animal source is believed to be native to Africa. Disease outbreaks in monkeys have occurred in the United States in research facilities. Several monkeys housed in separate cages became ill simultaneously. Laboratory workers working in these facilities were also infected and developed antibodies but never developed symptoms of the disease. Reston ebolavirus is known to have caused infections through aerosolization of secretions.

RT-PCR is used to identify the Ebola and Marburg viruses. Electron microscopy is also available in some research facilities. Cell culture is available in laboratories with BSL 4 facilities. Antibody production occurs after an Ebola virus infection, and an antigen-capture ELISA is available to detect IgM and IgG antibodies to Ebola virus.

Flaviviruses

The flaviviruses (family Flaviviridae; Table 66-9) include viruses that cause arbovirus diseases, such as yellow fever, dengue, West Nile viral encephalitis, and Japanese and St. Louis encephalitis. Hepatitis C virus (HCV) is a flavivirus but not an arbovirus. Flaviviruses are small, single-stranded, positive sense RNA, enveloped, icosahedral viruses. The name is derived from the Latin word flavus, which means yellow. The first disease identified in this group was yellow fever, which causes yellow jaundice in humans. Diseases in this viral group are transmitted to humans through the bite of an infected arthropod, usually the mosquito.

Yellow fever has been one of the great plagues throughout history. In 1900, thousands of individuals died during the construction of the Panama Canal. An army physician, Dr. Walter Reed, uncovered the source of the infection. In the jungle habitat, monkeys serve as the reservoir and the vector is a mosquito. This was the first virus clearly associated with transmission by a mosquito. The yellow fever virus also is the first flavivirus for which an effective vaccine has been developed. In urban outbreaks, humans can serve as the reservoir, as long as the mosquito vector is present.

The yellow fever virus primarily infects liver cells, resulting in fever, jaundice, and hemorrhage. Transmission through the mosquito bite is followed by a 3- to 6-day incubation period. The onset of symptoms is sudden and includes fever, rigors, headache, and backache. The patient’s clinical condition progresses rapidly, and the patient becomes intensely ill with nausea, vomiting, facial edema, dusky pallor, swollen, bleeding gums, and hemorrhagic tendencies with black vomit, melena (black, tarry feces), and ecchymoses (bruising). Mortality rates range from 5% to 50%; when death occurs, it is usually within 6 to 7 days following the onset of symptoms but rarely after 10 days. The characteristic yellow jaundice typically is seen in convalescing patients. Prevention in urban areas depends on elimination of the yellow fever vector, the mosquito, Aedes aegypti. The current vaccine is very effective at preventing infection.

Diagnosis of yellow fever infection is often a result of correlation of the patient’s clinical symptoms with the patient’s location and travel history. Laboratory testing on serum or cerebrospinal fluid (CSF) is available for detection of virus-specific antibodies or neutralizing antibodies. Serologic testing is also available using IgM-capture ELISA, microsphere-based immunoassays (MIAs), and IgG ELISA. In fatal cases of yellow fever, patient tissues may be sent to reference laboratories for nucleic acid amplification, histopathology, and cell culture.

The dengue virus is the most prevalent arbovirus in the world; more than 100 million people are infected annually. It is the leading cause of illness and death in the tropics and subtropics. The virus is endemic in Latin America, Puerto Rico, and Mexico. Most cases reported in the United States (more than 90%) are travel related. Humans are the main reservoir for this virus, and person-to-person transmission occurs through a mosquito vector. Dengue virus has four serotypes that cause a variety of clinical manifestations, including nonlethal fever, arthritis, and rash. Infection with one serotype confers immunity only to the infecting serotype. Subsequent infection with one of the three remaining serotypes results in immune-enhanced disease in the form of severe hemorrhagic fever or dengue shock syndrome. Of the more than 100 million cases of dengue fever, 250,000 cases result in dengue hemorrhagic fever, resulting in approximately 25,000 deaths annually. Dengue normally affects adults and older children. The infection begins with a sudden onset of fever, severe headache, chills, and general myalgia. Often a macropapular rash may be visible on the trunk of the body, which then spreads to the face and extremities. No vaccine is available for dengue. Laboratory diagnosis is based on the presence of virus-specific IgM antibody, a fourfold rise in specific IgG antibody, or a positive RT-PCR amplification for dengue genomic sequences.

Scores of other arthropod-borne flaviviruses, most transmitted by mosquitoes or ticks, cause encephalitis, hemorrhagic fever, or milder disease characterized by fever, arthralgia, and rash. West Nile virus (first isolated in the West Nile district of Uganda), a flavivirus closely related to the Japanese and St. Louis encephalitis viruses, is endemic in Africa, Israel, and Europe. West Nile virus has been endemic in the United States since 1999. Since the virus was identified in New York City in 1999, it has spread westward across the entire United States and into Canada, Mexico, Central America, South America, and some Caribbean islands. The virus accounts for the largest number of cases of viral encephalitis in the United States.

West Nile virus is maintained in a bird-mosquito cycle. Birds are the natural reservoir for the virus. Currently, 59 species of mosquitos and more than 300 species of birds are infected with the West Nile virus. Amplification of virus during warm months results in the death of bird hosts, most commonly crows, ravens, and jays. Bridge mosquitos (those that bite both humans and birds) are responsible for transmission to humans; as the viral populations in birds increases, more humans become infected. Interestingly, West Nile virus also has been transmitted person to person through blood transfusions, tissue transplantation, and in human breast milk. Infection is often accompanied by fever, leukopenia, and malaise and may progress to encephalitis.

Laboratory diagnosis typically involves detection of IgM antibody to West Nile virus in the patient’s serum or CSF. Several commercial kits are available for detection of West Nile IgM and IgG specific antibodies using ELISA or IFA methods. Nucleic acid amplification testing has been very successful in detecting the arbovirus from the tissues of fatal cases and has also been used to detect the virus from the tissues of birds. Additionally, molecular testing has used to detect West Nile virus in mosquito pools. West Nile mosquito surveillance has become increasingly important in the attempt to control this disease.

The hepatitis C virus causes hepatitis. Worldwide, an estimated 170 million people are HCV carriers, and about 4 million of those live in the United States. Acute infection with HCV progresses to a chronic infection in 50% to 90% of infected individuals (Figure 66-1). The acute infection with HCV often goes undiagnosed, because it is often asymptomatic. When clinical illness is present, it is generally mild. Chronic infection with HCV is an important cause of liver disease and is associated with the development of end-stage liver disease and hepatocellular carcinoma. The virus is transmitted predominantly by exposure to infected blood, such as during intravenous drug use and administration of contaminated blood products. The screening of blood products for HCV has eliminated the risk of transmission through contaminated blood products. Less efficient modes of transmission include sexual contact with infected partners, acupuncture, tattooing, and sharing of razors.

HCV disease is identified with screening antibody tests, anti-HCV EIA, confirmatory antibody testing, anti-HCV immunoblot, and RT-PCR. In addition, RT-PCR and quantitative bDNA (branched chain DNA) is used to quantitate virus in the blood to monitor viral therapy. Finally, viral genotyping using molecular techniques is available for identifying genotypes that do not respond appropriately to therapy. The full benefits of modern laboratory testing, including the application of molecular methods, has significantly improved the recognition, monitoring, and treatment of HCV disease.

Hepevirus

Hepatitis E virus (HEV) is the type species of the new genus Hepevirus, in the family Hepeviridae (Table 66-10). Previously classified in the family of caliciviruses, HEV is a small, nonenveloped virus with a single-stranded RNA genome. The only other member of this virus family is an avian HEV known to cause enlarged liver and spleen disease in chickens. Several genetic and antigenic variants or strains of HEV exist and are referred to as genotypes. The different viral strains are common to different geographic locations. Genotype 3 is the strain found in the United States. HEV has also been isolated from swine worldwide and from wild deer in Japan. This indicates that the potential for transmission from animal to humans, resulting in a zoonotic human infection.

HEV was discovered in Asia by a Russian virologist who volunteered to drink stool filtrates from a patient with an unidentified form of hepatitis. The virus is waterborne. The primary mode of transmission is the consumption of water contaminated with feces. HEV is not endemic in the United States and other developed areas of the world. HEV infection results in an acute and generally self-limiting viral hepatitis (inflammation of the liver). Most infected patients do not progress to a long-term carrier status. This virus is well established in developing countries as a cause of hepatitis clinically similar to infection with the hepatitis A virus (HAV). It differs from HAV in that the virus can cause an exceptionally high fatality rate among pregnant women. Fulminant hepatitis develops rapidly and is fatal in approximately 30% of women when infected during the third trimester of pregnancy. The reason for this high rate of mortality among pregnant women is not known. Women should take all possible precautions to avoid exposure to HEV while pregnant, including refraining from traveling to areas of the country where HEV is endemic, such as India and Pakistan.

HEV infection typically begins with nonspecific symptoms common to many viral illnesses, such as fever, headache, nausea, and stomach pain. One of the first signs of a potential hepatitis infection is dark urine, pale feces, yellow discoloration of the skin and sclera. However, not all patients develop jaundice. The liver of infected individuals typically is enlarged and tender.

Clinical diagnosis of HEV infection is important not only to control outbreaks, but also to clinical management of the disease. During patient diagnosis, it is imperative to rule out the other types of hepatitis that can cause a more serious form of disease. With HEV infection, liver function tests typically demonstrate increased levels of serum bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at the time of disease onset. The diagnosis is confirmed using serologic testing. High levels of both IgM and IgG antibodies are produced at disease onset. Although the high levels of IgG confer lifetime immunity to those infected with hepatitis A, whether the antibodies produced in HEV infection do the same is not known. A variety of commercial immunoassays are available that vary in sensitivity and specificity, primarily because of the antigenic variability of the virus. Nucleic acid testing is recommended to confirm positive serology results in areas where HEV is not endemic. Studies are underway to develop a vaccine against HEV, prompted by the highly successful immunization program against HAV.

Hepadnaviruses

Hepatitis B virus (HBV) (Table 66-11) is the prototype virus of the Hepadnaviridae family (hepa- from hepatitis and dna from the genome type). The virus has long been recognized as a significant cause of liver damage associated with morbidity and mortality. Other mammalian and avian hepadnaviruses are known to exist. Hepadnavirus is a pleomorphic, enveloped virus containing circular, partially double-stranded DNA that replicates through an RNA intermediate. Replication occurs by means of reverse transcription and then DNA replication.

Although a successful vaccine against HBV exists, the number of humans infected with HBV worldwide is nearly 400 million, and approximately 50 million new cases occur annually. Humans are the only source of the virus. Percutaneous exposure to blood or blood products is the primary route of transmission. However, the virus may also be contracted through perinatal or sexual contact. HBV is a relatively heat-stable virus and can retain its infectivity in drying blood and other bodily fluids for several days. HBV infection previously was associated with blood transfusion, but this is now rare because of the screening of blood products and vaccination program.

The incubation period for an acute HBV infection usually is 1 to 3 months but may be considerably longer. The initial symptoms of acute infection often are nonspecific, much like mild, flulike symptoms (Figure 66-2). Many cases are asymptomatic, especially in children. The infection presents as an acute or chronic hepatitis with a pathologic effect on the liver, resulting in self-limited or fatal outcomes. Fatal disease is most likely to occur in people co-infected with the hepatitis D virus (delta agent), a deficient RNA virus capable of replication in cells infected with HBV. Chronic HBV infection remains a significant worldwide cause of liver cirrhosis and hepatocellular carcinoma despite the availability of an effective vaccine.

Because of the generality of HBV symptoms and the similarities it shares with other causative agents of hepatitis, clinicians rely extensively on the laboratory for confirmation of the clinical diagnosis of acute or chronic infection and identification of the virus. Laboratory diagnosis typically uses immunoassays. Immunoassays are available for specific identification of viral antigens or antibodies (viral markers) in a patient’s blood. Several commercial types of assays exist. The most common type uses the EIA format. Most of the commercially available serologic assays demonstrate excellent specificity and sensitivity. HBV is not cultivatable in vitro.

Hepatitis B surface antigen (HBsAg) is the most reliable marker for identifying HBV infection. The antigen becomes evident in the patient’s serum weeks before any biochemical evidence associated with liver damage (biochemical liver assays may show only minimal elevation). HBsAg remains in the serum during the acute and chronic stages of hepatitis B. The presence of HBsAg 6 months after acute infection indicates that the patient is a chronic carrier. IgM (anti-HBcAg) to hepatitis B core antigen (HBcAg) appears early in the course of disease, during the acute infection. Anti-HBsAg (antibody to surface antigen) indicates the patient is in convalescence and has developed immunity. The presence of HBeAg (hepatitis B “e” antigen) indicates high infectivity and a chronic carrier state. The best indication of active viral replication and a high state of infectivity is the presence of HBV DNA in the serum. Viral DNA may be detected by a number of molecular tests, including PCR. A number of user-friendly molecular assays are now widely available. The molecular assays provide a quick turnaround time. Also, detection of HBV DNA in serum is used to resolve questionable serologic results, and quantitation is helpful for predicting the patient’s response to treatment.

Herpes Viruses

The word “herpes” is derived from the Greek word meaning “to creep” and was historically used to describe the spreading, ulcerative skin lesions typically seen in a herpes simplex virus (HSV) infection. Herpes viruses are large (150 to 200 nm), double-stranded DNA, enveloped viruses. The virion consists of four components, the nucleic acid core, the capsid, the tegument, and the envelope. The tegument, an asymmetric structure made of a fibrous-like material, surrounds the capsid and contains 20 different proteins. These proteins enter the host cell upon fusion of the envelope and cell membrane and initiate the viral replication cycle.

Eight human herpes group viruses have been described (Table 66-12). Herpes viruses are widely disseminated among animal species. However, the zoonotic forms of herpes do not infect humans, except for herpes B virus from nonhuman primates (not counted among the eight human herpes viruses). Herpes B virus causes a severe, usually fatal encephalitis in humans. Human herpes viruses include HSV types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). More recently detected herpes viruses include human herpes virus (HHV) types 6 (HHV-6), 7 (HHV-7), and 8 (HHV-8). HHV-6 and HHV-7 are lymphotropic viruses acquired early in life. HHV-8, Kaposi’s sarcoma–associated herpes virus (KSHV), causes a tumor of the connective tissue. HHV-6 and HHV-7 are associated with the childhood disease roseola (exanthem subitum). The disease is characterized by a short period of fever and a skin rash.

HSV-1 and HSV-2 share several viral characteristics, including a variable host range, a short replication cycle, rapid spread in cell culture, efficient destruction of infected cells, and the ability to establish latency in the sensory ganglia. These viruses affect individuals of all ages and are the cause of a wide variety of disease, including mucous membrane and skin lesions and ocular, visceral, and central nervous system (CNS) disease. HSV-1 and HSV-2 are transmitted during close personal contact; HSV-1 infection occurs at the oropharyngeal mucosa, and HSV-2 infection occurs at genital sites. Primary HSV-1 infection usually occurs by the time a child reaches the age of 5, and more than 50 million people in the United States are thought to have oral herpes. A subset of primary infections, 10% to 15%, actually produces clinical disease.

Although HSV-2 has been primarily linked to genital herpes, the incidence of genital herpes associated with HSV-1 infection has increased in the U.S. college population from 31% in 1993 to 78% in 2001. HSV-2 is the primary cause of genital herpes and is often associated with sexual promiscuity. Women are 45% more likely to become infected with HSV-2 than are men. The risk of contracting herpes from an infected male after a single sexual contact is 80%.

The defining characteristic associated with herpes infection is the reoccurrence of skin lesions following the primary infection. More than 50% of infected individuals have a recurrent episode of a lesion outbreak within 1 year following initial infection. HSV-1 is associated with mucosal lesions that resemble small vesicles that last 4 to 7 days. The lesions are referred to as herpes labialis, facialis, or febrilis; cold sores, or fever blisters. In women, HSV-2 produces vesicles on the mucosal membranes, labia, and vagina. In men, vesicles form on the shaft of the penis, the prepuce (foreskin), and the glans penis. Systemic symptoms often accompany the primary infection in women, including fever, headache, malaise, and generalized myalgias.

HSV-1 is the most commonly reported viral CNS infection and usually occurs as a result of viral neurotropic spread through the olfactory bulb. This type of infection often occurs in infants and immunocompromised patients. Without treatment, mortality rates associated with HSV infection may be as high as 80%. After appropriate treatment, the mortality rate typically is reduced to 15% in newborns and 20% in other patients. Even when treated, individuals who survive often suffer neurologic, lasting effects, experiencing difficulties in memory, cognition, and personality disorders.

Laboratory diagnosis of herpes infection is available using a variety of diagnostic methodologies. Cell culture traditionally has served as the “gold standard” for herpes virus identification. However, it is important to note that the success of the cell culture depends on the sample collection procedure and the quality of the specimen. The herpes lesion or vesicle should be punctured and the vesicular fluid absorbed with a swab, making sure to swab the base of the vesicle. Samples should be inoculated into cell culture within 1 hour after collection. If this is not possible, the swab should be placed in viral transport media and either refrigerated or frozen at −70°C to preserve the specimen until it can be properly processed and inoculated into cell culture. Herpes is readily grown in cell culture using A-549 or MRC-5 cell lines. The virus is fast growing and typically produces a characteristic rounding, refractile CPE within 1 to 2 days after inoculation of the cell culture. The virus also can be detected using direct antigen testing or nucleic acid amplification systems (PCR), and paired serologic assays of acute and convalescent serum specimens. Direct antigen detection is a rapid, sensitive, and inexpensive method for diagnosis. The antigen present in the lesion is mixed with HSV-specific antibody. If the viral antigen is present, it forms a complex with the antibody that can be identified using immunofluorescent (IF) or immunoperoxidase (IP) staining. This same reaction can be applied in an immunoassay, usually ELISA. Immunoassay offers the additional benefit of adaptability to automation. Nucleic acid testing for the herpes virus has become more widely used and is more sensitive than cell culture and antigen detection. Molecular amplification can be especially beneficial for rapid diagnosis and treatment of herpes viral encephalitis. Both qualitative and quantitative molecular assays exist for identification and diagnosis of herpes viral infection.

The varicella-zoster virus causes what is known as a “classic” childhood disease, chicken pox, and is characterized by the appearance of a maculopapular rash. Before the introduction of the vaccine for VZV, more than 90% of the adults in the United States demonstrated immunity to VZV as a result of childhood infection. Virus transmission is increased during the inclement months, because individuals remain indoors in proximity. The virus is transmitted person to person via respiratory secretions.

VZV infects the conjunctiva or mucosa of the upper respiratory tract and then travels to the lymph nodes. Four to 6 days after the initial infection, infected T cells enter the bloodstream and cause a primary viremia. The infected T cells invade the liver, spleen, and other organs, causing a second round of infection. A secondary viremia ensues, 14 days after the initial infection. This secondary wave infects cells in the skin, causing the characteristic vesicular rash of chicken pox. Symptoms at the onset of infection are usually general and include fever and malaise that appear before the onset of the maculopapular rash on the patient’s trunk and scalp. The lesions usually crust over in 1 to 2 days but do not resolve for approximately 3 weeks. After the acute viral replication in the skin, VZV affects the sensory ganglia in the CNS, where it establishes latency; that is, the virus “hides” in the CNS, which is not subject to vigorous immune surveillance. After a period of latency, the virus may initiate another acute infectious cycle. This reactivation produces the characteristic recurrent disease known as “shingles,” which occurs predominately in immunocompetent people over age 45. Shingles follows an anatomic route around the torso along the dorsal ganglia, as the virus spreads cell to cell along the neurons to epithelial cells in the skin. This condition results in vesicular lesions similar to those produced during the primary infection. Shingles may be accompanied by a painful condition known as postherpetic neuralgia. This condition causes a chronic, debilitating pain that can persist long after other symptoms of shingles have resolved. This pain is believed to be caused by VZV destruction of neurons.

Laboratory diagnosis is not recommended for uncomplicated cases of VZV infection in healthy children or adults. However, in certain situations, such as infection in an immunosuppressed patient or neonate, laboratory diagnosis of VZV may be beneficial.

The virus produces inclusions and giant cells. The traditional method for identifying VZV was to scrape the base of a fresh vesicular lesion and stain the scrapings with Tzanck, Giemsa, or hematoxylin-eosin stain to identify the inclusions or giant cells. This method was complicated by the fact that HSV also produces inclusion bodies. An additional rapid method for identification of VZV is direct identification of viral antigens. Samples of vesicle epithelial cells are collected and smears are prepared and stained with fluorescent, dye-conjugated, monoclonal antibodies to VZV and then observed with a fluorescent microscope. This is a fairly rapid method, because it can be performed within hours of receiving the specimen. However, interpretation and sensitivity can be questionable if not enough epithelial cells are collected.

VZV can grow in cell culture. It produces a characteristic CPE of small clusters of ovoid cells in fibroid cells such as MRC-5, HF, and A549. However, the virus grows slowly, and positivity of the culture may take 7 to 10 days. Shell vial cultures are a simplified method of detecting VZV compared with regular cell culture. Shell vials use cover slips with MRC-5 cells attached in a monolayer across the surface. After inoculation, the cover slip is fixed with acetone after 3 and 6 days of viral growth. The cover slip then is stained with fluorescein isothiocyanate– conjugated (FITC) monoclonal IgG antibody specific for VZV. Positive specimens exhibit a cytoplasmic, apple-green fluorescence when viewed under a fluorescent microscope. Serologic assays for VZV IgG and IgM antibodies are also available to determine the patient’s immune status. Several commercial ELISAs are available for detection and quantitation of VZV antibodies.

Molecular diagnostic testing for VZV is becoming increasingly popular and replacing conventional methods of identifying the virus. This is a result of improvements in diagnostic testing, such as rapid detection time and increased sensitivity and specificity associated with real-time PCR compared with conventional methods of antigen detection or cell culture. In addition, molecular methods can detect multiple human herpes viruses in a single clinical specimen, a technique referred to as multiplex PCR. An automated DNA microarray PCR method has been developed for high-throughput detection of multiple herpes viruses, including VZV, in clinical samples. Molecular diagnostics also can quantitate viral VZV DNA in the blood. Molecular testing is useful for monitoring patients considered high risk for severe VZV infection.

Epstein-Barr virus was discovered four decades ago during a search for the cause of Burkitt’s lymphoma, a disease that predominately affects children in Africa. EBV is responsible for the disease infectious mononucleosis (IM). The virus, which is transmitted in the saliva of infected patients, typically affects adolescents and young adults. The major symptoms include fever, sore throat, headache, malaise, and fatigue. Lymphadenopathy (swollen lymph nodes) and splenomegaly also may result during the disease. Mononucleosis typically is diagnosed through serologic methods that detect antibodies to EBV (Figure 66-3). Nonspecific heterophile antibodies (also referred to as Paul-Bunnell antibodies) appear early on during the disease, making the diagnosis difficult. Antibody production to the virus typically follows the classic immune response, resulting in specific IgM production followed by IgG production. Antibody to the viral capsid (IgM) appears within 4 weeks after infection. This is followed by IgG and IgA antibody to the early antigen (EA), indicating acute or recent infection. Both the EA-IgG and IgA may be undetectable after approximately 6 months. Some Anti-EA IgG antibodies may persist in the patient’s serum for life. These persistent antibodies typically are elevated in patients with Burkitt’s lymphoma. The final diagnostic serologic marker is the antibody to the nuclear antigen (EBNA) that appears within 1 month of infection and peaks approximately 6 to 12 months after infection. In addition to Burkitt’s lymphoma, other cancers have been associated with EBV infection (e.g., nasopharyngeal carcinoma), and the virus is recognized as an important agent in the development of lymphoma or other lymphoproliferative disorders in transplant recipients.

Molecular assays have become instrumental in the diagnosis of herpes viruses. Box 66-1 provides an outline for the basic procedure for EBV PCR amplification.

CMV infection is a common cause of congenital birth defects. The virus is included in the TORCH panel for disease screening in infants. (TORCH is an acronym for toxoplasma, rubella, CMV and HSV-1). Besides being the cause of congenital infection in infants, CMV has been found to cause an infectious mononucleosis–like illness in immunocompromised patients. The disease may be extremely serious in immunosuppressed organ transplant recipients. CMV may be identified using viral cell culture, serologic tests for IgM and IgG antibodies, direct antigen detection, and nucleic acid testing. Although the virus grows in cell culture using human fibroblasts, it is a slow-growing virus that requires 1 to 2 weeks of incubation before CPE is evident, and in some cases CPE may not be visible for a month. Centrifugation-amplified shell vials, a modification of conventional cell culture, can provide a diagnosis within 24 to 48 hours. CMV antigenemia (see Chapter 65 and Procedure 65-2) is routinely used to monitor therapy for CMV infection. A positive CMV antigenemia result is shown in Figure 66-4. Molecular qualitative and quantitative PCR amplification using analyte-specific reagents is available in some clinical laboratories. In addition, a fully automated quantitative CMV assay is available (COBAS AmpliPrep/COBAS TaqMan CMV Test, Roche Molecular Systems, Inc; Pleasanton, CA). Research studies indicate that nucleic acid-based methods are more sensitive for the detection of CMV in symptomatic and asymptomatic patients.

A unique feature of the herpes virus family is their “hallmark” characteristic of latency, or the virus’s ability to reside in the infected host while staying in a repressed state. Reactivation of the virus can be caused by various stimuli, including fever, emotional stress, exposure to UV light, or axonal injury. Recurrence of viral replication at subsequent times results in disease that may be present differentially as a result of the host’s immune response. Viruses in this family are capable of viral recurrence or reactivation. HSV-1 may reactivate, causing mucous membrane disease or life-threatening encephalitis. Encephalitis caused by HSV is the most common viral encephalitis, with 2.3 per million cases reported annually. HSV-2 reactivates, causing mucous membrane vesicles or aseptic meningitis. VZV reactivates as localized lesions (shingles). EBV reactivates, causing asymptomatic shedding of virus in the oropharynx or as disseminated disease in immunocompromised patients. As does EBV, CMV recurs symptomatically in compromised hosts as a pathogen in many tissues (e.g., heart, gastrointestinal tract, lung, brain). HHV-6 and HHV-7 also cause reactivation disease in compromised hosts.

Orthomyxoviruses

The influenza virus is a member of the family Orthomyxoviridae (Table 66-13). The members of this family are pleomorphic, spherical, enveloped, single-stranded, segmented, negative sense RNA viruses. Of all the respiratory viruses known to infect humans, influenza is the cause of the greatest number of serious acute illnesses; more than 200,000 hospitalizations and more than 30,000 deaths occur in the United States every year. Although three types of influenza viruses are known to infect humans (A, B, and C), type C usually causes subclinical infections and is not known to pose a threat to human health. The three genera Influenza virus A, Influenza virus B, and Influenza virus C can be distinguished based on the antigenic differences in the matrix protein (M) and the nucleoprotein (NP). Influenza virus A is further subdivided based on the major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Influenza A naturally infects many bird species, swine, seals, felines, and horses. Influenza B and C are only known to infect humans.

Projecting from the envelope of the virion are the two major surface glycoproteins, HA and NA. The HA proteins are rod-shaped spikes that enable viral attachment to sialic acid containing cellular receptors. Once attached to the receptors, the virus can initiate infection. The NA proteins are mushroom-shaped spikes. They facilitate the release of mature virions from infected cells and assist in viral movement through mucus to adjacent cells. Sixteen different HA molecules and nine different NA molecules have been identified. All of the different viral protein antigenic types can be found in the avian species. However, only H1, H2, H3, and N1 or N2 currently circulate in the human population. Strains of influenza A currently found in the human population are H1N1, H3N2, and H1N2. With the rise of the novel influenza A strain of 2009, it is important to separate the types of H1N1 that circulate through various groups of individuals. (This is discussed later in this chapter.) Influenza A H3N2 is a highly pathogenic virus. Infection with H3N2 results in greater mortality than infection with influenza A H1N1 or influenza B.

A unique property of influenza A and influenza B is the organization of the viral genome. It is composed of an eight-part, segmented RNA genome, each segment essentially serving as a single gene. This property, combined with the influenza viruses’ unique ability to alter their HA and NA antigens, results in the production of an antigenically different virus from year to year. This is termed antigenic drift. Antigenic drift is a continuous, gradual form of change in the viral genome during replication. Antigenic drift occurs in all viral influenza types.

Influenza A undergoes a seasonal antigenic drift every year, making the formulation of an effective vaccine challenging. Antigenic shift is a much more dramatic change in the viral genome and only occurs with influenza A viruses. Antigenic shift occurs when a circulating influenza A strain acquires a completely new or “novel” subtype. This phenomenon occurs when two different strains of influenza virus simultaneously infect a single host. During viral replication the segmented genome of the influenza virus may reassort, mixing segments from the different strains during the infection, resulting in a unique antigenic combination. Avian influenza and human influenza reassortment of genes have been the cause of several pandemics throughout history. Often swine act as the intermediate, or “mixing vessel,” for these reassortment events. Viruses from avian and human origin can infect and replicate in the swine respiratory epithelium. When these reassortments occur, a virus emerges against which the population does not have immune protection because of the new antigenic structure. When this happens, a pandemic can occur if the virus is able to sustain human-to-human transmission. A pandemic is a virulent human influenza virus that causes a global outbreak of serious illness, against which there is little natural immunity, and has sustained person-to-person transmission. Often pandemics result in high rates of human mortality with significant social, infrastructure, and economic consequences.

Six pandemics occurred during the past century. Of these the most devastating was the Spanish flu pandemic of 1918, which caused 25 million to 50 million deaths worldwide, more than 500,000 of them in the United States. The cause of this pandemic was the novel H1N1 virus, which emerged from a reassortment of human and avian influenza components. The survivors of an influenza pandemic develop immunity to the infecting strain. The virus then typically evolves into a “seasonal” circulating strain. Such is the case with the pandemic outbreak of influenza H1N1 in Mexico in 2009. This virus was a triple reassortment of human, avian, and swine viruses capable of human-to-human transmission. The triple reassortment was the result of an interaction between the recent North American H3N2 and H1N2 swine (avian, human, swine triple reassortment viruses) with a Eurasian avian-like swine virus. Although the mortality rate of this pandemic was not as significant as predicted, more than 214 countries and overseas territories reported laboratory-confirmed cases of pandemic influenza A H1N1 2009, resulting in approximately 18,449 deaths. These statistics may be an underestimate of the number of deaths resulting from this outbreak. Many individuals with influenza do not seek medical care, and a relatively small number of those who do are actually tested for influenza infection. Children and young adults, people with underlying health conditions, pregnant women and indigenous populations were more affected than the general population. The World Health Organization (WHO) officially declared the pandemic over in May, 2010. The virus continues to circulate as a “seasonal” strain throughout the world.

Enhanced surveillance for the next pandemic strain of influenza was preceded by the appearance of the avian influenza “bird flu” circulating in Asia. This influenza strain stems from the avian population. The virus is a highly pathogenic avian influenza that has reassorted multiple times with other avian influenza strains capable of causing disease in poultry and other birds. The first cases of human infection with “bird flu” were reported in Hong Kong, where 18 people became ill and six died. H5N1 influenza was identified as the cause. Infection control practices and prompt slaughter of infected domestic fowl halted the outbreak. Although the major outbreak seems to have been prevented, migratory birds have spread the virus along natural flyways to more than 30 countries. Despite the high prevalence of H5N1 among avian populations, human infection remains low, and the low transmissibility suggests a natural barrier to cross-species infection. Most human infections are acquired as a result of contact with infected poultry raised inside or outside the home. As of February, 2011, WHO reported 522 cases of H5N1 viral infection, with 309 associated deaths in 15 countries. The case fatality rate for this disease is close to 60%. This virus has significantly affected the worldwide economy. It has caused the death and destruction of more than 500 million wild and domestic birds worldwide and losses to the poultry industry of more than $10 billion.

Influenza normally is transmitted person to person through inhalation of aerosolized droplets of infected secretions. The incubation period is 1 to 4 days, with rapid onset of symptoms, including fever, nonproductive cough, sore throat, rhinitis, headache, malaise, and myalgia. The illness usually resolves within a week, although some symptoms may persist longer.

Bacterial co-infections are common with influenza, possibly because of viral NA-induced changes in the respiratory epithelium that allow increased bacterial adherence or decreased mucociliary clearance. Recently, clusters of fatal methicillin-resistant Staphylococcus aureus (MRSA) infections secondary to seasonal influenza A have been reported in otherwise healthy children and adults.

Testing for influenza can be completed by viral culture, detection of viral nucleic acid or antigen, and serology. Optimal testing requires proper collection and timing of specimens. Virus is shed 3 to 5 days after the onset of symptoms. Optimal specimens are collected from the posterior nasopharynx. The epithelium of the nasopharynx usually contains high titers of virus and large amounts of infected cells. A variety of other respiratory samples, including nasal aspirates, nasal wash, throat swabs, and throat washes, may be used for viral identification. The samples should be placed in viral transport media and may be stored at 4°C for up to 5 days. If the sample must be stored longer, it should be stored in a freezer at −70°C until processed.

Cell culture is available for influenza virus using a variety of cell lines. Primary monkey kidney (PMK) cell lines have demonstrated a consistent season-to-season isolation frequency of influenza virus. Sometimes the influenza viruses fail to produce a CPE in cell culture, requiring additional testing by hemadsorption with guinea pig red blood cells for viral detection. Follow-up confirmatory methods include assays (e.g., IFA). RT-PCR is rapidly replacing cell culture and is becoming the new gold standard for identification of respiratory viruses. The technique is effective when specimens are compromised, such as when they are collected late in the course of the disease or when appropriate collection and transportation requirements have not been met. Sensitivity has proven to be equal to or better than that of cell culture. Because the time from collection to detection is reduced compared to viral culture, nucleic acid testing likely will become more widely available for the detection of the respiratory virus causing infection.

In addition to immunization, antiviral treatment of influenza has proven to be effective in limiting the duration and severity of the disease. Treatment options are discussed in Chapter 67.

Papillomaviruses

Papilloma viruses are small, nonenveloped, circular, double-stranded DNA viruses. These viruses are abundant in nature and cause infections in humans, dogs, cattle, monkeys, and many other species. The Papillomaviridae family (Table 66-14) includes the human papillomaviruses (HPVs). HPVs cause human warts. They exhibit a tissue tropism for either cutaneous or mucosal tissue. The viruses have not been cultivated in cell culture, which prevents the production of type-specific antigens and corresponding typing antisera. HPVs are divided into more than 200 genotypes based on the viral DNA sequences; approximately 80 of those have been well characterized. Much attention has been focused on the more than 30 sexually transmitted genotypes and their role in the pathogenesis of cancer. The various HPV genotypes have differing cellular tropisms, resulting in defined variation in the clinical presentation of the warts. For example, HPV-1 is associated with plantar warts; HPV-2 and HPV-4 are associated with common warts of the hands; and HPV-6, HPV-11, and others are associated with genital warts. Fifteen to 20 types of HPV cause virtually all cases of cervical cancer, with types 16 and 18 causing more than 60% of cases. Type 16 is also responsible for a subset of cancers of the oropharynx and penile cancer in men (Table 66-14).

HPV infection is the most prevalent sexually transmitted viral disease in the United States; it is estimated that more than 65 million Americans are living with an incurable STD, such as HPV or HSV infection. Infection is detected using histopathologic or cytologic examination of cutaneous biopsy or cells, respectively, and DNA probe assays for identification of specific genotypes in infected epithelial cells. A single, liquid-based cytology sample can be used for cytology and genotyping. Several commercial HPV assays currently are available in the United States, including HC2 (Qiagen), Cervista HPR HR (Hologic), Roche Amplicor HPV Test (Roche Molecular Diagnostics), GenProbe Aptima HPV Test (GenProbe), Abbott RealTime High Risk HPV Test (Abbott Molecular), PreTect HPV-Proofer (Norchip) and NucliSENS Easy Q HPV v1 Test (bioMeriéux). Two vaccines for HPV are currently licensed by the FDA: Cervarix (Glaxo Smith Kline, United Kingdom and Gardasil (Merck & Co., Inc, Whitehouse Station, N.J.). The vaccines are a derivative of the protein viral coat and provide some protective immunity to HPV16.

Paramyxoviruses

The Paramyxoviridae family (Table 66-15) includes many pathogenic viruses. Many of these viruses are identified more often in young children, including measles, mumps, parainfluenza viruses, and respiratory syncytial virus (RSV). Recently, human metapneumovirus and Nipah virus (Nipah is the area in Malaysia where the virus first was isolated) have been recognized as disease-causing paramyxoviruses. Paramyxoviruses do not have a segmented genome, as do the orthomyxoviruses, and therefore do not undergo antigenic shift. Paramyxoviruses are spherical, enveloped RNA viruses, and all members of this group can cause respiratory disease.

Human parainfluenza viruses are important pathogens in children. Viral infection may present as either croup or other upper respiratory diseases in children and adults. The paramyxoviruses are second only to RSV in causing bronchiolitis and pneumonia in infants and young children. The parainfluenza virus has four subtypes; parainfluenza 1 is the most common cause of croup, and parainfluenza 3 is second in prevalence to RSV as a disease of infants and very young children. Most children have had an infection with parainfluenza 3 by 2 years of age. Parainfluenza 3 is most often associated with severe disease and fatalities. Not much is known about parainfluenza 4, which is difficult to grow in cell culture. Serologic studies have shown the virus to be as prevalent as parainfluenza 2.

Parainfluenza viral infection is acquired through inoculation of mucous membranes of the respiratory tract with infectious secretions transmitted on fomites or as large, droplet aerosols. Parainfluenza virus can live up to 10 hours on varying surfaces. Laboratory identification is accomplished through the use of cell culture, using primary or continuous cell lines, followed by confirmatory testing using IFA or other methods of antigen detection. Recently, a multiplex molecular diagnostic test that can differentiate the three major types of parainfluenza was approved for clinical use in the United States.

RSV causes bronchiolitis in young children and is the most significant cause of acute lower respiratory tract infection in children under 5 years of age worldwide. Each year in the United States, RSV is responsible for more than 100 deaths and approximately 60,000 to 100,000 hospitalizations. The virus contains a surface protein called F (fusion) protein. F protein mediates host cell fusion into syncytial cells, which are a hallmark of RSV infection and so named because of the CPE syncytia formation in monolayer cell culture. RSV immune serum prevents severe RSV bronchiolitis during the early months of life in susceptible newborns at risk for RSV disease and those with underlying medical conditions, especially in premature children with underdeveloped lungs. Diagnostic testing for RSV also is performed using cell culture and direct antigen detection. Amplified nucleic acid detection is becoming more readily available and desirable because of its increased sensitivity and specificity and faster turnaround time.

Mumps is an acute, self-limiting disease characterized by parotitis (inflamed salivary gland) accompanied by a high temperature (fever) and fatigue. The mumps virus is transmitted through droplets and contact with infected saliva. The measles virus causes an acute, generalized infection often accompanied by a characteristic rash. The hallmark rash of measles infection is referred to as Koplik’s spots, which are bluish white spots with a red halo located on the buccal or labial mucosa. These spots are found on the inner lip or opposite the lower molars in the mouth. The virus is transmitted from person to person through aerosols and infects the mucosal cells of the respiratory tract.

Measles is one of six classic childhood diseases capable of causing a rash or skin eruption (exanthem). The other diseases that cause an exanthem are scarlet fever (which is caused by a bacterium, Group A Streptococcus); rubella (German measles), referred to as atypical scarlet fever; erythema infectiosum (or fifth disease, caused by parvovirus B-19); and roseola (caused by HHV-6).

Since the introduction of the live attenuated childhood trivalent vaccine against measles, mumps, and rubella (MMR), cases of mumps have dropped by more than 99% and measles infections are rare in the United States and Europe. However, these viruses continue to circulate and remain a common illness in developing countries. Mortality rates from measles infections can be as high as 20% as a result of contributing factors such as poor hygiene and malnutrition. These viruses are often brought into the United States by travelers or people from other countries. The potential for an outbreak arises when infected individuals come in contact with unvaccinated individuals, and prompt laboratory investigation of suspect cases is required. Diagnostic testing for these viruses involves serologic analysis of patient serum for IgM and IgG antibodies and cell culture for virus detection and, recently, nucleic acid detection. For measles cell culture, the specimens of choice are respiratory or throat specimens; for mumps cell culture, buccal swabs collected from the inside of the cheek are recommended. These viruses are also shed in the urine; therefore, urine specimens can be examined for their presence.

Metapneumovirus is a newly discovered virus closely related to RSV. It has caused disease presumably throughout human history but has avoided detection in clinical specimens because it is difficult to grow in cell culture. In infections in children, this virus appears to be less common than RSV but more common than parainfluenza virus, making it an important, medically relevant infectious agent. The virus causes bronchiolitis and pneumonia in infants and most likely lower respiratory tract disease in older adults. In infants 6 to 12 months of age, infection with metapneumovirus is likely to show more lower airway involvement. The virus is considered the second or third most common cause of hospitalization for lower airway disease in pediatric patients. Like RSV, metapneumovirus is associated with winter epidemics that vary in severity from year to year. As stated previously, isolation of metapneumovirus from cell culture is difficult, because the virus is very slow to grow and often takes longer than 2 weeks to develop detectable CPE. Nucleic acid testing for viral RNA is becoming more widely available because of the reduced detection time and improved sensitivity of the assays.

Nipah virus is a recently discovered paramyxovirus capable of causing respiratory disease in pigs and acute, febrile encephalitis in humans. The first human outbreak was identified in 1999. The outbreak was a result of direct contact and viral transmission from diseased pigs and accounted for 265 human cases of viral infection and 108 deaths. Multiple outbreaks have been described in subsequent years. The reservoir for Nipah virus is presumed to be fruit bats, and pigs and other animals are intermediate hosts.

Parvoviruses

Parvoviruses (the Latin term parvus means small) have a wide distribution among warm-blooded animals (Table 66-16). Parvovirus B-19 is the single human pathogen among the Parvoviridae. The virus is a nonenveloped, icosahedral, single-stranded DNA virus that may appear spherical on electron microscopy. Because its replication in human cells is largely restricted to erythroid progenitor cells, adult bone marrow and fetal liver cells (the site of erythropoiesis during fetal development) are the major sites of viral replication. Important diseases associated with parvovirus B-19 infection are fifth disease (the fifth of the childhood exanthems), aplastic crisis in patients with underlying hemoglobinopathies, and fetal infection (hydrops fetalis) resulting from transplacental inoculation. Parvovirus causes a biphasic illness in humans. The first phase, marked fever, malaise, myalgia, and chills, corresponds to peak levels of virus and destruction of erythroblasts. This phase, when mild, may be overlooked or considered a nonspecific viral disease. The second phase involves rash and arthralgia, which occur after the virus has disappeared but at a time when parvovirus-specific antibody can be detected. This is consistent with the appearance of the rash caused by immune complex deposition in the capillaries of the skin. IgM antibodies appear within 7 days after infection, followed by IgG at approximately 14 days. Laboratory diagnosis is accomplished using parvovirus-specific IgM or virus-specific IgG antibody testing with paired acute and convalescent sera or by detection of viral DNA using PCR. Parvovirus cannot be cultivated in the typical cells available in clinical virology laboratories.

Picornaviruses

Picornaviruses (Table 66-17) are small (from the Italian word piccolo, meaning small), nonenveloped, single-stranded RNA viruses. They are among the simplest of the RNA viruses, with a highly structured capsid that has limited surface elaboration. This family of viruses includes the enteroviruses, rhinoviruses, and HAV. Enterovirus infections are among the most common human viral infections (Table 66-18), and although these infections often are mild, the viruses also can cause serious disease. Enteroviruses are responsible for a variety of diseases and conditions, including aseptic meningitis, paralytic poliomyelitis, and encephalitis, in addition to respiratory illness, myocarditis, and pericarditis. Enteroviruses are the most common cause of aseptic meningitis, an inflammation of the brain parenchyma, and have been isolated from more than 40% of patients with this disease.

Transmission Fecal-oral Disease Predominant virus in parentheses: polio (poliovirus), herpangina (coxsackie A), pleurodynia (coxsackie B), aseptic meningitis (many enterovirus types), hand-foot-mouth disease (coxsackie A), pericarditis and myocarditis (coxsackie B), acute hemorrhagic conjunctivitis (enterovirus 70), and fever, myalgia, summer “flu” (many enterovirus types), neonatal disease (echoviruses and coxsackie viruses) Detection Cell culture (PMK and HDF), PCR, and serology Treatment Supportive, pleconaril in development Prevention Avoid contact with virus; vaccination for polio Virus Hepatitis A virus (enterovirus type 72) Transmission Fecal-oral Disease Hepatitis with short incubation, abrupt onset, and low mortality; no carrier state Detection Serology Treatment Supportive Prevention Vaccine; prevent clinical illness with serum immunoglobulin Virus Rhinovirus (common cold virus) Characteristics Approximately 100 serotypes Transmission Contact with respiratory secretions Disease Common cold Detection Cell culture (usually not clinically necessary), RT-PCR Treatment Supportive Prevention Avoid contact with virus

Polyomaviruses

The polyomaviruses (Table 66-19) are small, nonenveloped, circular, double-stranded DNA viruses that have been isolated from many species, including humans. The first human viruses included the JC and BK viruses, named with the initials of the patients from whom the viruses were first isolated. Infection with these viruses usually occurs during childhood and has little clinical significance. These viral infections include latent states in the kidney and B lymphocytes and can result in symptomatic reactivation during periods of immune suppression.

Immunocompromised individuals almost always present with the pathologic effects of infections caused by the JC and BK viruses. JC virus reactivates, resulting in disease in the CNS; BK virus causes a hemorrhagic cystitis. Recently other viruses in this family have been discovered, including the KI virus, MC virus, and WU virus. Both the KI and WU viruses were detected independently through the use of molecular methods and in clinical specimens of respiratory secretions and stool specimens. The pathogenicity and prevalence of these viruses is not yet known. The MC virus, which causes Merkel cell carcinoma, is associated with a high percentage of tumors and has also been detected in respiratory specimens. In the late 1950s and early 1960s, millions of people were inadvertently exposed to a simian polyomavirus (SV40) as a result of administration of SV40-contaminated Salk polio vaccine. This virus has been shown to induce tumors in animals in the research laboratory and has since been periodically associated with several human tumors.

Laboratory detection of the JC virus is completed using PCR on CSF samples or electron microscopy of brain tissue. BK virus is detected using PCR or cytologic examination of urine. Because these are infrequent infections, testing is most likely to be referred to a reference laboratory.

Poxviruses

The poxviruses (Table 66-20) are the largest and most complex of all viruses. The virions consist of a double-stranded DNA genome. The virions appear as oval or brick-shaped structures 200 to 400 nm in length. Because of their large size, poxvirus virions may be visualized through a light microscope.

One of the most feared viruses of history, smallpox, is a member of this family. Smallpox played a crucial role in demonstrating the importance of vaccination to protect against disease. In 1798 Edward Jenner recognized that milkmaids previously infected with cowpox were immune to the disease of smallpox. This discovery led to the practice of inoculating humans against smallpox by using the actual organism (virus) responsible for the disease. Smallpox is known to infect only humans and exists as two distinct subtypes. Variola major, which caused the most severe disease (case fatality rate of 30%), occurred mainly in Asia; variola minor was associated with less severe disease and case fatality rates of 0.1% to 2%. As a result of an intensive vaccination campaign, WHO declared naturally occurring variola virus eradicated in 1980. The variola virus no longer circulates in nature. The virus is feared as a possible biologic weapon, and testing capability for this organism is maintained by hundreds of Laboratory Response Network (LRN) laboratories throughout the nation. All known stocks of the virus are held at two WHO collaborating laboratories: the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and the State Center of Virology and Biotechnology (VECTOR) in Kotsovo, Russia. WHO has requested destruction of the remaining stocks of this virus, but that has been postponed to evaluate the need for developing vaccines, rapid diagnostics, and antiviral therapy. Since the eradication of smallpox in 1980, most vaccination campaigns against this virus have stopped, and most of the world’s population lacks any protective immunity against this disease or any related poxviruses.

Besides the smallpox virus, 10 other poxviruses are capable of infecting humans. Except for the smallpox virus and the molluscum contagiosum virus, most of these are zoonoses, or infections that result from contact with animals. Fortunately, other than monkeypox and the eradicated smallpox virus, none of these viruses can sustain human-to-human transmission. The viruses normally are acquired through abrasions of the skin and contact with an infected animal, or in the case of human monkeypox, through the oropharynx or nasopharynx in addition to through abrasions on the skin. Poxvirus replicates in the epidermal cells and causes change in the cellular structure, characterized by the “pocks” on the skin. Poxvirus infection can take one of two courses: it can cause a localized infection at the site of inoculation, with little spread from the original site of inoculation, or it can cause a fulminant, systemic infection with spread of the virus throughout the body. The second type of infection is associated with variola virus (smallpox) and also monkeypox, and an increased mortality rate. Monkeypox is almost indistinguishable from smallpox infection except that it lacks the same level of mortality and transmissibility. The monkeypox virus is found in the tropical rain forests of Africa, and its host reservoir is one or more rodent species.

After the individual is exposed to the virus, symptoms of fever and headache occur first, followed by the development of a rash and lymphadenopathy. The rash typically first appears on the face, beginning as macules (small, round changes in skin color), progressing to papules (slightly elevated with no fluid) to vesicles (containing a bubble of fluid) and then pustules (containing purulent material consisting of necrotic inflammatory cells). Depending on the severity of the disease, the illness can last 2 to 4 weeks. Two clades of monkeypox exist, and the Congo Basin clade has the highest fatality rate (up to 12%). In 2003 the importation of rats as pets led to an outbreak of monkeypox in the United States, proving that international travel can be a significant portal of disease from anywhere and to anywhere in the world. RT-PCR (real-time) offers a rapid diagnostic identification tool for cases of monkeypox.

Another member of the poxvirus family is the molluscum contagiosum virus, which causes single or small clusters of lesions. Its only host is humans, and infection occurs either nonsexually, through direct contact or fomites, or sexually, through intimate contact. Usually a self-limiting disease in healthy individuals, molluscum contagiosum can cause a more severe form of disease in immunocompromised patients, resulting in large lesions, especially on the face, neck, scalp, and upper body. Laboratory diagnosis of molluscum contagiosum usually is through biopsy of the lesions and histologic examination. Molecular assays, such as traditional PCR, restriction fragment length polymorphism (RFLP), and real-time-PCR, are still under development.

Orf is another member of the poxvirus family and is transmitted from sheep to humans through human direct contact with infected sheep. This virus causes single or multiple nodules, usually on the hands. These nodules may be painful and may be accompanied by symptoms such as low-grade fever and lymph node swelling. The infection usually resolves in 4 to 6 weeks without further complication, although autoinoculation of the eye can have more serious consequences. An orf diagnosis is made through direct examination of the nodule, along with epidemiologic evidence of a recent history of contact with sheep or lambs. Continued development of PCR assays for identification of parapoxviruses will aid the diagnosis and identification of these viruses.

Reoviruses

The reoviruses (Table 66-21) were first isolated from respiratory and enteric specimens and therefore are referred to as respiratory-enteric-orphan viruses (reoviruses). The term “orphan” originally was included in the description of the virus as a result of the absence of an associated disease when the viruses were first described. Reoviruses infect most mammalian species and are readily detected in water contaminated with animal feces. Common human pathogens of this family include the rotavirus and the agent of Colorado tick fever. Rotaviruses are nonenveloped, double-stranded RNA viruses composed of three concentric protein shells, the outer shell, the inner shell, and the core. Based on the proteins present in these shells, rotavirus is further classified into seven distinct groups, A through G; groups A, B and C cause human disease. Rotavirus is now recognized as the major causative agent of infantile severe gastroenteritis throughout the world. Worldwide, rotavirus is responsible for more than 111 million cases per year, resulting in more than 2 million hospitalizations and 352,000 to 592,000 deaths. Gastroenteritis caused by rotavirus can occur in children of all ages but is most common in infants from 6 months to 3 years old. The disease is characterized by sudden onset of vomiting, followed by explosive, watery diarrhea and moderate to high fever, often accompanied by dehydration. The severity of the disease often is worse for children in developing countries because of malnutrition and limited or delayed health care. Rotaviruses are transmitted by the fecal-oral route, although airborne transmission has been suspected as the cause of nosocomial infections and outbreaks in nursing homes, hospitals, and day care centers. Rotavirus occurs more frequently in the winter months in temperate climates.

Many methods of laboratory testing are available for the diagnosis of rotavirus. Rotavirus can be detected directly in the stool using ELISA, latex agglutination, RT-PCR, cell culture, and electrophoretic separation of the viral genome or electron microscopy. The latex agglutination test offers rapid results with limited laboratory equipment, an advantage in developing countries where resources are limited. Rotavirus is difficult to cultivate from human specimens. Viral isolation is not normally attempted.

Retroviruses

The retrovirus family Retroviridae (Table 66-22) constitutes a large group of viruses that primarily infect vertebrates. They are enveloped RNA viruses, and each virion contains two identical copies of single-stranded RNA. The viral nucleic acid strands are surrounded by the structural proteins that form the nucleocapsid and the matrix shell. On the outer surface of the nucleocapsid and matrix protein is the lipid envelope derived from the host cell membrane. Proteins that mediate adsorption and penetration into the host cell membrane are inserted into the viral envelop. Retroviruses are unique, because they have the enzyme reverse transcriptase. Reverse transcriptase allows the viral RNA genome to be replicated into DNA and then RNA rather than directly into RNA.

Amino acid sequencing of the reverse transcriptase protein divides the retrovirus family into groups. The human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) are members of this family as are the human T cell lymphoma viruses types 1 and 2 (HTLV-1 and HTLV-2). HIV-1 (Figure 66-6) is the more aggressive virus and is responsible for the acquired immunodeficiency syndrome (AIDS) pandemic. The virus was first isolated in 1983, and a year later was proven to be associated with early and late stages of AIDS. HIV-2 was discovered in 1986 and is less pathogenic. AIDS is the end stage of a process in which the immune system and its ability to control infections and malignant proliferation is destroyed. The virus has an affinity for the CD4+ surface marker of T lymphocytes. As the number of CD4+ T lymphocytes decreases, the risk and severity of opportunistic infections increases. Some of the most common opportunistic infections associated with HIV infection include disseminated coccidioidomycosis, cryptococcosis, cryptosporidiosis, histoplasmosis, recurrent pneumonia, and pneumocystis pneumonia. Detection of the HIV antibody is the mainstay of clinical diagnosis. Repeatedly reactive antibody tests done with EIA should be confirmed using Western blot testing (Figure 66-7). Clinical management of infected individuals involves the use of highly active antiretroviral therapy (HAART) and depends on the measurement of CD4+ lymphocytes and the viral load. Molecular methods often are used to quantify the viral load. Diagnosis of HIV infection in babies born to HIV-positive mothers is problematic because of maternal IgG in the baby’s blood; therefore, PCR for identification of viral DNA or RNA is recommended. Genome sequencing is used to establish susceptibility to antiviral agents.

The risk of laboratory-acquired infections with these viruses is a critical consideration; the greatest caution must be exercised in handling any specimens capable of harboring a blood-borne agent. Infection occurs through contamination of the hand and mucous membranes of the eyes, nose, or mouth with infected blood or other body fluids. No evidence exists of airborne transmission. Proper personal protective equipment must always be worn, including a laboratory gown, good-quality gloves, and eye protection. Disposable, unbreakable plastic ware should always be used in the handling of blood or bodily fluids.

HTLV-1 is endemic in the Caribbean, Africa, South and Central America, Melanesia, and Japan. However, only a small percentage of people infected (fewer than 4%) develop symptoms and disease. Cell-to-cell contact and TAX-induced clonal expansion of infected cells are the major avenues for viral replication, making detection of the virus difficult. As a result, serologic detection has remained the gold standard for diagnosis. Molecular detection and the development of PCR assays are being investigated in research laboratories. The average time from infection to the development of adult T-cell leukemia is approximately 40 years.

Rhabdoviruses

Rhabdoviruses (Table 66-23) infect plants, arthropods, fish, and mammals. The virion consists of single-stranded RNA with a helical nucleocapsid surrounded by a lipid bilayer envelope. Spikelike projections approximately 10 nm long extend from the surface of the lipid bilayer. Electron microscopy has shown that the virion has a bullet-shaped or conical appearance. The rabies virus is a neurotropic virus that infects all mammals; with very few exceptions, infection terminates in the death of the infected mammal. The rabies virus is transmitted through the saliva of infected animals, usually by a bite. After inoculation, the virus may invade the peripheral nerves or nerve endings directly. Following infection of the nerve cells, the viral genome progresses centripetally transneuronally, through retrograde axoplasmal flow to the central nervous system. In the CNS it proceeds from first-order neurons to second-order neurons. the neurons are the site of viral replication, mainly in the brain and spinal cord; from there the virus spreads to peripheral nerves and to some nonnervous tissue, including the salivary glands. After a variable incubation period, human disease usually begins with generalized symptoms of malaise, fever, fatigue, anorexia, and headache. Frequently (and characteristically for this disease), symptoms include pain and sometimes “tingling” at the site of exposure, which can be the first “rabies-specific” symptom. After this prodromal phase, behavioral changes may start to manifest, followed by rapidly progressing neurologic symptoms that lead to coma and death.

Only six cases of survival of a rabies infection have been documented worldwide. These cases include patients who survived without any complications; other patients have experienced significant neurologic impairment. In 2004, Randy Willoughby developed a treatment protocol for rabies referred to as “The Milwaukee Protocol.” This protocol requires that the patient remain in a prolonged state of generalized anesthesia, anti-viral drugs, and supportive, life-sustaining care until the individual’s natural active immunity is capable of clearing and/or fighting the infection. Updated protocol and statistics related to patient treatment and survival are maintained by the Medical College of Wisconsin and can be accessed at mcw.edu/Pediatrics/Infectious Diseases/PatientCare/Rabies.htm

Animal rabies presents much as do human rabies cases. After the prodromal phase of the disease, a period of increased excitation occurs, with or without aggression. Clinical presentations of rabies often are described as “furious” or “dumb”; the furious type is associated with heightened aggression and agitation, and the dumb type with lethargy and paralysis.

Rabies is diagnosed by postmortem examination of brain tissue using a direct immunofluorescent assay. Specific sections of the brain are examined for the rabies antigen using fluorescent-tagged monoclonal antibodies and a fluorescent microscope. Prompt, accurate diagnosis of rabies infections in animals is important to ensure the success of postexposure prophylaxis for human victims of animal bites and injuries.

Togaviruses

The Togaviridae family (Table 66-24) includes rubella virus and the alpha viruses, a large group of mosquito-borne arboviruses. Rubella is found only in the human population and is transmitted through direct contact with nasopharyngeal secretions or by congenital transmission. Rubella, sometimes called the “German measles,” is usually a benign disease characterized by fever and rash. Before the trivalent vaccine, MMR (measles, mumps, and rubella), was developed, rubella was an epidemic disease. A risk associated with this disease is exposure and infection of pregnant women. The virus can infect the developing fetus, causing multiple congenital anomalies. Intrauterine infection during the first trimester may result in low birth weight, mental retardation, deafness, congenital heart disease, and neurologic defects. Infection that occurs later in pregnancy may result in splenomegaly or osteomyelitis, among other birth deficiencies. Fetal infection can be prevented through vaccination of all women before pregnancy.

In arbovirus infections, mosquitoes infect a vertebrate host (e.g., birds and rodents), the virus multiplies (amplifies) in this host and is picked up and passed along in subsequent mosquito bites. Humans are infected incidentally and are not amplifiers of the virus; rather, they are dead-end hosts, unable to pass on the virus to other humans or animals. Human disease varies from asymptomatic infection to fatal encephalitis and includes Eastern, Western, and Venezuelan equine encephalitides. Togavirus disease is diagnosed through detection of specific serum IgG and IgM antibodies. Virus isolation is not practical in clinical laboratories.

Miscellaneous Viruses

Additional viruses detected in humans include the astroviruses and potential agents of hepatitis, transfusion-transmitted virus (TTV), and hepatitis G virus (HGV). The astrovirus is a single-stranded RNA virus found in the gastrointestinal tract of many animals, including humans. Human astroviruses are ubiquitous in children, causing a minority of childhood diarrheas. Astroviruses are detected using electron microscopy.

TTV and HGV are DNA and RNA viruses, respectively. They are detected commonly in human blood specimens but have not yet been associated with any disease. HGV is a flavivirus, similar to HCV. TTV resembles a new group of animal viruses called circoviruses.

Interpretation of Laboratory Test Results

Interpretation of laboratory test results must be based on knowledge of the normal viral flora in the clinical specimen, the clinical findings, and the epidemiology of viruses. Serologic testing, in addition to virus detection assays, may be needed to support or refute the association of a virus isolate with a disease state.

Viruses in Tissue and Body Fluids

In general, detection of any virus in host tissues, CSF, blood, or vesicular fluid is significant. Recovery of adenovirus or mumps virus in urine is usually diagnostic of disease; in contrast, detection of CMV may merely reflect asymptomatic reactivation. Occasionally, enteroviruses are detected in urine as a result of fecal contamination, or HSV from symptomatic or asymptomatic infection of the external urogenital tract. Interpretation of these culture results requires correlation with clinical data. CMV viruria (virus in the urine) during the first 2 weeks of life establishes a diagnosis of congenital CMV infection, whereas detection at 4 weeks or later suggests intrapartum or postpartum acquisition.

Viruses in the Respiratory Tract

Detection of the measles, mumps, influenza, parainfluenza, and respiratory syncytial viruses is significant, because asymptomatic carriage and prolonged shedding is unusual. Conversely, HSV, CMV, and adenoviruses can be shed in the absence of symptoms for periods ranging from a few days to many months. Adenoviruses are detected commonly from asymptomatic infants and young children. Simultaneous detection of this virus from both throat and feces in febrile patients with respiratory syndromes increases the probability of association with illness. Isolation from throat but not feces has a lesser probability of association, and isolation from feces alone has the least diagnostic significance.

Viruses in the Eye

Detection of adenoviruses, HSV, VZV, and some enteroviruses from diseased cornea and conjunctiva usually establishes the etiology of the infection. Enterovirus type 70 is known to cause a particularly contagious form of viral conjunctivitis referred to as acute hyperemia conjunctivitis (AHC).

Detection of Epstein-Barr Virus

Disease caused by EBV is established by detecting antibody to multiple antigens (see Figure 66-3). Detection of antibody to viral capsid antigen, early antigen, and Epstein-Barr nuclear antigen is interpreted as shown in Table 66-25.

TABLE 66-25

Interpretation of Serology Results for Epstein-Barr Virus Infection

Clinical Situation	Heterophile Antibody	IgG-VCA	IgM-VCA	EA	EBNA
No past infection	Usually negative	–	–	–	–
Acute infection	Usually positive	+	+	+	–
Convalescence phase	+/–	+	+ or –	+ or –	+
Past infection	Usually negative	+	–	– or W+	+
Chronic or reactivation	Not useful	+	–	+	+

EA, Early antigen; EBNA, Epstein-Barr nuclear antigen; VCA, viral capsid antigen; W+, weakly positive.

Detection of Enteroviruses

Enteroviruses are most commonly found in asymptomatic infants and children, particularly during the late summer and early fall. Knowledge of the relative frequency of virus shedding is extremely helpful in assessing the significance of results of throat or stool cultures. The prevalence of enteroviruses in the stools of infants and toddlers may approach 30% during peak periods. Shedding of enteroviruses in the throat is relatively transient, usually 1 to 2 weeks, whereas fecal shedding may last 4 to 16 weeks. Thus, isolation of an enterovirus from the throat supports an etiology of a clinically compatible illness more than isolation from the feces alone. If live attenuated oral poliovirus vaccine is used, vaccine strains can be detected in stools of recently vaccinated children and their contacts (e.g., siblings). Typing identifies the enteroviruses as a poliovirus serotype and, in absence of clinical findings that suggest polio along with a setting of recent vaccination, the isolate can be considered “normal.”

Detection of Hepatitis Viruses

Disease caused by HAV is detected using serology tests specific for viral-induced IgM and IgG (see Figure 66-5; Table 66-26). In addition to clinical findings consistent with disease, the presence of HAV-specific IgM is diagnostic of current, active disease. HBV requires detection of antigen and antibody to multiple antigens to classify the disease type. HDV co-infection with HBV relies on detection of anti-HDV antibodies. Diagnostic tests for HCV include RT-PCR and antibody detection. PCR is used to detect early acute hepatitis C disease, because antibody detection tests may be negative. ELISA testing for antibody is used to detect chronic hepatitis C disease (see Figure 66-1). HCV RNA levels in serum, detected by a number of highly sensitive molecular biopsy methods, are used to differentiate patients who are likely to respond to therapy from those with lower response rates. All patients with sustained response to therapy became negative for HCV RNA within 6 months. HCV genotyping is used to identify genotypes more or less likely to respond to therapy. For example, patients with HCV genotype 1 have significantly lower response rates to therapy. Antibody tests are used to detect patients infected with hepatitis E; however, disease is rare in the United States.

TABLE 66-26

Serologic Profiles After Typical Hepatitis B Virus (HBV) Infection

HBsAg	Anti-HBsAg	HBeAg	Anti-HBeAg	Anti-HBcAg	Most Likely Interpretation
–	–	–	–	–	No (or very early) exposure to HBV
+	–	+/–	–	–	Early acute hepatitis B (HB)
+	–	+	–	+	Acute or chronic HB
+	–	–	+	+	Chronic HBV carrier state
–	–	–	+	+	Early recovery phase from acute HB
–	+	–	+	+	Recovery from HB with immunity
–	+	–	–	–	Distant HBV infection or HB vaccine

Detection of Varicella-Zoster Virus and Herpes Simplex Virus

Detection of VZV is always significant. Asymptomatic shedding does not appear to occur with this virus, as it does with other herpes viruses. Detection of HSV from cutaneous or mucocutaneous vesicles also is significant, implying primary or reactivation disease. HSV may be detected in respiratory secretions during asymptomatic “stress” reactivation unless typical vesicles or ulcers are present. HSV in stool usually represents either severe disseminated infection or infection of the anus or perianal areas. Isolation from any specimen from a newborn infant suggests potentially severe infection.

Detection of Cytomegalovirus

Interpretation of results of specimens containing CMV is most difficult. Primary CMV infection is usually asymptomatic and is commonly followed by silent reactivation of the latent virus throughout the patient’s life. CMV disease in immunocompromised patients can be life-threatening, and antiviral therapy may be warranted. Detection of CMV in urine or respiratory secretions, however, is not diagnostic of significant disease. Detection of CMV in tissue (e.g., lung) by culture or histopathology or in blood collected by venipuncture suggests an active role in disease. Detection of CMV antigenemia or DNA in the blood by a molecular method is highly suggestive of active disease, and quantitative results can be used to evaluate therapeutic intervention and the patient’s prognosis. Interpretation of the CMV antigenemia assay depends on the patient population and laboratory expertise. In general, detectable virus in peripheral leukocytes is seen with CMV disease. Disease severity is roughly proportional to the quantity of virus (i.e., the number of fluorescing cells. As the disease is treated and resolves, the number of positive cells decreases. Antigenemia levels should decrease to zero as the patient’s immune function is restored and antiviral therapy is introduced. The presence of virus-specific IgM or a fourfold increase in IgG antibodies may indicate disease. However, positive serology results must be interpreted with caution. False-positive IgM results have been attributed to infections caused by other viruses, such as EBV, and rises in both IgM and IgG may result from transfusions or immune globulin therapy.

Detection of Human Immunodeficiency Virus

The HIV-1 Western blot test provides a method of antibody-specific identification of several HIV antigens (see Figure 66-7). The presence of antibody to HIV p24 and to either gp41 or gp160 is sufficient to confirm HIV-1 infection. HIV-1 p24 antigen testing is used to detect acutely infected patients before the appearance of antibody. PCR testing for HIV is useful for newborns, whose maternal HIV antibody may confound interpretation of serology tests, and for all patients because detectable antibody may not be produced for months after primary infection. The quantitative plasma RNA test (viral load) is used to measure the amount of HIV in the blood. As many as 10 billion new HIV virions may be produced daily in the blood of untreated patients. Viral load testing has become an essential parameter in guiding decisions to begin or change antiviral therapy. Plasma HIV RNA can be quantified with various assays approved by the U.S. Food and Drug Administration, including the Roche Monitor RT-PCR (Roche Molecular Diagnostics), Bayer Versant HIV-1 (bDNA) Assay (Bayer Diagnostics), and NucliSense EasyQ HIV-1 (NASBA) Assay (bioMérieux). Viral load testing is performed at the time of diagnosis of HIV infection and periodically thereafter. Successful antiretroviral therapy should reduce plasma RNA to undetectable levels (less than 50 copies/mL).

Chapter Review

(1) Explain the transmissibility of a vector-borne virus. List some of the viruses transmitted through vectors and the disease associated with the virus.

(2) Human papillomavirus causes genital warts; what is a unique feature of the virus and what strains of this virus are frequently responsible for causing cancers?

(3) Explain the difference between antigenic shift and antigenic drift and describe how they affect yearly production of the influenza vaccine.

(4) Explain viral latency; also, list some of the viruses capable of it and the diseases associated with viral reactivation.

(5) Explain how an influenza pandemic can occur. What viral property makes genetic reassortment possible?

(6) What unusual clinical characteristic associated with SARS coronavirus infection distinguishes this virus from other respiratory viruses?

(7) How did the coronavirus acquire its name?

(8) Which historic individual studied “yellow fever” in the tropics? What was the significance of this study?

(9) List the six childhood rashes or exanthemas and the etiologic agents responsible.

(10) Two proteins on the surface of the influenza virus give this virus its unique properties; what are these proteins and what is their function?

(11) What are Koplik’s spots and for what viral disease are they diagnostic?