Alphaviruses (Togaviridae) and Flaviviruses (Flaviviridae)
Alan L. Schmaljohn
Disease occurs in either of two general forms, depending upon the virus: one is typified by fever, malaise, headache, and/or symptoms of encephalitis (e.g., eastern, western, or Venezuelan equine encephalitis viruses) and the other by fever, rash, and arthralgia (e.g., chikungunya, Ross River, Mayaro, and Sindbis viruses).
The enveloped virions are spherical, 60 to 70 nm in diameter with a positive-sense, monopartite, single-stranded RNA genome, ca. 11.7 kilobases long. The lipid-containing envelope has two (rarely three) surface glycoproteins that mediate attachment, fusion, and penetration. The icosohedral nucleocapsid contains capsid protein and RNA. Virions mature by budding through the plasma membrane.
Classification and Antigenic Types
Alphavirus is one of two genera in the family Togaviridae; Rubivirus (rubella virus), the other togavirus genus, is discussed in Chapter 55. The 27 alphaviruses are classified on the basis of antigenic properties. All alphaviruses share antigenic sites on the capsid and at least one envelope glycoprotein, but viruses can be differentiated by several serological tests, particularly neutralization assays.
Genomic RNA is capped and polyadenylated and serves as mRNA for nonstructural proteins (e.g., RNA-dependent RNA polymerase) which are encoded in the 5' two-thirds of the genome. Complementary (antisense) RNA, made from genomic RNA, serves as a template for progeny genomic RNA. A subgenomic mRNA representing the 3' one-third of the genome encodes the structural proteins.
Infection is transmitted via infected mosquitoes. In the vertebrate host, transient viremia and dissemination occur as virus is released from cells that later lyse. Infection with seroconversion in the absence of clinical disease is common, but disease can be incapacitating and, in cases of encephalitis, occasionally fatal. Virus is eliminated by the immune system but arthritis or central nervous system impairment may persist for weeks.
Initial resistance is conferred by nonspecific defenses such as interferon. Antibodies are important in recovery and resistance, and T-cell responses are also involved. Lasting protection is generally restricted to the same alphavirus and is associated with, but not solely attributable to, the presence of neutralizing antibodies.
Viruses are maintained in nature by mosquito-vertebrate-mosquito cycles. Restricted interactions between viruses, vector species, and vertebrate hosts tend to confine the geographic spread of alphaviruses. Occasionally, a virus may escape its usual ecological niche and cause widespread epizootics (Venezuelan equine encephalitis virus) or urban epidemics (chikungunya virus). Human infections are seasonal and are acquired in endemic areas.
Diagnosis is suggested by clinical evidence and by known risk of exposure to virus. Confirmation is typically by virus isolation and identification, or by a specific rise in IgG antibody, or the presence of IgM antibody.
Disease surveillance and virus activity in natural hosts are used to determine whether control measures will be undertaken to reduce populations of vector mosquitoes or to vaccinate hosts, especially horses. Human vaccines, where available, are used only in individuals at particularly high risk of exposure, such as laboratory workers.
Major syndromes and examples of causative flaviviruses include: encephalitis (St.Louis encephalitis, Japanese encephalitis, Powassan, and tick-borne encephalitis viruses), febrile illness with rash (dengue virus), hemorrhagic fever (Kyasanur Forest disease virus and sometimes dengue virus), and hemorrhagic fever with hepatitis (yellow fever virus).
Virions are spherical and 40-50 nm in diameter with a positive-sense, nonsegmented, single-stranded RNA genome of ca. 10.9 kilobases. The lipid-containing envelope has one surface glycoprotein that mediates attachment, fusion, and penetration, and an internal matrix protein. The nucleocapsid contains capsid protein and RNA. Virions mature at intracytoplasmic membranes.
Classification and Antigenic Types
Classification within the genus is based upon antigenic properties. Flaviviruses share one or more common antigenic sites, but viruses can be differentiated by several serological tests, particularly neutralization assays.
Genomic RNA is capped (not polyadenylated) and serves as mRNA for all proteins. Structural proteins are encoded at the 5' end of the genome, and nonstructural proteins (e.g., RNA-dependent RNA polymerase) are encoded in the 3' two-thirds. Complementary (antisense) RNA, made from genomic RNA, serves as a template for progeny genomic RNA.
Infection is initiated by the bite of an infected mosquito or tick. Virus disseminates during lytic infection of cells, causing viremia. Infection and seroconversion in the absence of apparent disease are common, but case fatality rates can be high. Virus is eliminated (with rare exception) by the immune system. In dengue hemorrhagic shock syndrome, disease is thought to be exacerbated by preexisting immunity to a related flavivirus (i.e., immune enhancement).
Initial resistance can be conferred by a variety of nonspecific defenses. Antibodies are demonstrably important in recovery and resistance, and T-cell responses are also evident. Lasting protection is generally restricted to the same flavivirus, and is associated with neutralizing antibodies.
Viruses are maintained in nature by transmission in mosquito-vertebrate-mosquito or tick-vertebrate-tick cycles. With yellow fever and dengue viruses, humans are important intermediate hosts during urban epidemics. Human infections are seasonal and are acquired in endemic areas.
Diagnosis is suggested by clinical evidence and by known risk of exposure to virus. It is confirmed by virus isolation and identification. Alternatively, a specific rise in antibody titer may confirm diagnosis, but for individuals immune to more than one flavivirus, it may be difficult to serologically discriminate the more recent infection due to some type of cross reactivity.
Surveillance of disease activity and of virus in natural hosts is used to determine whether control measures will be undertaken to reduce populations of vector mosquitoes. A safe and effective live-attenuated vaccine exists for yellow fever, and inactivated-virus vaccines are available for Japanese encephalitis and tick-borne encephalitis.
At least 27 alphaviruses and 68 flaviviruses have been recognized, approximately one-third of which are medically important human pathogens. They vary widely in their basic ecology; each virus occupies a distinct ecologic niche, often with restricted geographic and biologic distribution. As shown in Tables 54-1 and 54-2, alphaviruses and flaviviruses can cause various syndromes, ranging from benign febrile illnesses to severe systemic diseases with hemorrhagic manifestations or major organ involvement. The neurotropic alphaviruses and flaviviruses can produce severe destructive central nervous system disease with serious sequelae. Several alphaviruses (chikungunya, Mayaro, and Ross River) cause painful arthritis that persists for weeks or months after the initial febrile illness. Yellow fever virus has unique hepatotropic properties that cause a clinically and pathologically distinct form of hepatitis with a hemorrhagic diathesis. The dengue viruses, which cause more human illness than all other members of their family, may produce a serious, sometimes fatal, immunopathologic disease in which shock and hemorrhage occur. Hepatitis C virus (Chapter 70) may be a flavivirus.
Alphavirus is one of the two genera in the family Togaviridae; the other genus (Rubivirus) has rubella virus (Chapter 55) as its only member. Flavivirus, once classified in the Togaviridae, now constitutes one of three genera in the family Flaviviridae; the other two genera are Pestivirus and "Hepatitis C-like viruses". Pestivirus includes animal pathogens (bovine viral diarrhea and hog cholera viruses) that are of considerable economic importance, but contains no known human pathogens. Hepatitis C virus is described in Chapter 70. All alphaviruses and flaviviruses that cause disease in humans are arthropod-borne viruses (arboviruses). In the original classification scheme based on antigenic relationships, alphaviruses and flaviviruses were termed group A and group B arboviruses, respectively.
Most alphaviruses and flaviviruses survive in nature by replicating alternately in a vertebrate host and a hematophagous arthropod (mosquitoes or, for some flaviviruses, ticks). Arthropod vectors acquire the viral infection by biting a viremic host, and after an extrinsic incubation period during which the virus replicates in the vector's tissues, they transmit virus through salivary secretions to another vertebrate host. Virus replicates in the vertebrate host, causing viremia and sometimes illness. The ability to infect and replicate in both vertebrate and arthropod cells is an essential quality of alphaviruses and flaviviruses. The principal vertebrate hosts for most are various species of wild mammals or birds. The natural zoonotic cycles that maintain the virus do not usually involve humans. However, a few viruses (yellow fever virus, dengue virus types 1, 2, 3 and 4 and chikungunya virus) can be transmitted in a human-mosquito-human cycle. As a result of being pathogenic for humans and capable of transmission in heavily populated areas, these viruses can cause widespread and serious epidemics. Because of their high transmission potential, these viruses are major public health problems in many tropical and subtropical regions of the world where appropriate mosquito vectors are present. Because some of these agents are dangerous human pathogens and are highly infectious, special containment and safety precautions in the laboratory are required.
Pathogenesis and Clinical Manifestations
Human illness caused by alphaviruses (Figure 54-1) is exemplified by agents that produce three markedly different disease patterns. Chikungunya virus is the prototype for those causing an acute (3- to 7-day) febrile illness with malaise, rash, severe arthralgias, and sometimes arthritis. O'nyong'nyong, Mayaro, and Ross River viruses, which are closely related (antigenically) to chikungunya virus, cause similar or identical clinical manifestations; Sindbis viruses cause similar but milder diseases known as Ockelbo (in Sweden), Pogosta (Finland), or Karelian fever (Russia). Virus introduced by the bite of an infected mosquito replicates and causes a viremia coincident with abrupt onset of fever, chills, malaise, and joint aches. The specific site of viral replication is unknown. The viremia subsides in 3 to 5 days, and antiviral antibodies appear in the blood within 1 to 4 days of the onset of symptoms. A macular-papular rash typically develops around the third to fifth day of illness, when the patient is defervescing. The migratory arthralgia, which is so characteristic of these viral diseases, involves mainly the small joints and occurs more prominently in adults than children. In more severe cases the involved joints are swollen and tender, and rheumatic signs and symptoms may persist for weeks or months following the acute illness.
FIGURE 54-1 Pathogenesis of alphaviruses.
The pathogenesis of eastern equine encephalitis and western equine encephalitis virus infection of humans (as well as of equines) similarly involves percutaneous introduction of virus by a vector (Figure 54-1) and development of viremia; however, the majority of human infections with these viruses are either asymptomatic or present as a nonspecific febrile illness or aseptic meningitis. The ratio of neurologic disease per human infection is estimated for eastern equine encephalitis as 1:23. For western equine encephalitis this ranges from about 1:1000 in adults to nearly 1:1 in infants, respectively. Symptoms usually begin with malaise, headache, and fever, followed by nausea and vomiting. Over the next few days the symptoms intensify, and somnolence or delirium may progress into coma. Seizures, impaired sensorium, and paralysis are common. The severity of neurologic involvement and sequelae is greater with decreasing age. Histopathologic findings resulting from neuronal invasion and replication are similar to those of most other acute viral encephalitides, and include inflammatory cell infiltration, perivascular cuffing, and neuronal degeneration. All regions of the brain may be affected.
Venezuelan equine encephalitis virus infection in humans routinely produces an acute febrile illness with pronounced systemic symptoms, whereas the central nervous system disease occurs only infrequently and usually is much less severe than in eastern and western equine encephalitis. Following an incubation period of 2 to 6 days, patients typically develop chills, high fever, malaise, and a severe headache. A small percentage of human infections (less than 0.5% in adults and up to 4% in children, but probably varying with virus subtype) will progress to neurologic involvement with lethargy, somnolence or mild confusion, and possibly nuchal rigidity. Seizures, ataxia, paralysis, or coma herald more severe central nervous system invasion. Overt encephalitis is more commonly seen in infected children, where case fatalities range as high as 35% in comparison to 10% for adults. However, for those who survive encephalitic involvement, neurologic recovery is usually complete.
Virions are spherical, 60 to 70 nm in diameter, with an icosahedral nucleocapsid enclosed in a lipid-protein envelope. Alphavirus RNA is a single 42S strand of approximately 4 x 106 daltons that is capped and polyadenylated. Alphavirus genomes that have been sequenced in their entirety are approximately 11.7 kilobases long. Virion RNA is positive sense: it can function intracellularly as mRNA, and the RNA alone has been shown experimentally to be infectious. The single capsid protein (C protein) has a molecular weight of approximately 30,000 daltons. The alphavirus envelope consists of a lipid bilayer derived from the host cell plasma membrane and contains two viral glycoproteins (E1 and E2) of molecular weights of 48,000 to 52,000 daltons. A small third protein (E3) of molecular weight 10,000 to 12,000 daltons remains virion-associated in Semliki Forest virus but is dispatched as a soluble protein in most other alphaviruses. The only proteins in the envelopes of alphaviruses are the viral glycoproteins, each anchored in the lipid at or near their C-terminus. On the virion surface, E1 and E2 are closely paired, and together form trimers that appear as "spikes" in an orderly array.
Classification and Antigenic Types
Classification is based upon antigenic relationships. Viruses have been grouped into seven antigenic complexes; typical species in four medically important antigenic complexes are Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis, and Semliki Forest viruses. Genome sequence information typically obtained after viral RNA has been amplified by polymerase chain reaction (PCR) is used with increasing frequency in the identification and classification of new viruses. The capsid protein induces antibodies, some of which are widely cross-reactive within the genus by complement fixation and fluorescent-antibody tests. Anti-capsid antibodies do not neutralize infectivity or inhibit hemagglutination. The E2 glycoprotein elicits and is thought to be the principal target of neutralizing antibodies; however, some neutralizing antibodies react with E1. Similarly, hemagglutination-inhibiting antibodies may react with either E2 or E1. Hemagglutination-inhibiting antibodies cross-react, sometimes extensively, among alphaviruses. Such cross-reactivity is attributable to the E1 glycoprotein, the amino acid sequences of which are more highly conserved among alphaviruses than those of E2. Neutralization assays are virus-specific, and species or subtypes are defined principally on the basis of neutralization tests.
Alphaviruses attach to cells, probably via interactions between E2 and a poorly defined family of cellular receptors found on many vertebrate and invertebrate cells. Entry is thought to take place in mildly acidic endosomal vacuoles where glycoprotein spikes undergo conformational rearrangements and an acid-dependent fusion event (principally a function of E1) delivers genomic RNA to the cell cytoplasm. Viral replication occurs in the cytoplasm. Initial translation of virion RNA produces a polyprotein that is proteolytically cleaved into an RNA polymerase. Transcription of the virion RNA through a negative-strand RNA intermediate produces a 26S positive-strand mRNA which encodes only the structural proteins, as well as additional 42S RNA, which is incorporated into progeny virions. Translation from the 26S mRNA (which represents the 3' one-third of genomic RNA) produces a polyprotein that is cleaved proteolytically into three proteins: C, PE2, and E1; PE2 is subsequently cleaved into E2 and E3. Envelope proteins formed by posttranslational cleavage are glycosylated and translocated to the plasma membrane. Virion formation occurs by budding of preformed icosahedral nucleocapsids through regions of the plasma membrane containing E1 and E2 glycoproteins (Figure 54-2).
FIGURE 54-2 Morphogenesis of alphaviruses.
Differences in susceptibility between individuals and species are not easily ascribed to specific immune responses, and a variety of non-specific defense mechanisms may be important. Alphaviruses are efficient inducers of interferon, the production of which probably plays a role in modulating or resolving infections. Antibodies are important in disease recovery and resistance. The appearance of neutralizing antibodies in serum coincides with viral clearance, and immune serum can diminish or prevent alphavirus infection. Although their precise roles are not clearly established, T-cell responses are also demonstrable and may contribute substantially to immunity. Lasting protection is generally restricted to the same alphavirus, and is associated with (but not solely attributable to) the presence of neutralizing antibodies. Cross-reactive immunity among different alphaviruses is sometimes observed in the absence of cross-neutralizing antibodies. In experimental animals, such immunity can be mediated by cytolytic nonneutralizing antibodies. The role of T cells is less clear but has been inferred from cytotoxic and other effector activities in vitro that may be alphavirus specific or cross-reactive.
Eastern and western equine encephalitis viruses are important in the United States. Both are maintained in natural ecologic cycles involving birds and, principally, bird-feeding mosquitoes such as Culiseta melanura. Eastern equine encephalitis (EEE) virus is enzootic in fresh water swamps in the eastern United States; it causes sporadic equine and rare human cases. Small human outbreaks may occur (Figure 54-3). Since Cs. melanura mosquitoes usually do not feed on humans, transmission to horses and humans is potentiated when less fastidious Aedes species feed upon an adequate natural reservoir of infected birds. EEE virus outbreaks are typically recognized upon the occurrence of severe equine or human encephalitis in a discrete geographic area. Western equine encephalitis (WEE) virus is widespread in the United States and Canada and has been responsible for outbreaks of equine and human disease in western and southwestern states. Its principal vector, Culex tarsalis, is a common mosquito, especially in irrigated regions.
FIGURE 54-3 Alphavirus transmission. Virus abbreviations: Chik, chickungunya; RR, Ross River; May, Mayaro; ONN, O'nyong-nyong; SIN, Sindbis; EEE, eastern equine encephalitis; VEE, Venezuelan equine encephalitis.
Eight or more antigenic subtypes of Venezuelan equine encephalitis (VEE) virus exist; they have differing virulence and epidemic potentials. At least 10 different species of mosquitoes, including Culex and Aedes species, may transmit VEE virus, and vector competence varies for enzootic versus epizootic subtypes. Birds do not seem to play an important reservoir role in nature. The enzootic, less pathogenic strains are maintained in mosquito-rodent-mosquito cycles. Enzootic subtypes of relatively low equine virulence exist in South and Central America and Florida. Enzootic strains are ecologically restricted to cycles between small mammals and mosquitoes. Sporadic and sometimes severe human cases have been described. In contrast to other alphavirus encephalitides, epizootic strains of VEE are mainly amplified in horses, so that equine cases occur prior to reports of human disease. Although the reservoir in nature for the epizootic/epidemic strains of VEE virus (IA, IB, IC) has long been baffling, recent evidence suggests that these may arise via mutation from enzootic strains of ID subtype. In this manner, these strains have caused massive equine epizootics with associated human epidemics. A 1969 to 1972 epizootic involved several Central American countries and spread through Mexico to Texas.
Chikungunya virus exists in Africa in a forest cycle involving
baboons and other primates and forest species of mosqui
toes. It can also be transmitted in a human-mosquito-human cycle by Aedes aegypti. This mode of transmission has caused massive epidemics in Africa, India, and Southeast Asia. The virus is endemic throughout much of south and Southeast Asia. The antigenically similar Mayaro virus exists in the Amazon Basin. Its cycle involves new world primates and hematophagous mosquitoes and causes outbreaks of human disease through exposure to the forest cycle. Ross River virus is endemic in Australia and has spread in epidemic form to several islands of the Western Pacific.
Diagnosis of alphavirus infection is suggested by clinical evidence and known risk of exposure to virus. It can be confirmed only by laboratory tests. Infection by one of the viruses of the chikungunya-Mayaro complex may be difficult to distinguish from many clinically similar illnesses such as rubella, dengue, phlebotomus fever, enterovirus infection, and scrub typhus.
Encephalitis from one of the alphaviruses must be suspected on epidemiologic grounds. Risk for disease is increased relative to arthropod contact near swampy forested areas during the summer, and encephalitic illness of horses in the surrounding locale is an important indication of ongoing transmission of an alphavirus. In conjunction with laboratory serologies, the geographic locale and patient's travel history are of major importance in diagnosing an arboviral encephalitis. Non-togaviral etiologies of encephalitis include lymphocytic choriomeningitis virus, echo, coxsackie, polio, herpes simples, rabies, mumps, and Colorado tick fever. Cerebrospinal fluid examination, to include viral cultures, is critical in differentiating bacterial from viral infections, and infectious from noninfectious etiologies. In some instances it is necessary to institute chemotherapy for possible treatable infecting organisms and await definitive laboratory diagnostic tests. Laboratory diagnosis can be established by isolating virus from the blood during the viremic phase or by antibody determination. A variety of serologic tests, especially neutralization, but also enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition, complement fixation, and reactivities with appropriate monoclonal antibodies, are used by public health laboratories to diagnose alphavirus infections. Testing by ELISA for specific IgM is particularly useful in discriminating recent infection with one alphavirus from previous exposure to another alphavirus. An increasing number of laboratories have the capacity to diagnose alphavirus infections by detection of viral RNA (e.g. using polymerase chain reaction, PCR) or proteins (e.g., immunohistochemistry) in frozen or fixed tissues.
Control of alphavirus diseases in the United States is based on surveillance of disease and virologic activity in natural hosts and, when necessary, on control measures directed at reducing populations of vector mosquitoes. These measures include control of larvae and adult mosquitoes, sometimes by using ultra-low-volume aerial spray techniques. In some areas, insecticide resistance (for example, resistant C tarsalis) is a major limitation to effective control. Inactivated vaccines are used to protect laboratory workers from eastern, western, and Venezuelan equine encephalitis viruses. An effective live attenuated Venezuelan equine encephalitis vaccine has been employed extensively in equines as an epidemic control measure, and a similar vaccine is used to protect laboratory workers. A live attenuated chikungunya vaccine has proven safe and immunogenic in investigational human trials.
Pathogenesis and Clinical Manifestations
Flaviviruses vary widely in their pathogenic potential and mechanisms for producing human disease. However, it is useful to consider them in three major categories: those associated primarily with the encephalitis syndrome (prototype: St. Louis encephalitis), with fever-arthralgia-rash (prototype: dengue fever), or with hemorrhagic fever (prototype: yellow fever).
Human infection with both mosquito-borne and tick-borne flaviviruses is initiated by deposition of virus through the skin via the saliva of an infected arthropod (Fig 54-4). Virus replicates locally and in regional lymph nodes and results in viremia. In most human infections with St. Louis encephalitis (SLE) and Japanese encephalitis (JE) viruses, there is either no apparent disease or a nonspecific febrile illness with headache. The infection resolves, and lasting immunity is produced. However, central nervous system invasion may develop and present as aseptic meningitis or encephalitis. For SLE the ratio of apparent to inapparent infection ranges from 1:800 to 1:100 with increasing age, whereas for JE it averages from 1:200 to 1:300 depending upon age, prior immunity and virus strain differences. Clinical manifestations of encephalitis due to SLE, JE, or Murray Valley encephalitis (MVE) virus begin as fever, headache, and stiff neck, and progress to an altered level of consciousness and focal neurologic deficits (e.g., tremors, pathologic reflexes, cranial nerve palsies, nystagmus, ataxia). Paralysis is more commonly seen with JE and MVE. Seizures occur in about 10% of SLE and JE cases, and constitute a poor prognostic sign in adults. The case fatality rate ranges from 2% in young adults to over 20% in the elderly. Neurologic sequelae are common in older age groups afflicted with SLE, and in children recovering from JE. Viral replication in neural and glial tissue produces a severe inflammatory process, evidenced by extensive neuronal degeneration and lymphocytic infiltrates. The pathogenic potential of St. Louis encephalitis virus and several other flaviviruses such as Japanese encephalitis (JE), Murray Valley encephalitis (MVE), and tick-borne encephalitis (TBE) depends entirely on their neurovirulence (i.e., their ability to invade and replicate in the central nervous system). For example, the Far Eastern type of TBE (Russian spring summer encephalitis, or RSSE) has a more gradual onset but greater case-fatality rate (20% vs. less than 5%), as well as more severe and frequent neurologic sequelae, than the central European type.
FIGURE 54-4 Pathogenesis of flaviviruses.
In contrast to the encephalitis-producing agents, yellow fever virus produces severe systemic disease with relatively high frequency. The case:infection ratio ranges from approximately 1:2 to 1:20 in those with and without prior heterologous flavivirus immunity, respectively. Viral replication occurs in reticuloendothelial cells in many organs and in the parenchyma of the liver, adrenal glands, heart, and kidneys. High concentrations of virus are present in the blood and involved organs. Characteristic liver damage from infection of hepatic cells is midzonal necrosis and intracellular eosinophilic deposits called Councilman bodies. Liver function tests become markedly abnormal, and icterus is often severe. Prerenal azotemia as well as acute tubular necrosis may give rise to oliguria and acid-base disturbances. Myocardial fiber degeneration and myocarditis may contribute to general circulatory collapse. Coagulation defects, probably resulting from both liver damage and disseminated intravascular coagulation, are major manifestations and are associated with the severe gastrointestinal hemorrhages characteristic of yellow fever. The clinical course of yellow fever is that of an acute illness lasting 1 week or more, and ranges from a nonspecific, mild febrile illness to classic disease with severe hemorrhagic and hepatic involvement. The initial onset is abrupt with fever, myalgia, headache, vomiting, and minor gingival hemorrhage or epistaxis lasting for about 3 days. Then a brief day of improvement may precede fulminant illness manifested as severe toxicity, jaundice, extensive mucosal and gastrointestinal hemorrhage, azotemia, and shock. Death may occur within 5-10 days; case fatality rates are 10%-50%.
Dengue viruses of all four serotypes cause three distinct syndromes: classic dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Although caused by the same viruses, dengue and dengue hemorrhagic fever are pathogenetically, clinically, and epidemiologically distinct. Dengue viruses appear to replicate in macrophages at the site of the mosquito bite, in regional lymph nodes, and then throughout the reticuloendothelial system. Viremia is concurrent with clinical illness. Virus is present in the serum and in association with circulating monocytes. Severe leukopenia often is present. Dengue fever is characterized by sudden onset of systemic toxicity, fever, headache, vomiting, and severe myalgia or bone pain of escalating intensity. Either coincident with or following remittance of fever on days 3 to 5 of the illness, there appears a maculopapular or morbilliform rash on the trunk which spreads to the limbs and face. This phase of the illness is often accompanied by recrudescence of fever, lymphadenopathy, granulocytopenia, and thrombocytopenia. Minor mucocutaneous bleeding is occasionally manifested by petechiae, epistaxis, menorrhagia, and a positive tourniquet test. Dengue fever lasts 3 to 9 days, is self-limiting, and is rarely associated with serious sequelae.
In contrast, the clinical course of dengue hemorrhagic fever is characterized by an initial stage of fever, rash, and anorexia (lasting 3 to 5 days) followed by a shock phase in which hepatomegaly, hypotension, and a hemorrhagic diathesis occur. Complement activation and thrombocytopenia typically take place at the onset of the shock phase and reverse spontaneously after a period that ranges from hours to a few days. Dengue hemorrhagic fever results from additional pathogenetic processes not present in classical dengue fever; the most important of which are diffuse capillary leak with hemoconcentration, thrombocytopenia, and disseminated intravascular coagulation.
In dengue shock syndrome, the decreased plasma volume which results from increased vascular permeability causes clinical shock that, if uncorrected, may lead to acidosis, hyperkalemia, and death. Ninety percent of dengue hemorrhagic fever cases occur in children experiencing multiple infections with dengue. There are four dengue serotypes that can be discriminated by neutralization assays, and persons can be infected serially or even simultaneously by two different serotypes.
Dengue hemorrhagic fever and dengue shock syndrome arise via immunopathologic mechanisms following sequential infection of an individual with these heterologous, antigenically-related serotypes (dengue-1, -2, -3, or -4). In infants, the presence of subprotective levels of maternal anti-dengue antibody has been reported to be a factor. "Immune enhancement" is thought to play a major role in pathogenesis, whereby both homologous and heterologous antibodies binding to dengue virus can markedly enhance infection of macrophages in vitro via cellular Fc receptors. Several antigenic determinants for infection-enhancing antibodies have been found on the envelope glycoprotein. Thus, it has been postulated that cross-reacting antibodies from a previous dengue infection or maternal anti-dengue antibodies in infants enhance the entry of virus into macrophages. The increased viral replication in the macrophages then contributes to the complement activation, vascular permeability, and clotting abnormalities observed in patients, through the release of products from infected macrophages. These products may be released by increased destruction of infected macrophages. An alternative or expanded view of the "immune enhancement" phenomenon incriminates cross-reactive T cells as important mediators of immunopathology. It is speculated that T cells exacerbate the antibody-enhanced destructive cascade, as they vigorously respond to (and then destroy) antigen-presenting cells, with concomitant release of cytokines by both T cells and damaged macrophages.
In the great majority of flavivirus infections, virus is cleared by the immune system. However, persistence in neurological tissue has been noted with tick-borne encephalitis viruses, and recent reports of recurrent encephalitic bouts in children have been associated with JE virus recovery from peripheral blood mononuclear cells.
Flavivirus virions are spherical, ca. 50 nm in diameter, and consist of a nucleoprotein capsid enclosed in a lipid envelope. The RNA is a single 40S (ca. 10.9 kilobases) positive-sense strand and is capped at the 5' end, but, unlike alphaviruses, has no poly A segment at the 3' end. The virion has a single capsid protein (C) that is approximately 13,000 daltons. The envelope consists of a lipid bilayer, a single envelope protein (E) of 51,000-59,000 daltons, and a small nonglycosylated protein (M) of approximately 8,500 daltons. Only E, which is glycosylated in most flaviviruses, is clearly demonstrable on the virion surface.
Classification and Antigenic Types
Classification within the genus is based upon antigenic relationships and genetic relationships are becoming increasingly important in classification. Viruses have been grouped into several antigenic complexes typified, for example, by dissimilar viruses such as dengue, tick-borne encephalitis, St. Louis encephalitis, and yellow fever viruses. Although classification was not intentionally based upon vectors or diseases, the tick-borne flaviviruses important in human disease are aligned with tick-borne encephalitis virus in a single antigenic group, while several encephalitogenic mosquito-borne viruses (St. Louis encephalitis, Japanese encephalitis, Kunjin, Murray Valley encephalitis, and West Nile viruses) make up another group.
All flaviviruses are antigenically related by sharing common or similar antigenic determinants on C and E proteins. The single envelope glycoprotein, E, is the viral hemagglutinin; antibodies against E are involved in virus neutralization and hemagglutination inhibition. The antigenic determinants that induce neutralizing antibody are specific, and species or subtypes of flaviviruses are distinguished principally by neutralization tests. Hemagglutination inhibition tests reveal a broad range of cross-reactions among the flaviviruses. Monoclonal antibody studies reveal genus, group, and virus-specific epitopes on the envelope glycoprotein. The nonstructural proteins also are antigenic, and at least one nonstructural protein, NS-1, contains both virus-specific and cross-reactive epitopes.
The mechanism by which flaviviruses enter cells probably involves an interaction between the E protein and cellular receptors, followed by a post-attachment fusion event that occurs in acidic intracytoplasmic vacuoles. Naked genomic RNA is infectious if introduced into the cytoplasm. The genomic RNA is capped but not polyadenylated; it serves as mRNA for all proteins. Structural proteins are encoded at the 5' end of the genome, and nonstructural proteins (e.g., NS-1 and RNA-dependent RNA polymerase) are encoded in the 3' two-thirds. Complementary (negative-sense) RNA, made from genomic RNA, serves as a template to generate genomic RNA. Replication occurs in the cytoplasm.
Virions are formed in perinuclear regions of the cytoplasm in association with Golgi or smooth membranes (Figure 54-5). Virions appear within cytoplasmic vacuoles and appear to exit the cell as vacuoles fuse with the plasma membrane. Unlike alphaviruses, no evidence of budding has been seen in flavivirus-infected cells, and the mechanisms of virion assembly and release remain obscure.
FIGURE 54-5 Morphogenesis of flaviviruses.
Flavivirus proteins arise by co- or post-translational cleavage of the polyprotein encoded by the genome. The E protein mediates attachment and penetration; it also has hemagglutinating activity. The C protein associates with RNA to form a nucleocapsid. The M protein is membrane-associated and is thought to serve a matrix function, linking capsid and envelope. The precursor of M (prM) is cleaved late in viral morphogenesis and is thought to stabilize the E protein during early events of viral assembly and transport. The nonstructural glycoprotein NS-1 is not incorporated into virions but is found in endoplasmic reticulum, at the cell surface, and also in a soluble form. The function of this protein has not been defined but is likely involved in virion morphogenesis. Other nonstructural proteins form the RNA-dependent RNA polymerase.
Nonspecific host factors are responsible for heritable patterns of susceptibility and resistance in mice, and interferon may play a role in resolving infections. As with many viruses, neonates and children tend to be particularly susceptible. However, natural defenses against central nervous system invasion by St. Louis encephalitis virus are most effective in children and much less effective in the elderly, resulting in higher disease-to-infection ratios in older persons. Antiviral antibody has an important protective role in host defenses against flaviviruses: recovery from infection usually coincides with the appearance of neutralizing antibodies, and prophylactically administered immune serum can prevent or diminish infection. Virus-specific cytotoxic and helper T cell activities are also demonstrable. Heterologous immunity is not usually protective, even though prominent serologic cross-reactions by hemagglutination inhibition and complement fixation tests are present. Indeed, heterologous antibody increases the infectivity of dengue viruses for human macrophages in vitro.
Lasting protection is generally restricted to the immunizing flavivirus, and is associated with neutralizing antibodies directed against the E protein. Antibodies directed against NS-1, in the presence of complement or Fc receptor-bearing cells, can mediate destruction of flavivirus-infected cells and thereby contribute to humoral immunity.
The flaviviruses constitute a highly diverse genus, and their ecology is similarly varied and complex. Only the basic concepts are considered here. The mosquito-borne encephalitis viruses (St. Louis encephalitis, Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses) exist primarily as viruses of birds and are transmitted by Culex mosquitoes that feed readily on birds (Figure 54-6). St. Louis encephalitis virus is maintained in nature in an avian cycle; in temperate areas, the virus is maintained through the winter in infected hibernating adult mosquitoes. In the United States, human epidemics of St. Louis encephalitis are preceded by increased virus dissemination among wild birds along with increased vector populations and vector infection rates. St. Louis encephalitis virus is widespread in the United States and causes periodic outbreaks in California, Texas, the Ohio-Mississippi Valley, and the southeast. In Asia, Japanese encephalitis virus occupies an ecologic niche similar to St. Louis encephalitis virus in the western hemisphere, with one major difference: it infects swine, in which it causes high viremias. Because Asian vectors of Japanese encephalitis virus feed readily on swine, these animals are an efficient amplifying host for this virus. As a result, Japanese encephalitis epi demics in regions where swine are present have been frequent and severe. This virus is a major public health concern in Japan, China, India, and Southeast Asia.
FIGURE 54 -6 Flavivirus transmission. Virus abbreviations: DEN, dengue; KFD, Kyasanur Forest Disease; OMSK, Omsk hemorrhagic fever; WN, West Nile, YF, yellow fever; SLE, St. Louis encephalitis; JE, Japanese encephalitis; POW, Powssan, RSSE, Russian spring-summer encephalitis; MVE, Murray Valley encephalitis; ROC, Rocio.
The tick-borne flaviviruses are maintained by tick-mammal cycles and by transovarian transmission in ticks. Humans are infected with this subgroup of flavivirus through the bite of infected ticks, and thousands of cases may occur annually in the region of the Eurasian continent between central Europe and western Siberia. A closely-related virus transmitted by Ixodes ticks, termed Powassan virus, produces sporadic cases of encephalitis in the eastern parts of Canada and the U.S.
Yellow fever virus in Africa and South America has two distinct epidemiologic patterns: sylvan and urban. Sylvan (jungle) yellow fever is maintained among canopy-dwelling monkeys and tree-hole breeding mosquitoes (Aedes spp. in Africa, and Haemagogus spp. in South America). This pattern of human disease occurs sporadically or in small outbreaks, and initially only in persons exposed to forest mosquitoes. Urban yellow fever is transmitted in a highly efficient human-mosquito-human cycle by the urban mosquito Aedes aegypti. The extensive re-expansion of A aegypti's habitat in the Americas has raised the specter of potential epidemics of yellow fever, similar to those occurring in several U.S. cities (e.g., Philadelphia, New York, New Orleans) in the late 19th and early 20th centuries. Currently in the Americas, yellow fever is confined to the Amazon basin and adjacent savannah forest, whereas yellow fever epidemics with high mortality rates continue to occur with alarming frequency in tropical Africa.
Dengue fever is the most common flavivirus infection among humans, with extensive distribution of virus serotypes through the tropics and warm temperate areas of Africa, Asia, Australia, Oceania, India, and the Americas. The incidence of disease corresponds to the worldwide dispersion of the principal vector, A aegypti, which serves to maintain the virus in a human-mosquito-human cycle. The resurgence of this arthropod vector has resulted in recent large urban outbreaks of dengue fever in South America. Other Aedes sp of the subgenus Stegomyia are implicated as vectors in Asia and the Pacific region. Epidemiologic patterns are highly varied but may be described as epidemic, endemic, or hyperendemic. In endemic regions, dengue fever occurs principally in children and often is unrecognized. In Southeast Asia where all four serotypes are continuously transmitted, epidemics of dengue hemorrhagic fever are a major cause of death among children, involving thousands of cases and a 3 to 10 percent case fatality rate.
It is critical that compatible epidemiologic factors be aligned with clinical features in order to make the diagnosis of a flaviviral disease, which can then only be confirmed by laboratory tests (e.g., serology, virus isolation, or PCR). The clinical manifestations of encephalitis from either an alphavirus or a flavivirus infection do not usually suggest a specific etiologic agent. Furthermore, infectious virus is not easily demonstrable in the cerebrospinal fluid and is no longer present in the blood. Virus usually can be isolated only by brain biopsy or from the brain at autopsy. Thus, serologic tests showing an antibody rise are most practical for diagnosis. Epidemic arboviral fevers that are accompanied by rash and arthralgia and which may be confused with dengue include Chikungunya, O'nyong-nyong, West Nile, Sindbis, Mayaro, and Ross River diseases. Yellow fever must be differentiated from viral hepatitis, falciparum malaria, or drug-induced hepatic injury. Both yellow fever and dengue hemorrhagic fever can be difficult to distinguish from other viral hemorrhagic fevers, such as those caused by Arenaviridae (Lassa, Junin, Machupo, Guanarito, and Sabia), Filoviridae (Marburg and Ebola), and Bunyaviridae (Congo-Crimean hemorrhagic fever, Rift Valley Fever).
Interpretation of serologic data obtained by hemagglutination-inhibition, complement-fixation, and fluorescent antibody tests is difficult in most tropical areas where several flaviviruses are endemic. In primary infections, the virus neutralization test provides virus-specific confirmation. If a patient has had previous flavivirus infections, cross-reactions make even neutralization test results difficult or impossible to interpret. Demonstration of specific IgM in the cerebrospinal fluid by antibody-capture immunoassay can be an excellent way to diagnose flaviviruses encephalitis. In yellow fever, dengue, and dengue hemorrhagic fever, virus is present in the blood for 4 or even 5 days after onset of fever. Virus isolation in mammalian or insect cell culture is the classical method of choice for diagnosis, though genotypic detections and analyses (e.g., using PCR) provide good alternatives. The earlier the specimen is obtained for isolation, the higher the likelihood of success.
Control of disease caused by flaviviruses is based on vaccines for some viruses and on vector control. At present, formalinized (killed) virus vaccines are used to prevent Japanese encephalitis and tick-borne encephalitis in endemic regions. The live attenuated 17D yellow fever vaccine is an effective and safe vaccine, and is widely used in South America and Africa.
Urban epidemics of yellow fever in the Americas were controlled by containment and, in some regions, eradication of the A aegypti vectors. Vector control is not feasible to prevent jungle yellow fever; therefore, vaccines are widely used in affected regions.
Live attenuated vaccines for dengue have been developed and used in Thailand, and experimental subunit vaccines have been made through recombinant DNA technologies. It is important that a dengue vaccine confer appropriate immunity to all four serotypes so as to avoid potentiating the immunopathologic phenomenon of dengue hemorrhagic fever upon subsequent exposure to wild-type heterologous strains. Vector control, including destroying larval habitats and spraying insecticide to kill adult mosquitoes, is the only widely available means for controlling dengue and dengue hemorrhagic fever.
St. Louis encephalitis is managed in the United States by vector control, including water drainage and aerial ultra-low-volume spraying of insecticides in populated areas with epidemic potential. No vaccine is available. Surveillance of St. Louis encephalitis virus activity in wild bird populations and vectors is used to monitor the risk of epidemics and to guide vector control requirements.
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