Chapter 54:  Alphaviruses

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.

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.

Structure

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 × 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.

Multiplication

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).

Host Defenses

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.

Epidemiology

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.

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 mosquitoes. 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

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

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.