Initial isolations of alphaviruses were accomplished by intracerebral inoculation into suckling mice—a host very susceptible to infection with most alphaviruses.³³⁵,⁶⁶⁸,⁶⁸⁹,⁷⁸¹,⁷⁸²,⁸⁶⁷,⁸⁸⁴ Many alphaviruses can also be isolated and propagated efficiently in primary chick embryo fibroblasts (CEFs) and in various continuous mammalian cell lines such as human epithelial (HeLa, MRC5) cells, baby hamster kidney (BHK) cells, monkey kidney (Vero) cells, and mouse fibroblast and neuroblastoma cells¹⁰⁸,⁵⁸¹ (Fig. 23.3).

Most alphaviruses will form plaques on susceptible mammalian or avian cells under an agar overlay. Mosquito cell lines also support replication, although often without cytopathic effect (CPE).⁶³⁵,⁷⁶¹,⁷⁶³,⁸⁰³ The first plaque assay of an animal virus was performed using WEEV on CEF cells,¹⁹⁰ and plaque assay remains a convenient and sensitive way to quantify infectious virus. Plaque size has been used to differentiate strains and is determined by the type of overlay used, by relative virus binding to negatively charged sulfated polysaccharides present in the overlay, and by replication efficiency.⁸⁷,⁹⁶,⁷⁷⁵

All alphaviruses are related and share common antigenic sites, as revealed by HI and complement fixation (CF) tests with polyclonal immune sera¹⁰⁷ and by cytotoxic T-cell lysis of infected cells.⁴⁶⁵,⁵⁵⁰ Antigenic cross-reactivities may confer some cross protection and interfere with sequential alphavirus immunizations.¹⁰⁶,¹⁸¹,²⁷⁷,⁴⁶⁵,⁵⁰⁸ These cross-reactivities formed the basis for the original classification into the group A arboviruses and continue to be a valuable means for initial identification and classification of alphaviruses.¹⁰⁷ Closely related viruses within a serogroup form a complex. Seven broad antigenic complexes have been identified within the alphavirus serogroup: Barmah Forest, eastern equine encephalitis (EEE), Middelburg (MID), Ndumu (NDU), Semliki Forest, Venezuelan equine encephalitis (VEE), and western equine encephalitis (WEE).⁴²⁷,⁶²⁶,⁸⁰⁶ A few viruses remain unclassified and will probably form one or more additional complexes (see Table 23.1). The Barmah Forest, EEE, MID, and NDU complexes each contain only a single virus, whereas the Semliki Forest, VEE, and WEE complexes include several viruses. Viruses within each complex can be subtyped using reactivity with MAbs, kinetic HI, or neutralization assays.⁹⁸

Antibodies to E1 are more likely to cross-react with other alphaviruses than are antibodies to E2.⁶⁷,³⁴³,⁸⁹² This is
consistent with the documented sequence conservation in the E1 protein. Competitive binding assays using MAbs have identified approximately seven epitopes on the E1 glycoproteins of SINV, SFV, WEEV, and VEEV.⁶⁷,³⁴³,⁵¹⁹,⁶⁶²,⁷⁰³ Most E1 epitopes are not exposed on the virion surface but are present on the surface of infected cells or on acid-exposed virions.¹⁵,²⁸²,³²⁰,⁵¹⁹,⁷⁰³ These transitional epitopes map to domain III in a region buried at the spike interfaces⁸⁴⁰ (Fig. 23.4). The in vitro biological activities of antibodies to E1 include HI, neutralization of virus infectivity, and inhibition of fusion.¹⁵,⁶⁷,²⁸²,³⁴³,⁷⁰³ Neutralizing epitopes map to domains I, II, and III.²⁸²,⁴⁵⁵

Antibodies to E2 are usually alphavirus specific. Competitive binding assays using MAbs identify four to five epitopes on the E2 glycoproteins of SINV, SFV, RRV, and VEEV.⁶⁷,³⁹⁴,⁵¹⁹,⁵⁸⁶,⁶⁶² In vitro biological activities of antibodies to E2 include HI, neutralization of virus infectivity, and blocking of virus binding to the cell surface.⁶⁶² Many anti-E2 MAbs have both neutralizing and HI activity, suggesting that these functions overlap.²⁸² Neutralization escape mutants, naturally occurring variants, lgt11 expression libraries, site-directed mutagenesis, and recombinant viruses have been used to identify amino acids contributing to the various epitopes on E2 and have identified two major neutralizing sites⁷⁰⁴,⁷⁵⁸,⁸⁵¹ that have been mapped onto the crystal structure of the E1-E2 heterodimer and trimerized spike and are in domain B⁸,²⁸²,³⁶⁷,⁴⁵⁵,⁸⁴⁰ (see Fig. 23.4). This is an exposed hydrophilic region that often includes an N-linked carbohydrate. There are linear, as well as conformational, determinants in this region because these MAbs frequently react in Western blots and recognize l fusion proteins, and antibodies to peptides from this region are protective against challenge.⁸⁵¹

The second neutralizing epitope on E2 appears to be primarily conformational and is in domain A. This region is responsible for binding to HS and is obscured if pE2 is not cleaved.⁹⁶,⁶⁷⁵,⁸⁴⁰ Visualization by cryo-electron microscopy of the binding of HS and MAbs to this epitope on SINV and RRV identifies the domain A knob on the glycoprotein spike.⁷⁴²,⁹⁰²

Alphaviruses, which replicate in arthropods, birds, reptiles, fish, and mammals, derive from a single unknown protoalphavirus and are part of the alphavirus superfamily of viruses. Viruses in this superfamily, including many RNA plant viruses, have a similar genetic organization and replicase proteins but diverse coat proteins.⁴¹⁷,⁷⁶⁶ Among alphaviruses, amino acids important in secondary structure (e.g., cysteines and those close to one another in adjacent β sheets and a helices) have been conserved for the glycoproteins E1, E2, and E3, consistent with a similar three-dimensional structure of the virion for all.²¹⁹ The most variable regions of the genome are in the C-terminus of nsP3 and the N-terminus of C.²¹⁹ Highly conserved regions in the nsP1 and nsP4 genes have allowed development of primers to detect a broad range of alphaviruses by reverse transcription polymerase chain reaction (RT-PCR).²⁰¹,²⁹⁰ Sequence information from the entire genome generally groups the viruses similarly to that derived by antigenic analysis (Fig. 23.5) and has detected at least one recombination event.²⁹⁷ Criteria for species demarcation of alphaviruses combine genetic, ecological, and antigenic information. Species generally have distinct transmission cycles and differ by more than 23% at the nucleotide level and 10% in amino acid sequence when E1 genes are compared.

The origin of the alphaviruses is unclear. Partial genome sequencing has suggested origins both in the Americas and in the Old World.¹⁰⁷,²⁶⁷,⁴³⁴,⁶²⁶,⁸⁵⁹ Recently, comparison of whole genome sequences from all known alphaviruses has suggested an origin from the louse-borne aquatic alphaviruses²¹⁹ (see Fig. 23.5). All of these scenarios require repeated movement across the globe to explain the current virus distributions.

Like other RNA viruses, alphaviruses undergo genetic change primarily by accumulation of point mutations in the genomic RNA; however, deletions and duplications also occur.¹,²¹⁷ Mutation occurs at a rate that is slower (1–7 × 10−⁴ substitutions/nucleotide/year) than is estimated for other RNA viruses,⁹³,¹⁴⁶ presumably because fitness must be maintained in both insect vectors and vertebrate hosts.¹⁴⁷ Recombination
between alphaviruses can be demonstrated in vitro but is infrequent and usually puts the chimeric virus at a replicative disadvantage.⁹³,¹⁴⁶ However, successful recombination has occurred at least occasionally in nature because WEEV resulted from recombination between EEE- and Sindbis-like viruses (see Fig. 23.5) at the junction of E3 and E2, an event that is estimated to have occurred thousands of years ago.²¹⁹,⁴²¹,⁷⁶⁶

Alphaviruses replicate in and are transmitted horizontally by a wide range of invertebrate, primarily mosquito, species. However, each virus usually has a principal or preferred vector for the enzootic cycle. Most alphaviruses can infect various vertebrates but have birds, mammals, or fish as their primary amplifying and reservoir hosts (see Table 23.1). The specific invertebrate vector and vertebrate host used by an alphavirus will contribute significantly to determining the geographic distribution of that virus. Experiments modeling evolution in vitro show that fewer mutations accumulate if replication alternates between vertebrate and invertebrate cells, although diversity, fitness, and adaptability are greater with serial passage.¹⁴⁹,¹⁵⁰,¹⁵³,²⁷⁶,⁸⁵⁶ Experiments employing in vivo passage show that serial passage in mosquitoes increases mosquito infection and that passage in vertebrates produces higher viremias; however, alternately passaged viruses do not change fitness.¹⁴⁹ It is hypothesized that short transmission seasons and host mobility influence alphavirus genetic diversity and evolution in a geographic region.¹⁴⁶,⁸⁵⁸ Viruses using avian enzootic hosts
(e.g., EEEV, WEEV, SINV) extend over wide geographic regions and evolve as a few highly conserved genotypes, whereas viruses using mammalian enzootic hosts with a more limited range of dispersal (e.g., RRV, VEEV) evolve within multiple geographically restricted genotypes.⁴⁹⁰,⁶⁸⁸

Some strains of alphaviruses associated with epidemics or epizootics are antigenically and biologically distinguishable from enzootic strains. Phylogenetic evidence indicates that, at least for VEEV and WEEV, the virulent epizootic strains evolve by mutation from avirulent viruses being maintained in the enzootic cycle.²³,⁶¹,⁶²⁹,⁶⁵⁷

The primary mode of alphavirus transmission to vertebrates is through the bite of an infected insect, most often a mosquito. Mosquitoes salivate during feeding and deposit virus-infected saliva extravascularly.⁸¹⁹ Saliva virus titers are highest early after the mosquito is infected and decline, along with transmission rates, after 1 to 2 weeks; however, mosquitoes remain infected for life.⁵²⁵,⁸³³ The high-mannose glycans on virus from mosquitoes inhibits induction of IFN by myeloid DCs,⁷¹⁸ and proteins in saliva further facilitate transmission by skewing the host cellular immune response toward Th2 cytokines.⁷⁹⁴

The initial sites of virus replication vary with the virus and host. Mice have received the most extensive study. After subcutaneous inoculation, viruses may infect skeletal muscle or fibroblasts at the local site (e.g., EEEV, WEEV, SFV, RRV, SINV, and Getah virus) or be taken up by and infect Langerhans cells in the skin (e.g., VEEV)²⁸⁷,³²⁵,⁴⁶⁶,⁵⁵³ (Fig. 23.6). Langerhans cells and DCs transport virus to lymph nodes draining the site of inoculation that also may become infected.²⁴²,⁴⁸⁷ In vitro, human DCs are susceptible to infection with VEEV but not to infection with CHIKV or EEEV²⁴¹,⁵⁷³,⁷⁰²,⁷⁰⁷,⁷⁵³; thus, the importance of DC infection after mosquito inoculation is likely to differ with the infecting virus.

Alphaviruses induce a substantial plasma viremia in their amplifying hosts and in hosts susceptible to disease (Fig. 23.7). The ability to mount and sustain a viremia depends on the continued efficient production of virus, delivery of virus into the vascular system, and slow clearance from the blood. Animal studies have shown that small plaque viruses are generally less virulent because they are cleared more rapidly from the circulation than are large plaque viruses.³⁵⁴,³⁶⁰,⁶²³ This phenomenon is related to the ability of small plaque viruses to bind HS and thus to be rapidly removed from the circulation by the highly sulfated glycosaminoglycans in the liver.⁹⁷ Ability to invade target organs depends in part on the duration and height of the viremia but also on other characteristics of the virus important for tissue invasion.⁴⁸⁴

Viruses that replicate initially in skeletal muscle and lymph nodes near the site of inoculation often spread through the bloodstream to more distant skeletal muscles and other lymphatic tissues. In addition, cardiac myocytes, osteoblasts, brain and spinal cord neurons, and brown fat cells are secondary sites of replication for many alphaviruses in mice⁹,⁴⁶⁶,⁵³⁵,⁵⁵⁴ (see Fig. 23.6). Getah virus causes polymyositis.³²⁵,⁵⁵⁴,⁷¹³ EEEV, WEEV, SFV, and SINV cause encephalitis⁴⁴,²⁴²,³⁵⁰,⁴⁶⁶,⁸³⁸; RRV and CHIKV cause myositis and arthritis²⁴⁴,⁵⁴³,⁹⁰⁷; and VEEV causes lymphoid depletion and encephalitis.²⁶⁰,³⁵⁹,⁸⁴⁵

In humans, the skin is a target for alphaviruses that cause a rash,²³⁰,⁴⁹³ the joints for alphaviruses that cause arthritis,²²⁹,³²⁸,⁵⁴³,⁷⁷² muscle for alphaviruses that cause myalgia,⁵⁴³,⁵⁹⁰,⁹⁰⁷ and the nervous system for alphaviruses that cause encephalitis.²⁴⁵,⁷⁶⁰ RRV and SINV have been recovered from skin biopsies.⁴²³,⁴²⁴,⁴⁹³ RRV replicates in skin basal epidermal and eccrine duct epithelial cells.²³⁰ CHIKV is found in fibroblasts and macrophages.³²⁸,⁴²⁸ Human synovial cells support RRV infection in vitro,³⁷³ and RRV RNA is detected in synovial biopsy specimens.⁷⁴⁷ Joint fluid taken from humans with acute arthritis has
not yielded infectious virus; however, viral antigen and RNA can be detected in macrophages, and these cells support RRV and CHIKV replication in vitro.³²⁸,³⁷³,⁸⁵⁴

The mechanism by which encephalitic alphaviruses enter the CNS is not entirely clear. Neuroinvasiveness is a component of virulence that varies between viruses and virus strains.¹⁸⁸ Murine studies have shown infection or transport by cerebrovascular endothelial cells,³⁹⁶,⁶⁰²,⁷⁴⁸ infection of choroid plexus epithelial cells,⁴⁶⁶ infection of olfactory neurons,¹³³,⁶⁷⁰,⁷⁹²,⁸³⁷ and transport by peripheral nerves.¹⁵² Once within the CNS, virus can spread cell to cell or through the cerebrospinal fluid (CSF).³⁴⁹,⁵⁸⁴,⁷⁹² For most encephalitic alphaviruses, the targeted cell within the CNS is the neuron,³⁵⁰,⁴⁶⁶,⁵⁸⁴ where cellular protein synthesis is suppressed⁸⁰⁴ and damage can be severe and irreversible. In mice that recover from neuronal infection, infectious virus is cleared but viral RNA persists.²²³,⁴⁴⁶,⁵²¹,⁸²¹ SFV, RRV, and VEEV can cause persistent infection of microglial and oligodendroglial cells leading to demyelination.⁹⁴,¹⁶⁰,²²⁶,⁷¹⁵

A common feature of alphaviruses is their transmission by insects and maintenance in a natural cycle of replication in vertebrate and invertebrate hosts. Arthropod vectors become infected by feeding on a viremic host, are able to transmit the virus 4 to 10 days later (external incubation), and remain persistently infected. Maintenance of this cycle requires an amplifying host that develops a viremia of sufficient magnitude to infect feeding mosquitoes. For many alphaviruses, humans are dead-end hosts unable to infect mosquitoes efficiently. However, human-mosquito-human transmission has been important in epidemics of RRV, o’nyong nyong virus (ONNV), and CHIKV-induced polyarthritis,⁴⁷⁸,⁶⁸⁹,⁷⁹⁰ and horse-mosquito-horse transmission is important in epizootics of VEE.⁶⁵⁹,⁷⁷¹

Other modes of transmission are occasionally important. Horses infected with VEEV may shed virus in nasal, eye, and mouth secretions, as well as in urine and milk, resulting in the potential for transmission by the respiratory route.¹¹⁸,⁴⁰⁵ Aerosol transmission of VEEV, CHIKV, and Mayaro virus has occurred in laboratory settings,¹¹⁸,³⁷⁸,⁴³⁹,⁷⁸⁸,⁷⁹⁹ and aerosolized VEEV has been developed as an agent of biological warfare.⁷²⁸ EEEV persists in the feather follicles of infected pheasants, and transmission among penned pheasants can occur through feather picking and cannibalism.⁶⁹⁷ Person-to-person transmission has not been documented.⁴³⁹,⁶⁵⁹

WEEV, EEEV, and VEEV—the first alphaviruses to be cultured—came to the attention of virologists because they caused fatal disease in horses; these viruses remain important equine pathogens.¹⁸²,⁵²²,⁷⁸⁶ EEEV and WEEV cause encephalitis
in horses, whereas VEEV causes severe respiratory disease associated with leukopenia; encephalitis may or may not be present. Getah virus causes an urticarial rash and hind leg edema in horses.⁷¹⁷ WEEV, EEEV, and Highlands J virus (HJV) cause disease in domesticated birds such as chickens, pigeons, pheasants, turkeys, and emus.⁴²,¹⁶⁷,²¹³,²²¹,⁴⁷⁶,⁸²²,⁸⁶⁸ The alphaviruses associated with arthritis in humans have not been recognized as important causes of disease in domestic animals.

Good small animal models exist for the encephalitogenic alphaviruses but are less satisfactory for study of the arthritogenic alphaviruses. In mice, alphaviruses generally infect lymphatic tissue, muscle, brown fat, brain, and spinal cord; however, the extent and relative importance of infection at these sites differs among these viruses. For instance, RRV and Getah virus cause primarily myositis, VEEV causes reticuloendothelial infection, and WEEV and EEEV cause encephalitis with neurons as the main target cells.⁹,⁵²⁶,⁵⁵⁴ In mice infected with relatively avirulent strains of SFV, RRV, and VEEV, the acute encephalitic phase is accompanied by infection of oligodendroglial cells and demyelination.⁹⁴,⁷¹⁵ For all alphaviruses, fatal disease in mice is usually associated with CNS infection even if encephalitis is not a manifestation of the human infection. For instance, SINV infection of mice is studied as a model for acute viral encephalitis, although SINV causes arthritis and rash, not encephalitis, in humans.³⁴⁹,⁴⁸⁹,⁸²⁰ Specifics of these animal model systems are discussed with the individual viruses.

Virulence is a measure of the ability of the virus to cause fatal disease; for alphaviruses, this usually reflects the severity of neurologic disease. Outcome is influenced by characteristics of both the host and the virus. An early virus determinant of virulence is induction of IFN and susceptibility to IFN-mediated inhibition of replication. Viruses that induce IFN and are susceptible to IFN are generally attenuated.¹²,⁹²,¹⁵⁸,²³⁵,²⁴³,⁷³⁴,⁷⁵⁶,⁸⁸⁰ Most alphaviruses show an age-dependent susceptibility to disease.⁹,⁷⁵,²⁹⁴,⁵⁵⁴,⁵⁸⁴,⁷¹³ Resistance increases with maturation and is associated with decreased virus replication in tissues at the site of virus inoculation and in target tissues (e.g., brain), not with changes in induction of IFN or the ability of infected mice to mount a virus-specific immune response.²⁸⁰,²⁸⁷,⁵⁵⁴ The ability of a virus strain to cause fatal disease or a particular complication of infection also often depends on the genetic background of the host¹⁸⁶,⁷⁵⁹,⁷⁹²,⁸¹⁴; however, the specific genetic determinants of susceptibility are just beginning to be identified.⁷⁹³ Avirulent alphavirus strains may replicate poorly even in newborn animals, whereas virulent strains can usually replicate well and cause disease in adult and newborn animals. The role of the response to IFN is unclear, although older mice increase ISG12 whereas young mice do not.⁴²⁹

For encephalitic alphaviruses, another viral determinant of virulence is their ability to enter the CNS efficiently (neuroinvasiveness). Many alphavirus strains can cause fatal disease after intracerebral or intranasal inoculation but not after subcutaneous or intraperitoneal inoculation. The duration of viremia often correlates with virulence with virulent strains sustaining longer viremias than avirulent strains.³⁵⁵,³⁵⁷,³⁶⁰ Peripheral replication, viremia, neuroinvasiveness, and neurotropism (ability to replicate in CNS cells) all contribute to virulence.

The viral determinants of virulence have been most extensively studied in murine models for SINV, SFV, RRV, and VEEV infections. Viruses with altered virulence have been selected after chemical mutagenesis,⁴⁸,⁸⁵ by passage in tissue culture,⁵⁸,¹⁶⁶,³⁹⁵,⁷⁸³ by passage in mice,²⁸⁴,⁵¹⁷,⁷⁸³ by isolation of MAb escape mutants⁸⁴² or plaque variants,³⁵⁴ and by manipulation of complementary DNA (cDNA) virus clones.¹⁶⁶,⁴⁸⁵,⁷⁷⁹,⁸¹⁵ Nucleotide and amino acid changes affecting virulence have been mapped to the 5′ NTR, nsP1, nsP2, nsP3, E1, and E2. Virulence determinants in the glycoproteins map to receptor-binding regions of the E2 A and B domains⁸⁴⁰ (see Fig. 23.4). Specifics are covered in the sections dealing with each of these viruses.

There is substantial evidence that alphaviruses can persist after appearance of an immune response and clearance of infectious virus from the circulation and from tissue.⁴⁴⁷ Pathologic examination of CNS tissue from human cases of progressive WEEV months to years after resolution of acute encephalitis has shown an active inflammatory process.⁵⁷⁵,⁵⁷⁷ Viral RNA and proteins can be detected in the nervous system long after recovery of mice from SINV or SFV-induced encephalitis and for several weeks in the joints of humans with RRV-induced arthritis.¹⁸⁶,²⁸⁵,³⁹⁶,⁴⁴⁷,⁵²¹,⁷⁴⁷,⁸²¹ It is postulated that this persistence of RNA is attributable to failure of the virus or the immune system to eliminate the infected cells. Interestingly, passive antibody protection predisposes to persistent infection and the late onset of progressive disease.³⁹⁹,⁷¹²

Alphaviruses can be transmitted transplacentally. This has been documented in mice for RRV, SFV, VEEV, and Getah virus³,³⁰,³⁵,⁵²⁴,⁷⁵⁵ and in humans for RRV, WEEV, VEEV, and CHIKV.⁶,²³¹,³⁸⁰,⁷²³ In mice, the virus infects the placenta, where it is able to persist and spread to the fetus despite the development of maternal antibody. The outcome of fetal infection depends on the timing of infection relative to transfer of maternal antiviral IgG to the fetus. Fetuses are protected if transfer occurs prior to infection; however, transfer of antibody after fetal infection does not mediate recovery.⁵²⁴ In monkeys, congenital infection with VEEV induces malformations of the brain and eye.⁴⁷¹ In humans, no abnormalities were observed in infants infected with RRV at 11 to 19 weeks gestation; however, earlier infection may lead to fetal death.⁶ Epidemics of VEE are associated with increases in spontaneous abortion.⁶⁵⁹,⁸⁶² No effect on pregnancy outcome was identified during the CHIKV outbreak on Reunion Island.²³¹

Posterior midgut epithelial cells are the initial sites of infection,⁷⁷,⁵⁵⁸,⁶¹²,⁷⁰⁸,⁸⁶⁴ and infection is facilitated when virus in the serum is concentrated next to these cells as the blood meal clots⁸⁶⁶ (Fig. 23.8). Susceptibility of mosquitoes to alphavirus infection is determined in large part by the ability of the virus to infect midgut epithelial cells.³³⁴,³⁹⁷,⁷¹⁰ Changes both in the virus and vector can affect this interaction.⁸⁸⁹ WEEV rapidly fuses with microvillar membrane preparations from Cx. tarsalis mosquitoes, and both WEEV and CHIKV bind better to membranes from susceptible mosquitoes than to membranes from refractory mosquitoes,³³⁴,⁵⁴⁶ which is consistent with a role for viral structural proteins as determinants of vector specificity, midgut infection, and dissemination.⁶¹²,⁸¹³,⁸³²,⁸³³ The high-affinity laminin receptor A is a receptor for VEEV and SINV on the surface of C6/36 larval Ae. albopictus cells,⁴⁷⁴,⁸⁵⁰ and proteins of 60 and 38 kDa have been identified as putative receptors for CHIKV on brush-border membranes from Ae. aegypti.⁵⁴⁶

Host cell membrane cholesterol levels affect the efficiency of alphavirus entry.⁴⁷³ Mosquitoes, like other insects, do not synthesize cholesterol and obtain sterols needed for reproduction and development from dietary blood. Cholesterol-independent mutants of SFV replicate better than parental SFV in cholesterol-depleted C6/36 cells and in adult Ae. albopictus mosquitoes.¹⁶