Impact of alphavirus 3'UTR plasticity on mosquito transmission.

Alphaviruses such as chikungunya and western equine encephalitis viruses are important human pathogens transmitted by mosquitoes that have recently caused large epidemic and epizootic outbreaks. The epidemic potential of alphaviruses is often related to enhanced mosquito transmission. Tissue barriers and antiviral responses impose bottlenecks to viral populations in mosquitoes. Substitutions in the envelope proteins and the presence of repeated sequence elements (RSEs) in the 3'UTR of epidemic viruses were proposed to be specifically associated to efficient replication in mosquito vectors. Here, we discuss the molecular mechanisms that originated RSEs, the evolutionary forces that shape the 3'UTR of alphaviruses, and the significance of RSEs for mosquito transmission. Finally, the presence of RSEs in the 3'UTR of viral genomes appears as evolutionary trait associated to mosquito adaptation and emerges as a common feature among viruses from the alphavirus and flavivirus genera.

Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests.

Fourteen viruses closely related to the Fleming strain of western equine encephalitis (WEE) virus were cross-tested by serum dilution-plaque reduction neutralization. The results demonstrate that strains McMillan, R-43738, AG80-646, BeAr 102091, and Y62-33 are subtypes or varieties of western equine encephalitis virus strain Fleming. Ockelbo, Kyzylagach, and Babanki are subtypes of the prototype strain (EgAr 339) of Sindbis virus. Fort Morgan and Buggy Creek viruses are closely related to each other, whereas Highlands J and Aura viruses are distinct from other members of this antigenic complex. There appear to be parallels between geographic distribution and antigenic relatedness. We hypothesize that birds, the principal vertebrate hosts for these viruses, spread the progenitor viruses north and south and from continent to continent. Viruses of the WEE complex with lesser antigenic differences may develop in discrete ecologic conditions.

Characterization of Fort Morgan virus, an alphavirus of the western equine encephalitis virus complex in an unusual ecosystem.

An alphavirus isolated from nestling Cliff Swallows (Petrochelidon pyrrhonota) and House Sparrows (Passer domesticus) and from cimicid bugs (Oeciacus vicarius) in eastern Colorado, for which we propose the name Fort Morgan (FM) virus, is sensitive to the action of sodium deoxycholate, unstable at pH 2.0-4.0, and demonstrates no characteristics of temperature-sensitive mutants. Unpassaged field strains are nonpathogenic, or of low pathogenicity, for suckling mice; however, plaque-purified FM virus is pathogenic for a variety of laboratory hosts. By hemagglutination-inhibition (HI), complement-fixation, and neutralization tests, cross-reactions were observed between FM virus and members of the western equine encephalitis (WEE) virus antigenic complex. Short-incubation HI tests indicated that the new isolate shared closer antigenic relationships with WEE complex virus strains from the eastern United States (Highlands J virus) than with other WEE complex viruses. On the basis of these serological findings, as well as characterization of the structural polypeptides and oligonucleotides, we suggest that FM is a distinct virus belonging to the WEE antigenic complex. A reconsideration of the taxonomy of the WEE complex and discussion of the epizoologic significance of FM virus are presented.

Identification of Highlands J virus from a Florida horse.

A virus, strain 64A-1519, isolated from the brain of a horse dying of encephalitis in Florida in 1964, was identified as western equine encephalomyelitis (WEE) virus. Recently, we used polyclonal and monoclonal immune reagents to identify this isolate by comparing it to 2 strains of WEE virus and to Highlands J (HJ) virus in hemagglutination-inhibition, immunofluorescent antibody, and plaque-reduction neutralization tests. These tests demonstrate that strain 64A-1519 is a strain of HJ virus distinct from WEE virus.

Limited potential for mosquito transmission of genetically engineered, live-attenuated western equine encephalitis virus vaccine candidates.

Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.

Biological characteristics of an enzootic subtype of western equine encephalomyelitis virus from Argentina.

In order to expand our knowledge on the biological characteristics of an enzootic South American subtype of western equine encephalomyelitis (WEE) virus, strain AG80-646, we inoculated guinea pigs, rabbits, newborn chickens and Vero and chick embryo cell cultures with this and other WEE and Wee-related viruses. AG80-646 was found apathogenic for guinea pigs even when inoculated intracranially (i.e.) or intraperitoneally (i.p.), and the animals did not develop viraemia. AG80-646 killed rabbits and the animals developed high viraemia (peak titer was 7.0 log PFU/0.1 ml). These data and previous serological evidence led us to look for a mammal as a natural host. AG80-646 was found lethal for newborn chickens inoculated subcutaneously (s.c.) (peak viraemia titer was 6.6 log PFU/0.1 ml). AG80-646 produced plaques (diameter 0.8-1.0 mm) in Vero and chick embryo cells 3-4 days post infection (p.i.) A comparison of AG80-646 with other WEE complex virus strains led to the following observations: (1) The lethality for guinea pigs was high for the two epizootic Argentinian strains, Cba 87 and Cba CIV 180, zero for the two enzootic strains, AG80-646 and BeAr 10315 (virus Aura), and intermediate for the Russian strain Y62-33 (low by i.c. route and zero by i.p. route); (2) AG80-646 was more virulent for rabbits inoculated i.p. than the three epizootic strains Cba 87, Cba CIV 180 and McMillan; (3) AG80-646 was less virulent for new-born chickens than the Argentinian epizootic strain Cba CIV 180; (4) The viraemia level correlated always with the strain virulence in each animal host. This study provides tools for the differentiation of WEE complex viruses and strains in the future ecological work on WEE in South America.

[Similarities and differences between western equine encephalomyelitis viruses with respect to genes for nonstructural protein NSP2 and structural proteins C and E2]. / Skhodstvo i razlichie virusov zapadnogo éntsefalomielita loshadei po genam nestrukturnogo belka NSP2 i strukturnykh belkov C i E2.

Genetic relationships of geographical isolates of the members of WEE virus serocomplex (McMillan, Fort Morgan, Highlands J, and Y62-33) were assessed by the polymerase chain reaction (PCR) and restriction analysis of the PCR products. Oligonucleotide primers (21 nucleotides in length) were chosen for NSP2, nucleocapsid C, and E2-E1 protein genes based on the known primary structure of the McMillan 16310-5614 genome (L. Uryvayev et al., 1994, 1995). These primers were shown to differentiate well the WEE and SV-like strains of the serocomplex. Y62-33 virus (Udmurtia, Russia) was identical to McMillan strain in three studied regions of NSP2, C, and E2-E1 genes. NSP2 gene could be detected in all the studied geographical isolates and was characterized by the same restriction patterns as endonucleases; it appeared to be the most conservative. The structural genes were less conservative. Fort Morgan virus (Colorado, USA) genome reliably differed from McMillan virus (California, USA) and was negative in PCR with primers to C and E2 gene regions. Highlands J genome (Florida, USA) was positive in PCR with the primers to E2-E1 gene regions but differed from McMillan strain by the nucleocapsid gene. An additional comparative PCR analysis of the C-E2 region in the McMillan and Highlands J genomes showed some, but not complete identity. The origin of these two viruses might be due to the selection of different forms of recombinant viruses. A good correlation of structural genes in PCR and the infectivity neutralization test was noted with the primers and polyclonal antibodies to the closely related strains. High specificity of PCR permits a more accurate detection of the virus origin and relationships.

Single-dose, fast-acting vaccine candidate against western equine encephalitis virus completely protects mice from intranasal challenge with different strains of the virus.

Western equine encephalitis virus (WEEV) causes a fatal infection of the central nervous system in humans and horses. However, neither human vaccine nor antiviral drug is available. We found previously that immunization of mice with two doses of an adenovirus-vectored WEEV vaccine, Ad5-WEEV, confers complete protection against homologous WEEV challenge. In this paper, we report that a single-dose injection of Ad5-WEEV completely protected mice against both homologous and heterologous strains of WEEV at 1 week after immunization. In addition, mice immunized with Ad5-WEEV were protected when challenged at 13 weeks after a single-dose immunization. Therefore, the protection conferred by Ad5-WEEV is rapid, cross-protective, and long-lasting. These results warrant further development of Ad5-WEEV into an emergency vaccine that can be used during a natural outbreak or a bioterrorism attack.

Infectivity variation and genetic diversity among strains of Western equine encephalitis virus.

Variation in infectivity and genetic diversity in the structural proteins were compared among eight strains of Western equine encephalitis virus (WEEV) to investigate WEEV virulence at the molecular level. A lethal intranasal infectivity model of WEEV was developed in adult BALB/c mice. All eight strains examined were 100 % lethal to adult mice in this model, but they varied considerably in the time to death. Based on the time to death, the eight strains could be classified into two pathotypes: a high-virulence pathotype, consisting of strains California, Fleming and McMillan, and a low-virulence pathotype, comprising strains CBA87, Mn548, B11, Mn520 and 71V-1658. To analyse genetic diversity in the structural protein genes, 26S RNAs from these eight strains were cloned and sequenced and found to have > 96 % nucleotide and amino acid identity. A cluster diagram divided the eight WEEV strains into two genotypes that matched the pathotype grouping exactly, suggesting that variation in infectivity can be attributed to genetic diversity in the structural proteins among these eight strains. Furthermore, potential amino acid differences in some positions between the two groups were identified, suggesting that these amino acid variations contributed to the observed differences in virulence.

Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses.

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses.

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.