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.     

Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses

Although alphaviruses have been extensively studied as model systems for the structural organization of enveloped viruses, no structures exist for the phylogenetically distinct eastern equine encephalomyelitis (EEE)-Venezuelan equine encephalomyelitis (VEE) lineage of New World alphaviruses. Here we report the 25-A structure of VEE virus, obtained from electron cryomicroscopy and image reconstruction. The envelope spike glycoproteins of VEE virus have a T=4 icosahedral arrangement, similar to that observed in Old World Sindbis, Semliki Forest, and Ross River alphaviruses. However, VEE virus has pronounced differences in its nucleocapsid structure relative to nucleocapsid structures repeatedly observed in Old World alphaviruses.     

Western equine and St. Louis encephalomyelitis

In a clinical review of 50 cases of western equine and 16 cases of St. Louis encephalomyelitis in humans it was noted that fever, headache, lethargy, drowsiness, tremor and stiffness of the neck were the most frequent signs or symptoms initiating the illness. The great majority of patients recovered without residual effect. These two diseases of the central nervous system can only be differentiated on an immunological basis but may be suspected during seasonal periods in geographical areas where these virus infections are known to exist. Neuropathological studies were done in four cases of human western equine and two cases of St. Louis encephalomyelitis. The primary point of attack by the virus is the cell body, the lesions being concentrated in the striate body, diencephalon, the brain stem and cerebellum. All histo-anatomical findings (nerve cell destruction, microglial nests, small isolated and confluent areas of necrosis and perivascular round cell infiltration) are secondary to the injury of the nerve cell body caused by the neurotropic virus.     

Virologic and serologic survey for eastern equine encephalomyelitis and certain other viruses in colonial bats of New England.

To test the hypothesis that hibernating colonial bats serve as an overwintering reservoir host of eastern equine encephalomyelitis (EEE) and certain other arthropod-borne viruses in southern New England, 1128 bats of 4 species were collected from 1966 through 1976. Blood and tissue samples and ectoparasites from these bats were tested in suckling mice, wet chicks, and/or chick-embryo tissue cultures for virus. Rabies, the only virus isolated, was recovered from the brain, salivary glands, and brown fat of an apparently healthy adult male Myotis keenii found hibernating in western Massachusetts.     

Eastern equine encephalomyelitis virus in experimentally infected bats.

Colonial bats (Myotis supp. and Eptesicus sp.) were infected with eastern equine encephalomyelitis virus by subcutaneous inoculation or by the bite of infected mosquitoes. Bats were maintained in an environment simulating conditions encountered in hibernacula or in summer maternal colonies. Virus was detected in the blood of hibernating bats at irregular intervals over a 42-day observation period; viremia perhaps was influenced by the amount of disturbance (arousal) involved in the blood sampling process. Target organs included brown fat, spleen, lung, kidneys, pancreas, and liver. Neutralizing antibody was not detected in sera collected from these bats between days 4 and 42 post-inoculation. In nonhibernating bats, virus was recovered from mammary glands, brown fat, pancreas, lungs, kidneys, and liver, in addition to blood. Attempts to infect bats orally or to transmit virus to suckling mice by the bite of viremic bats were unsuccessful. Virus was transmitted from viremic chickens to E. fuscus by the bite of Culiseta melanura and Aedes aegypti.     

Seroprevalence of eastern equine encephalomyelitis virus in birds and larval survey of Culiseta melanura Coquillett during an interepizootic period in central Ohio.

From June through August in 1999 and 2000, we conducted an avian serosurvey for eastern equine encephalomyelitis (EEE) virus at Killbuck Marsh Wildlife Area (KMWA), a focus of infection in central Ohio. We also monitored abundance of the suspected enzootic vector, Culiseta melanura Coquillett, in Brown's Lake Bog, an adjacent wetland. Of the 363 birds of 30 species sampled in 1999, three gray catbirds (Dumetella carolinensis) were positive for antibodies to EEE virus, representing 1.2% of the avian samples and 4.2% of the gray catbirds sampled. Given these results and the abundance of gray catbirds at this site, this species became the focus of our sampling efforts in 2000. However, none of the 109 samples collected from 98 catbirds in 2000 was positive for the virus. Culiseta melanura adults were monitored using resting boxes and CDC CO, light traps at both sites in 1999. Culiseta melanura larvae were monitored in 1999 and 2000 at Brown's Lake Bog, the closest known source of this species, approximately 5km from the avian serosurvey site. We suggest that dry conditions reduced the breeding and abundance of Cs. melanura in 2000 and possibly the transmission of EEE virus at KMWA.     

Packaging signals in alphaviruses.

Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This implies the existence of an encapsidation or packaging signal which would be responsible for selectivity. Previously, we had identified a region of the Sindbis virus genome that interacts specifically with the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the 132-mer is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome. For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene. This is the same region that was proposed to contain the packaging signal for Semliki Forest virus genomic RNA.     

Liquid-phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus.

Ozone, in a liquid-phase application, was evaluated as a residue-free viral inactivant that may be suitable for use in an arboviral research laboratory. Commonly used sterilizing agents may leave trace residues, be flammable or explosive, and require lengthy periods for gases or residues to dissipate after decontamination of equipment such as biological safety cabinets. Complete liquid-phase inactivation of Venezuelan equine encephalomyelitis virus was attained at 0.025 mg of ozone per liter within 45 min of exposure. The inactivation of 10(6.5) median cell culture infective doses (CCID50 of Venezuelan equine encephalomyelitis virus per milliliter represented a reduction of 99.99997% of the viral particles from the control levels of 10(7.25-7.5) CCID50/ml. A dose-response relationship was demonstrated. Analysis by polynomial regression of the logarithmic values for both ozone concentrations and percent reduction of viral titers had a highly significant r2 of 0.8 (F = 63.6; df = 1, 16). These results, together with those of Akey (J. Econ. Entomol. 75:387-392, 1982) on the use of ozone to kill a winged arboviral vector, indicate that ozone is a promising candidate as a sterilizing agent in some applications for biological safety cabinets and other equipment used in vector studies with arboviruses.