INTRODUCTION
As a member of the Alphavirus genus in the Togaviridae family, Venezuelan equine encephalitis virus (VEEV) is an enveloped virus with a non-segmented, positive-sense RNA genome of approximately 11.5 kb containing a 5′ methylguanylate cap and a 3′ polyadenylate tail.1 The viral genome is translated by cellular components upon arrival in the cytoplasm.2 The 5′ two-thirds of the genome encode four non-structural proteins (nsP1-4) that form a protein complex required for viral replication and the 3′ one-third of the genome, expressed from a 26S subgenomic message, encodes three structural proteins, the capsid and E2 and E1 envelope glycoproteins2 that are involved in packaging of the viral genome and envelopment to produce infectious viral particles.
Alphaviruses in the VEE antigenic complex3 are classified as epidemic/epizootic, equine-virulent strains isolated during epidemics and equine epizootics (subtypes IAB, IC), or enzootic (subtypes ID-IF, II-VI), equine-avirulent strains that circulate continuously among rodents in sylvatic habitats and only occasionally cause human disease. Periodic VEE outbreaks, primarily in Latin America, have involved up to hundreds of thousands of humans and equines. Recent outbreaks in South America4,5 and Mexico6 emphasize the continuing role of VEEV as a re-emerging human and equine pathogen, and the potential use of VEEV as a biologic weapon has recently received renewed attention.7,8
Major VEEV epidemics have always been associated with the ability of epizootic strains (subtypes IAB and IC) to exploit equines as highly efficient amplifying hosts.9–11 Following infection with these epizootic VEEV strains, horses, donkeys, and mules develop high-titered viremia associated with lymphopenia/leukopenia, which leads to central nervous system invasion and fatal encephalitis (neurologic) disease in 19–83% of the cases.12,13 In contrast to epizootic strains, enzootic VEEV is rarely associated with clinical disease in equines and produces low-level or undetectable equine viremia, which is generally insufficient for efficient infection of mosquito vectors.14 Following infection with both enzootic and epizootic VEEV, humans experience acute febrile illness that sometimes progresses to clinical encephalitis including disorientation, ataxia, mental depression, and convulsions in up to 14% of clinically ill individuals, especially children.10 Neurologic sequelae following VEE are also common in children and have been described in rats.15,16
The pathogenesis of VEEV appears to be determined by multiple factors that include the structure of the E2 envelope glycoprotein spikes on the surface of virions and the ability to replicate in specific tissues.17–19 However, little information is available about the role of non-E2 genes in VEEV pathogenesis and their potential to influence virulence of natural isolates, either in vitro or in vivo.19,20 Considering its multifaceted importance, improved understanding of VEEV biology, particularly the role of the replicative nonstructural proteins in pathogenesis, is critical.
The combination of reverse genetics approaches and the identification of closely related enzootic (subtype ID) and epizootic (subtype IC) VEEV strains has provided new opportunities to identify mutations that allow enzootic variants to generate epizootic strains via small numbers of mutations.12,21,22 To exploit these opportunities, a small animal model for equine disease that responds differentially to enzootic versus epizootic VEEV strains was used. Mice and hamsters are equally susceptible to enzootic and epizootic strains and have uniformly fatal disease following peripheral infection with subtype IC and ID strains.12 Guinea pigs, the best model identified to date, have more variable susceptibility to fatal VEE, and survive infection with enzootic strains longer than infection with epizootic, subtype IAB and IC variants.20,23,24 They have served as a model for preliminary VEEV pathogenesis studies because 1) equine virulence of VEEV strains correlates to some degree with virulence in guinea pigs; 2) in contrast to horses, guinea pigs of known exposure history (e.g., unvaccinated) are available; 3) experiments with guinea pigs are safer to perform than those with equines in Animal Biosafety Level-3 (ABSL-3) containment; and 4) equine experiments with sufficient numbers to obtain statistically significant data are extremely costly. Guinea pigs have been used previously to map virulence determinants of epizootic strains to structural and nonstructural protein genes.20
To determine the role of the envelope glycoprotein genes versus non-envelope proteins and sequences in VEE pathogenesis, we created reciprocal epizootic IC/enzootic ID chimeric viruses and tested them in the guinea pig model. Our results demonstrate the importance of both envelope and non-envelope genes and/or cis-acting elements in the epizootic-specific pathogenesis in guinea pigs.
MATERIALS AND METHODS
Cell cultures.
Vero and baby hamster kidney (BHK) cells were obtained from the American Type Culture Collection (Manassas, VA). They were propagated in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS), and gentamicin (10 μg/mL).
Plasmids.
Recombinant plasmids encoding cDNA copies of the genomes of relevant epizootic and enzootic VEEV strains were constructed and described previously.22,25 The epizootic VEEV subtype IC strain 3908 was isolated from the blood of a febrile human patient during an epidemic in Zulia State, Venezuela on September 16, 1995.5 It was passaged only once in C6/36 cells before cDNA clone (pM1-3908) production.25 Enzootic VEEV subtype ID strain ZPC 738 was isolated from a sentinel hamster on September 24, 1997, in Zulia State, Venezuela,21 and was subjected to one passage in Vero cells prior to cDNA clone (pM1-738) construction.22 Chimeras (Figure 1) were generated by reciprocal replacement of the cDNA fragments located between Afl II and Snab I restriction enzyme sites. The final plasmids contained an SP6 promoter, a 5′-untranslated region (UTR), nsP1-4, a subgenomic 26S promoter, and 3′-UTR sequences of the backbone virus; the last 111 amino acids in the capsid, the complete PE2 and 6K genes, and the first 108 amino acids of the E1 gene were derived from the heterologous virus strain to generate chimeras. The replacement of the desired genome regions was confirmed via sequencing.
In vitro transcriptions and transformations.
Plasmid DNA from chimeric clones pM1-738/3908-PE2 and pM1-3908/738-PE2 was linearized with Not I and Mlu I restriction enzymes (New England Biolabs, Beverly, MA), respectively, and RNA was transcribed in the presence of m7G(5′)ppp(5′)A cap analog (New England Biolabs) with SP6 RNA polymerase (In-vitrogen, Carlsbad, CA), followed by electroporation of BHK-21 cells. Viruses were harvested 24 hour after electroporation, aliquoted, and titered. Viruses generated by electroporation were used without additional cell passage.
Infectious center assay.
Immediately after electroporation with 1 μg of transcribed RNA, BHK-21 cells were diluted 101-,102-, 103-, and 104-fold in MEM supplemented with 5% FBS and seeded on Vero cell monolayers. After a one-hour incubation at 37°C, cells were covered with MEM containing 1.0% Noble agar (Sigma, St. Louis, MO) and incubated for 48 hours at 37°C, followed by fixation with 10% formaldehyde, and staining with a 20% methanol, 0.25% crystal violet solution. In vitro synthesized RNA from the Sindbis virus infectious clone, pTOTO 1101,26 was used as a positive control to compare the efficiency of electroporation and virus recovery.
Analysis of virus replication in vitro.
Vero cells were used to measure viral replication rates of parental and chimeric rescued viruses. Cells from the same passage were seeded into 12-well tissue culture plates and infected at a multiplicity of infection (MOI) of 1.0 plaque-forming units (PFU)/cell. Plates were incubated at 37°C for one hour, then washed three times with phosphate-buffered saline (PBS), followed by the addition of 1 mL of MEM containing 2% FBS. A sample of the supernatant was immediately collected (zero time point) followed by incubation at 37°C for 42 hours. Additional aliquots for virus titration were collected at 6, 10, 24, and 42 hours post-infection.
Plaque size assay.
Vero cells were seeded into six-well Costar plates (Corning, Corning, NY) and allowed to grow to confluency. Serial 10-fold dilutions of virus in MEM supplemented with 1% FBS were adsorbed to the monolayers for one hour at 37°C. A 2-mL overlay consisting of MEM with 2% Noble agar (Sigma) was added to each well, and the cells were incubated at 37°C for 48–72 hours. Agar was removed and the cells were fixed with 10% formaldehyde and stained with a 20% methanol, 0.25% crystal violet solution. The diameters of 20 plaques were measured for determination of average plaque size.
Immunofluorescence.
Immunofluorescence using mono-clonal antibodies (MAbs) recognizing specific epitopes within the E2 envelope glycoprotein was performed as described previously.27 The MAb 1A1B-9 is specific for enzootic VEEV strains including subtype ID, but does not react with epizootic subtype IC viruses; conversely, MAb 1A3A-5 binds to IC viruses, but not ID viruses. Vero cell monolayers prepared on cover slips in 12-well plates were infected with 1,000 PFU of parental and chimeric viruses for 12–24 hours, and fixed with 85% ice-cold acetone at 4°C. The MAbs were diluted 1:400 and incubated for one hour at 37°C with acetone-fixed monolayers. After washing, cells were stained with a secondary goat anti-mouse antibody (diluted 1:1000) for one hour at 37°C, conjugated to fluorescein isothiocyanate (Sigma), and visualized with a fluorescent microscope.
Hemagglutination (HA) assays.
The HA activity of viruses rescued from parental and chimeric cDNA clones was determined as described previously.28 Briefly, an identical amount (PFU) of each virus was resuspended in saline (pH 9.0). The ability of serial dilutions of the extracted virus to agglutinate goose red blood cells at pH 6.0–7.0 was then determined.
Infection of guinea pigs.
Ten to 11-week-old female out-bred guinea pigs (Hartley) were obtained from Harlan (Indianapolis, IN). Animals were maintained on guinea pig chow (LabDiet 5025; Purina Mills, LLC, St. Louis, MO) and allowed to acclimate to the ABSL-3 laboratory for one week prior to subcutaneous infection with 1,000 PFU of parental and chimeric VEEV strains in the right medial thigh. Animals were observed twice a day for signs of infection; average survival time (AST) was determined. Guinea pigs were bled daily from the saphenous vein; viremia was determined via plaque assay on Vero cells.
Pathology.
Tissue samples, including the brain, bone marrow, draining lymph node, and spleen, were collected at 24 and 48 hours after inoculation for virus detection and/or histology. Following anesthesia, animals were perfused with PBS and tissue samples were either homogenized and frozen or fixed in 4% buffered formalin for 24 hours, and stored in 70% ethanol for 12 hours. Tissue samples for histologic examination were then embedded in paraffin, sectioned (5 μm), and mounted on slides. After staining with hematoxylin and eosin, slides were examined by light microscopy. Organs for infectious virus assay were dissected, weighed, homogenized (except bone marrow samples, which were only diluted) as 10–50% suspensions in MEM, and stored at −80°C until titration by plaque assay.
Statistical analysis.
One-way analysis of variance with the Mann-Whitney multiple comparisons test and the Kruskal-Wallis multiple comparisons test were performed for AST and plaque sizes, respectively. The Mann-Whitney multiple comparisons test was performed for viral tissue titers.
RESULTS
Parental and chimeric infectious clones.
The epizootic subtype IC virus rescued from the pM1-3908 cDNA clone, produced after a single C6/36 passage of the field-isolated virus, is indistinguishable from the parent virus in mice29 and is highly pathogenic for equines; experimental infection of horses with this virus resulted in high-titered viremia and encephalitis (Bowen R, Colorado State University, Fort Collins, CO, unpublished data). The enzootic ID virus rescued from the pM1-738 cDNA clone, made from the closely related enzootic strain ZPC738, is indistinguishable from the parent virus in vitro and in vivo.25 The parent ZPC738 strain12 and virus rescued from the infectious clone (Bowen R, unpublished data) are also apathogenic for equines. There are approximately 1% amino acid sequence differences between the two parental viruses (Table 1).
Genetic studies with subtype IC VEEV strain SH3, which was responsible for a small epizootic in 1992 and its close enzootic relative, subtype ID VEEV strain 66637, have suggested that mutations in the E2 envelope glycoprotein, which increase its positive charge, can generate an epizootic-like serotype and plaque phenotype from an enzootic progenitor.21,29 Similar E2 mutations were associated with the emergence of VEEV in Mexico in 1993 and 1996.29 Therefore, we sought to determine the role of the envelope glycoproteins, particularly E2, in the 1995 emergence of epizootic VEE. Chimeric VEEV strains were designed to distinguish between the effects on the development of the epizootic phenotype of mutations in the envelope protein genes and those in other regions of the genome. The chimeras contained the C-terminal 111 amino acids in the capsid, the complete PE2 (the E2 precursor protein that includes the small E3 protein that is cleaved and removed before virus maturation) and 6K (a small gene linking the E2 and E1) genes, and the N-terminal 108 amino acids of the E1 envelope glycoprotein of the epizootic parent in the backbone of the enzootic parent or vice versa (Figure 1). This structural gene region encoded a total of seven amino acids that differ between strains 3908 and ZPC738: six in the PE2 envelope glycoprotein precursor gene and one in the E1 glycoprotein. In particular, the E2 E201K and T213K differences were identified via phylogenetic analyses as strongly associated with epizootic emergence.29 Two additional amino acid differences located in the C-terminus of the E1 gene, one of which is conservative and the other which lies within the transmembrane domain and is probably unrelated to virulence, were not predicted to be important for emergence of strain 3908 (i.e., these amino acid residues are found in some enzootic strains) and were therefore not included in our swapped region (Table 1).
The RNA derived from in vitro transcription from both chimeric cDNA clones demonstrated comparable infectivity to parental and Sindbis control (Toto1101) RNA in the infectious center assay. Both chimeras produced approximately 5 log10 infectious centers per microgram of RNA compared with 5, 5.5, and 4 log10 for the epizootic, enzootic and Sindbis control, respectively. This indicated that no deleterious errors were inadvertently introduced into the chimeric clones and no compensatory mutations were required for their viability.
In Vero cells, strain 3908 virus exhibited faster replication at the early time points examined, with cell culture supernatant titers approximately 2 log10 PFU/ml higher than the enzootic and chimeric infectious clones after 6–10 hours of infection. However, virus from all clones replicated to similar final titers (Figure 2). Strain ZPC738 and the chimeric viruses produced similar replication kinetics in Vero cells at the same MOI.
In vitro markers.
Martin and others30 used plaque size on Vero cells overlaid with unpurified agar as a marker of the epidemiologic phenotype of VEEV strains, and previous work in our laboratory has confirmed the usefulness of this simple in vitro marker.20,21 In this study, the epizootic infectious clone virus 3908 produced smaller plaques (mean ± SD plaque diameter = 1.0 ± 0.2 mm) compared with the enzootic ZPC738 strain (3.6 ± 0.4 mm; P < 0.001). The mean plaque diameters of the reciprocal envelope chimeras were intermediate. Replacement of the envelope gene region in the strain ZPC738 with the epizootic gene (ZPC738/3908-PE2 chimera) significantly reduced the mean ± SD plaque size to 2.6 ± 0.3 (P < 0.01), compared with the enzootic parent. The reciprocal chimera (3908/ZPC738-PE2) generated by replacing the epizootic envelope with that from the enzootic strain resulted in a mean ± SD plaque size of 2.5 ± 0.3 mm that was significantly larger than the epizootic parent (P < 0.001) and smaller (P < 0.001) compared with the enzootic parent, indicating that both PE2 and non-PE2 genome regions determine plaque size. Plaque size on Vero cells overlaid with unpurified agar is thought to be determined by interactions of positive charge E2 glycoprotein-residues on the surface of the virion with poly-anions in the agar.29 Our results indicate that residues within the non-envelope regions effect plaque size by an unknown mechanism, possibly related to viral replication efficiency.
Parental and chimeric viruses produced from the cDNA clones reacted as expected in immunofluorescence assays using MAbs. Positive staining was detected in strain 3908-infected Vero cells using the 1A3A-5 (IAB/IC epizootic-specific) MAb, but not the enzootic-specific 1A1B-9 MAb; the ZPC738 virus-infected cells reacted only with the IAIB-9 MAb. Antigenicity of the envelope chimeras was specific to the PE2 gene of the parent donor, with the ZPC738/3908-PE2 showing epizootic-specific reactivity and 3908/ZPC738-PE2 chimera reacting only with the enzootic-specific MAb.
Comparisons of infectious virus titers and HA activity.
To ensure that comparative pathogenesis studies were not influenced by differences in the plaque-forming efficiency of the VEEV strains and variants tested, we used both infectious and non-infectious assays to quantify the amount of virus used for guinea pig inoculations. Equal PFU amounts (approximately 7.4-7.5 log10) of infectious virus exhibited similar HA activity, a measure of the total quantity of envelope proteins in the virus preparations. Parental and chimeric viruses agglutinated red blood cells at the same pH (6.4) with identical titers (1:32). These data indicated that all viruses produced from cDNA clones had comparable particle:PFU ratios.
Guinea pig virulence.
Ten- to eleven-week-old female out-bred guinea pigs, six per virus, were infected subcutaneously with 1,000 PFU of parental and chimeric viruses. Experimental infections resulted in 100% mortality regardless of the virus strain used (Figure 3 and Table 2); however, guinea pigs infected with the parental viruses experienced different pathogenesis. Strain 3908-infected guinea pigs developed high titered viremia (see below) and extensive viral replication in various tissues (see below), followed by a shock-like death characterized by hypothermia, renal, hepatic and intestinal congestion as well as pulmonary edema and alveolar hemorrhages (mean ± SD AST = 2.9 ± 0.6 days; other data not shown). Conversely, strain ZPC738-infected animals developed lower levels of viremia, neurologic disease, and experienced prolonged survival (mean ± SD AST = 5.5 ± 1.0 days). Both chimeric strains generated intermediate survival times; guinea pigs infected with the chimeric virus containing epizootic nonstructural proteins and the enzootic PE2-E1 region exhibited an AST that was longer but not significantly different from those infected with the epizootic strain 3908 (Table 2). The introduction of the PE2-E1 region of epizootic origin into the enzootic backbone also resulted in a shorter but not a significant change in AST from the enzootic strain. Animals infected with the chimera containing the epizootic PE2-E1 in the enzootic backbone (ZPC738/3908-PE2) developed neurologic symptoms previously described for animals infected with the enzootic strain, rather than the shock-like syndrome characteristic of epizootic strain infection. These results strongly suggested that non-envelope genes affect guinea pig virulence.
In vivo replication.
Guinea pigs, six per virus, were bled daily for three days for viremia determination (Figure 4); blood collection at later times was precluded by the mortality caused by some strains. All animals developed peak viremia approximately 48-hours post-infection. By 24-hours post-infection, epizootic strain 3908-infected guinea pigs developed a viremia that was approximately 2 log10 PFU/mL higher than enzootic strain ZPC738-infected animals; this difference was maintained throughout the three days assayed. By 72-hours post-infection, viremia in ZPC738-infected guinea pigs was decreasing, while it remained high in animals infected with the epizootic strain and both chimeras. The chimeras generated viremia levels intermediate between the two parental viruses, and both envelope and non-envelope genes appeared to determine viremia magnitude.
Evaluation of VEEV titers in tissues showed marked differences between parent and chimeric virus strains. Following infection, four animals per virus strain were killed daily to study early VEEV dissemination. Brains, bone marrow (titration only), draining lymph nodes, and spleens were collected at 24 and 48 hours after infection for titration and histology. Twenty-four-hours post-infection, epizootic strain 3908 replicated to higher titers within these tissues compared with the enzootic virus ZPC738 (Table 3). The chimeric viruses exhibited less efficient replication during the first 24 hours, and the strain containing the envelope genes from the enzootic strain (3908/ZPC738-PE2) produced significantly lower viral replication in the bone marrow, draining lymph node and spleen (P = 0.03), compared with the epizootic parent. The chimera containing the epizootic envelope genes replicated consistently to higher titers than the reciprocal chimera, and to significantly higher titers in the draining lymph node and the spleen (P = 0.03), compared with the enzootic parent. By 48-hours post-infection, there were similar titers of virus in the lymph node, bone marrow, and spleen for 3908, ZPC738/ 3908-PE2, and 3908/ZPC738-PE2-infected animals. Titers in the brain were approximately 1–2 logs higher for 3908 compared with ZPC738 and the envelope chimeras. In summary, both tissue tropism and replication levels differed in parent viruses and were differentially altered in the chimeras.
There was destruction of lymphoid tissue in the spleens and draining lymph nodes of all animals (parental viruses, Figure 5). In general, the first lesions were detected in the lymphoid tissue, where large amounts of necrosis and depletion of lymphoid cells were present through days 1–3 post-infection. However, on day 3 the spleen was highly congested and depleted of lymphoid cells in animals infected with strains 3908 and 3908/ZPC738-PE2, which did not survive long enough to show the potential for recovery of this tissue. In some of these animals, we also detected bacterial invasion of the liver associated with focal necrosis that is commonly seen following lymphoid depletion. However, no specific bacterial staining was performed to identify the bacteria. All of these animals also developed alveolar lung edema.
In contrast to the epizootic and 3908/ZPC738-PE2 strains, ZPC738- and ZPC738/3908-PE2-infected guinea pigs initially developed similar necrosis within lymphoid tissue. However, full recovery and even hyperplasia of the lymphoid tissue was detected prior to the development of the encephalitic phase. Neither lung edema nor liver necrosis was detected in these animals, but periportal infiltration of mononuclear cells in the liver and generalized meningoencephalitis was present at the point of the death.
These results indicate the importance of nonstructural proteins of epizootic origin in the development of the peracute, virus-induced, shock-like death in guinea pigs.
DISCUSSION
During the past decade VEE epizootics/epidemics have affected hundred of thousands of humans and equines, and recent epizootics highlight their continued threat of emergence. Additionally, these viruses are potential biologic weapons because they are readily available in nature, relatively easy to manipulate, highly infectious by aerosol, and can cause a debilitating, sometimes fatal illness.8 Phylogenetic evidence supports the origins and emergence of epizootic VEEV strains from enzootic subtype ID progenitors. However, the molecular mechanism(s) and determinants of equine amplification responsible for epidemics remain enigmatic.
Epizootic VEEV strains are equine-virulent, and infection of horses results in high-level viremia, lymphopenia associated with necrosis of lymphoid tissue, and fatal encephalitis.13 Enzootic VEEV strains are rarely associated with clinical disease in infected equines. However, seroconversion occurs, indicating their ability to productively infect horses.12
Laboratory mice, the most widely used animal model for VEE,12,17,31,32 develop a biphasic disease characterized by early replication in the periphery, culminating in central nervous system invasion, an inflammatory response within the brain, and death from encephalitis. However, mice are unsuitable for studies of VEEV epizootic potential because they do not reproduce the equine virulence and viremia phenotypes characteristic of enzootic versus epizootic strains.12 Hamsters also suffer fatal disease due to both enzootic and epizootic strains, and they die of septic shock, which is preventable by antibiotic treatment, prior to the development of encephalitis.33,34
In contrast, guinea pigs respond with different disease manifestations after infection with epizootic versus enzootic strains, as reflected by different pathologic changes and survival times. Infection with the epizootic 3908 strain was characterized by replication in lymphoid and neuronal tissues, depletion of lymphoid cells in the spleen, and shock-like death before the development of clinical encephalitis. This disease was similar to VEE in hamsters, which die 2–3 days after infection with the development of septic-like shock due to extensive damage of lymphoid tissues associated with ulcerations of the gut and bacteremia.35,36 Infection with epizootic strains, such as 3908 used in our study, produce similar pathology and identical survival time as described in hamsters.
Although detailed clinical and pathologic studies on guinea pig infections have not been reported, we speculate that the cause of guinea pig death was virus-initiated septic shock as a result of necrosis of lymphoid tissue. This speculation is further supported by the fact that our pathologic findings correlate with those described in hamsters. Additional experiments are needed to confirm this hypothesis. It should be noted, however, that the guinea pig model is imperfect because these animals, unlike equines, die following infection with some enzootic VEEV strains, including subtype ID variants that are believed to be the progenitors of epizootic strains.37 Nevertheless, the significant differences in AST after infection with enzootic versus epizootic strains makes guinea pigs the only useful small animal identified to date for estimating equine amplification and virulence potential of natural VEEV isolates.
The epizootic phenotype of VEEV in equines also correlates with the selective lymphotropism and lymphopenia early in the disease. The VEEV strains that do not produce strong and extended lymphopenia and necrosis of lymphoid tissues in equines do not produce high viremia and are usually less virulent (Bowen R, Paessler S, unpublished data).
Previous studies have described genetic changes associated with attenuation of VEEV in vivo, such as mutations in the structural proteins or the 5′-UTR. These mutations have been identified either artificially through virus adaptation to replication in cell cultures19 or through site-directed mutagenesis.38 The main mechanisms suggested for this artificial attenuation are the enhanced affinity for heparan sulfate associated with faster clearance of the virus from tissues or blood, and/or higher susceptibility to interferon (IF)-type I antiviral activity as shown for the TC-83 vaccine strain.19,39 Grieder and others40 showed that a single mutation in the E2 gene, resulting in the amino acid change of Glu to Lys at codon 76, is sufficient to restrict VEEV dissemination in a murine model.
While genetic determinants responsible for artificial attenuation of epizootic VEEV have been described, they are not related to the natural emergence of the epizootic phenotype. To better assess the role of mutations in the epizootic phenotype, we have begun exploiting the most closely related (≤ 1% sequence divergence) enzootic and epizootic VEEV strains isolated during our field studies. Here, we used recombinant genetics to create reciprocal chimeras between the epizootic strain 3908 (subtype IC) and the enzootic strain ZPC738 (subtype ID). Both chimeric VEE viruses were intermediate in their disease syndrome presentation, suggesting that both envelope glycoprotein and non-envelope mutations act in concert to generate the epizootic phenotype via their influences on pathogenesis. The envelope protein genes appeared to primarily influence 1) development of viremia levels and 2) the level of infection in lymphoid tissues, especially early in infection. However, the non-envelope and/or cis-acting sequence elements of VEEV also played a role in viremia development, and depletion of lymphoid cells within lymphoid tissue. In addition, our data suggest that the non-envelope genes were the sole determinants of average survival time.
Our data agree with recent studies performed with other alphaviruses. Sindbis virus neuroinvasiveness has been mapped to both the 5′ UTR and the E2 protein.41–43 Changes in Sindbis virus nsP2 lead to altered suppression/induction of IFN-type I production and virus attenuation in vivo and in vitro.44 We also recently demonstrated the importance of the nonstructural proteins in attenuation of the SIN/83 chimeric SINV/VEEV candidate vaccine strain that, while replicating efficiently in vitro, is strongly attenuated in six-day-old mice after peripheral or intracranial inoculation.45 Amino acid changes in several nonstructural proteins of Semliki Forest virus affect neurovirulence,46–49 as well as the structural proteins49 including E2 protein.50,51
In summary, we have demonstrated experimentally the importance of both envelope and non-envelope sequences for VEEV pathogenesis in guinea pigs. The VEEV envelope proteins may be primarily responsible for the ability of the virus to bind to and infect lymphoid cells. However, the outcome of the infection within lymphoid tissues may also depend on non-envelope genome regions and their interaction with host factors. Further studies are underway to confirm our findings in equines and to map the individual genes responsible for the devastating replication within equine lymphoid tissues.
Amino acid differences between Venezuelan equine encephalitis virus subtype IC strain 3908 and subtype ID strain ZPC738
| Gene or location in viral genome* | Amino acid position(s) | Strain 3908 | Strain ZPC738 | Number of nucleotide differences in the untranslated regions |
|---|---|---|---|---|
| * UTR = untranslated region; ns = nonstructural; E = envelope. | ||||
| †Amino acid positions in the swapped portions of the chimeric strains. | ||||
| 5′ UTR | 6 | |||
| nsP1 | 106 | Glu | Gly | |
| 475 | His | Arg | ||
| 487 | Ile | Val | ||
| 517 | Phe | Val | ||
| nsP2 | 122 | Thr | Ile | |
| 454 | Asp | Glu | ||
| 572 | Gln | Arg | ||
| 589 | Thr | Ala | ||
| 591 | Arg | Cys | ||
| 647 | Lys | Thr | ||
| 752 | His | Leu | ||
| nsP3 | 61 | Val | Ala | |
| 62 | Thr | Ala | ||
| 339-45 | Thr-Pro-Glu-Ser- Pro-Ala-Glu | — | ||
| 354 | Glu | — | ||
| 355 | Gln | Glu | ||
| 358 | Leu | Pro | ||
| 368 | Met | Thr | ||
| 403 | Ser | Pro | ||
| 431 | Asp | Glu | ||
| 440 | Val | Ala | ||
| 450 | Arg | Ser | ||
| 455 | Arg | Leu | ||
| 469 | Pro | Leu | ||
| 480 | Pro | Ser | ||
| 483 | His | Pro | ||
| 484 | Ser | Cys | ||
| 489 | Arg | Gly | ||
| nsP4 | 176 | Ser | Ala | |
| 253 | Val | Ile | ||
| 513 | Ser | Thr | ||
| 537 | Val | Ala | ||
| Capsid | 86 | Gln | Pro | |
| 100 | Ala | Val | ||
| 108 | Pro | Thr | ||
| E3 | 18† | Glu | Gln | |
| E2 | 117† | Gly | Asn | |
| 179† | Ile | Val | ||
| 193† | Val | Ala | ||
| 201† | Lys | Glu | ||
| 213† | Lys | Thr | ||
| E1 | 72† | Asn | Try | |
| 352 | Ala | Val | ||
| 416 | Gly | Arg | ||
| 3′ UTR | 9 | |||
Survival time of guinea pigs infected with parental and chimeric viruses
| Virus strain | Mean ± SD survival time (days) | P value compared to strain 3908† | P value compared to strain ZPC738† |
|---|---|---|---|
| * NA = not applicable. | |||
| † Preliminary analysis with one-way analysis of variance demonstrated a statistically significant difference in average survival time overall (P < 0.001). The Mann-Whitney multiple comparisons test (P values shown above) was used to investigate differences among strains. | |||
| 3908 | 2.9 ± 0.6 | NA* | < 0.001 |
| ZPC738 | 5.5 ± 1.0 | < 0.001 | NA |
| ZPC738/3908-PE2 | 4.4 ± 0.9 | < 0.05 | < 0.05 |
| 3908/ZPC738-PE2 | 3.4 ± 0.5 | > 0.05 | < 0.001 |
Virus titers in guinea pig tissues at 24 and 48 hours post-infection (PI)
| Titer (log10 PFU/mL)* | |||
|---|---|---|---|
| Tissue | Virus strain | 24 hours PI (Mean ± SD) | 48 hours PI (Mean ± SD) |
| * PFU = plaque-forming units. | |||
| †Indicates a significant difference compared with strain 3908; P < 0.05. | |||
| ‡Indicates a significant difference compared with strain ZPC738; P < 0.05. | |||
| Brain | 3908 | 4.9 ± 0.6 | 6.1 ± 0.4 |
| ZPC738 | 3.6 ± 0.9 | 4.8 ± 0.6 | |
| 738/3908-PE2 | 4.5 ± 0.6 | 5.1 ± 0.7 | |
| 3908/738-PE2 | 3.7 ± 0.7 | 5.1 ± 0.9 | |
| Bone marrow | 3908 | 8.5 ± 0.6 | 8.2 ± 0.4 |
| ZPC738 | 6.5 ± 0.6 | 6.8 ± 0.3 | |
| 738/3908-PE2 | 7.8 ± 0.4 | 8.0 ± 0.8 | |
| 3908/738-PE2 | 6.2 ± 0.4† | 8.1 ± 0.7 | |
| Lymph node | 3908 | 7.2 ± 0.5 | 7.9 ± 0.4 |
| ZPC738 | 5.0 ± 0.4 | 5.4 ± 0.7 | |
| 738/3908-PE2 | 7.1 ± 0.8‡ | 7.9 ± 0.4 | |
| 3908/738-PE2 | 5.8 ± 0.4† | 7.8 ± 0.8 | |
| Spleen | 3908 | 7.3 ± 0.1 | 8.6 ± 0.4 |
| ZPC738 | 5.4 ± 0.7 | 6.9 ± 0.4 | |
| 738/3908-PE2 | 7.0 ± 0.4‡ | 7.8 ± 0.4 | |
| 3908/738-PE2 | 5.2 ± 0.3† | 8.0 ± 0.7 | |
Schematic representation of parental infectious clones of Venezuelan equine encephalitis virus and reciprocal envelope chimeras. Envelope chimeras were generated via restriction digestion of parental clones with Afl II and Snab I at genomic nucleotide positions 8031 and 10298, and contain the last 111 amino acids in the capsid (C), the complete envelope precursor (PE2) and 6K genes, and the first 108 amino acids of the envelope (E1) gene of the epizootic parent in the backbone of the enzootic parent and vice versa. ns = nonstructural.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Schematic representation of parental infectious clones of Venezuelan equine encephalitis virus and reciprocal envelope chimeras. Envelope chimeras were generated via restriction digestion of parental clones with Afl II and Snab I at genomic nucleotide positions 8031 and 10298, and contain the last 111 amino acids in the capsid (C), the complete envelope precursor (PE2) and 6K genes, and the first 108 amino acids of the envelope (E1) gene of the epizootic parent in the backbone of the enzootic parent and vice versa. ns = nonstructural.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Schematic representation of parental infectious clones of Venezuelan equine encephalitis virus and reciprocal envelope chimeras. Envelope chimeras were generated via restriction digestion of parental clones with Afl II and Snab I at genomic nucleotide positions 8031 and 10298, and contain the last 111 amino acids in the capsid (C), the complete envelope precursor (PE2) and 6K genes, and the first 108 amino acids of the envelope (E1) gene of the epizootic parent in the backbone of the enzootic parent and vice versa. ns = nonstructural.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Analysis of chimeric virus replication in Vero cells. Vero cells were infected with virus produced from the 3908, ZPC738, ZPC738/3908-PE2, and 3908/ZPC738-PE2 cDNA clones at a multiplicity of infection of 1.0 plaque-forming units (PFU)/cell. At the indicated times, aliquots were collected and virus titers were determined by plaque assay. The epizootic parent (3908) had a mean ± SD plaque diameter of 1.0 ± 0.2 mm, which was significantly smaller than that of the enzootic parent (ZPC738) (3.6 ± 0.4 mm), while the mean ± SD diameters of the two chimeric viruses were intermediate (2.6 ± 0.3 mm and 2.5 ± 0.3 mm) for ZPC738/3908-PE2 and 3908/ZPC738-PE2, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Analysis of chimeric virus replication in Vero cells. Vero cells were infected with virus produced from the 3908, ZPC738, ZPC738/3908-PE2, and 3908/ZPC738-PE2 cDNA clones at a multiplicity of infection of 1.0 plaque-forming units (PFU)/cell. At the indicated times, aliquots were collected and virus titers were determined by plaque assay. The epizootic parent (3908) had a mean ± SD plaque diameter of 1.0 ± 0.2 mm, which was significantly smaller than that of the enzootic parent (ZPC738) (3.6 ± 0.4 mm), while the mean ± SD diameters of the two chimeric viruses were intermediate (2.6 ± 0.3 mm and 2.5 ± 0.3 mm) for ZPC738/3908-PE2 and 3908/ZPC738-PE2, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Analysis of chimeric virus replication in Vero cells. Vero cells were infected with virus produced from the 3908, ZPC738, ZPC738/3908-PE2, and 3908/ZPC738-PE2 cDNA clones at a multiplicity of infection of 1.0 plaque-forming units (PFU)/cell. At the indicated times, aliquots were collected and virus titers were determined by plaque assay. The epizootic parent (3908) had a mean ± SD plaque diameter of 1.0 ± 0.2 mm, which was significantly smaller than that of the enzootic parent (ZPC738) (3.6 ± 0.4 mm), while the mean ± SD diameters of the two chimeric viruses were intermediate (2.6 ± 0.3 mm and 2.5 ± 0.3 mm) for ZPC738/3908-PE2 and 3908/ZPC738-PE2, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Mean ± SD average survival time (AST) and mortality of guinea pigs infected with parental and chimeric viruses. Infected guinea pigs were checked twice a day for signs of morbidity and or mortality. Infection resulted in 100% mortality regardless of the virus strain used. However, animals infected with the epizootic parent had an AST of 2.9 ± 0.6, which was significantly lower than those infected with the enzootic parent (5.5 ± 1.0), while animals infected with the chimeric viruses had intermediate ASTs (4.4 ± 0.9 and 3.4 ± 0.5) with ZPC738/3908 PE2-E1 and 3908/ZPC738 PE2-E1, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Mean ± SD average survival time (AST) and mortality of guinea pigs infected with parental and chimeric viruses. Infected guinea pigs were checked twice a day for signs of morbidity and or mortality. Infection resulted in 100% mortality regardless of the virus strain used. However, animals infected with the epizootic parent had an AST of 2.9 ± 0.6, which was significantly lower than those infected with the enzootic parent (5.5 ± 1.0), while animals infected with the chimeric viruses had intermediate ASTs (4.4 ± 0.9 and 3.4 ± 0.5) with ZPC738/3908 PE2-E1 and 3908/ZPC738 PE2-E1, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Mean ± SD average survival time (AST) and mortality of guinea pigs infected with parental and chimeric viruses. Infected guinea pigs were checked twice a day for signs of morbidity and or mortality. Infection resulted in 100% mortality regardless of the virus strain used. However, animals infected with the epizootic parent had an AST of 2.9 ± 0.6, which was significantly lower than those infected with the enzootic parent (5.5 ± 1.0), while animals infected with the chimeric viruses had intermediate ASTs (4.4 ± 0.9 and 3.4 ± 0.5) with ZPC738/3908 PE2-E1 and 3908/ZPC738 PE2-E1, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Viremia in guinea pigs infected with Venezuelan equine encephalitis virus strains. Following infection, guinea pigs were bled once a day from the saphenous vein for three days. Serum was separated from whole blood and titered as outlined in the Materials and Methods. PFU = plaque-forming units.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Viremia in guinea pigs infected with Venezuelan equine encephalitis virus strains. Following infection, guinea pigs were bled once a day from the saphenous vein for three days. Serum was separated from whole blood and titered as outlined in the Materials and Methods. PFU = plaque-forming units.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Viremia in guinea pigs infected with Venezuelan equine encephalitis virus strains. Following infection, guinea pigs were bled once a day from the saphenous vein for three days. Serum was separated from whole blood and titered as outlined in the Materials and Methods. PFU = plaque-forming units.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Hematoxylin and eosin–stained spleens from guinea pigs infected with Venezuelan equine encephalitis virus (VEEV) strains. A, Twenty-four hours after infection with epizootic strain 3908 showing normal spleen architecture and mild congestion. B, Mild depletion of lymphoid cells from white pulp 48 hours after infection with strain 3908. C, Terminal stage of infection exhibiting severe necrosis and lymphodepletion within the spleen of a guinea pig infected with strain 3908. D, Spleen of guinea pig 24 hours after infection with enzootic VEEV showing mild congestion and physiologic architecture of the organ. E, Necrosis and lymphodepletion within the spleen 72 hours after infection with strain ZPC738. F, Terminal stage of the disease on the fifth day after infection with enzootic VEEV strain ZPC738 from a guinea pig with clinical encephalitis, showing reactivated white pulp in the spleen and the complete recovery of this organ. (Magnification × 40.) This figure appears in color at www.ajtmh.org.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Hematoxylin and eosin–stained spleens from guinea pigs infected with Venezuelan equine encephalitis virus (VEEV) strains. A, Twenty-four hours after infection with epizootic strain 3908 showing normal spleen architecture and mild congestion. B, Mild depletion of lymphoid cells from white pulp 48 hours after infection with strain 3908. C, Terminal stage of infection exhibiting severe necrosis and lymphodepletion within the spleen of a guinea pig infected with strain 3908. D, Spleen of guinea pig 24 hours after infection with enzootic VEEV showing mild congestion and physiologic architecture of the organ. E, Necrosis and lymphodepletion within the spleen 72 hours after infection with strain ZPC738. F, Terminal stage of the disease on the fifth day after infection with enzootic VEEV strain ZPC738 from a guinea pig with clinical encephalitis, showing reactivated white pulp in the spleen and the complete recovery of this organ. (Magnification × 40.) This figure appears in color at www.ajtmh.org.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Hematoxylin and eosin–stained spleens from guinea pigs infected with Venezuelan equine encephalitis virus (VEEV) strains. A, Twenty-four hours after infection with epizootic strain 3908 showing normal spleen architecture and mild congestion. B, Mild depletion of lymphoid cells from white pulp 48 hours after infection with strain 3908. C, Terminal stage of infection exhibiting severe necrosis and lymphodepletion within the spleen of a guinea pig infected with strain 3908. D, Spleen of guinea pig 24 hours after infection with enzootic VEEV showing mild congestion and physiologic architecture of the organ. E, Necrosis and lymphodepletion within the spleen 72 hours after infection with strain ZPC738. F, Terminal stage of the disease on the fifth day after infection with enzootic VEEV strain ZPC738 from a guinea pig with clinical encephalitis, showing reactivated white pulp in the spleen and the complete recovery of this organ. (Magnification × 40.) This figure appears in color at www.ajtmh.org.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.330
Authors’ addresses: Ivorlyne P. Greene, Johns Hopkins University, School of Public Health, Department of Molecular Microbiology and Immunology, 615 N. Wolfe St., WS114, Baltimore, MD 21205; Slobodan Paessler, Michael Anishchenko, and Darci R. Smith, Center for Biodefense and Emerging Infectious Diseases, and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. Aaron C. Brault, Department of Pathology, Microbiology and Immunology, Center for Vector-Borne Diseases, School of Veterinary Medicine, University of California, Davis, CA 95616. Ilya Frolov, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-0609. Scott C. Weaver, Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-0609, Telephone : 409-747-0758, Fax : 409-747-2415, E-mail : sweaver@utmb.edu.
Acknowledgments: We thank Amelia Travassos da Rosa for technical assistance and John Roehrig (Centers for Disease Control and Prevention, Fort Collins, CO) for providing monoclonal antibodies.
Financial support: Ivorlyne P. Greene was supported by National Institutes of Health (NIH) minority supplemental grant AI-10984, Ivorlyne P. Greene and Darci R. Smith were supported by the Centers for Disease Control and Prevention Vector-Borne Infectious Disease Training Program 417760. Slobodan Paessler was supported by the NIH Emerging and Tropical Diseases T32 Training grant AI-107536. Aaron C. Brault was supported by the James L. McLaughlin Fellowship Fund and the NIH Emerging and Tropical Diseases T32 Training grant AI-107526. This research was supported by NIH grants AI-39800 and AI-48807.
REFERENCES
- 1↑
Kinney RM, Johnson BJ, Welch JB, Tsuchiya KR, Trent DW, 1989. The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83. Virology 170 :19–30.
- 2↑
Strauss JH, Strauss EG, 1994. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58 :491–562.
- 3↑
Young NA, Johnson KM, 1969. Antigenic variants of Venezuelan equine encephalitis virus: their geographic distribution and epidemiologic significance. Am J Epidemiol 89 :286–307.
- 4↑
Rico-Hesse R, Weaver SC, de Siger J, Medina G, Salas RA, 1995. Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America. Proc Natl Acad Sci U S A 92 :5278–5281.
- 5↑
Weaver SC, Salas R, Rico-Hesse R, Ludwig GV, Oberste MS, Boshell J, Tesh RB, 1996. Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348 :436–440.
- 6↑
Oberste MS, Fraire M, Navarro R, Zepeda C, Zarate ML, Ludwig GV, Kondig JF, Weaver SC, Smith JF, Rico-Hesse R, 1998. Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico. Am J Trop Med Hyg 59 :100–107.
- 7↑
Hawley RJ, Eitzen EM Jr, 2001. Biological weapons–a primer for microbiologists. Annu Rev Microbiol 55 :235–253.
- 8↑
Rosenbloom M, Leikin JB, Vogel SN, Chaudry ZA, 2002. Biological and chemical agents: a brief synopsis. Am J Ther 9 :5–14.
- 9↑
Walton TE, Grayson MA, 1988. Venezuelan equine encephalomyelitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Volume IV. Boca Raton, FL: CRC Press, 203–231.
- 11↑
Aguilar PV, Greene IP, Coffey LL, Medina G, Moncayo AC, Anishchenko M, Ludwig GV, Turell MJ, O’Guinn ML, Lee J, Tesh RB, Watts DM, Russell KL, Hice C, Yanoviak S, Morrison AC, Klein TA, Dohm DJ, Guzman H, Travassos da Rosa AP, Guevara C, Kochel T, Olson J, Cabezas C, Weaver SC, 2004. Endemic Venezuelan equine encephalitis in northern Peru. Emerg Infect Dis 10 :880–888.
- 12↑
Wang E, Bowen RA, Medina G, Powers AM, Kang W, Chandler LM, Shope RE, Weaver SC, 2001. Virulence and viremia characteristics of 1992 epizootic subtype IC Venezuelan equine encephalitis viruses and closely related enzootic subtype ID strains. Am J Trop Med Hyg 65 :64–69.
- 13↑
Walton TE, Alvarez O, Buckwalter RM, Johnson KM, 1973. Experimental infection of horses with enzootic and epizootic strains of Venezuelan equine encephalomyelitis virus. J Infect Dis 128 :271–282.
- 14↑
Sudia WD, Newhouse VF, Henderson BE, 1971. Experimental infection of horses with three strains of Venezuelan equine encephalomyelitis virus. II. Experimental vector studies. Am J Epidemiol 93 :206–211.
- 15↑
Leon CA, 1975. Sequelae of Venezuelan equine encephalitis in humans: a four year follow-up. Int J Epidemiol 4 :131–140.
- 16↑
Garcia-Tamayo J, Carreno G, Esparza J, 1979. Central nervous system alterations as sequelae of Venezuelan equine encephalitis virus infection in the rat. J Pathol 128 :87–91.
- 17↑
Aronson JF, Grieder FB, Davis NL, Charles PC, Knott T, Brown K, Johnston RE, 2000. A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus. Virology 270 :111–123.
- 18
Davis NL, Brown KW, Greenwald GF, Zajac AJ, Zacny VL, Smith JF, Johnston RE, 1995. Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1. Virology 212 :102–110.
- 19↑
Kinney RM, Chang GJ, Tsuchiya KR, Sneider JM, Roehrig JT, Woodward TM, Trent DW, 1993. Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5′-noncoding region and the E2 envelope glycoprotein. J Virol 67 :1269–1277.
- 20↑
Powers AM, Brault AC, Kinney RM, Weaver SC, 2000. The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants. J Virol 74 :4258–4263.
- 21↑
Wang E, Barrera R, Boshell J, Ferro C, Freier JE, Navarro JC, Salas R, Vasquez C, Weaver SC, 1999. Genetic and phenotypic changes accompanying the emergence of epizootic subtype IC Venezuelan equine encephalitis viruses from an enzootic subtype ID progenitor. J Virol 73 :4266–4271.
- 22↑
Anishchenko M, Paessler S, Greene IP, Aguilar PV, Carrara A, Weaver SC, 2004. Generation and characterization of closely related epizootic and enzootic infectious cDNA clones for studying interferon sensitivity and emergence mechanisms of Venezuelan equine encephalitis virus. J Virol 78 :1–8.
- 23↑
Cupp EW, Scherer WF, Lok JB, Brenner RJ, Dziem GM, Ordonez JV, 1986. Entomological studies at an enzootic Venezuelan equine encephalitis virus focus in Guatemala, 1977–1980. Am J Trop Med Hyg 35 :851–859.
- 24↑
Cupp EW, Scherer WF, Ordonez JV, 1979. Transmission of Venezuelan encephalitis virus by naturally infected Culex (Melanoconion) opisthopus. Am J Trop Med Hyg 28 :1060–1063.
- 25↑
Brault AC, Powers AM, Weaver SC, 2002. Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein. J Virol 76 :6387–6392.
- 26↑
Rice CM, Levis R, Strauss JH, Huang HV, 1987. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol 61 :3809–3819.
- 27↑
Kinney RM, Butrapet S, Chang GJ, Tsuchiya KR, Roehrig JT, Bhamarapravati N, Gubler DJ, 1997. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230 :300–308.
- 28↑
Lennette EH, Lennette DA, Lennette ET, 1995. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. Washington, DC: American Public Health Association.
- 29↑
Brault AC, Powers AM, Holmes EC, Woelk CH, Weaver SC, 2002. Positively charged amino acid substitutions in the E2 envelope glycoprotein are associated with the emergence of Venezuelan equine encephalitis virus. J Virol 76 :1718–1730.
- 30↑
Martin DH, Dietz WH, Alvaerez O Jr, Johnson KM, 1982. Epidemiological significance of Venezuelan equine encephalomyelitis virus in vitro markers. Am J Trop Med Hyg 31 :561–568.
- 31↑
Gleiser CA, Gochenour JWS, Berge TO, Tigertt WD, 1961. The comparative pathology of experimental Venezuelan equine encephalitis infection in different animal hosts. J Infect Dis 110 :80–97.
- 32↑
Jackson AC, SenGupta SK, Smith JF, 1991. Pathogenesis of Venezuelan equine encephalitis virus infection in mice and hamsters. Vet Pathol 28 :410–418.
- 33↑
Gorelkin L, Jahrling PB, 1975. Virus-initiated septic shock. Acute death of Venezuelan encephalitis virus-infected hamsters. Lab Invest 32 :78–85.
- 34↑
Gorelkin L, Jahrling PB, 1974. Pancreatic involvement by Venezuelan equine encephalomyelitis virus in the hamster. Am J Pathol 75 :349–362.
- 35↑
Jahrling PB, 1976. Virulence heterogeneity of a predominantly avirulent Western equine encephalitis virus population. J Gen Virol 32 :121–128.
- 36↑
Jahrling PB, Scherer F, 1973. Histopathology and distribution of viral antigens in hamsters infected with virulent and benign Venezuelan encephalitis viruses. Am J Pathol 72 :25–38.
- 37↑
Scherer WF, Chin J, 1977. Responses of guinea pigs to infections with strains of Venezuelan encephaltis virus, and correlations with equine virulence. Am J Trop Med Hyg 26 :307–312.
- 38↑
Bernard KA, Klimstra WB, Johnston RE, 2000. Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. Virology 276 :93–103.
- 39↑
Spotts DR, Reich RM, Kalkhan MA, Kinney RM, Roehrig JT, 1998. Resistance to alpha/beta interferons correlates with the epizootic and virulence potential of Venezuelan equine encephalitis viruses and is determined by the 5′ noncoding region and glycoproteins. J Virol 72 :10286–10291.
- 40↑
Grieder FB, Davis NL, Aronson JF, Charles PC, Sellon DC, Suzuki K, Johnston RE, 1995. Specific restrictions in the progression of Venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins. Virology 206 :994–1006.
- 41↑
Dubuisson J, Lustig S, Ruggli N, Akov Y, Rice CM, 1997. Genetic determinants of Sindbis virus neuroinvasiveness. J Virol 71 :2636–2646.
- 42
Tucker PC, Lee SH, Bui N, Martinie D, Griffin DE, 1997. Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells. J Virol 71 :6106–6112.
- 43↑
Dropulic LK, Hardwick JM, Griffin DE, 1997. A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication. J Virol 71 :6100–6105.
- 44↑
Frolova EI, Fayzulin RZ, Cook SH, Griffin DE, Rice CM, Frolov I, 2002. Roles of nonstructural protein nsP2 and alpha/beta interferons in determining the outcome of Sindbis virus infection. J Virol 76 :11254–11264.
- 45↑
Paessler S, Fayzulin RZ, Anishchenko M, Greene IP, Weaver SC, Frolov I, 2003. Recombinant Sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic. J Virol 77 :9278–9286.
- 46↑
Fazakerley JK, Boyd A, Mikkola ML, Kaariainen L, 2002. A single amino acid change in the nuclear localization sequence of the nsP2 protein affects the neurovirulence of Semliki Forest virus. J Virol 76 :392–396.
- 47
Ahola T, Kujala P, Tuittila M, Blom T, Laakkonen P, Hinkkanen A, Auvinen P, 2000. Effects of palmitoylation of replicase protein nsP1 on alphavirus infection. J Virol 74 :6725–6733.
- 48
Tuittila M, Hinkkanen AE, 2003. Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence. J Gen Virol 84 :1525–1533.
- 49↑
Tarbatt CJ, Glasgow GM, Mooney DA, Sheahan BJ, Atkins GJ, 1997. Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus. J Gen Virol 78 :1551–1557.
- 50↑
Santagati MG, Maatta JA, Roytta M, Salmi AA, Hinkkanen AE, 1998. The significance of the 3′-nontranslated region and E2 amino acid mutations in the virulence of Semliki Forest virus in mice. Virology 243 :66–77.
- 51↑
Santagati MG, Maatta JA, Itaranta PV, Salmi AA, Hinkkanen AE, 1995. The Semliki Forest virus E2 gene as a virulence determinant. J Gen Virol 76 :47–52.