.png)
INTRODUCTION
Venezuelan equine encephalitis virus (VEEV) is an alphavirus (family Togaviridae, genus Alphavirus) and a member of the VEE complex, which comprises six distinct antigenic subtypes (I–VI). Generally, human and equine pathogenicity correlate with the antigenic subtype. Specifically, major human epidemics and equine epizootics have been associated with subtypes IAB and IC.1–5 Enzootic transmission is generally associated with subtypes ID and IE, which are usually less virulent in horses.6 In contrast, the epizootic subtypes IAB and IC are highly pathogenic in horses, with case fatality rates of up to 83% reported.6 The most recent major outbreak occurred in 1995 in Venezuela and Colombia in which 75,000–100,000 human cases occurred, with more than 300 fatal encephalitis cases.7 This epidemic was exclusively associated with VEEV subtype IC.8 However, the epidemiologic partition of these subtypes is not exclusive. For example, in 1993, equine disease was associated with VEEV-IE in Mexico9; in the period between 1993 and 1995, human cases of VEEV-ID–associated disease occurred in Peru.10 Thus, identification of the VEEV subtype is critical for epidemiologic surveillance, including the serological monitoring of sentinel rodents and horses to determine the potential for equine-associated amplification, for tracking of deliberate VEEV dissemination as a bioterrorist agent, or for assessment of vaccine efficacy.
Currently, VEEV infection is diagnosed principally by direct detection, e.g., nucleic acid11–13 or virus isolation from acute-phase serum or spinal fluid14 or by serological assay, e.g., detection of VEEV-specific IgM in the cerebrospinal fluid using IgM capture enzyme-linked immunosorbent assay (ELISA) or using monoclonal antibody-based antigen-capture ELISA15–19 at the time of clinical encephalitis. The plaque reduction neutralization test (PRNT), which like IgM-ELISA is useful in distinguishing VEEV from other alphaviruses, does not accurately identify the infecting VEEV serotype. Recently, a VEEV-specific blocking ELISA was described that also identified serotype-specific antibodies to VEEV in well-characterized serum from VEEV-infected humans, horses, and mice.20
Currently, diagnostic laboratories that produce VEEV antigens, reference sera, or viral stocks for biologic assays are required to perform the work in BSL-3 facilities; these facilities also must be registered and approved for the use of select agents. Although the TC83 vaccine strain can be handled in BSL-2 facilities, it can only be used to identify VEEV-IAB subtype infections. In addition, for their own protection, laboratory personnel working with VEEV should be vaccinated with TC83, which is administered solely by the U.S. Army. This reduces the capability of nonselect agent laboratories to diagnose cases and to respond to natural outbreaks or deliberate introduction of VEEV as a weapon of bioterrorism.
To increase the safety of laboratory workers as well as to enable larger numbers of laboratories to perform diagnostic work in the event of emergency, we created chimeric alphaviruses that are highly attenuated, that are consequently not classified as select agents, and that can potentially be used in BSL-2 facilities. We believe these viruses may enable state and local public health laboratories to do future diagnostic work without handling the wild-type pathogen, thereby eliminating the need to transport or distribute virulent VEEV.
Alphavirus (Sindbis)-based recombinant viruses engineered to express VEEV structural proteins are attenuated in mice and hamsters.21,22 Thus, recombinant Sindbis-VEE virus technology offers the prospect of detecting VEEV without the use of viral particles or antigens prepared from virulent virus stocks. The structural proteins of VEEV are comprised of the capsid and the envelope glycoproteins E2 and E1,23,24 which project from the virion as protein spikes formed by E1/E2 heterodimers.25–28 The E2 protein is the principal target of neutralizing antibodies in the acute phase of infection, and VEEV serotypes are conferred by E2-specific antibody recognition.29 Therefore, we hypothesized that specific antigen-antibody recognition would occur in serological assays using Sindbis-VEE viruses that express VEEV structural proteins, thereby permitting the diagnosis of VEEV infection. We constructed recombinant Sindbis/VEE viruses (varieties IAB, IE, ID, and IF) using the TRD, TC83 (the attenuated vaccine derivative of TRD),21,22 ZPC738,21,22 68U201, and 78V-3531 strains of VEEV (Figure 1). Previous studies demonstrated that inoculation of mice with the SIN/83, SIN/ZPC, or SIN/TRD chimeras did not result in pathogenicity, e.g., observable clinical disease, tissue pathology, or death of animals.21,22 In this study, we also evaluated the safety of the recombinant SIN/68U201 and SIN/78V-3531 viruses in mice. We then tested the ability of chimeric SIN/VEE viruses for use in accurately identifying VEEV infection of horses, humans, or rodents using three serological assays: plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) test, and complement fixation (CF) test.
MATERIALS AND METHODS
Cell culture.
BHK-21 cells, provided by Dr. Paul Olivo (Washington University, St. Louis, MO), were maintained at 37 °C in minimum essential medium (MEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and vitamins.
Viruses.
Sindbis strain AR339 (SINV339)30 and the attenuated VEEV strain, V3526 (a derivative of the virulent TRD strain, in which lethal mutations were introduced into the PE2 furin cleavage site of an infectious cDNA clone, followed by selection of a second-site suppressor mutation in the E1 glycoprotein gene),31,32 which were used as controls in animal infection experiments, were provided by Dr. Robert Johnston and Dr. William Klimstra (University of North Carolina, Chapel Hill, NC). VEEV strain 66637 (variety ID, accession no. AF004458),33 MX01-32 (variety IE, acc. no. AY823298), 3908 (variety IC, acc. no. U55350),13 (GenBank) and 64A99 (IE) viruses were used for experimental infection of horses. IQT1724 (variety ID, acc. no. AF004464) and AR339 were used to generate antisera and antigens for CF tests and HI assays.33
Plasmid constructs.
All of the plasmids were constructed by standard recombinant DNA techniques, as described previously.21,22 Chimeric SIN83, SIN/TRD, SIN/ZPC, SIN/ 68U201, and SIN/78V-3531 viruses were designed to encode Sindbis nonstructural proteins and structural proteins derived from the TC8334,35 (acc. no. L01442), Trinidad donkey (TRD, acc. no. L01442),35 ZPC738 (acc. no. AF100566),36,37 68U201 (acc. no. U34999),38,39 and 78V-3531 (acc. no. AF075257)40,41 strains of VEEV, respectively. Maps and sequences of the plasmids are available from the authors upon request.
Animals.
Pregnant female NIH Swiss mice were purchased from Harlan (Indianapolis, IN). Newborn mice were maintained for 6 days after birth in an Animal Biosafety Level-3 (ABSL-3) facility prior to experimental infection. All animal infections using virulent VEEV, SINV339, and chimeric SIN/ VEE viruses were carried out at in ABSL-3 facilities in accordance with animal protocols approved by the UTMB Animal Care and Use Committee.
Virus stock.
Plasmids were purified using standard protocols by centrifugation in cesium chloride gradients. They were linearized using the XhoI restriction enzyme site immediately downstream from the poly(A) sequences. RNAs were synthesized using SP6 RNA polymerase (Invitrogen) in the presence of a cap analogue. The yield and integrity of transcripts were monitored by agarose gel electrophoresis in non-denaturing conditions. For electroporation, aliquots of transcription reactions were used without additional purification, and RNAs were transfected into BHK-21 cells.42 Viruses were harvested after development of cytopathic effects, usually at 24 hours after electroporation. Viral titer was determined as previously described.21,22
Animal infections.
Six-day-old female NIH Swiss mice were infected via subcutaneous route with 1 × 105 PFU of VEEV strain V3526, SINV strain AR339 (SINV339), or the following chimeric SIN/VEE viruses: SIN/83, SIN/68U201, or SIN/78V-3531. Animals were monitored daily for 28 days, and mortality was recorded.
Plaque reduction neutralization test (PRNT).
Previously characterized sera were obtained from the following sources: (1) humans in a VEEV-IE–endemic area of Mexico20; (2) humans vaccinated with TC83 (identification nos. X01–X06); (3) experimentally infected horses43; (4) experimentally infected mice20,44; or (5) defined monoclonal antibodies (mAb) to VEEV-IAB: mAb 3B4C-445 and 1A3A-9.36 Serum samples were used for PRNT performed on BHK-21 monolayers, as follows. The chimeric SIN/VEE or parental virus (IAB/C, ID, or IE) was incubated for 1 hour at 37°C with serial 2-fold dilutions of sera ranging from 1:20 to 1:1280 for serum or to 1:20,480 for mAbs. Subsequently, cell monolayers were incubated with serum/virus mixtures for 1 hour at 37°C, overlaid with 0.5% agarose, maintained for 36 hour at 37°C, and stained with crystal violet.46 The PRNT titer is presented as the reciprocal of the serum dilution capable of neutralizing 80% of PFU of the parental or chimeric virus.
Hemagglutination inhibition (HI) test.
HI antibodies to antigens from the indicated chimeric SIN/VEE or parental virus were measured by a standard method.46 Briefly, antigens for the HI were prepared from mosquito (C6/36) cells infected with the indicated chimeric (SIN/68U201, SIN/ZPC, or SIN/ TRD) or parental (SIN AR339 strain or VEEV IQT1724 strain) viruses, and, as controls, antigens from uninfected mosquito cells. All were extracted using acetone. Serial dilutions of serum, also extracted in acetone, were mixed with 4 units of antigen. After overnight incubation at 4°C, goose erythrocytes suspended at a dilution of 1:200 at the optimal pH for each antigen were added. The mixture was left for ≈ 15 minutes at room temperature. The HI titers of the serum are reported as the reciprocal of the highest dilution at which hemagglutination was inhibited.
Complement fixation (CF) test.
Antigens for CF tests were prepared as described for the HI test, but were not extracted using acetone. CF tests were performed by a microtechnique46 using 2 units of guinea pig complement. Antigen, serum inactivated at 60°C for 20 minutes, and complement were incubated together at 4°C overnight. Subsequently, a mixture of sheep red blood cells and rabbit antiserum was incubated for 30 minutes at 37°C. The titer was recorded as the reciprocal of the highest dilution giving 3+ or 4+ hemolysis on a scale of 0 (negative = complete hemolysis) to 4+ (positive = no hemolysis).47
RESULTS
Chimeric SIN/VEE viruses are attenuated in 6-day-old mice.
The constructs we have used in this study are shown in Figure 1. In prior studies, we demonstrated the safety of chimeric viruses SIN83, SIN/ZPC, and SIN/TRD in mice and hamsters.21,22 In this study, we used the same murine infection model to evaluate the safety of newly constructed chimeric viruses, SIN/68U201 and SIN/78V-3531, containing the structural protein-encoding regions from virulent IE and IF subtypes, respectively. For comparison, we evaluated the attenuated VEEV vaccine strain, V3526, which was created from a cDNA clone of the virulent Trinidad Donkey (TRD) strain.31 Subcutaneous infection of 6-day-old NIH Swiss mice with 1 × 105 PFU of chimeric SIN/68U201 or SIN/78V-3531 virus did not result in death in any of the infected mice over a 16-day period, whereas the attenuated VEEV vaccine strain 3526 (V3526) and the prototype Sindbis strain AR339 (SINV339) were uniformly lethal at 4 and 6 days following infection, respectively. As was shown previously21,22 and also confirmed in this study, 100% of SIN/83-infected 6-day-old mice survived. Thus, chimeric SIN/VEE viruses or viral antigens prepared from these chimeras were further evaluated as alternative reagents in VEEV-specific serological assays, e.g., PRNT, HI assays, and CF tests.
Serum antibody of VEEV-infected horses and humans neutralizes chimeric SIN/VEE viruses.
We assessed the ability of well-characterized serum from naturally and experimentally infected humans and horses to neutralize the chimeric SIN/ VEE viruses in comparison to the parental VEE virus using PRNT (Table 1). Sera 503, 525, 545, and 168 tested positive for VEEV using the parental as well as the chimeric viruses, with titers in the range of 160–320 or 20–640, respectively, for the corresponding subtype. Serum 505 tested positive overall at the lowest dilution tested (1:20) against the parental 68U201 strain, which is the strain that circulates in the area where the sample was obtained; however, this sample tested negative at this dilution against the chimeric SIN/68U201 virus (Table 1A). Thus, although generally 1- to 2-fold differences in titer were detected using the parental versus chimeric virus titer, testing of replicate 1- to 2-fold serum dilutions is recommended for this assay. Among the TC83 vaccinees tested, three of six serum samples tested (X01, X02, and X05) were strongly positive, with reciprocal 80% PRNT titers ranging from 80 to 320 when tested with either the parental or chimeric virus. Two of the six serum samples (X03 and X04) tested weakly positive against the subtype matched parental viruses, with reciprocal titers ranging from 20 to 40, and one of the six serum samples (X04) tested negative using the chimeric viruses. Among the six, serum from one TC83 vaccinee (X06) tested negative with parental as well as the chimeric viruses. Our results further show that chimeric SIN/VEE viruses are neutralized at a similar dilution as the parental viruses, with no more than 2-fold variation in the PRNT titer compared with the corresponding parental VEEV. For all TC83 vaccinees that tested positive in this PRNT, the ability of sera to neutralize ID virus subtype ZPC or recombinant SIN/ZPC was marginally less efficient compared with the IAB type, with titers 2- to 4-fold lower than for IAB virus neutralization.
Serum from horses infected with VEEV-IE tested positive in PRNT, with 2- to 128-fold differences in titer between subtypes. Further, titers observed for parental and chimeric virus were either similar or higher when using the chimeric viruses. Although titers were more variable for the VEEV-IC–infected horse sera (20–640 for parental virus; 20–1280 for the chimeric virus), comparable titers were observed for the matching subtypes. For serum obtained from two different horses at 8 weeks (Horse 2, T2) and 14 weeks (Horse 3, T3) following VEEV-ID infection (Horse 2, Horse 3), PRNT was positive with similar titers obtained for the parental and chimeric virus, but, as expected, was negative at 0 weeks (Horse 1, T1). For horses infected with VEEV subtype IAB (EqIABa, EqIABb), the chimeric viruses consistently generated higher titers than the parental strains and typically had higher homologous than heterologous titers.
To confirm the utility of SIN/VEE viruses in testing of sera from VEEV-infected rodents, we evaluated sera from NIH Swiss mice experimentally infected with ZPC738 (serotype ID) in PRNT (Table 1C). Our results indicate that all mice were VEEV-antibody–positive, as expected, when tested with all the parental viruses (TC83, ZPC738, and 68U201). In assays with TC83 virus (IAB subtype), the titers were lower overall, ranging from 40 to 640. For the matching mouse serum sample, 2- to 4-fold differences were observed between the titers that neutralized the parental IAB-type versus ID-type virus. Positive PRNT results were also obtained using the chimeric SIN/VEE viruses. In PRNT assays with the infecting subtype, ZPC738, titers ranged from 160 to 1280. In matching serum samples, the parental and chimeric virus produced identical results for the majority of the sera (5/7 tested) with no greater than 2-fold difference between titer in assays using the corresponding parental versus chimeric viruses, i.e., TC83 versus SIN/TRD. PRNT was positive using E2-specific antisera 3B4C-4 and 1A3A-9, with the highest titer against SIN/TRD and SIN/68U201, respectively.
Chimeric SIN/VEE viruses can be used to evaluate the reactivity of defined VEEV-specific serum in standard serological assays.
We further tested the ability of VEEV-specific antiserum to recognize the antigens of chimeric SIN/VEE viruses using two standard serological assays, HI and CF test. The results of HI testing are shown in Table 2. In HI, SINV-specific antiserum tested positive against SINV antigens at a titer of 2,560; in contrast, HI results were negative when the SINV antiserum was tested against antigens from VEEV-ID or any of the chimeric SIN/VEE virus types. Conversely, VEEV-specific antiserum tested positive at a titer of 5,120 against antigens from VEEV-ID and all chimeric SIN/VEE viruses, whereas the titer was 32-fold lower (titer of 80) against SINV antigens. As expected, the media-only control was negative when tested with either the SINV- or VEEV-specific antiserum. VEEV-negative serum, which was tested as an additional negative control, did not show reactivity with SINV, VEEV, or any of the chimeric viruses. These HI results indicate that antigen recognition of the chimeric SIN/VEE viruses tested in this study are highly VEEV-specific, and thus, these recombinant viruses can be used as antigen equivalents of the virulent parental VEE viruses in serological assays to evaluate VEEV-positive sera.
The results of CF testing are shown in Table 3. In CF test, VEEV-specific antiserum tested positive at titers ranging from 512 to 1,024 when evaluated against VEEV-ID or the chimeric SIN/VEE virus antigens at dilutions of ≥ 1:8; however, the titer was substantially lower (32- to 64-fold; 1:16) when VEEV antiserum was tested against the same dilution of SINV antigens. On the contrary, SINV-specific antiserum tested positive in CF test against SINV antigens but showed no reactivity against antigens from VEEV or any of the chimeric SIN/VEE viruses. No positive reactions were observed for the media-only control when tested against either the VEEV or SINV antiserum; likewise, no positive reactions were obtained for VEEV-negative serum tested against antigen from any of the parental or chimeric SIN/VEE viruses. Thus, as demonstrated using the HI assay, CF test using antigens from chimeric SIN/VEE viruses yield identical results to that obtained using antigens prepared from virulent parental VEE viruses.
DISCUSSION
Our study of VEEV vaccine candidates suggests that virulence in mice may be correlated to the safety profile in humans. The SIN/VEE chimeric viruses we have used for diagnostic assay are less virulent in mice and hamsters than the TC83 and V3526 strains, as shown in this report as well as in previous studies in our laboratory.21,22 TC83 has been extensively used to vaccinate at-risk humans as well as horses during epizootics34 despite its infrequent association with febrile, systemic illness and other adverse effects.48 Indeed, TC83 vaccination results in residual virulence in mice; it is uniformly lethal for C3H/HeN mice after intracerebral inoculation, and produces clinical illness in BALB/c and C3H/HeN mice up to 14 days after subcutaneous inoculation.49 In 6-day-old NIH Swiss mice, TC83 was also uniformly lethal at 6 days following subcutaneous inoculation.21,22 The prototypical virulent Sindbis virus, strain AR339 (SINV339),30 which is pathogenic for neonatal CD-1 mice,50 was also virulent in our animal model system employing 6-day-old NIH Swiss mice. Nevertheless, the pathogenesis of VEEV variants is animal model-dependent. Thus, although the V3526 strain, a derivative of the virulent TRD strain,31,32 was attenuated in subcutaneously infected adult C57BL/6 mice, Syrian hamsters,51 and non-human primates (macaques),52 in our murine model, V3526 was more virulent than SINV339. In contrast, all mice survived infection with the SIN/VEE chimeras—SIN/68U201 and SIN/78V-3531—when monitored for 28 days. Thus, chimeric SIN/VEE viruses have improved safety relative to the TC83 and V3526 strains of VEEV.
SIN/VEE chimeras were useful alternatives to the virulent parental VEE viruses for antiserum production in mice and for serological assays. Provided that these chimeras retain the antigenic structure conferred by the parental VEEV structural proteins, we hypothesized that it would be advantageous to use these attenuated viruses in place of the virulent parental VEE viruses in VEEV-specific serological assays. Our study shows that alphaviruses can be manipulated in a similar manner to closely related flavivirus chimeras, which have been used successfully as safe diagnostic reagents.53 The PRNT, CF, and HI results reported here suggest that recombinant SIN/VEE viruses may be useful for evaluating the efficacy of TC83 and other vaccine candidates currently in development. As sera from mice experimentally infected with virulent ZPC738 can be evaluated efficiently via PRNT, we foresee that these recombinant viruses will be useful in future surveillance of sentinel rodents for VEEV infection in endemic areas.
Notably, we demonstrate that the chimeric viruses serve effectively as surrogates for the virulent parental viruses in PRNT, HI, and CF tests. The results of PRNT, CF, and HI tests indicate that these recombinant SIN/VEE viruses can be used to assess whether sera from infected humans, horses, and rodents contain antibodies that recognize VEEV. In PRNT, positive results were obtained for infected sera tested against all parental VEEV and matching chimeric SIN/VEE viruses, indicating that neutralizing antibody is likely raised to an epitope shared by all three subtypes. Thus, PRNT using either the parental or chimeric viruses could not effectively and consistently distinguish the infecting VEEV subtype (Table 1), as reported by others.20 Similarly, HI and CF tests with defined VEEV-antiserum recognized VEEV in tests using antigens from either the parental or chimeric SIN/VEE virus, with identical titers for nearly all viruses used (Tables 2 and 3). The ability to differentiate between antigenic subtypes IAB, IC, ID, and IE would be advantageous for epidemiologic and diagnostic analysis and furthermore, may lead to a better understanding of the pathogenesis of VEE subtypes. We anticipate that these chimeric SIN/VEE viruses21,22 can be used for a variety of serological tests and can be used to produce subtype-specific reagents, as previously shown for anti-ID and for anti-IAB sera21,22 because all of these chimeric viruses are capable of inducing production of neutralizing antibodies in mice, hamsters, and some of them in equines (data not shown). Finally, these chimeras will facilitate mechanistic studies, e.g., host cell infection by enveloped viruses, structural studies of VEEV proteins, in addition to enabling diagnostics without the requirement for special shipping permits and establishment of high-level biosafety facilities, thereby avoiding additional regulatory impasses to the study of VEEV.
Neutralization of VEEV and recombinant SIN/VEE viruses by serum from VEEV-infected humans, horses, and rodents
| (A) Human serum | |||||||
|---|---|---|---|---|---|---|---|
| 80% PRNT titer | |||||||
| Parental VEEV virus | Chimeric SIN/VEE virus | ||||||
| Serum no.* | Blocking ELISA20 | TC83 (IAB) | ZPC738 (ID) | 68U201 (IE) | SIN/TRD | SIN/ZPC | SIN/68U201 |
| 503a | IE | − | 20 | 320 | − | 40 | 20 |
| 525a | IE | 160 | 320 | 320 | 320 | 320 | 640 |
| 545a | IE | 40 | 80 | 160 | 40 | 80 | NT |
| 168a | IE | 80 | 40 | 160 | 80 | 40 | 80 |
| 505a | − | − | − | 20 | − | − | − |
| X01c | NT | 160 | 40 | NT | 320 | 40 | − |
| X02b | NT | 160 | 40 | NT | 80 | 20 | NT |
| X03b | NT | 40 | 20 | NT | 20 | − | − |
| X04b | NT | 40 | 20 | NT | − | − | − |
| X05b | NT | 160 | 80 | NT | 160 | 80 | − |
| X06b | NT | − | − | NT | − | − | − |
| (B) Horse serum | ||||||||
|---|---|---|---|---|---|---|---|---|
| 80% PRNT titer | ||||||||
| Parental virus | Chimeric SIN/VEE virus | |||||||
| Serum no.† | dpi | Blocking ELISA20 | TC83 (IAB) | ZPC738 (ID) | 68U201 (IE) | SIN/TRD | SIN/ZPC | SIN/68U201 |
| 744d | 8 | IC | 640 | 20 | NT | 640 | 20 | 80 |
| 968d | 15 | IC | 80 | 640 | NT | 80 | 640 | 1280 |
| 876d | 15 | IC | 80 | 80 | NT | 80 | 80 | − |
| DP1d | 14 | IE | 640 | 1280 | 640 | 640 | 1280 | 1280 |
| DP2d | 14 | IE | 640 | 640 | 640 | 640 | 640 | 1280 |
| DP3d | 14 | IE | 640 | 640 | 640 | 320 | 640 | 1280 |
| DP4d | 14 | IE | 160 | 640 | 640 | 160 | 640 | 1280 |
| DP29d | 14 | IE | 80 | 160 | 640 | 160 | 160 | 1280 |
| Horse 1e | 0 | ID | − | − | NT | − | − | − |
| Horse 2e | 8 | ID | 320 | 640 | NT | 320 | 640 | 640 |
| Horse 3e | 14 | ID | 80 | 640 | NT | 160 | 640 | 640 |
| T1e | 0 | ID | − | − | NT | − | − | − |
| T2e | 8 | ID | 40 | 160 | NT | 40 | 160 | 320 |
| T3e | 14 | ID | 20 | 80 | NT | 40 | 80 | 160 |
| EqIEf | 21 | NT | NT | NT | 80 | 20 | 320 | 2560 |
| EqIABag | 21 | NT | 40 | NT | NT | 640 | 2560 | 640 |
| EqIABbg | 21 | NT | 160 | NT | 40 | 320 | 640 | 160 |
| (C) Mouse serum | ||||||
|---|---|---|---|---|---|---|
| 80% PRNT titer | ||||||
| Parental VEEV virus | Chimeric SIN/VEE virus | |||||
| Serum no.‡ | TC83 (IAB) | ZPC738 (ID) | 68U201 (IE) | SIN/TRD | SIN/ZPC | SIN/68U201 |
| * Place and year serum sample was obtained: (a) Mexico, 200120; TC83 vaccinee in (b) 2002 or (c) 2003. NT, not tested due to limited serum amount; −, negative. | ||||||
| † Horses were experimentally infected with VEEV strains (d) MX01-32 (IE), (e) 66637 (ID), and (f) 64A99 (IE) or (g) TRD (IAB) and bled at the indicated day following infection. For Horse 1, Horse 2, and Horse 342 and for T1, T2, and T3, serum was obtained from the same horse at different time points, as indicated. “0 dpi” indicates pre-convalescent serum sample; dpi, days post infection; NT, not tested due to limited serum availability; −, negative. | ||||||
| ‡ NIH Swiss mice infected with ZPC738, designated by “h”. Defined anti-VEEV (IAB) monoclonal antibodies (mAb),45,36 designated by “i”. NT, not tested due to limited serum availability. | ||||||
| MA1h | 640 | 1280 | NT | 640 | 1280 | 2560 |
| MA2h | 640 | 1280 | NT | 640 | 1280 | 2560 |
| MA3h | 640 | 1280 | NT | 640 | 2560 | 2560 |
| MA4h | 160 | 160 | NT | 320 | 160 | 160 |
| MB1h | 40 | 640 | NT | 80 | 640 | 160 |
| MC1h | 160 | 640 | NT | 160 | 640 | 1280 |
| MC2h | 640 | 320 | NT | 640 | 320 | 160 |
| 3B4C-4 mAbi | NT | NT | NT | > 20,480 | 640 | 20 |
| 1A3A-9 mAbi | NT | NT | NT | 10,240 | 5120 | > 20,480 |
Results of hemagglutination inhibition (HI) test
| HI titer* | ||||||
|---|---|---|---|---|---|---|
| Antigens (4 units) | ||||||
| Chimeric SIN/VEE virus‡ | Parental virus§ | |||||
| Antiserum† | SIN/68U2011 | SIN/ZPC2 | SIN/TRD3 | SIN | VEEV (ID) | Cell control |
| * HI titers are expressed as the reciprocal of the highest serum dilution giving a positive result. 0 = < 1:10. | ||||||
| † SINV-specific antiserum was hyperimmune ascitic fluid obtained from mice immunized with SINV strain AR339; VEEV-specific antiserum was immune ascitic fluid obtained from mice infected with VEEV strain TC83; control serum was ascitic fluid obtained from uninfected mice. | ||||||
| ‡ The coding region for structural genes was derived from the following VEEV strains: 68U201 (subtype IE1), ZPC738 (subtype ID2), and TRD (subtype IAB3), as shown in Figure 1. For chimeric SIN/VEE viruses, the replicative machinery was derived from the Sindbis (SIN) virus Tota1101 strain.54 | ||||||
| § SIN and VEEV-ID antigens were obtained from the AR339 and IQT1724 strains, respectively. | ||||||
| SIN T-33609 | 10 | 0 | 0 | 2560 | 0 | 0 |
| VEEV T-34257 | 5120 | 5120 | 5120 | 80 | 5120 | 0 |
| Control | 0 | 0 | 0 | 0 | 0 | |
Results of complement fixation (CF) tests
| CF titer* Antigens | ||||||
|---|---|---|---|---|---|---|
| Chimeric SIN/VEE virus‡ | Parental virus§ | |||||
| Antiserum† | SIN/68U2011 | SIN/ZPC2 | SIN/TRD3 | SIN | ZPC738 | Cell control |
| * CF titers are expressed as the reciprocal of the highest positive serum dilution/antigen dilution. 0, 100% hemolysis at all dilutions tested. | ||||||
| † SINV-specific antiserum was hyperimmune ascitic fluid obtained from SINV-infected mice; VEEV-specific antiserum was immune ascitic fluid obtained from VEEV-infected mice; control serum was ascitic fluid obtained from uninfected control mice. | ||||||
| ‡ The coding region for structural genes was derived from the following VEEV strains: 68U201 (subtype IE1), ZPC738 (subtype ID2), and TRD (subtype IAB3), as shown in Figure 1. For chimeric SIN/VEE viruses, the replicative machinery was derived from the Sindbis (SIN) virus Tota1101 strain.54 | ||||||
| § SIN and VEEV-ID antigens were obtained from the AR339 and IQT1724 strains, respectively. | ||||||
| SIN T-33609 | 0 | 0 | 0 | 1024/> 32 | 0 | 0 |
| VEEV T-34257 | 512/≥8 | ≥1024/≥8 | ≥1024/≥8 | 16/8 | ≥1024/≥8 | 0 |
| Control | 0 | 0 | 0 | 0 | 0 | |
Characteristics of parental and chimeric SIN/VEE viruses. Schematic representation of parent and chimeric viruses is shown. For all chimeras, the nonstructural protein-encoding portions are derived from the SINV genome.21,22 Structural proteins for the chimeric SIN83, SIN/TRD, SIN/ZPC, SIN/68U201, and SIN/78V-3531 viruses were derived from the TC83, Trinidad donkey (TRD), ZPC738, 68U201, and 78V-3531 strains of VEEV, respectively. Horizontal arrows indicate the location of the subgenomic promoter in the viral genome. The serological subtype of the parental virus is listed.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.774
Characteristics of parental and chimeric SIN/VEE viruses. Schematic representation of parent and chimeric viruses is shown. For all chimeras, the nonstructural protein-encoding portions are derived from the SINV genome.21,22 Structural proteins for the chimeric SIN83, SIN/TRD, SIN/ZPC, SIN/68U201, and SIN/78V-3531 viruses were derived from the TC83, Trinidad donkey (TRD), ZPC738, 68U201, and 78V-3531 strains of VEEV, respectively. Horizontal arrows indicate the location of the subgenomic promoter in the viral genome. The serological subtype of the parental virus is listed.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.774
Characteristics of parental and chimeric SIN/VEE viruses. Schematic representation of parent and chimeric viruses is shown. For all chimeras, the nonstructural protein-encoding portions are derived from the SINV genome.21,22 Structural proteins for the chimeric SIN83, SIN/TRD, SIN/ZPC, SIN/68U201, and SIN/78V-3531 viruses were derived from the TC83, Trinidad donkey (TRD), ZPC738, 68U201, and 78V-3531 strains of VEEV, respectively. Horizontal arrows indicate the location of the subgenomic promoter in the viral genome. The serological subtype of the parental virus is listed.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.774
Address correspondence to Slobodan Paessler, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019. E-mail: slpaessl@utmb.edu
Authors’ addresses: Haolin Ni, Nadezhda E. Yun, Michele A. Zacks, Scott C. Weaver, Robert B. Tesh, Amelia P. Travassos da Rosa, and Slobodan Paessler, Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-1019, Telephone: +1 (409) 747-0764, Fax: +1 (409) 747-0762, E-mail: slpaessl@utmb.edu. Ann M. Powers, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522. Ilya Frolov, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019.
Acknowledgments: The authors thank Richard Bowen (Colorado State University, Fort Collins, CO) for providing equine serum samples and John Roehrig (Centers for Disease Control and Prevention, Fort Collins, CO) for providing monoclonal antibodies.
Financial support: Slobodan Paessler was supported by a National Institutes of Health K08 award (AI059491). This work was also supported by a grant from the National Institute of Allergy and Infectious Diseases through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (U54 AI057156), by NIH grants awarded to S.C.W. (AI39800, AI48807) and R.B.T. (RO1-AI30027), and by an NIH contract (N01-AI25489).
REFERENCES
- 1↑
Young NA, Johnson KM, 1969. Antigenic variants of Venezuelan equine encephalitis virus: their geographic distribution and epidemiologic significance. Am J Epidemiol 89 :286–307.
- 2↑
Young NA, Johnson KM, Gauld LW, 1969. Viruses of the Venezuelan equine encephalomyelitis complex. Experimental infection of Panamanian rodents. Am J Trop Med Hyg 18 :290–296.
- 3↑
Young NA, 1972. Serologic differentiation of viruses of the Venezuelan encephalitis (VE) complex. Proc. Workshop-Symposium on Venezuelan Encephalitis Virus. Washington, D.C.: Pan American Health Organization, 84–89.
- 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 USA 92 :5278–5281.
- 5↑
Walton TE, Grayson MA, 1988. Venezuelan equine encephalomyelitis. Monath TP, ed. The Arboviruses: Epidemiology and Ecology, Vol IV. Boca Raton, Florida: CRC Press, 203–231.
- 6↑
Weaver SC, Anishchenko M, Bowen R, Brault AC, Estrada-Franco JG, Fernandez Z, Greene I, Ortiz D, Paessler S, Powers AM, 2004. Genetic determinants of Venezuelan equine encephalitis emergence. Arch Virol Suppl (18):43–64.
- 7↑
Rivas F, Diaz LA, Cardenas VM, Daza E, Bruzon L, Alcala A, De la Hoz O, Caceres FM, Aristizabal G, Martinez JW, Revelo D, De la Hoz F, Boshell J, Camacho T, Calderon L, Olano VA, Villarreal LI, Roselli D, Alvarez G, Ludwig G, Tsai T, 1997. Epidemic Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis 175 :828–832.
- 8↑
Weaver S, Salas R, Rico-Hesse R, Ludwig G, Oberste S, Smith J, Cardenas J, Marino O, Barreto A, Boshell J, Fernandez Z, Godoy O, Rueda E, Guaqueta A, Roberts B, Camacho T, de Calderon L, Tesh RB, 1996. Nueva emergencia de la encefalitis equina venezolana epidemica en Suramerica. Biomedica (Bogota) 16 :78–79.
- 9↑
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.
- 10↑
Watts DM, Callahan J, Rossi C, Oberste MS, Roehrig JT, Wooster MT, Smith JF, Cropp CB, Gentrau EM, Karabatsos N, Gubler D, Hayes CG, 1998. Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region. Am J Trop Med Hyg 58 :35–40.
- 11↑
Powers AM, Brault AC, Shirako Y, Strauss EG, Kang W, Strauss JH, Weaver SC, 2001. Evolutionary relationships and systematics of the alphaviruses. J Virol 75 :10118–10131.
- 12
Linssen B, Kinney RM, Aguilar P, Russell KL, Watts DM, Kaaden OR, Pfeffer M, 2000. Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses. J Clin Microbiol 38 :1527–1535.
- 13↑
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. Lancet 348 :436–440.
- 14↑
Johnston RE, Peters CJ, 1996. Alphaviruses. Fields BN, Knipe DM, Howley PM, eds. Virology. Third edition. New York: Lippincott-Raven, 843–898.
- 15↑
Calisher CH, el-Kafrawi AO, Al-Deen Mahmud MI, Travassos da Rosa AP, Bartz CR, Brummer-Korvenkontio M, Haksohusodo S, Suharyono W, 1986. Complex-specific immunoglobulin M antibody patterns in humans infected with alphaviruses. J Clin Microbiol 23 :155–159.
- 16
CDC. Centers for Disease Control information on arboviral encephalitides. http://www.cdc.gov/ncidod/dvbid/arbor/arbdet.htm. Accessed November 7, 2005.
- 17
Deresiewicz RL, Thaler SJ, Hsu L, Zamani AA, 1997. Clinical and neuroradiographic manifestations of eastern equine encephalitis. N Engl J Med 336 :1867–1874.
- 18
Martin DA, Muth DA, Brown T, Johnson AJ, Karabatsos N, Roehrig JT, 2000. Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J Clin Microbiol 38 :1823–1826.
- 19↑
Sahu SP, Alstad AD, Pedersen DD, Pearson JE, 1994. Diagnosis of eastern equine encephalomyelitis virus infection in horses by immunoglobulin M and G capture enzyme-linked immunosorbent assay. J Vet Diagn Invest 6 :34–38.
- 20↑
Wang E, Paessler S, Aguilar PV, Smith DR, Coffey LL, Kang W, Pfeffer M, Olson J, Blair PJ, Guevara C, Estrada-Franco J, Weaver SC, 2005. A novel, rapid assay for detection and differentiation of serotype-specific antibodies to Venezuelan equine encephalitis complex alphaviruses. Am J Trop Med Hyg 72 :805–810.
- 21↑
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.
- 22↑
Paessler S, Ni H, Petrakova O, Fayzulin RZ, Yun N, Anishchenko M, Weaver SC, Frolov I, 2006. Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses. J Virol 80 (6):2784–2796.
- 23↑
Rice CM, Strauss JH, 1981. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci USA 78 :2062–2066.
- 24↑
McKnight KL, Simpson DA, Lin SC, Knott TA, Polo JM, Pence DF, Johannsen DB, Heidner HW, Davis NL, Johnston RE, 1996. Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes. J Virol 70 :1981–1989.
- 25↑
Strauss JH, Wang KS, Schmaljohn AL, Kuhn RJ, Strauss EG, 1994. Host-cell receptors for Sindbis virus. Arch Virol Suppl 9 :473–484.
- 26
Strauss JH, Strauss EG, 1994. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58 :491–562.
- 27
Shirako Y, Strauss JH, 1994. Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. J Virol 68 :1874–1885.
- 28↑
Schlesinger S, Schlesinger MJ, 2001. Togaviridae: The Viruses and Their Replication. Knipe DM, Howley PM, eds. Fields’ Virology. Fourth edition. New York: Lippincott, Williams and Wilkins, 895–916.
- 29↑
Roehrig JT, Bolin RA, Hunt AR, Woodward TM, 1991. Use of a new synthetic-peptide-derived monoclonal antibody to differentiate between vaccine and wild-type Venezuelan equine encephalomyelitis viruses. J Clin Microbiol 29 :630–631.
- 30↑
Taylor RM, Hurlbut HS, Work TH, Kingston JR, Frothingham TE, 1955. Sindbis virus: a newly recognized arthropod-transmitted virus. Am J Trop Med Hyg 4 :844–862.
- 31↑
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.
- 32↑
Hart MK, Caswell-Stephan K, Bakken R, Tammariello R, Pratt W, Davis N, Johnston RE, Smith J, Steele K, 2000. Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate. Vaccine 18 :3067–3075.
- 33↑
Powers AM, Oberste MS, Brault AC, Rico-Hesse R, Schmura SM, Smith JF, Kang W, Sweeney WP, Weaver SC, 1997. Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis from a single genotype of enzootic subtype ID virus. J Virol 71 :6697–6705.
- 34↑
Berge TO, Banks IS, Tigertt WD, 1961. Attenuation of Venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea pig heart cells. Am J Hyg 73 :209–218.
- 35↑
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.
- 36↑
Roehrig JT, Bolin RA, 1997. Monoclonal antibodies capable of distinguishing epizootic from enzootic varieties of subtype I Venezuelan equine encephalitis viruses in a rapid indirect immunofluorescence assay. J Clin Microbiol 35 :1887–1890.
- 37↑
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.
- 38↑
Oberste MS, Parker MD, Smith JF, 1996. Complete sequence of Venezuelan equine encephalitis virus subtype IE reveals conserved and hypervariable domains within the C terminus of nsP3. Virology 219 :314–320.
- 39↑
Scherer WF, Dickerman RW, Ordonez JV, 1970. Discovery and geographic distribution of Venezuelan encephalitis virus in Guatemala, Honduras, and British Honduras during 1965–68, and its possible movement to Central America and Mexico. Am J Trop Med Hyg 19 :703–711.
- 40↑
Calisher C, Kinney R, de Souza Lopes O, Trent D, Monath T, Francy D, 1982. Identification of a new Venezuelan equine encephalitis virus from Brazil. Am J Trop Med Hyg 31 :1260–1272.
- 41↑
Kinney RM, Pfeffer M, Tsuchiya KR, Chang GJ, Roehrig JT, 1998. Nucleotide sequences of the 26S mRNAs of the viruses defining the Venezuelan equine encephalitis antigenic complex. Am J Trop Med Hyg 59 :952–964.
- 42↑
Bredenbeek PJ, Frolov I, Rice CM, Schlesinger S, 1993. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol 67 :6439–6446.
- 43↑
Kolokoltsov AA, Wang E, Colpitts T, Weaver SC, Davey RA, 2006. Pseudotyped viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus. Am J Trop Med Hygiene 75 (4):702–709.
- 44↑
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.
- 45↑
Roehrig JT, Hunt AR, Kinney RM, Mathews JH, 1988. In vitro mechanisms of monoclonal antibody neutralization of alphaviruses. Virology 165 :66–73.
- 46↑
Beaty BJ, Calisher CH, Shope RE, 1989. Arboviruses. Schmidt NJ, Emmons RW, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. Sixth edition. Washington, D.C.: American Public Health Association, 797–855.
- 47↑
Ratterree MS, Gutierrez RA, Travassos da Rosa AP, Dille BJ, Beasley DW, Bohm RP, Desai SM, Didier PJ, Bikenmeyer LG, Dawson GJ, Leary TP, Schochetman G, Phillippi-Falkenstein K, Arroyo J, Barrett AD, Tesh RB, 2004. Experimental infection of rhesus macaques with West Nile virus: level and duration of viremia and kinetics of the antibody response after infection. J Infect Dis 189 :669–676.
- 48↑
Alevizatos AC, McKinney RW, Feigin RD, 1967. Live, attenuated Venezuelan equine encephalomyelitis virus vaccine. I. Clinical effects in man. Am J Trop Med Hyg 16 :762–768.
- 49↑
Ludwig GV, Turell MJ, Vogel P, Kondig JP, Kell WK, Smith JF, Pratt WD, 2001. Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice. Am J Trop Med Hyg 64 :49–55.
- 50↑
Klimstra WB, Ryman KD, Bernard KA, Nguyen KB, Biron CA, Johnston RE, 1999. Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome. J Virol 73 :10387–10398.
- 51↑
Pratt WD, Davis NL, Johnston RE, Smith JF, 2003. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 21 :3854–3862.
- 52↑
Reed DS, Lind CM, Lackemeyer MG, Sullivan LJ, Pratt WD, Parker MD, 2005. Genetically engineered, live, attenuated vaccines protect nonhuman primates against aerosol challenge with a virulent IE strain of Venezuelan equine encephalitis virus. Vaccine 23 :3139–3147.
- 53↑
Pugachev KV, Guirakhoo F, Mitchell F, Ocran SW, Parsons M, Johnson BW, Kosoy OL, Lanciotti RS, Roehrig JT, Trent DW, Monath TP, 2004. Construction of yellow fever/St. Louis encephalitis chimeric virus and the use of chimeras as a diagnostic tool. Am J Trop Med Hyg 71 :639–645.
- 54↑
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.