Awareness of the importance of laboratory-based surveillance for arboviruses in the United States has increased since the introduction of West Nile virus in 1999. In response, state public health laboratories have been called on to do more comprehensive testing for arboviruses, and as a result, the numbers of samples collected and tested and the number of different arboviruses routinely tested for have increased substantially. Subsequently, laboratory capacity has been enhanced through training and technical support, and many public health laboratories now have the capacity to do their own confirmatory testing by PRNT.
Because of the high rates of mortality associated with human NA EEEV infection and the sporadic nature of the outbreaks, laboratory-based surveillance is essential for detection of EEE cases, as well as vector control response. However, in the United States, use of all wt EEEV strains is restricted to select agent-registered laboratories with BSL-3 containment facilities, which has become a barrier to increasing diagnostic laboratory capacity, particularly in regard to the PRNT, which requires the use of live virus. Therefore, alternatives to using restricted viruses that require BSL-3 containment facilities have been developed for use in public health diagnostic laboratories, which often do not have these facilities. SINV/VEEV was previously shown to perform as well as wt VEEV for this purpose for diagnosis of suspected cases of VEEV complex alphavirus (13
). We evaluated the performance of two SINV/EEEV strains in the diagnostic laboratory setting to determine if they could replace the wt EEEVs in the PRNT. Chimeric NA EEEVs were neutralized similarly to wt NA EEEVs in sera from experimentally infected horses and mice and from suspected human EEE cases, as well as from EEEV vaccinees. There was 100% serostatus agreement between the wt and chimeric EEEVs in all the types of samples, indicating that the anti-EEEV neutralizing antibodies in the test sera reacted effectively to both the wt and chimeric EEEV strains. Antibody recognition and neutralization are specific to the EEEV structural proteins of the chimeric viruses. Our results confirmed that sera contain no antibodies directed against alphavirus (in this case, SINV) nonstructural proteins, and there was no evidence that the SINV nonstructural protein genes present in the chimeras influenced the serological results.
The small differences in PRNT endpoint titers that we detected between wt and chimeric EEEV strains were unexpected because the structural protein compositions of these viruses should be identical. Although this difference could occasionally result in a low-titer seropositive sample testing negative with the chimeric SINV/EEEV, we believe that this occurrence will be rare, because in natural EEEV infections, human and equine titers are usually well above the cutoff of 20 (Tables and ). One of the criteria for laboratory confirmation of recent arbovirus infection is a 4-fold or greater rise in virus-specific antibody titer in serum collected during the acute and convalescent phases of illness (4
), and the paired sera included in the study had the typical rise in titers of antibodies against both the wt and chimeric challenge EEEVs. Although a very small paired sample set was tested, SINV/EEEV would likely be neutralized similarly with other appropriately timed specimens.
The difference in titer could be more important for testing seroconversion in vaccinees, where titers generally remain low because equine and human EEE vaccines are inactivated. However, equine titers are not generally tested in response to vaccination, and the U.S. Army Special Immunizations Program, which administers the human vaccine being tested under an investigational new drug application, is equipped to handle wt EEEV.
The reasons for the slightly reduced seroreactions to SINV/EEEV compared to wt EEEV are unknown. It is possible that protein-RNA interactions in some way determine the structure of alphaviruses and that the small differences in antibody neutralization that we detected are due to slight conformational changes in the chimeric viruses caused by unnatural interactions between the SINV portion of the chimeric RNA genome and the EEEV proteins. Structural studies of the chimeras and wt EEEV strains using cryo-electron microscopy are needed to test this hypothesis. However, despite slightly higher endpoint neutralization titers of antibodies against the wt NA EEEV compared to the SINV/NA EEEV, for diagnostic purposes, the titers of antibodies against both viruses were sufficiently high. It is possible that in certain cases, when serum samples are collected at very early or late time points after infection, neutralizing antibody titers might fall below the LLOQ of the PRNT by using the chimeric EEEVs, resulting in false negatives. However, in our opinion, this small chance is outweighed by the enhanced safety and reduced regulatory requirements for use of the chimeras.
Among EEEV subtypes and lineages, the North American subtype (subtype I) is primarily associated with human illness. This EEEV lineage occurs in eastern North America, the Caribbean, and Mexico. The SA EEEVs, which include lineages II to IV, are restricted to South and Central America (2
). Although the SA EEEVs are not often associated with human illness (1
), they do cause equine disease, occasionally involving large numbers of horses. There are considerable antigenic and sequence differences between the SA and NA EEEV lineages, and as shown in , there is little cross-reactivity of neutralizing antibodies to the challenge SA and NA EEEVs. Therefore, to increase sensitivity where SA and NA EEEVs may cocirculate, such as in southern Mexico, it is essential to test sera by PRNT using both SINV/NA EEEV and SINV/SA EEEV.
In summary, the chimeric SINV/EEEV strains appear to produce qualitatively identical results in the critical PRNT required to diagnose EEEV infections in humans and domestic animals. The use of these attenuated, chimeric viruses will facilitate diagnostic testing of EEE in public health laboratories that do not have the high-level biosafety facilities and select agent certification required for working with wt EEEV. Their use will therefore enhance arboviral disease surveillance as well as improve biodefense preparedness.