Rift Valley fever virus (RVFV) is a member of the family Bunyaviridae
and as such is an enveloped virus that has a negative stranded RNA genome consisting of three fragments, aptly named S (small), M (medium) and L (large). The S segment codes for two proteins, a nucleocapsid protein that coats the viral genome in the virion, and a non-structural protein (NSs). The NSs protein is especially interesting, in that it is a filamentous nuclear protein[1
], expressed by a virus that replicates and assembles in the cytoplasm of infected cells. The NSs protein is known to be involved in altering the host immune response because the virulence of viruses lacking a functional NSs is attenuated in mice, and these viruses are potent inducers of IFN α/β, unlike the wild type (WT) virus [2
]. The M segment of the genome codes for two viral glycoproteins that are on the surface of the virion, as well as a nonstructural protein (NSm) that has unknown function. Finally, the L segment of the virus encodes the viral RNA polymerase.
RVFV is a mosquito-borne virus that causes significant morbidity and mortality in humans and livestock and is considered to be a bioterrorism threat agent. It was first identified in the 1930's in Kenya after isolation from a sheep in the Rift Valley [5
]. It is present throughout Africa, and has also caused outbreaks in Madagascar off the Eastern coast of Africa as well as in Yemen and Saudi Arabia [6
The virus is transmitted to humans by contact with infected livestock, usually through the butchering or the birthing process, or by the bite of an infected mosquito. Infected individuals typically have a mild disease consisting of fever, malaise, and myalgia; a very small percentage of these individuals will develop severe disease manifested as hepatitis, encephalitis, retinitis or hemorrhagic fever, which are the hallmarks of RVFV clinical disease. The overall fatality rate is estimated at 0.5-1%. However, in patients whose clinical illness is sufficiently severe to bring them to the attention of medical personnel, it has been reported to be as high as 29%, as was seen in the Kenya 2006-2007 outbreak [7
RVFV is also a significant veterinary pathogen that affects livestock, such as cattle, goats, and sheep. Up to 90% mortality has been reported in newborn animals and as high as 30% in adult animals [8
]. Consistent with its degree of pathogenicity in juvenile animals, RVFV is also extremely abortigenic; 40-100% of pregnant animals will abort during an outbreak [9
]. Furthermore, livestock caretakers are exposed to virus in the process of caring for sick and dying animals, especially since amniotic fluid contains high quantities of virus.
There is a clear need for development of a safe efficacious vaccine to prevent these naturally occurring large scale outbreaks of severe disease in livestock and humans in the affected regions. The sporadic and explosive nature of these outbreaks makes vaccination control efforts challenging. It is very difficult in resource limited areas of Africa or the Middle East to sustain annual vaccination for a disease that appears infrequently. On the other hand, it is impossible to effectively vaccinate in the face of a rapidly moving ongoing epizootic. In addition, the regulatory hurdles and enormous expense to advancement of a human use vaccine make it unlikely that a product which targets poorly defined human populations in rural Africa and the Middle East would get developed. It has been observed that virus amplification cycles in livestock frequently precede human cases by 3-4 weeks, and play a critical role in the early stages of an outbreak. These highly viremic animals serve as an excellent source of direct contamination of humans, as well as a blood meal source for mosquitoes which can transmit the virus to humans. Recently, satellite derived data and rainfall measurements have proven to be effective predictors of time periods and geographical regions at high risk of experiencing RVF epizootics [10
]. A viable strategy for control of RVF may be to use these predictive methods for targeted application of an inexpensive efficacious livestock vaccine which could prevent livestock epizootics, limit the vertebrate host virus amplification cycle and thereby also prevent human epidemics. Due to export restrictions and other regulatory issues, acceptance of such a vaccine would require development of a companion diagnostic assay that could differentiate between infected and vaccinated animals (DIVA).
There is currently no licensed vaccine available for use in the US or Europe and vaccine options in Africa and the Middle East are limited. A formalin inactivated RVFV vaccine has limited availability in the US for protection of military personnel and laboratory workers [11
]. Two live attenuated viruses have been tested in various animals as potential vaccine strains. A mutagen-attenuated strain (MP12) and the live attenuated Smithburn strain have been tested in pregnant ewes and lambs, as well as in pregnant, fetal, neonatal and adult bovids. The results of these studies with live vaccines are varied, in some instances showing no clinical illness and the development of neutralizing antibody titers as well as protection from challenge [18
], and in other studies showing the viruses to be abortogenic and teratogenic [22
]. Therefore neither of these virus strains appears to be an ideal candidate for a vaccine strain because of their questionable safety profiles, in addition to their lack of DIVA capability.
In recent years, a reverse genetics system has become available for RVFV, thereby facilitating studies of viral pathogenesis and the development of specifically attenuated vaccine strains [24
]. This system has been used to generate viruses that are missing the NSs protein, the NSm protein, or both. These live attenuated vaccine candidates provide complete protection with a single administration in the highly sensitive Wistar-Furth rat model [26
]. ΔNSm/ΔNSs virus infected rats demonstrate a strong antibody response to the N protein, but as expected, no antibody response to the NSs protein. In contrast, rats infected with WT virus demonstrate an antibody response to both the N and NSs proteins by immunofluorescence analysis. The ΔNSm/ΔNSs virus has immense potential as a vaccine for use in the model proposed above where predictive methods guide targeted vaccine strategies to prevent livestock epizootics. Not only is the exact genetic makeup of this virus known, since it was generated from cloned cDNA, but it is more attenuated than the currently available attenuated strains, MP12 and Smithburn. The ΔNSm/ΔNSs virus bypasses the problem of possible reversion to virulence by having two large deletions, one on the M segment and one on the S segment of the genome. In addition, unlike the currently available attenuated strains, the ΔNSm/ΔNSs vaccine meets the DIVA requirement by virtue of the missing NSs protein.
In this study, we build upon the observation that infection with the mutant virus can be distinguished from infection with the WT virus by immunofluorescence analysis. We describe the generation of an ELISA that can distinguish infected from vaccinated animals. This companion assay can easily be performed in a rudimentary laboratory setting and would be ideal for use the in resource poor countries where RVFV is prevalent.