Interventions Against West Nile Virus,Rift Valley Fever Virus,and Crimean-Congo Hemorrhagic Fever Virus: Where Are We?

Abstract

ARBO-ZOONET is an international network financed by the European Commission's seventh framework program. The major goal of this initiative is capacity building for the control of emerging viral vector-borne zoonotic diseases, with a clear focus on West Nile virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus. To evaluate the status quo of control measures against these viruses, an ARBO-ZOONET meeting was held in Istanbul, Turkey, from 19 to 20 November 2009. The symposium consisted of three themes: (1) vaccines: new and existing ones; (2) antivirals: existing and new developments; and (3) antivector vaccines. In addition, a satellite workshop was held on epidemiology and diagnosis. The meeting brought together foremost international experts on the subjects from both within and without the ARBO-ZOONET consortium. This report highlights selected results from these presentations and major conclusions that emanated from the discussions held.

Introduction

G lobalization and climate change increase the risk of vector-borne viral disease incursions into previously unaffected areas. Particularly, outbreaks of zoonotic viruses can have serious socioeconomic consequences. This is underscored by the incursion and subsequent rapid spread of West Nile virus (WNV) in North America and the establishment of new endemic foci in Europe (Blitvich 2008, Barzon et al. 2009). WNV is a flavivirus of the Flaviviridae family (Fauquet et al. 2005). The virus is transmitted by various mosquito species of the Culex genus and is maintained in the environment in an enzootic mosquito–bird–mosquito cycle. WNV infections occur mostly in birds, whereas horses and humans are considered incidental hosts (Kramer et al. 2007). Before the 1990s WNV was associated with mild asymptomatic disease in horses and humans and was therefore not considered an important human or animal pathogen (Hayes 2001, Blitvich 2008). In the mid-1990s outbreaks of severe WNV neuroinvasive disease in Europe and the Mediterranean region came as a complete surprise (Blitvich 2008). In 1999, WNV was found to be the causative agent of a human encephalitis case in New York City, representing the first occurrence of WNV in the Western Hemisphere (Blitvich 2008). Within 4 years, the virus spread to all 48 contiguous states. This rapid spread is believed to result from evolution of the WNV into a new genotype that replicates considerably faster in mosquitoes compared to the genotype that was initially introduced (Moudy et al. 2007). By virtue of its adaptive capability, an inherent property of RNA viruses, WNV has been remarkably successful in adapting to a new habitat and has emerged as a serious pathogen for humans, horses, and birds.

Another example of an emerging vector-borne zoonotic virus is Rift Valley fever virus (RVFV). RVFV belongs to the Phlebovirus genus of the Bunyaviridae family (Fauquet et al. 2005, Bird et al. 2009). This membrane-enveloped negative-strand RNA virus contains a segmented genome that is comprised of the large (L), medium (M), and small (S) segment. The L segment encodes the viral RNA-dependent RNA polymerase. The M segment encodes the structural glycoproteins Gn and Gc and a number of nonstructural proteins designated NSm (Schmaljohn 2001, Gerrard and Nichol 2007). The S segment encodes the nonstructural protein NSs and the nucleocapsid protein (Schmaljohn 2001).

RVFV was first identified in 1931 as the causative agent of enzootic hepatitis of sheep in Kenya (Findlay and Daubney 1931) and has since then spread across most of the African continent and more recently emerged on the Arabian peninsula (Al-Hazmi et al. 2003, Madani et al. 2003). Similar as WNV, RVFV has demonstrated its ability to establish outside its original habitat. However, whereas WNV transmission is limited to two mosquito genera (Culex and Aedes spp.) (Sardelis et al. 2001), RVFV can be transmitted by at least five mosquito genera (Smithburn et al. 1949, Fontenille et al. 1998), many of which are not confined to the African continent. This explains the fear for RVFV incursions into other parts of the world, including Europe (Moutailler et al. 2008) and the United States (Turell et al. 2008). Importantly, RVFV has a much broader effective host-range compared to WNV, capable of causing severe disease in sheep, goat, cattle, water buffalo, and humans.

Epidemics of RVF are characterized by the so-called abortion storms of gestating sheep. Lethality rates of adult sheep and cattle are estimated at 20% and 10%, respectively, whereas RVFV infection of few-week-old ruminants, particularly lambs, can result in a 100% fatality rate (Coetzer 1977; (for a review, see Easterday 1965). The virus can be transmitted to humans via infected mosquitoes or via direct contact with contaminated animal products. Most human infections are mild, and often manifest as a selflimiting febrile illness that resembles influenza. RVFV infection can, however, result in severe complications, including fulminant hepatitis, encephalitis, and retinitis with visual impairment or hemorrhagic fever (McIntosh et al. 1980, Al-Hazmi et al. 2003). Although the fatality rate of RVFV infections in humans is historically reported to be below 1%, recent outbreaks have demonstrated considerably higher fatality rates, exceeding 30% (WHO 2007a, 2007b, 2008).

Although the mortality rate of RVFV can be considerable, Crimean Congo hemorrhagic fever virus (CCHFV) is another member of the Bunyaviridae family, which is potentially even more deadly for humans. Many vertebrates are susceptible to CCHFV infection, among which are cattle, sheep, goat, horses, and swine, as well as smaller wildlife species such as hedgehogs and hares (Hoogstraal 1979). In contrast to WNV and RVFV, CCHFV does not seem to cause clinical illness in animals other than humans and infant mice. In humans, CCHFV infection can result in severe hemorrhagic fever (Swanepoel et al. 1989) with a case fatality that ranges widely from 5% to 70% (Centers for Disease Control and Prevention 1983, Ergönül and Whitehouse 2007).

CCHFV is maintained in the environment in a vertebrate–tick transmission cycle. The virus is transmitted to humans via hard-bodied ticks (Ixodidae family) or by the contact with contaminated blood products (Hoogstraal 1979). It has been well established that ticks from the Hyalomma genus play a major role in the epidemiology of CCHF. The distribution of CCHFV-infected cases in Europe, Africa, and Asia coincides well with the distribution of Hyalomma ticks (Hoogstraal 1979, Ergönül 2006). Despite its broad host range and its presence in >30 countries, CCHFV has historically caused only small outbreaks (Ergönül and Whitehouse 2007). In the past decade, however, outbreaks of CCHFV have occurred more frequently, predominantly in Turkey, Iran, and the Balkans (Ergönül 2006, Ergönül and Whitehouse 2007). In Turkey, since 2002, around 5000 laboratory-confirmed cases with overall fatality rate of 5% were reported.

The symposium on interventions against WNV, RVFV, and CCHFV was organized by Prof. Dr. Rob Moormann and Prof. Dr. Önder Ergönül, workpackage leaders of the ARBO-ZOONET (Ahmed et al. 2009) workpackage 6 coordination action: “Intervention strategies.” The ARBO-ZOONET symposium consisted of three themes: (1) vaccines: new and existing ones; (2), antivirals: existing and new developments; and (3) antivector vaccines. In addition, a satellite workshop was held on epidemiology and diagnosis. There were in total 23 oral presentations: 11 in theme 1, 7 in theme 2, 2 in theme 3, and 3 in the satellite workshop. Of these presentations, 10 were provided by ARBO-ZOONET members and 13 presentations were provided by invited external experts.

Most presentations in theme 1 were focused on vaccine development against RVFV. This is explained by the fact that effective veterinary vaccines for the prevention of WNV are commercially available and a promising vaccine for use in humans is currently being tested in a phase II clinical trial (see below). CCHFV vaccine development was not discussed, since a robust animal model essential for the development of such vaccines has only just become available. Presentations in theme 2 were primarily focused on experiences with the antiviral agent ribavirin for the treatment of patients with CCHFV infections and the development of novel antiviral agents for the treatment of CCHF and RVF. Finally, two presentations in theme 3 focused on the status and recent advances in antivector vaccine development. This report summarizes recent progress made by foremost international scientists in establishing intervention strategies against three of the most important emerging vector-borne viral diseases.

Theme 1: Vaccines

Vaccines for the control of WNV

There are currently three WNV vaccines for applications in horses commercially available in the United States and one in Europe. Vaccines based on adjuvanted, formalin-inactivated whole-virus named West Nile Innovator^® and Duvaxyn^® were commercialized by Fort Dodge Animal Health (FDAH) in the United States and Europe, respectively. The West Nile Innovator vaccine was applied in the United States on a large scale from 2002. Although the number of human WN fever cases did not decline between 2002 and 2003, vaccination with the Innovator vaccine could have contributed to the significant reduction of equine cases in this period (Granwehr et al. 2004). Since then, the following second-generation vaccines have come to market: a chimeric vaccine based on the Yellow fever virus 17D vaccine strain named PreveNile™ was commercialized by Intervet (Schering-Plough Animal Health/Merck), and a canarypox-vectored vaccine (RecombiTEK) was commercialized by Merial (El Garch et al. 2008). During the symposium the results obtained from vaccination challenge trials in horses with the RecombiTEK vaccine were presented by Dr. Jean-Christophe Audonnet (Merial) (Siger et al. 2004). The Recombitek^® vaccine is based on an ALVAC vector that produces the prM-E polyprotein region of WNV strain NY99 (Minke et al. 2004, El Garch et al. 2008). Horses received a single vaccination and were challenged using WNV-infected mosquitoes 26 days later. Whereas 8 out of 10 nonvaccinated horses developed viremia after challenge, only 1 out of 9 vaccinated horses developed viremia (Siger et al. 2004). Importantly, vaccination with the ALVAC vector does not result in failure of subsequent vaccinations with the same vector.

It is worthwhile to mention that a DNA vaccine (FDAH) for the prevention of WNV in horses was licensed by the USDA in 2005, representing the first license granted for a DNA vaccine for use in animals. A DNA vaccine for human application was also developed and a phase I clinical trial demonstrated that this vaccine induces neutralizing antibodies (Martin et al. 2007). Further, the ChimeriVax technology that was used to produce the PreveNile vaccine for horses was also used to produce a vaccine for human use, named ChimeriVax West Nile (Monath et al. 2006, Guy et al. 2010). This vaccine was found to be safe and immunogenic in humans and is currently being tested in a phase II clinical trial (Guy et al. 2010). From this, it can be concluded that effective vaccines for the prevention of WNV in horses are available and a vaccine for application in humans is expected to become available in the current decade.

Vaccines for the control of RVFV

Live-attenuated RVFV vaccines

In 1949, the RVFV was attenuated by passage in suckling mouse brain (Smithburn 1949), resulting in the neurotropic Smithburn strain. This virus is still used today as a live-attenuated vaccine for the prevention of RVFV in African livestock. Although the vaccine can provide long-lasting immunity after a single vaccination, the vaccine can cause fetal aberrations and abortions in gestating ruminants (Botros et al. 2006, Kamal 2009). Inactivated whole-virus vaccines are considered safe for animals of all age groups and have been used to vaccinate people with increased risk of contracting RVFV infection (Pittman et al. 1999, Frank-Peterside 2000). Unfortunately, however, inactivated RVFV vaccines developed thus far have proven to be less efficacious than live-attenuated vaccines and repeated vaccination is required to obtain immunity (Barnard and Botha 1977, Barnard 1979, Harrington et al. 1980, Frank-Peterside 2000).

With the aim to produce a safer alternative for the live Smithburn vaccine, the RVFV was previously passaged in the presence of the mutagen 5-fluorouracil (Morrill et al. 1987) to attenuate the virus by mutation. The resulting mutant virus, named MP12, was shown to protect sheep and cattle from challenge with virulent RVFV and appeared to be safe, even for pregnant animals (Morrill et al. 1991, 1997a, 1997b). In a later study, however, vaccination of ewes in the first trimester of pregnancy caused fetal malformations (Hunter et al. 2002). The biological safety of the MP12 vaccine therefore remains questionable, explaining the need for human and veterinary vaccines that optimally combine efficacy and safety.

It is important to note that the migration of RVFV to new virgin soils could result in unpredictable rapid spread and severity of disease, as occurred after the first introductions of RVFV in Egypt (Meegan 1979) and the Arabian peninsula (Al-Hazmi et al. 2003, Madani et al. 2003). A vaccine that is to be used to control future RVFV epidemics should, therefore, provide swift and long-lasting immunity after a single vaccination.

During the symposium, several new approaches for the development of RVFV vaccines were discussed. The vaccine that is the furthest in development is the live-attenuated Clone 13 vaccine. The Clone 13 virus originates from a plaque-purified clone of RVFV, which was found to contain a large deletion in the small (S) genome segment. This deletion was shown to disable the biological functions of the nonstructural protein NSs (Muller et al. 1995). The NSs protein is an important virulence factor by functioning as an antagonist of type I interferon (IFN) (Bouloy et al. 2001) and by promoting posttranscriptional downregulation of double-stranded RNA-dependent protein kinase (Habjan et al. 2009b, Ikegami et al. 2009a, 2009b). Previous work demonstrated that Clone 13 is highly immunogenic, but strongly attenuated in mice (Muller et al. 1995). This experimental vaccine was recently evaluated for safety and efficacy in sheep, and the results of this trail were presented by Dr. Michèle Bouloy (Institut Pasteur, France). The experiment demonstrated that the Clone 13 vaccine generates immunity in pregnant ewes without causing clinical signs such as fever, abortion, or malformations of the fetus. All vaccinated ewes appeared to be completely protected from a lethal dose of RVFV and no abortions occurred. Importantly, shedding of the vaccine virus was not detected. After these promising results, the registration of the Clone 13 vaccine in South Africa is currently under way.

Viruses with segmented genomes, such as RVFV, have the ability to exchange genome fragments. Reassortment of genome segments of live-attenuated vaccine viruses with circulating field strains could result in new virus variants with potentially undesirable properties. Evidence for reassortment of RVFVs in the field was reported earlier (Sall et al. 1999). Considering this, the participants of the symposium discussed, and agreed on the added value of attenuating mutations on multiple genome segments. Several recently developed live-attenuated experimental vaccines in fact fulfill this requirement. A Clone 13 derivative named R566 was developed, which contains the S segment of Clone 13 and the M and L segments of the MP12 virus (Bouloy and Flick 2009). Although promising preliminary results obtained from vaccination studies with R566 were already described (Bouloy and Flick 2009), the protective efficacy of the R566 virus is not yet reported.

The availability of genetic modification systems (i.e., reverse-genetics) for Bunyaviruses has greatly facilitated our understanding of the biology of these viruses and has enabled the development of new candidate vaccines. The first Bunyavirus that was produced using reverse-genetics was Bunyamwera virus (Bridgen and Elliott 1996). RVFV was successfully produced from cDNA several years later (Ikegami et al. 2006, Gerrard et al. 2007, Billecocq et al. 2008, Habjan et al. 2008). A presentation by Prof. Dr. Richard M. Elliott (University of St. Andrews, Scotland, United Kingdom) demonstrated the remarkable power of reverse-genetics technology. The work of Prof. Dr. Elliott and coworkers has resulted in important new insights into the biology of bunyaviruses (Lowen et al. 2005, Eifan and Elliott 2009, Shi and Elliott 2009) and has opened up new ways to produce novel live-attenuated vaccines (Lowen et al. 2005). Of particular interest were the novel ideas presented to produce bunyaviruses that are incapable of reassorting with field viruses by modifying untranslated regions.

By making use of RVFV reverse-genetics, another experimental vaccine was developed that contains attenuating mutations on multiple genome segments. In this experimental vaccine, the NSs-encoding gene of the S genome segment is replaced by enhanced green-fluorescent protein (eGFP) and the NSm-encoding region of the M genome segment is deleted (Bird et al. 2008). The results of vaccination challenge experiments in rats with the so-called rRVF-▵NSs:eGFP-▵NSm strain were presented during the symposium by Dr. Stuart Nichol (Centers for Disease Control and Prevention [CDC], Atlanta, GA). This live-attenuated candidate vaccine was shown to protect rats against a challenge with the virulent parental virus (Bird et al. 2008). An additional advantage of the rRVF-▵NSs:eGFP-▵NSm strain is its potential use as a vaccine that enables the serological Differentiation between Infected and Vaccinated Animals (DIVA).

Past outbreaks of diseases that are notifiable to the World Organization for Animal Health (i.e., Office International des Epizooties [OIE]) have made clear the added value of vaccines with DIVA properties. DIVA vaccines enable the identification of infected animals in a vaccinated population and thereby minimize economic damage imposed by animal transport restrictions. There was clear consensus among the participants of the meeting that future vaccines against OIE-notifiable diseases, such as RVF, should preferentially fulfill the DIVA criterion. Clone 13, the R566, and the recombinant rRVF-▵NSs:GFP-▵NSm virus lack the NSs protein. Although being a nonstructural protein, it was demonstrated during the symposium that RVF-infected rats, goats, and humans develop anti-NSs antibodies, and an ELISA was recently developed that can be used to detect these antibodies (McElroy et al. 2009). Although these promising findings suggest that vaccines lacking the NSs protein can possibly be used as DIVA vaccines, extensive analysis of ruminant and human sera must be performed to substantiate this assumption.

RVFV vaccines based on viral vectors

Besides the efforts that are being made to produce new live-attenuated vaccines by attenuating the RVFV, other promising approaches are based on subunits of the virus. The RVFV glycoproteins Gn and Gc are known to induce protective immunity, and several types of experimental vaccines based on these glycoproteins have been produced (Schmaljohn et al. 1989, Wallace et al. 2006, Gorchakov et al. 2007, Lorenzo et al. 2008, Heise et al. 2009, Mandell et al. 2009). One promising approach that was presented at the symposium by Nitin Bhardwaj, D.V.M., M.S. (University of Pittsburgh, Pittsburgh, PA) is based on using an alphavirus replicon for the in vivo production of the RVFV glycoprotein Gn. Similar as other recently published alphavirus replicon-based RVFV vaccines (Gorchakov et al. 2007, Heise et al. 2009), this experimental vaccine was shown to protect mice from a lethal dose of RVFV. Although these results are promising, future experiments should be performed in the RVFV target species (e.g., sheep) to establish the true efficacy of alphavirus-based RVFV vaccines.

Another experimental vector vaccine that was presented at the symposium by Prof. Dr. Rob Moormann (Central Veterinary Institute of Wageningen UR [CVI-WUR], Lelystad, The Netherlands) is based on Newcastle disease virus (NDV). NDV is an avian paramyxovirus that causes disease in poultry. The vector vaccine that was presented is based on an NDV vaccine virus that is used in the field to prevent NDV in poultry and is therefore considered environmentally highly safe. An important additional advantage of using NDV as a vaccine vector is the lack of pre-existing immunity against the vector in mammals. By making use of reverse-genetics, an NDV vaccine virus was created that produces the RVFV Gn glycoprotein. A pilot experiment demonstrated the ability of this vaccine virus to induce neutralizing antibodies in calves (Kortekaas et al. 2010). Future experiments will focus on determining the protective efficacy of this vaccine candidate in vaccination challenge trials.

DNA vaccines and subunit vaccines

In two presentations, the ability to confer protection against RVFV by DNA vaccination was reported. DNA vaccines offer several advantages. They are easily and quickly produced, they are extremely stable, and DNA vaccines against different diseases can potentially be combined in a singe inoculum. Despite these benefits, the protective efficacy of DNA vaccines is generally less than other types of vaccines, and many efforts are therefore being made to improve their efficacy (Kutzler and Weiner 2008). A particularly promising approach is based on the use of molecular adjuvants. Conjugation of the C3d fragment of complement factor C3 to an antigen can stimulate the immune response against this antigen (Dempsey et al. 1996). A presentation by Nitin Bhardwaj (University of Pittsburgh, PA, USA) demonstrated that three inoculations with a plasmid encoding the Gn glycoprotein fused to three copies of C3d provided protection in mice. A presentation by Dr. Hani Boshra (CISA-INIA, Valdeolmos, Spain) demonstrated partial protection of mice by vaccination with a plasmid encoding the nucleocapsid protein fused to the lysosome integral membrane protein II. These recent developments in DNA vaccination for the prevention of RVFV are certainly promising and their efficacy in ruminants should therefore be determined in future experiments.

Subunit vaccines based on purified proteins

In a presentation by Prof. Dr. Friedemann Weber (University of Freiburg, Germany), the efficient production of RVFV-like particles (VLPs) by mammalian cells was reported (Habjan et al. 2009a) and it was demonstrated that three inoculations with these VLPs confer protection against RVFV in mice (Naslund et al. 2009). In a presentation by Matthijn de Boer (CVI-WUR), the production of VLPs by insect cells was reported and it was demonstrated that a double vaccination with these VLPs protects mice from a lethal dose of RVFV. Moreover, this work demonstrated that water-in-oil-adjuvanted VLPs induce even sterilizing immunity (de Boer et al. 2010). The major advantage of subunit vaccines that are based on purified protein is their intrinsic biological safety, rendering these vaccines particularly useful for applications as vaccines for human use. Clearly, future experiments should focus on determining the protective efficacy of VLPs when administered in ruminants and nonhuman primates, preferably after a single vaccination.

Considering the recent accomplishments in RVFV vaccine development, it can be expected that safe and effective vaccines for both humans and livestock will become available in the near future.

Vaccines for the control of CCHFV

There have been very few reports dealing with vaccine development against CCHFV. Although CCHFV is clearly emerging, outbreaks of CCHF have so far remained sporadic and limited in size, explaining the apparent lack of interest from industry in the development of CCHF vaccines. The development of CCHF vaccines should thus be funded by grants made available by governments, international institutions, or nongovernmental organizations. Funding should be substantial, since vaccine development against CCHFV requires access to biosafety level-4 (BSL-4) laboratories and the availability of highly trained personnel qualified to work with the virus.

CCHFV vaccine development has also been held back by the lack of an animal model. A presentation by Dr. Dennis Bente (Public Health Agency of Canada, Winnipeg, MB, Canada) made clear that a robust CCHFV animal model has now become available. It was recently established that the IFN pathway plays an important role in controlling CCHFV infections (Andersson et al. 2004, 2006, 2008). Bente et al. (unpublished data) used mice that are incapable of responding to IFN due to a homozygous disruption of the stat-1 gene and demonstrated that CCHFV infection is uniformly lethal in these mice, even when a low viral dose is used. Clinical signs, viremia, tissue tropism, cellular immune responses, clinical pathology, and histopathology were studied in great detail. The data presented convincingly demonstrated the suitability of the stat-1 knockout mouse model for studying CCHFV pathology and provides an essential tool for the development and evaluation of vaccine candidates and antivirals.

As presented by Dr. Stuart Dowall, the Health Protection Agency (HPA; Porton Down, Salisbury, Wiltshire, United Kingdom) is aiming to adapt the CCHFV to mice and to develop a disease model in rhesus macaques. Further, the HPA aims to develop a wide range of diagnostic tools to study pathology and immunology of CCHFV infections. These tools will be invaluable for the development and testing of new antiviral agents and vaccines.

One of the very few vaccines ever developed for the prevention of CCHF was produced already in 1974 in Bulgaria. The history and use of this vaccine, which is based on thermochemical-inactivated CCHFV, was presented by Dr. Iva Christova (National Centre for Infectious and Parasitic Diseases, Sofia, Bulgaria). Scientific publications about the efficacy of this vaccine is limited. Now that an animal model is available, the efficacy of this vaccine can be thoroughly evaluated.

Theme 2: Antivirals

There are currently no effective drugs available in the clinic for the treatment of patients suffering from WN fever. Several studies have demonstrated the effectiveness of ribavirin in inhibiting WNV growth in vitro (Jordan et al. 2000, Anderson and Rahal 2002, Morrey et al. 2002). However, ribavirin does not seem to be effective against viruses from the flavivirus genus in animal models (Huggins 1989, Malinoski et al. 1990). In accordance with this, a retrospective evaluation of ribavirin treatment during a WNV outbreak in Israel in 2000 demonstrated no correlation with improved survival (Chowers et al. 2001).

Dr. Markus Keller (Friedrich-Loeffler Institut, Riems, Germany), presented studies that were aimed to identify novel targets for antiviral agents against WNV. These studies aim to elucidate the role of integrins and other membrane proteins in attachment of WNV to host cells. Dr. Keller and coworkers demonstrated that WNV is able to infect cells that are devoid of the proposed cellular receptor of WNV (αvβ3 integrin) (Chu and Ng 2004), suggesting that integrins may not be optimal targets for intervention strategies and that future work should focus on identifying alternative targets.

Two presentations focused on the development of novel antiviral therapies for RVFV and CCHFV. The first presentation (Helen Karlberg, Swedish Institute for Infectious Disease Control, Solna, Sweden) reported a study where three different kinds of IFN-α-based antiviral agents were compared. More specifically, the antiviral activity of two recombinant forms of IFN-α, Roferon^® (rIFN-2a) and Intron A^® (rIFN-2b), was compared with the activity of Multiferon™, an antiviral agent that contains several naturally occurring IFN-α subtypes. It was demonstrated that Multiferon has antiviral activity against both RVFV and CCHFV in vitro at low concentrations, which was superior to the activity of recombinant IFN-α-based antiviral agents. The second presentation, provided by Sara Åkerström (Swedish Institute for Infectious Disease Control, Solna, Sweden), dealt with the antiviral activity of S-nitroso-N-acetylpenicillamine, a nitric oxide donor. This compound was shown to inhibit replication of CCHFV, but not of RVFV. In future experiments, the in vivo antiviral activity of these new antiviral compounds should be evaluated.

The use of the antiviral drug ribavirin for the treatment of patients suffering from RVF or CCHF was discussed. Ribavirin was shown to be effective against RVFV both in vitro and in animal models (Peters et al. 1986, Garcia et al. 2001) and was used to treat patients with severe RVF during a large outbreak in Saudi Arabia. Although ribavirin treatment seemed to be effective against hemorrhagic diathesis with hepatitis, it did not prevent the neurological complications associated with RVFV infections (Bouloy and Flick 2009, pers. comm.). The apparent ineffectiveness of ribavirin against neuroinvasive disease caused by WNV or RVFV suggests that the passage of ribavirin across the blood–brain barrier is insufficient to counteract viruses that replicate beyond this barrier. Much effort is currently being made to develop novel antiviral agents against WNV (Perera et al. 2008, Diamond 2009) and RVFV (Bouloy and Flick 2009), but discussing these novel approaches is beyond the scope of this report.

Ribavirin treatment is effective against CCHFV infection in vitro (Watts et al. 1989) and was further demonstrated to reduce infant mouse mortality (Tignor and Hanham 1993). The experiences with ribavirin treatment of human cases in the clinic in Turkey were presented by Prof. Dr. Önder Ergönül (Marmara University, Istanbul, Turkey). In clinical practice, ribavirin treatment was found to be beneficial, especially when administered during the early phase of infection (Ergönül 2008, Tasdelen Fisgin et al. 2009). The conclusions presented by Prof. Dr. Ergönül are in line with several other studies reported in literature, which report the beneficial effect of ribavirin in CCHF patients (Mardani et al. 2003, Ergönül et al. 2004, Ergönül 2006, Ozkurt et al. 2006, Izadi and Salehi 2009, Sharifi-Mood et al. 2009, Tasdelen Fisgin et al. 2009).

However, because these studies were observational in nature, based on a limited number of cases, and were not placebo-controlled or randomized, a statistically significant effect of ribavirin treatment on the mortality rate could never be demonstrated. Prof. Dr. Ergönül noted that the treatment effect of ribavirin is strongly dependent on many variables, of which the most important ones are (1) the number of days that pass from onset of illness until the moment when ribavirin treatment is initiated; (2) the severity of disease and the protopathic bias; (3) the severity of gastrointestinal symptoms, and (4) the duration of ribavirin use (Ergönül 2006, 2008). To determine statistical significance of a ribavirin treatment effect, these variables should be carefully controlled. Control of these variables with appropriate placebo-control is, however, considered unethical, leading to the conclusion that statistical significance of the ribavirin treatment effect may never be established.

Although ribavirin is currently the drug of choice for the treatment of CCHFV infections, there is a clear need for more effective antiviral drugs, which preferentially lack the undesirable side effects of ribavirin. So far, the development of novel antiviral agents against CCHFV is greatly complicated by the fact that the virus must be handled under BSL-4 containment. A presentation by Dr. Éric Bergeron (CDC) demonstrated the successful establishment of a CCHFV minigenome assay, which can be used to screen antiviral agents outside of the BSL-4 laboratory (Bergeron et al. 2010). Dr. Bergeron demonstrated that ribavirin inhibited minigenome replication with efficacy comparable to in vitro studies with live CCHFV. Efforts will be made to render this system suitable for the high-throughput screening of novel antiviral agents against CCHFV. Of note, one potential novel antiviral target was already identified as the subtilisin kexin isozyme-1/site-1 protease (Bergeron et al. 2007).

Theme 3: Antivector Vaccines

Virgin soil epidemics caused by vector-borne viruses can be effectively controlled by vaccination against the pathogen, and antiviral drugs can be useful adjuncts in some situations. However, upon the incursion of a completely new pathogen, it is unlikely that suitable vaccines or antiviral agents will be readily available. Vaccines that target the insect vector are attractive alternatives, and offer the additional advantage that the control measures can potentially be used to control a variety of viruses that share the insect vector. In Theme 3, two presentations were held. The first presentation dealing with antitick vaccines was provided by Dr. Peter Willadsen (CSIRO Livestock Industries, Brisbane, Australia) and the second presentation dealing with antimosquito vaccines was provided by Dr. Paulo Almeida (Universidade Nova de Lisboa, Lisbon, Portugal).

Ticks can be controlled by the mass application of acaricides. Repeated acaricide application, however, results in drug-resistant ticks and considerable environmental impact (Kunz and Kemp 1994). The feasibility of vaccinating pigs and cattle with tick antigens was first described by Allen and Humphreys (1979). In the field of antivector vaccines, the biggest success story tells about vaccines based on the Bm86 antigen from the tick Boophilus microplus, which were developed in the early 90s (Willadsen et al. 1995). B. microplus is considered the most important tick parasite of livestock in the world, decreasing milk production and reducing weight gain in cattle as well as transmitting economically important protozoal parasites such as Babesia spp. and Anaplasma spp. The Bm86 antigen is naturally present on microvilli of tick gut cells (Gough and Kemp 1993) and was identified as a promising antigen by vaccination of cattle with protein fractions of B. microplus (Willadsen et al. 1989). In the mid-1990s, two Bm86-based vaccines were commercialized, one in Latin America (Gavac) and one in Australia (TickGARD). Uptake of antibodies induced by these vaccines by the tick results in lysis of cells from the tick gut and thereby reduces the number of ticks feeding on cattle (for a review, see Willadsen 2004). The Bm86 vaccine has also been shown to reduce the incidence of babesiosis in vaccinated cattle. Another success story describes the vaccination with a tick salivary protein named 64TRP, which was shown to prevent the transmission of tick-borne encephalitis virus (TBEV) (Labuda et al. 2006). Importantly, vaccination with the 64TRP vaccine was equally effective as a conventional TBEV vaccine in providing protection, and its transmission-blocking effect was even superior to that obtained by the conventional vaccine (Labuda et al. 2006). The accomplishments achieved so far with antitick vaccines are very promising and are expected to be greatly improved by the future discovery of new targets by molecular and systems biology approaches.

The second presentation in Theme 3 dealt with antimosquito vaccines. It is important to state first that the development of vaccines against ticks has been far more successful than the development of vaccines against insect vectors. There are two major explanations for this that are briefly discussed. First, ticks are in contact with the host's immune system for days to weeks, whereas hematophagous insects are in contact with the host for minutes at the most, rendering insects less vulnerable for immune attack. The second reason results from a distinct difference in physiology between ticks and insects. Digestion in ticks occurs largely intracellular. The gut of the tick is a friendly environment, being at near-neutral pH and largely free of proteases. In contrast, digestion in insects occurs in the gut, which is a hostile environment, filled with proteases that are able to break down ingested antibodies. These two reasons explain the generally low susceptibility of hematophagous insects to the immune system of the host and thereby the relative difficulty of producing effective vaccines against these vectors.

The studies that were presented by Dr. Paulo Almeida were aimed at developing vaccines against Anopheles stephensi, the vector of Plasmodium berghei. P. berghei is a species of malaria parasite that is infectious for laboratory rodents and is easily maintained and transmitted by A. stephensi in the laboratory. Almeida and Billingsley demonstrated that repeated inoculation of mice with midgut extracts resulted in a significant reduction of mosquito survival and fecundity (Almeida and Billingsley 1998, 2002), although reproducibility was a concern in these studies. It was concluded that innovative molecular biological tools should be employed to identify new effective targets in the mosquito. In this respect, it is interesting to point to a recent study in which a new approach termed reverse antigen screening was successfully employed to identify a novel target in a sand fly vector. Vaccination with this salivary protein was shown to protect against leishmaniasis in a hamster model (Gomes et al. 2008), demonstrating that salivary proteins of insects, similar as earlier reported for ticks (Labuda et al. 2006), are promising vaccine targets. The availability of new molecular tools such as RNA interference and microarrays will undoubtedly result in the identification of promising new candidates (Willadsen 2001).

Satellite Workshop on Epidemiology and Diagnosis

The satellite workshop on epidemiology and diagnosis consisted of three presentations. The first presentation (Dr. Vittorio Sambri, University of Bologna, Bologna, Italy) provided a comprehensive overview of the epidemiology of WNV in Europe and subsequently focused on the spread and mode of surveillance of WNV in Northern Italy in 2008 and 2009 (Barzon et al. 2009). The data presented suggest that the virus is expanding its habitat in Italy. The integrated way of surveillance of WNV in horses, birds, mosquitoes, and humans was highlighted. It was reported that WNV was detected in mosquitoes, birds, and horses weeks before WNV was detected in humans. Integrated surveillance thus allows for early warning of people at risk, and the timely establishment of personal control measures in newly infected areas.

Dr. Catherine Cétre-Sossah (CIRAD, Montpellier, France) presented data on the recent circulation of RVFV on Mayotte, a French island located west of Madagascar in the Indian Ocean. A retrospective study demonstrated RVFV-specific IgG antibodies in 304 bovine sera that were collected in 2007 and 2008 from all over the island. Subsequent serosurveillance of five goat herds performed with RVFV-specific IgG and IgM ELISAs provided evidence of recent circulation of RVFV on Mayotte. It was concluded, however, that the risk of RVFV epidemics and epizootics in Mayotte is considered low because of the small population of ruminants.

The final presentation was provided by Nadine Litzba (Robert Koch Institut, Berlin, Germany). The presentation focused on biochip technology as a new serological test for CCHF. This indirect immunofluorescence test for IgM and IgG detection uses cells that produce recombinant CCHFV glycoproteins and nucleocapsid proteins that are fixed on biochips. The biochip system shows promise but needs more extensive validation.

Concluding Remarks

The main aim of this ARBO-ZOONET symposium was the identification of research gaps. In the field of WNV intervention strategies, the most important research gap is the lack of effective antiviral agents. Developing novel antiviral agents should, thus, be a priority in the coming years.

In the field of RVFV intervention strategies the availability of antivirals is of even higher importance considering the continuous large impact of RVF on human health in Africa and the threat of incursions into previously unaffected areas. Further, although promising results were obtained with new candidate RVFV vaccines in laboratory rodents, the focus in this field should be on the establishment of vaccine efficacy in the target species of RVFV.

The potential high impact of RVFV outbreaks on the economy as a direct consequence of imposed trade restrictions makes clear that future RVFV vaccines should preferentially fulfill the DIVA criterion. Two approaches in this field were discussed. First, experimental vaccines were developed that lack the RVFV N protein (de Boer et al. 2010, Kortekaas et al. 2010). These vaccines can potentially be used as DIVA vaccines when accompanied by a commercially available N ELISA. Second, experimental ELISAs that detect antibodies against the NSs protein can potentially be used as DIVA tests to accompany any candidate vaccine that lacks the NSs protein (McElroy et al. 2009). Thus, future experiments should not only elaborate on the protective efficacy of candidate vaccines in livestock, but also investigate their potential use as DIVA vaccines when accompanied by the appropriate ELISA.

Until recently, the major research gaps in CCHFV intervention strategies were the lack of an animal model and a minigenome system, both important tools for the evaluation of vaccine candidates and antiviral agents. As reported during the symposium, both systems are now available, two major strikes in the battle against this important pathogen. Efforts should be made to optimally exploit these valuable systems in the years to come.

In the field of antivector vaccine development, combining target antigens and the use of novel targets such as salivary proteins hold promise for the future control of both ticks and insects. Besides the need for more extensive research on the effect of current vaccines on the vectors and the outcome of disease, it was concluded that future studies should also focus on the effect of antivector vaccines on pathogen transmission. It is clear that the complexity of this area of research requires strong interdisciplinary efforts of molecular virologists and entomologists as well as extensive use of novel technologies that define the postgenomics era.

Footnotes

Acknowledgments

The authors would like to thank the participants of the ARBO-ZOONET symposium for their contribution: Michèle Bouloy, Richard Elliott, Stuart Nichol, Friedemann Weber, Nitin Bhardwaj, Gema Lorenzo, Hani Boshra, Matthijn de Boer, Iva Christova, Jean-Christophe Audonnet, Peter Willadsen, Paulo Almeida, Helen Karlberg, Sara Åkerström, Éric Bergeron, Dennis Bente, Stuart Dowall, Markus Keller, Vittorio Sambri, Catherine Cêtre-Sossah, and Nadine Litzba.

The ARBO-ZOONET Coordination Action Project is financed by the Food, Agriculture, and Fisheries, and Biotechnology program of the European Commission, Brussels, Belgium, through Coordination Action Project No. 211757.

Disclosure Statement

No competing financial interests exist.

References

Ahmed

, Bouloy

, Ergönül

, Fooks

et al. International network for capacity building for the control of emerging viral vector-borne zoonotic diseases: ARBO-ZOONET. Euro Surveill, 2009; 14pii:19160.

Al-Hazmi

, Ayoola

, Abdurahman

, Banzal

et al. Epidemic Rift Valley fever in Saudi Arabia: a clinical study of severe illness in humans. Clin Infect Dis, 2003; 36:245–252.

Allen

, Humphreys

. Immunisation of guinea pigs and cattle against ticks. Nature, 1979; 280:491–493.

Almeida

, Billingsley

. Induced immunity against the mosquito Anopheles stephensi (Diptera: Culicidae): effects of cell fraction antigens on survival, fecundity, and Plasmodium berghei (Eucoccidiida: Plasmodiidae) transmission. J Med Entomol, 2002; 39:207–214.

Almeida

, Billingsley

. Induced immunity against the mosquito Anopheles stephensi Liston (Diptera: Culicidae): effects on mosquito survival and fecundity. Int J Parasitol, 1998; 28:1721–1731.

Anderson

, Rahal

. Efficacy of interferon alpha-2b and ribavirin against West Nile virus in vitro. Emerg Infect Dis, 2002; 8:107–108.

Andersson

, Bladh

, Mousavi-Jazi

, Magnusson

et al. Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus. J Virol, 2004; 78:4323–4329.

Andersson

, Karlberg

, Mousavi-Jazi

, Martinez-Sobrido

et al. Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response. J Med Virol, 2008; 80:1397–1404.

Andersson

, Lundkvist

, Haller

, Mirazimi

. Type I interferon inhibits Crimean-Congo hemorrhagic fever virus in human target cells. J Med Virol, 2006; 78:216–222.

10.

Barnard

. Rift Valley fever vaccine—antibody and immune response in cattle to a live and an inactivated vaccine. J S Afr Vet Assoc, 1979; 50:155–157.

11.

Barnard

, Botha

. An inactivated rift valley fever vaccine. J S Afr Vet Assoc, 1977; 48:45–48.

12.

Barzon

, Squarzon

, Cattai

, Franchin

et al. West Nile virus infection in Veneto region, Italy, 2008–2009. Euro Surveill, 2009; 14pii:19289.

13.

Bergeron

, Albarino

, Khristova

, Nichol

. Crimean-Congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus RNA polymerase function. J Virol, 2010; 84:216–226.

14.

Bergeron

, Vincent

, Nichol

. Crimean-Congo hemorrhagic fever virus glycoprotein processing by the endoprotease SKI-1/S1P is critical for virus infectivity. J Virol, 2007; 81:13271–13276.

15.

Billecocq

, Gauliard

, Le May

, Elliott

et al. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses. Virology, 2008; 378:377–384.

16.

Bird

, Albarino

, Hartman

, Erickson

et al. Rift valley fever virus lacking the NSs and NSm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals. J Virol, 2008; 82:2681–2691.

17.

Bird

, Ksiazek

, Nichol

, Maclachlan

. Rift Valley fever virus. J Am Vet Med Assoc, 2009; 234:883–893.

18.

Blitvich

. Transmission dynamics and changing epidemiology of West Nile virus. Anim Health Res Rev, 2008; 9:71–86.

19.

Botros

, Omar

, Elian

, Mohamed

et al. Adverse response of non-indigenous cattle of European breeds to live attenuated Smithburn Rift Valley fever vaccine. J Med Virol, 2006; 78:787–791.

20.

Bouloy

, Flick

. Reverse genetics technology for Rift Valley fever virus: current and future applications for the development of therapeutics and vaccines. Antiviral Res, 2009; 84:101–118.

21.

Bouloy

, Janzen

, Vialat

, Khun

et al. Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs. J Virol, 2001; 75:1371–1377.

22.

Bridgen

, Elliott

. Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci U S A, 1996; 93:15400–15404.

23.

Centers for Disease Control and Prevention. Viral hemorrhagic fever: initial management of suspected and confirmed cases. MMWR Morb Mortal Wkly Rep, 1983; 32:27S–38S.

24.

Chowers

, Lang

, Nassar

, Ben-David

et al. Clinical characteristics of the West Nile fever outbreak, Israel, 2000. Emerg Infect Dis, 2001; 7:675–678.

25.

Chu

, Ng

. Interaction of West Nile virus with alpha v beta 3 integrin mediates virus entry into cells. J Biol Chem, 2004; 279:54533–54541.

26.

Coetzer

. The pathology of Rift Valley fever. I. Lesions occurring in natural cases in new-born lambs. Onderstepoort J Vet Res, 1977; 44:205–211.

27.

de Boer

, Kortekaas

, Antonis

, Kant

et al. Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge. Vaccine, 2010; 28:2330–2339.

28.

Dempsey

, Allison

, Akkaraju

, Goodnow

et al. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science, 1996; 271:348–350.

29.

Diamond

. Progress on the development of therapeutics against West Nile virus. Antiviral Res, 2009; 83:214–227.

30.

Easterday

. Rift valley fever. Adv Vet Sci, 1965; 10:65–127.

31.

Eifan

, Elliott

. Mutational analysis of the Bunyamwera orthobunyavirus nucleocapsid protein gene. J Virol, 2009; 83:11307–11317.

32.

El Garch

, Minke

, Rehder

, Richard

et al. A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse. Vet Immunol Immunopathol, 2008; 123:230–239.

33.

Ergönül

. Crimean-Congo haemorrhagic fever. Lancet Infect Dis, 2006; 6:203–214.

34.

Ergönül

. Treatment of Crimean-Congo hemorrhagic fever. Antiviral Res, 2008; 78:125–131.

35.

Ergönül

, Celikbas

, Dokuzoguz

, Eren

et al. Characteristics of patients with Crimean-Congo hemorrhagic fever in a recent outbreak in Turkey and impact of oral ribavirin therapy. Clin Infect Dis, 2004; 39:284–287.

36.

Ergönül

, Whitehouse

. Crimean-Congo Hemorrhagic Fever: A Global Perspective. Dordrecht, The Netherlands: Springer, 2007.

37.

Fauquet

, Mayo

, Maniloff

, Desselberger

et al. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. Academic Press, London: an imprint of Elsevier, 2005.

38.

Findlay

, Daubney

. The virus of Rift Valley fever or enzoötic hepatitis. Lancet, 1931; ii:1350–1351.

39.

Fontenille

, Traore-Lamizana

, Diallo

, Thonnon

et al. New vectors of Rift Valley fever in West Africa. Emerg Infect Dis, 1998; 4:289–293.

40.

Frank-Peterside

. Response of laboratory staff to vaccination with an inactivated Rift Valley fever vaccine—TSI-GSD 200. Afr J Med Med Sci, 2000; 29:89–92.

41.

Garcia

, Crance

, Billecocq

, Peinnequin

et al. Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds. J Clin Microbiol, 2001; 39:4456–4461.

42.

Gerrard

, Bird

, Albarino

, Nichol

. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection. Virology, 2007; 359:459–465.

43.

Gerrard

, Nichol

. Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins. Virology, 2007; 357:124–133.

44.

Gomes

, Teixeira

, Oliveira

et al. Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Proc Natl Acad Sci U S A, 2008; 105:7845–7850.

45.

Gorchakov

, Volkova

, Yun

, Petrakova

et al. Comparative analysis of the alphavirus-based vectors expressing Rift Valley fever virus glycoproteins. Virology, 2007; 366:212–225.

46.

Gough

, Kemp

. Localization of a low abundance membrane protein (Bm86) on the gut cells of the cattle tick Boophilus microplus by immunogold labeling. J Parasitol, 1993; 79:900–907.

47.

Granwehr

, Lillibridge

, Higgs

, Mason

et al.

West Nile virus: where are we now?

Lancet Infect Dis, 2004; 4:547–556.

48.

Guy

, Guirakhoo

, Barban

, Higgs

et al. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine, 2010; 28:632–649.

49.

Habjan

, Penski

, Spiegel

, Weber

. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus. J Gen Virol, 2008; 89:2157–2166.

50.

Habjan

, Penski

, Wagner

, Spiegel

et al. Efficient production of Rift Valley fever virus-like particles: the antiviral protein MxA can inhibit primary transcription of bunyaviruses. Virology, 2009a. 385:400–408.

51.

Habjan

, Pichlmair

, Elliott

, Overby

. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase. J Virol, 2009b. 83:4365–4375.

52.

Harrington

, Lupton

, Crabbs

, Peters

et al. Evaluation of a formalin-inactivated Rift Valley fever vaccine in sheep. Am J Vet Res, 1980; 41:1559–1564.

53.

Hayes

. West Nile virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci, 2001; 951:25–37.

54.

Heise

, Whitmore

, Thompson

, Parsons

et al. An alphavirus replicon-derived candidate vaccine against Rift Valley fever virus. Epidemiol Infect, 2009; 137:1309–1318.

55.

Hoogstraal

. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol, 1979; 15:307–417.

56.

Huggins

. Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev Infect Dis, 1989; 11:S750–S761.

57.

Hunter

, Erasmus

, Vorster

. Teratogenicity of a mutagenised Rift Valley fever virus (MVP 12) in sheep. Onderstepoort J Vet Res, 2002; 69:95–98.

58.

Ikegami

, Narayanan

, Won

, Kamitani

et al. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR. Ann N Y Acad Sci, 2009a. 1171:E75–E85.

59.

Ikegami

, Narayanan

, Won

, Kamitani

et al. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation. PLoS Pathog, 2009b. 5:e1000287.

60.

Ikegami

, Won

, Peters

, Makino

. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J Virol, 2006; 80:2933–2940.

61.

Izadi

, Salehi

. Evaluation of the efficacy of ribavirin therapy on survival of Crimean-Congo hemorrhagic fever patients: a case-control study. Jpn J Infect Dis, 2009; 62:11–15.

62.

Jordan

, Briese

, Fischer

, Lau

et al. Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis, 2000; 182:1214–1217.

63.

Kamal

. Pathological studies on postvaccinal reactions of Rift Valley fever in goats. Virol J, 2009; 6:94.

64.

Kortekaas

, Dekker

, de Boer

, Weerdmeester

et al. Intramuscular inoculation of calves with an experimental Newcastle disease virus-based vector vaccine elicits neutralizing antibodies against Rift Valley fever virus. Vaccine, 2010; 28:2271–2276.

65.

Kramer

, Li

, Shi

. West Nile virus. Lancet Neurol, 2007; 6:171–181.

66.

Kunz

, Kemp

. Insecticides and acaricides: resistance and environmental impact. Rev Sci Tech, 1994; 13:1249–1286.

67.

Kutzler

, Weiner

. DNA vaccines: ready for prime time? Nat Rev Genet, 2008; 9:776–788.

68.

Labuda

, Trimnell

, Lickova

, Kazimirova

et al. An antivector vaccine protects against a lethal vector-borne pathogen. PLoS Pathog, 2006; 2:e27.

69.

Lorenzo

, Martin-Folgar

, Rodriguez

, Brun

. Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep. Vaccine, 2008; 26:5255–5262.

70.

Lowen

, Boyd

, Fazakerley

, Elliott

. Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences. J Virol, 2005; 79:6940–6946.

71.

Madani

, Al-Mazrou

, Al-Jeffri

, Mishkhas

et al. Rift Valley fever epidemic in Saudi Arabia: epidemiological, clinical, and laboratory characteristics. Clin Infect Dis, 2003; 37:1084–1092.

72.

Malinoski

, Hasty

, Ussery

, Dalrymple

. Prophylactic ribavirin treatment of dengue type 1 infection in rhesus monkeys. Antiviral Res, 1990; 13:139–149.

73.

Mandell

, Koukuntla

, Mogler

, Carzoli

et al. A replication-incompetent Rift Valley fever vaccine: Chimeric virus-like particles protect mice and rats against lethal challenge. Virology, 2009; 397:187–198.

74.

Mardani

, Jahromi

, Naieni

, Zeinali

. The efficacy of oral ribavirin in the treatment of Crimean-Congo hemorrhagic fever in Iran. Clin Infect Dis, 2003; 36:1613–1618.

75.

Martin

, Pierson

, Hubka

, Rucker

et al. A West Nile virus DNA vaccine induces neutralizing antibody in healthy adults during a phase 1 clinical trial. J Infect Dis, 2007; 196:1732–1740.

76.

McElroy

, Albarino

, Nichol

. Development of a RVFV ELISA that can distinguish infected from vaccinated animals. Virol J, 2009; 6:125.

77.

McIntosh

, Russell

, dos Santos

, Gear

. Rift Valley fever in humans in South Africa. S Afr Med J, 1980; 58:803–806.

78.

Meegan

. The Rift Valley fever epizootic in Egypt 1977–78. 1. Description of the epizootic and virological studies. Trans R Soc Trop Med Hyg, 1979; 73:618–623.

79.

Minke

, Siger

, Karaca

, Austgen

et al. Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge. Arch Virol Suppl, 2004; 18:221–230.

80.

Monath

, Liu

, Kanesa-Thasan

, Myers

et al. A live, attenuated recombinant West Nile virus vaccine. Proc Natl Acad Sci U S A, 2006; 103:6694–6699.

81.

Morrey

, Smee

, Sidwell

, Tseng

. Identification of active antiviral compounds against a New York isolate of West Nile virus. Antiviral Res, 2002; 55:107–116.

82.

Morrill

, Carpenter

, Taylor

, Ramsburg

et al. Further evaluation of a mutagen-attenuated Rift Valley fever vaccine in sheep. Vaccine, 1991; 9:35–41.

83.

Morrill

, Jennings

, Caplen

, Turell

et al. Pathogenicity and immunogenicity of a mutagen-attenuated Rift Valley fever virus immunogen in pregnant ewes. Am J Vet Res, 1987; 48:1042–1047.

84.

Morrill

, Mebus

, Peters

. Safety and efficacy of a mutagen-attenuated Rift Valley fever virus vaccine in cattle. Am J Vet Res, 1997a. 58:1104–1109.

85.

Morrill

, Mebus

, Peters

. Safety of a mutagen-attenuated Rift Valley fever virus vaccine in fetal and neonatal bovids. Am J Vet Res, 1997b. 58:1110–1114.

86.

Moudy

, Meola

, Morin

, Ebel

et al. A newly emergent genotype of West Nile virus is transmitted earlier and more efficiently by Culex mosquitoes. Am J Trop Med Hyg, 2007; 77:365–370.

87.

Moutailler

, Krida

, Schaffner

, Vazeille

et al. Potential vectors of Rift Valley fever virus in the Mediterranean region. Vector Borne Zoonot Dis, 2008; 8:749–753.

88.

Muller

, Saluzzo

, Lopez

, Dreier

et al. Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment. Am J Trop Med Hyg, 1995; 53:405–411.

89.

Naslund

, Lagerqvist

, Habjan

, Lundkvist

et al. Vaccination with virus-like particles protects mice from lethal infection of Rift Valley Fever Virus. Virology, 2009; 385:409–415.

90.

Ozkurt

, Kiki

, Erol

, Erdem

et al. Crimean-Congo hemorrhagic fever in Eastern Turkey: clinical features, risk factors and efficacy of ribavirin therapy. J Infect, 2006; 52:207–215.

91.

Perera

, Khaliq

, Kuhn

. Closing the door on flaviviruses: entry as a target for antiviral drug design. Antiviral Res, 2008; 80:11–22.

92.

Peters

, Reynolds

, Slone

, Jones

et al. Prophylaxis of Rift Valley fever with antiviral drugs, immune serum, an interferon inducer, and a macrophage activator. Antiviral Res, 1986; 6:285–297.

93.

Pittman

, Liu

, Cannon

, Makuch

et al. Immunogenicity of an inactivated Rift Valley fever vaccine in humans: a 12-year experience. Vaccine, 1999; 18:181–189.

94.

Sall

, Zanotto

, Sene

, Zeller

et al. Genetic reassortment of Rift Valley fever virus in nature. J Virol, 1999; 73:8196–8200.

95.

Sardelis

, Turell

, Dohm

, O'Guinn

. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus. Emerg Infect Dis, 2001; 7:1018–1022.

96.

Schmaljohn

, Parker

, Ennis

, Dalrymple

et al. Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins. Virology, 1989; 170:184–192.

97.

Schmaljohn

, Hooper

. Bunyaviridae: the viruses and their replication. Fields

B.N.

, Knipe

D.M.

, Howley

P.M.

, Griffin

D.E.

Fields Virology. Philadelphia: Lippincott Williams & Wilkins, 2001.

98.

Sharifi-Mood

, Metanat

, Ghorbani-Vaghei

, Fayyaz-Jahani

et al. The outcome of patients with Crimean-Congo hemorrhagic fever in Zahedan, southeast of Iran: a comparative study. Arch Iran Med, 2009; 12:151–153.

99.

Shi

, Elliott

. Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins. J Gen Virol, 2009; 90:297–306.

100.

Siger

, Bowen

, Karaca

, Murray

et al. Assessment of the efficacy of a single dose of a recombinant vaccine against West Nile virus in response to natural challenge with West Nile virus-infected mosquitoes in horses. Am J Vet Res, 2004; 65:1459–1462.

101.

Smithburn

. Rift Valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine. Br J Exp Pathol, 1949; 30:1–16.

102.

Smithburn

, Haddow

, Lumsden

. Rift Valley fever; transmission of the virus by mosquitoes. Br J Exp Pathol, 1949; 30:35–47.

103.

Swanepoel

, Gill

, Shepherd

, Leman

et al. The clinical pathology of Crimean-Congo hemorrhagic fever. Rev Infect Dis, 1989; 11,Suppl 4:S794–S800.

104.

Tasdelen Fisgin

, Ergönül

, Doganci

, Tulek

. The role of ribavirin in the therapy of Crimean-Congo hemorrhagic fever: early use is promising. Eur J Clin Microbiol Infect Dis, 2009; 28:929–933.

105.

Tignor

, Hanham

. Ribavirin efficacy in an in vivo model of Crimean-Congo hemorrhagic fever virus (CCHF) infection. Antiviral Res, 1993; 22:309–325.

106.

Turell

, Dohm

, Mores

, Terracina

et al. Potential for North American mosquitoes to transmit Rift Valley fever virus. J Am Mosq Control Assoc, 2008; 24:502–507.

107.

Wallace

, Ellis

, Espach

, Smith

et al. Protective immune responses induced by different recombinant vaccine regimes to Rift Valley fever. Vaccine, 2006; 24:7181–7189.

108.

Watts

, Ussery

, Nash

, Peters

. Inhibition of Crimean-Congo hemorrhagic fever viral infectivity yields in vitro by ribavirin. Am J Trop Med Hyg, 1989; 41:581–585.

109.

WHO. Outbreak news. Rift Valley fever, Madagascar. Wkly Epidemiol Rec, 2008; 83:157.

110.

WHO. Outbreak news. Rift Valley fever, Sudan-update. Wkly Epidemiol Rec, 2007a. 82:417–418.

111.

WHO. Outbreaks of Rift Valley fever in Kenya, Somalia and United Republic of Tanzania. December 2006–April 2007. Wkly Epidemiol Rec, 2007b. 82:169–178.

112.

Willadsen

. Anti-tick vaccines in “Ticks, Disease and Control.” Parasitology, 2004; (Suppl),129:S367–S388.

113.

Willadsen

. The molecular revolution in the development of vaccines against ectoparasites. Vet Parasitol, 2001; 101:353–368.

114.

Willadsen

, Bird

, Cobon

, Hungerford

. Commercialisation of a recombinant vaccine against Boophilus microplus. Parasitology, 1995; 110,Suppl:S43–S50.

115.

Willadsen

, Riding

, McKenna

, Kemp

et al. Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus. J Immunol, 1989; 143:1346–1351.