The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

Rift Valley fever.

Rift Valley fever (RVF) is a mosquito-borne zoonotic viral disease affecting domestic and wild ruminants, camels and humans. The causative agent of RVF, the RVF virus (RVFV), has the capacity to cause large and severe outbreaks in animal and human populations and to cross significant natural geographic barriers. Rift Valley fever is usually inapparent in non-pregnant adult animals, but pregnant animals and newborns can be severely affected; outbreaks are characterised by a sudden onset of abortions and high neonatal mortality. The majority of human infections are subclinical or associated with moderate to severe, non-fatal, febrile illness, but some patients may develop a haemorrhagic syndrome and/or ocular and neurological lesions. In both animals and humans, the primary site of RVFV replication and tissue pathology is the liver. Outbreaks of RVF are associated with persistent high rainfalls leading to massive flooding and the emergence of large numbers of competent mosquito vectors that transmit the virus to a wide range of susceptible vertebrate species. Outbreaks of RVF have devastating economic effects on countries for which animal trade constitutes the main source of national revenue. The propensity of the virus to spread into new territories and re-emerge in traditionally endemic regions, where it causes large outbreaks in human and animal populations, presents a formidable challenge for public and veterinary health authorities. The presence of competent mosquito vectors in RVF-free countries, the wide range of mammals susceptible to the virus, altering land use, the global changes in climate, and increased animal trade and travel are some of the factors which might contribute to international spread of RVF.

Identification of human linear B-cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus.

A peptide library was used to screen for regions containing potential linear B-cell epitope sites in the glycoproteins and nucleoprotein of Crimean-Congo haemorrhagic fever virus (CCHFV) in an enzyme-linked immunosorbent assay (ELISA). The library consisted of 156 peptides, spanning the nucleoprotein and mature GN and GC proteins in a 19-mer with 9-mer overlap format. Using pooled serum samples from convalescent patients to screen the library, six peptides were identified as potential epitope sites. Further testing of these six peptides with individual patient sera identified two of these peptides as probable epitope sites, with peptide G1451-1469 reacting to 13/15 and peptide G1613-1631 to 14/15 human sera. These peptides are situated on the GC protein at amino acid positions 1451-1469 (relative to CCHFV isolate SPU103/97) (TCTGCYACSSGISCKVRIH) and 1613-1631 (FMFGWRILFCFKCCRRTRG). Identified peptides may have application in ELISA for diagnostic or serosurveillance purposes.

Rift valley Fever in Kruger national park: do buffalo play a role in the inter-epidemic circulation of virus?

Rift Valley fever (RVF) is a zoonotic mosquito-borne virus disease of livestock and wild ruminants that has been identified as a risk for international spread. Typically, the disease occurs in geographically limited outbreaks associated with high rainfall events and can cause massive losses of livestock. It is unclear how RVF virus persists during inter-epidemic periods but cryptic cycling of the virus in wildlife populations may play a role. We investigated the role that free-living African buffalo (Syncerus caffer caffer) might play in inter-epidemic circulation of the virus and looked for geographic, age and sex patterns of Rift Valley fever virus (RVFV) infection in African buffalo. Buffalo serum samples were collected (n = 1615) in Kruger National Park (KNP), South Africa, during a period of 1996-2007 and tested for antibodies to RVF. We found that older animals were more likely to be seropositive for anti-RVFV antibody than younger animals, but sex was not correlated with the likelihood of being anti-RVFV antibody positive. We also found geographic variation within KNP; herds in the south were more likely to have acquired anti-RVFV antibody than herds farther north - which could be driven by host or vector ecology. In all years of the study between 1996 and 2007, we found young buffalo (under 2 years of age) that were seropositive for anti-RVFV antibody, with prevalence ranging between 0 and 27% each year, indicating probable circulation. In addition, we also conducted a 4-year longitudinal study on 227 initially RVFV seronegative buffalo to look for evidence of seroconversion outside known RVF outbreaks within our study period (2008-2012). In the longitudinal study, we found five individuals that seroconverted from anti-RVFV antibody negative to anti-RVFV antibody positive, outside of any detected outbreak. Overall, our results provide evidence of long-term undetected circulation of RVFV in the buffalo population.

Next-generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment.

Crimean Congo haemorrhagic fever virus (CCHFV) is a bunyavirus with a single-stranded RNA genome consisting of three segments (S, M, L), coding for the nucleocapsid protein, envelope glycoproteins and RNA polymerase, respectively. To date only five complete genome sequences are available from southern African isolates. Complete genome sequences were generated for 10 southern African CCHFV isolates using next-generation sequencing techniques. The maximum-likelihood method was used to generate tree topologies for 15 southern African plus 26 geographically distinct complete sequences from GenBank. M segment reassortment was identified in 10/15 southern African isolates by incongruencies in grouping compared to the S and L segments. These reassortant M segments cluster with isolates from Asia/Middle East, while the S and L segments cluster with strains from South/West Africa. The CCHFV M segment shows a high level of genetic diversity, while the S and L segments appear to co-evolve. The reason for the high frequency of M segment reassortment is not known. It has previously been suggested that M segment reassortment results in a virus with high fitness but a clear role in increased pathogenicity has yet to be shown.

Human cases of Sindbis fever in South Africa, 2006-2010.

Sindbis virus (SINV), the prototype positive-sense RNA alphavirus, causes febrile arthritis and is present throughout Afro-Eurasia. Little is known of the epidemiology of Sindbis fever due to insufficient surveillance in most endemic countries. The epidemiological features of Sindbis fever in humans in South Africa are described here based on a retrospective study of suspected arbovirus cases submitted for laboratory investigation from 2006 to 2010. Cases were detected annually mostly during the late summer/early autumn months and an increase in cases was noted for 2010, coinciding with an outbreak of Rift Valley fever. Cases were reported most often from the central plateau of South Africa and involved mostly males. No severe or fatal cases were reported and cases were associated with febrile arthralgia as commonly reported for SINV infection. Further surveillance is required to reveal the true extent of the morbidity of Sindbis fever in South Africa.

A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants.

Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies.

Generation and characterization of monoclonal antibodies against Rift Valley fever virus nucleoprotein.

Due to the unpredictable and explosive nature of Rift Valley fever (RVF) outbreaks, rapid and accurate diagnostic assays for low-resource settings are urgently needed. To improve existing diagnostic assays, monoclonal antibodies (MAbs) specific for the nucleocapsid protein of RVF virus (RVFV) were produced and characterized. Four IgG2a MAbs showed specific binding to denatured nucleocapsid protein, both from a recombinant source and from inactivated RVFV, in Western blot analysis and in an enzyme-linked immunosorbent assay (ELISA). Cross-reactivity with genetically related and non-related arboviruses including Bunyamwera and Calovo viruses (Bunyaviridae family), West Nile and Dengue-2 viruses (Flaviviridae family), and Sindbis and Chikungunya viruses (Togaviridae family) was not detected. These MAbs represent a useful tool for the development of rapid diagnostic assays for early recognition of RVF.

Stability of a formalin-inactivated Rift Valley fever vaccine: evaluation of a vaccination campaign for cattle in Mozambique.

In Africa and the Arabian Peninsula, outbreaks of Rift Valley fever (RVF) are characterized by abortions in gestating animals and high mortality rates among domestic ruminants. An immunization program using a formalin-inactivated vaccine was initiated in Mozambique in 2002 to control RVF in cattle. In this intervention, the vaccine must be transported for more than a week within the country before it can be administered to the animals, and it is practically impossible to maintain low storage temperatures during that time. Here, we evaluated the influence of transportation conditions on the efficacy of the vaccine. Sixty-three previously unvaccinated and RVF virus seronegative cattle were divided into four groups, which were given vaccine that had been stored for 1 week at 4°C (n=9, group A), at 25°C (n=8, group B), or alternating between 4 and 25°C (n=8, group C), or under the temperature conditions ordinarily occurring during transportation within Mozambique (n=38, group D). The antibody responses induced were monitored for 6-9 months and in some animals up to 21 months. Two immunizations (3 weeks apart) with the formalin-inactivated vaccine induced a long-lasting neutralizing antibody response that was still detectable up to 21 months later. The antibody titers in the animals did not differ significantly between the temperature-assigned vaccine groups A, B, and C, whereas they were significantly higher in group D. These results show that the formalin-inactivated RVF virus vaccine is stable, and, importantly, it is not adversely affected by the variation in temperature that ordinarily occurs during transport within Mozambique.

A comparison of the susceptibility of the biting midge Culicoides imicola to infection with recent and historical isolates of African horse sickness virus.

The susceptibility of Culicoides (Avaritia) imicola Kiefer (Diptera: Ceratopogonidae) to 21 isolates representing all nine known serotypes of African horse sickness virus (AHSV), recovered from clinical cases of the disease in South Africa during 1998-2004, was compared with its susceptibility to approximately 40-year-old isolates stored at the Agricultural Research Council-Onderstepoort Veterinary Institute. Field-collected C. imicola were fed through a chicken skin membrane on sheep blood spiked with one of the virus isolates to a concentration in the range of 5.6-7.5 log (10)TCID(50)/mL. After 10 days incubation at 23.5 degrees C, five of the nine historical serotypes (AHSV-1, -2, -3, -7 and -9) could not be isolated from C. imicola. All nine serotypes were recovered for the 21 recent isolates, for 16 of which the virus recovery rates were higher than for the corresponding historical isolates. These results emphasize the need to assess the oral susceptibility of local Culicoides populations to viruses in circulation during outbreaks in order to estimate their vector potential.

The oral susceptibility of South African field populations of Culicoides to African horse sickness virus.

Twenty-two isolates of African horse sickness virus (AHSV), representing its distinct serotypes, geographical and historical origins, were fed to three populations of South African livestock-associated Culicoides spp. (Diptera, Ceratopogonidae). Infective blood meals included 12 recent isolates, nine historical reference strains and one live attenuated vaccine strain serotype 7 (AHSV-7) of the virus. Field-collected midges were fed through a chicken-skin membrane on sheep blood spiked with one of the viruses, which concentrations ranged from 5.4 to 8.8 log(10)TCID(50)/mL of blood. After 10 days incubation at 23.5 degrees C, AHSV was isolated from 11 Culicoides species. Standard in vitro passaging of AHSV-7, used for the preparation of live attenuated vaccine, did not reduce its ability to infect Culicoides species. Virus recovery rates in orally infected Culicoides midges differed significantly between species and populations, serotypes, isolates and seasons. Significant variations in oral susceptibility recorded in this study emphasize a complex inter-relationship between virus and vector, which is further influenced by multiple intrinsic and extrinsic factors. As it is not possible to standardize all these factors under laboratory conditions, conclusive assessment of the role of field-collected Culicoides midges in the transmission of orbiviruses remains problematic. Nevertheless, results of this study suggest the potential for multi-vector transmission of AHSV virus in South Africa.

Laboratory safe detection of nucleocapsid protein of Rift Valley fever virus in human and animal specimens by a sandwich ELISA.

A safe laboratory procedure, based on a sandwich ELISA (sAg-ELISA), was developed and evaluated for the detection of nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) in specimens inactivated at 56 degrees C for 1h in the presence of 0.5% Tween-20 (v/v) before testing. Polyclonal capture and detection immune sera were generated respectively in sheep and rabbits immunized with recombinant NP antigen. The assay was highly repeatable and specific; it detected strains of RVFV from the entire distributional range of the disease, isolated over a period of 53 years; no cross-reactivity with genetically related African phleboviruses or other members of the family Bunyaviridae was observed. In specimens spiked with RVFV, including human and animal sera, homogenates of liver and spleen tissues of domestic ruminants, and Anopheles mosquito homogenates, the sAg-ELISA detection limit ranged from log(10)10(2.2) to 10(3.2) TCID(50)/reaction volume. The ELISA detected NP antigen in spiked bovine and sheep liver homogenates up to at least 8 days of incubation at 37 degrees C whereas infectious virus could not be detected at 48h incubation in these adverse conditions. Compared to virus isolation from sera from RVF patients and sheep infected experimentally, the ELISA had 67.7% and 70% sensitivity, and 97.97% and 100% specificity, respectively. The assay was 100% accurate when testing tissues of various organs from mice infected experimentally and buffalo foetuses infected naturally. The assay was able to detect NP antigen in infective culture supernatants 16-24h before cytopathic effects were observed microscopically and as early as 8h after inoculation with 10(5.8) TCID(50)/ml of RVFV. This ability renders the assay for rapid identification of the virus when its primary isolation is attempted in vitro. As a highly specific, safe and simple assay format, the sAg-ELISA represents a valuable diagnostic tool for use in less equipped laboratories in Africa, and for routine differential diagnosis of viral hemorrhagic fevers.

An alphavirus replicon-derived candidate vaccine against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus (genus Phlebovirus) associated with severe disease in livestock and fatal encephalitis or haemorrhagic fever in a proportion of infected humans. Although live attenuated and inactivated vaccines have been used in livestock, and on a limited scale in humans, there is a need for improved anti-RVFV vaccines. Towards this goal, Sindbis virus replicon vectors expressing the RVFV Gn and Gc glycoproteins, as well as the non-structural nsM protein, were constructed and evaluated for their ability to induce protective immune responses against RVFV. These replicon vectors were shown to produce the RVFV glycoproteins to high levels in vitro and to induce systemic anti-RVFV antibody responses in immunized mice, as determined by RVFV-specific ELISA, fluorescent antibody tests, and demonstration of a neutralizing antibody response. Replicon vaccination also provided 100% protection against lethal RVFV challenge by either the intraperitoneal or intranasal route. Furthermore, preliminary results indicate that the replicon vectors elicit RVFV-specific neutralizing antibody responses in vaccinated sheep. These results suggest that alphavirus-based replicon vectors can induce protective immunity against RVFV, and that this approach merits further investigation into its potential utility as a RVFV vaccine.

Genetic relationship in southern African Crimean-Congo haemorrhagic fever virus isolates: evidence for occurrence of reassortment.

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. Reassortment of CCHF genome segments has been shown to occur in nature. We therefore investigated the genetic relationship of southern African isolates using partial sequence data for each RNA segment, S, M and L, and comparing the tree topologies constructed using a neighbour joining method. A total of 21 southern African isolates were studied. The incongruencies which were identified in S, M and L sequence datasets involved group switching implying reassortment for 15 isolates. A higher fatality rate occurred in patients infected with isolates which had apparently acquired M segments from a group in which predominantly Asian strains are usually found. This suggests that reassortment may affect the pathogenicity of the virus.

Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid detection of Rift Valley fever virus in clinical specimens.

This paper reports on the development and validation of a real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) targeting the genomic large RNA segment of Rift Valley fever virus (RVFV). The set of six designed RT-LAMP primers identified strains of RVFV isolated in geographically distinct areas over a period of 50 years; there was no cross-reactivity with other genetically related and unrelated arboviruses. When testing serial sera and plasma from sheep experimentally infected with wild-type RVFV, there was 100% agreement between results of the RT-LAMP, a TaqMan-based real-time PCR, and virus isolation. Similarly, the assay had very high levels of diagnostic sensitivity and specificity when testing various clinical specimens from humans and animals naturally infected with the virus during recent outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 min. As a highly accurate, rapid, and very simple nucleic acid detection format, the RT-LAMP has the potential to be used in less-well-equipped laboratories in Africa and as a portable device during RVF outbreaks in remote areas, and it can be a valuable tool for the differential diagnosis of viral hemorrhagic fevers.

Epidemiology and pathogenicity of African bat lyssaviruses.

Lyssaviruses belonging to all four known African Lyssavirus genotypes (gts) have been reported and isolated from SouthAfrica over the past few decades. These are: (1) Duvenhage virus (gt4), isolated again in 2006 from a human fatality; (2) Mokola virus (gt3), isolated irregularly, mostly from cats; (3) Lagos bat virus (gt2) continually isolated over the past four years from Epomophorus fruit bats and from incidental terrestrial animals and (4) Rabies virus (gt1) - with two virus biotypes endemic in mongoose and in canid species (mostly domestic dogs, jackals and bat-eared foxes), respectively. Only two of these are associated with bats in Southern Africa, viz. Duvenhage virus and Lagos bat virus (gts 4 and 2). For both these genotypes the authors have embarked on a programme of comparative study of molecular epidemiology. Duvenhage virus nucleoprotein nucleotide sequence analysis indicated a very low nucleotide diversity even though isolates were isolated decades apart. In contrast, individual isolates of Lagos bat virus were found to differ significantly with respectto nucleoprotein gene nucleotide sequence diversity as well as in pathogenicity profiles.

Prevalence of antibodies against Rift Valley fever virus in Kenyan wildlife.

Rift Valley fever virus (RVFV) is an arbovirus associated with periodic outbreaks, mostly on the African continent, of febrile disease accompanied by abortion in livestock, and a severe, fatal haemorrhagic syndrome in humans. However, the maintenance of the virus during the inter-epidemic period (IEP) when there is low or no disease activity detected in livestock or humans has not been determined. This study report prevalence of RVFV-neutralizing antibodies in sera (n=896) collected from 16 Kenyan wildlife species including at least 35% that were born during the 1999-2006 IEP. Specimens from seven species had detectable neutralizing antibodies against RVFV, including African buffalo, black rhino, lesser kudu, impala, African elephant, kongoni, and waterbuck. High RVFV antibody prevalence (>15%) was observed in black rhinos and ruminants (kudu, impala, buffalo, and waterbuck) with the highest titres (up to 1:1280) observed mostly in buffalo, including animals born during the IEP. All lions, giraffes, plains zebras, and warthogs tested were either negative or less than two animals in each species had low (or= 1:80. These data provide evidence that wild ruminants are infected by RVFV but further studies are required to determine whether these animals play a role in the virus maintenance between outbreaks and virus amplification prior to a noticeable outbreak.

Use of a molecular epidemiological database to track human rabies case histories in South Africa.

The KwaZulu Natal and Eastern Cape provinces of South Africa have experienced a serious dog rabies epidemic over the past three decades. Towards a better understanding of this epidemic, we have previously analysed nucleotide sequences of 142 rabies virus specimens that were obtained from these regions during 2003-2004 and provided a molecular description of the geographical distribution of rabies viral variants in the affected provinces. Here, as an extension, we studied five human cases that occurred during 2002-2003 and demonstrated the use of the sequence database in tracking unknown human rabies case histories. We were able to identify the geographical origin of viruses responsible for each human infection and in one case obtained evidence that suggested a non-bite transmission of rabies virus from an infected dog to a child. We argue for the value of this information in surveillance and epidemiological study and in the follow-up and management of potential exposures.

Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species.

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.