EPIZONE és la Xarxa d’Excel·lència per al Diagnòstic i Control de les Malalties Epizoòtiques. Investigadors del CReSA van participar en la seva tercera reunió anual amb cinc comunicacions, mostrant experiments recents en virus de la pesta porcina africana, febre aftosa, llengua blava i febre de la Vall del Rift.

EPIZONE (finançat pel Sisè Programa Marc de la UE) té com a objectiu desenvolupar una xarxa de científics per a millorar la investigació en la preparació, prevenció, detecció i control de malalties epizoòtiques a Europa.

Les contribucions científiques del CReSA es descriuen a continuació:

LA IMMUNITZACIÓ AMB UN BACULOVIRUS RECOMBINANT QUE CODIFICA UNA FUSIÓ DE TRES ANTÍGENS DE VIRUS DE LA PESTA PORCINA AFRICANA (VPPA) SOTA EL CONTROL DEL PROMOTOR CMV, ESPECIFIC DE MAMÍFER, PROTEGEIX ALS PORCS ENFRONT A UN DESAFIAMENT  SUBLETAL AMB VPPA

GM Keil JM Argilaguet, E Pérez-Martín, JM Escribano, F Rodríguez.

Comunicació oral presentada pel Dr. Fernando Rodríguez.

Resum
Previous studies in our lab have shown that fusion of the soluble fraction of the ASFV haemmaglutinin (sHA) to two immunodominat ASFV antigens (PQ) enhanced both the humoral and cellular responses induced in pigs upon DNA vaccination. Unfortunately, no protection against alethal challenge with ASFV was afforded. In order to enhance the immune responses induced against these antigens, we decided to use a novel strategy for immunization which is based on the in vivo inoculation of recombinant baculovirus carrying mammalian cell-active expression cassettes (BacMam technology). Recombinant baculoviruses encoding sHAPQ under the control of the mammalian CMV promoter (same mammalian promotor used in the DNA vaccination protocols) were isolated. The resulting BacMam BacCMV-sHAPQ, very efficiently expressed in sHAPQ in mammalian cells upon in vitro transduction. Initialimmunization experiments demonstrated that two of three pigs vaccinated with BacMAM-sHAPQ were fully protected from viremia after in vivo challlenge  with a sub-lethal dose of ASFV. While protection against viremia perfectly matched with the presence of a large number of specific IFNsecretory T-cells in the blood of the animals at day 10 after challenge, the presence of high T-cell precursors before challenge seemed to correlate with total protection from disease symptoms such as fever. Further experiments will be performed in the future aiming to study the potential of these vaccines to confer protection against a much more aggressive challenge with a lethal dose of ASFV.

INFECCIÓ EXPERIMENTAL D'OVELLES EUROPEES AMB EL VIRUS DE LA FEBRE DE LA VALL DEL RIFT

FX Abad, N Busquets, F Rodriguez, D Solanes, M Domingo, A Brun.

Comunicació oral presentada pel Dr. Mariano Domingo.

Resum
Rift Valley Fever Virus (RVF) is a zoonotic disease showing episodic outbreaks in Sub Saharan countries, Egypt and the Arabian Peninsula eventually associated to climatic conditions. The disease is caused by an arbovirus competent in many mosquito species present in most European countries. Many European regions have optimal habitats in which the virus could persist in the event of an unwanted introduction of the virus within European boundaries.

Increasing our understanding of the disease is therefore highly desirable to increase our preparedness to fight this important disease. In this study, researchers of the CReSA tried to establish a reproducible infection model of RVF in a European sheep breed. Groups of 4 sheep of 6-8 weeks old were inoculated subcutaneously with 105 TCID50 of each isolate. Viremia titers were estimated by real time PCR and presence of viable virus confirmed by virus isolation. Different tissues were analysed for specific pathological changes by histopathology and/or immunhistochemistry. Leukocyte populations were also studied looking for immune correlates of infection. Shedding of RVFV and horizontal transmission to non-infected, in contact lambs was also investigated.

LA PROTECCIÓ INDUÏDA PER UNA VACUNA D’ADN QUE CODIFICA MINIGENS DEL VIRUS DE LA FEBRE AFTOSA (VFA) ES CORRELACIONA AMB LA INDUCCIÓ D’UNA RESPOSTA CEL·LULAR CONTRA UN EPÍTOP CONSERVAT

JM Argilaguet, E Pérez-Martín, M Pérez-Filgueira, JM Escribano, F Sobrino, B Borrego, F Rodríguez.

Pòster.

Resum
Vaccination against FMDV is still controversial, due to the problems associated to classical vaccines based on chemically inactivated virus. Because of its many advantages, DNA vaccination appears as one of the most promising choices, although for large animals the capability of inducing strong immune responses must be improved. In this work we present preliminary experiments showing the protection afforded by a DNA vaccine strategy; previously reported to enhance the immunogenicity of the vaccine encoded-antigens, focus on targeting FMDV epitopes to antigen presenting cells (APCs).

We analysed the response induced in pigs immunized with pCMV-APCH I-BTT, a DNA plasmid encoding previously characterised FMDV B- and T-cell epitopes fused to APCH I, a single chain variable fragment (scFv) of an antibody that recognizes the Class II swine leukocyte antigen (SLA II). Immunization of pigs with this construct, even after one single shot, resulted in full protection for 50% of the animals, in which no signs of disease or viral replication were observed, and in partial protection for the rest of the pigs receiving the same plasmid. Protection seemed to correlate with the strong induction of specific T-cells against a conserved T-cell epitope prior to FMDV-challenge that secrete IFN in response to specific stimulation, as well as the rising of a fast neutralizing activity after viral challenge.

The fact that this protection was achieved in the absence of anti-FMDV antibodies before viral challenge, drive the attention to the crucial role played by cellular responses in protection against FMD. On the other hand, the possibility of inducing protective cellular responses against highly conserved T-cell epitopes open the possibility of generating “universal” vaccines against a highly variable virus such as FMDV, one of the main problems currently struggling the development of effective FMDV vaccines.

GENERACIÓ DE VACUNES D’ADN CO-EXPRESSANT EPÍTOPS DE VIRUS DE LA FEBRE AFTOSA I PROTEÏNES ANTIAPOPTÒTIQUES

SG Iz, SID Gurhan, M Doskaya, B Borrego, F Rodriguez.

Pòster.

Resum
Foot and Mouth Disease (FMD) is one of the most contagious animal diseases, acusing important economic losses. Foot and Mouth Disease virus (FMDV) is a member of the aphthovirusgenus of Picornaviridae family and the causative agent of FMD. Natural hosts of FMDV are cattle, sheep, goats, swine, buffalo and all other ruminants. FMDV is epidemic in most parts of the world and unexpected outbreaks occurs frequently. The highly contagious nature of the disease makes necessary to combat it with several strategies.

Conventional inactivated whole virus FMD vaccine that has been used confers high protection rates but that protection lasts short time. Conventional vaccines has some additional problems: on one hand it doesn’t allow serological distinction between infected and vaccinated animals and on the other hand there are serious risks of virus release and/or insufficient inactivation of the virus from the FMD Reference Laboratory and/or vaccine production plants as recently reported for one of the most recent FMD outbreaks in United Kingdom.  Therefore, development of new generation of efficient and safer vaccines is a necessity to overcome all the problems above described.

The aim of the study is to construct DNA vaccine plasmids co expressing FMDV antigens and anti-apoptotic proteins. FMDV antigens used in this study are major B and T-cell FMDV epitopes of FMDV C and FMDV O1K isolate. Borrego et al., have shown that DNA vaccines based on B and T epitopes of FMDV C isolate can protect mice even in the absence of specific antibodies at the time of challenge. Anti-apoptotic proteins used in this study were Bcl-xL of the host animal and the 3CD gene region of FMDV. While the addition of either Bcl-xL or the FMDV-3CD gene should increase the half-life of the antigen presenting cells co-expressing the B and T FMDV epitopes thus enhancing the specific immune responses induced, the induction of a specific response against 3CD might also contribute to protect the host against the FMDV chalenge. Preliminary studies about the vaccine generation and their expression upon transient in vitro transfection will be presented.

ÚS DE fHA DE BORDETELLA BRONCHISEPTICA COM A ADJUVANT PER A MILLORAR LA VACUNACIÓ AMB ADN EN PETITS I GRANS ANIMALS

S Okay, V Aragon, R Rosell, J Pujols, G Özcengiz, F Rodriguez.

Pòster

Resum

DNA vaccination is one of the most promising strategies to develop recombinant vaccines either alone, or in protocols of prime-boost. However, one of the main criticisms comes from its low immunogenicity in large animal species and/or against some specific antigens. 

In this work, we explores a new strategy based on fusing antigens to the Bordetella bronchiseptica fitohemaglutinin (fHA), an adhesion molecule capable to bind to Antigen Presenting Cells (APCs), including from mucosa, from many different animal origins. Upon DNA vaccination in vivo, fused antigens should be carried out to the sites where fHA have their antigen receptors: mainly in macrophages, dendritic cells and epithelial cells.

Once demonstrated the capability of fHA-GFP fusion proteins to bind to porcine epithelial cells in vitro, we decided to compare the immune responses induce by a DNA vaccine (pCMV-PQ) encoding two African swine fever virus (ASFV) genes expressed in tandem (PQ), with two DNA constructs encoding same genes as fusions with two different domains from the fHA: pCMV-fHAsPQ and pCMV-fHAlPQ. Preliminary results obtained using non-syngeneic mice (NIH-swiss mice) clearly demonstrated the adjuvant efficacy of fHA. The fusion of either one of the fHA domains to PQ enhanced both the antibody and the cellular responses induced in mice. We are currently extending these studies, on one hand, to pigs, natural host of ASFV and on the other hand to one of the most threatening virus affecting ruminants: Bluetongue virus (BTV).

The selection of these two viruses obey to several reasons: i) both are diseases of obligatory declaration and between the most threatening viruses for animal health, as listed by the OIE and ii) finding and adjuvant capable to work not only in mice but also in two animal species such porcine and ovine and against diseases and provoked by viruses as different as ASFV (double stranded DNA virus) and BTV (negative stranded RNA virus) would mean the definitive consolidation of DNA vaccines as an alternative strategy to control animal diseases.

ESTUDI EPIDEMIOLÒGIC DE LA LLENGUA BLAVA EN EL SUD D’ESPANYA DURANT L’ANY 2007

I García, A Allepuz, MA González, J Casal, S Napp, A Carbonero, C Borge, A Arenas

Póster.

After the epidemic of BTV-4 in 2004, which affected Andalusia (Southern Spain), a new serotype (BTV-1) appeared in this region in 2007. During the following months, the virus spread quickly and 7,916 outbreaks were detected in Spain in 2007. Still, most of the outbreaks (56%) were detected in Andalusia.

In this communication we present the results of the epidemiological and spatial study of the outbreaks that affected Andalusia during 2007.

Data on BT outbreaks in domestic animals in Andalusia were obtained from the Spanish Ministry of Agriculture, Fisheries and Food. Information relative to epidemiological data (general characteristics of the farms, clinical sings, mortality and morbidity), were collected from the affected farms through personal interview with farmers.

A total of 4,436 outbreaks of BTV-1 were confirmed during that year: 3,162 in sheep flocks, 113 in goat flocks, 7 in cattle herds and 1,154 in mixed farms (sheep, goat and/or cattle in the same farm). The temporal distribution of the outbreaks presented a clear peak of infection between October and November.

The prevalence of positive farms to the RT-PCR analysis was 17.7%, and the within farm prevalence was 16.8%. The mortality rate in the affected farms was 8.6%. The most common clinical signs were: fever, depression, lethargy, facial edema, and salivation (observed in more than 70% of the infected farms). Lesions in oral mucosa, lameness and dyspnea were also observed.

The infected farms were located mainly in the western Andalusia, which coincided with the areas with both highest ruminant census and highest captures of C.imicola. Spatial models are being built in order to analyze the spatial distribution of the disease and more detailed results will be presented during the meeting.