Argentinean agid test for diagnosis of equine infectious anemia: six years of history

Equine infectious anemia (EIA) is a disease of high economic impact on the equine industry worldwide. Since horses are frequent travelers, EIA falls under strict regulatory control programs in many countries. In Argentina the national animal health authority (SENASA) states that all horses imported, moving within the country, or congregating at public assemblies must have a negative EIA report conducted within the previous 2 months. The agent causing EIA is a RNA virus from the Retroviridae family and its major capsid protein named p26 is the most immunogenic protein in the viral particle. Thus, the detection of specific antibodies directed to p26 is the aim of most diagnosis tests available in the world. The agar gel immunodifusion (AGID) is the officially accepted method to certify the diagnosis of EIA in Argentina. Since 2009 IncuINTA was working on the scaling up and production of the KIT AIE IDGA RP26, an Argentinean AGID test entirely developed in the laboratory containing a recombinant p26 protein to detect EIA antibodies in horses’ serum. Until 2015 IncuINTA produced two pilot batches and six commercial batches (one per year) containing from 24000 determinations in 2011 to 39600 determinations in 2015. Since the product was launched in 2011, the sales were increased 109%. Up to date we have placed on the market 170640 determinations. As expected, the number of laboratories buying the KIT AIE IDGA RP26 was also increasing through time being 26 in 2011 and 36 in 2015. This number of clients represents 17% of the 207 laboratories authorized by SENASA to diagnose EIA in Argentina. These laboratories are located mostly in Buenos Aires, Santa Fe, Entre Ríos, Formosa, La Pampa, Rio Negro, Cordoba, Corrientes, Salta and Tucum an provinces. Until 2009 there was no Argentinean EIA test available in our market being the imported ones very expensive. IncuINTA, which is a R&D laboratory, could scale up, produce and sell the KIT AIE IDGA RP26 during six consecutive years. After this success, IncuINTA perspective is to increase the number of batches each year to be able to attend the demand of most diagnosis laboratories in the country.Fil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Asenzo, G.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Vena, M. M.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parreño, V.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Wigdorovitz, A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina10th International Equine Infectious Diseases ConferenceBuenos AiresArgentinaUniversity of Kentuck

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells PALABRAS CLAVE

Abstract Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 g of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. PALABRAS CLAVE Glucoproteína E2; Anticuerpo de cadena simple; Vacuna a subunidad; Baculovirus; Desarrollo de una vacuna de subunidad BVDV mejorada basada en la glucoproteína E2 fusionada a un anticuerpo de cadena simple que se dirige a células presentadoras de antígeno Resumen El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versió

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

Foot-and-mouth disease:overview of motives of disease spread and efficacy of available vaccines

Control and prevention of foot and mouth disease (FMD) by vaccination remains unsatisfactory in endemic countries. Indeed, consistent and new FMD epidemics in previously disease-free countries have precipitated the need for a worldwide control strategy. Outbreaks in vaccinated animals require that a new and safe vaccine be developed against foot and mouth virus (FMDV). FMDV can be eradicated worldwide based on previous scientific information about its spread using existing and modern control strategies

Detection of BLV in frozen semen samples by PCR assay

Detection of BLV in frozen semen samples by PCR assay Dus Santos Maria Jose; Rodriguez Sabrina; Wigdorovitz Andrés; Trono Karina Instituto de Virología, CICVyA, INTA-Castelar. CC 77. Buenos Aires, Argentina. Detection of BLV in frozen semen samples by PCR assay María José Dus Santos, Sabrina Rodriguez, Andrés Wigdorovitz and Karina Trono. Instituto de Virología, CICVyA, INTA-Castelar. CC 77. Buenos Aires, Argentina [email protected] The sanitary and economic impact of BLV infection is associated with the interference in the international movement of cattle and their germ plasm. Although experimental data support the improbability that semen from BLV-positive bulls could infect recipient cows, restriction for commercialization of semen from infected animals is still present. The objective of this work was to standardize a PCR assay to diagnose the presence of BLV genome and to describe the pattern of BLV detection in frozen semen samples. The developed methodology involves the amplification of an internal fragment of gag gene and posses a limit of detection of 60 viral particles. Additionally, a biological test in susceptible sheep was performed in order to evaluate the transmission of BLV genome by semen from seropositive animals. This data strongly suggest that semen from seropositive bulls that resulted negative by PCR can be used for artificial insemination (AI), accompanied by proper collection protocols. Frozen semen samples from 30 seropositive bulls were analyzed. It was possible to detect proviral DNA in 118 out of 545 samples. It was important to note that BLV genome detection occurred in several collections but in an alternated way with non detection periods. On the other hand, in 4 seropositive bulls, it was not possible to detect BLV genome in all the samples analyzed. The development of this PCR assay constitutes a valuable diagnostic tool to determine the BLV-free status of semen used for AI. Moreover, the results suggest that BLV could present an intermittent pattern of excretion

Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus

Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV

Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1

We previously demonstrated that the immunization of cattle with a synthetic peptide representing the amino acid sequence of foot and mouth disease virus (FMDV) type O1 Campos VP1 residues 135-160 (p135-160), containing immunodominant T and B epitopes, was able to induce a strong neutralizing antibody (NA) response. The epitope mapping of p135-160 identified T and B epitopes in the area restricted to amino acid residues 135-144 (Zamorano et al. 1994, Virology 201; 1995, Virology 212). We are now reporting that, although immunization with a synthetic peptide covering amino acids 135-144 (p135-144) failed to elicit an anti-FMDV response, a synthetic peptide representing a tandem duplication of the VP1 epitope 135-144 (p135-144 x 2) was very efficient in inducing a strong NA response in cattle. Both the antibody and T cell responses elicited by p135-144 x 2 were highly specific for the VP1 135-144 sequence since no reactivity was detected against synthetic peptides representing the 140-160 sequence of VP1. Additionally, both responses to B and T epitopes were long lasting in the immunized cattle. These results constitute a good example of the improvement of the immune response by rational handling of precisely identified B and T epitopes. To our knowledge, this is the shortest native amino acid sequence to induce a significant NA response to FMDV in cattle