Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

High Doses of Inactivated African Swine Fever Virus Are Safe, but Do Not Confer Protection against a Virulent Challenge.

African swine fever (ASF) is currently the major concern of the global swine industry, as a consequence of which a reconsideration of the containment and prevention measures taken to date is urgently required. A great interest in developing an effective and safe vaccine against ASF virus (ASFV) infection has, therefore, recently appeared. The objective of the present study is to test an inactivated ASFV preparation under a vaccination strategy that has not previously been tested in order to improve its protective effect. The following have been considered: (i) virus inactivation by using a low binary ethyleneimine (BEI) concentration at a low temperature, (ii) the use of new and strong adjuvants; (iii) the use of very high doses (6 × 109 haemadsorption in 50% of infected cultures (HAD50)), and (iv) simultaneous double inoculation by two different routes of administration: intradermal and intramuscular. Five groups of pigs were, therefore, inoculated with BEI- Pol16/DP/OUT21 in different adjuvant formulations, twice with a 4-week interval. Six weeks later, all groups were intramuscularly challenged with 10 HAD50 of the virulent Pol16/DP/OUT21 ASFV isolate. All the animals had clinical signs and pathological findings consistent with ASF. This lack of effectiveness supports the claim that an inactivated virus strategy may not be a viable vaccine option with which to fight ASF.

Development of a multiplex assay for antibody detection in serum against pathogens affecting ruminants.

Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually.

Genetic variation among African swine fever genotype II viruses, eastern and central Europe.

African swine fever virus (ASFV) was first reported in eastern Europe/Eurasia in 2007. Continued spread of ASFV has placed central European countries at risk, and in 2014, ASFV was detected in Lithuania and Poland. Sequencing showed the isolates are identical to a 2013 ASFV from Belarus but differ from ASFV isolated in Georgia in 2007.

Global gene expression analysis in skin biopsies of European red deer experimentally infected with bluetongue virus serotypes 1 and 8.

Bluetongue virus (BTV) is a double-stranded RNA virus transmitted by blood-feeding biting midges of the genus Culicoides to wild and domestic ruminants, causing high morbidity and variable mortality. The aim of this study was to characterize differential gene expression in skin biopsies of red deer (Cervus elaphus) hinds experimentally infected with BTV serotypes 1 and 8. Skin biopsies were collected from BTV-1 and BTV-8 experimentally infected and control hinds at 14 and 98 days post-infection (dpi). Global gene expression profile in response to BTV infection was characterized at 14 dpi using a bovine microarray together with real-time RT-PCR analysis of differentially expressed genes at 14 and 98 dpi. Eighteen genes were upregulated and three were downregulated in response to virus infection, with no significant differences between BTV-1 and BTV-8 infected hinds. Seven unique genes, six upregulated (ISG15, PSMB8, PSMB9, BOLA, C1qA, C4) and one downregulated (FOS) were over-represented after conditional test for biological process gene ontology, which affected five molecular pathways (RIG-1, proteasome, MHC-1, complement, TLR) implicated in host immune response. BTV infection had a minor and transient effect on gene expression in hinds, as shown by the very few genes that were differentially expressed in response to infection at 14 dpi, most of which had similar expression levels between infected and uninfected animals at 98 dpi. These results suggested that red deer could control BTV infection with little effect on host molecular pathways.

Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences.

Porcine teschoviruses (PTVs) have been previously shown to be the most abundant cytopathic viruses found in swine feces. In the present study, the diversity of PTVs was studied, using PTV isolates collected between 2004 and 2009 in a wide territory in Spain. In order to characterize genetically the isolates, phylogeny reconstructions were made using maximum likelihood and Bayesian inference methods, based on the 1D (VP1) gene, and including sequences available in public databases. The phylogenetic trees obtained indicated that PTVs present 12 main lineages, 11 corresponding to the PTV serotypes described to date, and one lineage distinct from the rest. The geographic distribution of the different lineages does not seem to be strongly associated to particular territories, and co-circulation of multiple lineages was found in the same geographic areas. Nevertheless, some spatial structuring of the viral populations studied is indicated by the differences found between Spanish samples with respect to other European countries. A coalescent-based approach indicated that mutation may have been the main factor in originating the genetic diversity observed in the VP1 gene region. This study revealed a high diversity of teschoviruses circulating in the pig populations studied, and showed that molecular analysis of the complete VP1 protein is a suitable method for the identification of members of the porcine teschovirus group. However, further analyses are needed to clarify the geographical structuring of the different PTV populations.

Evaluation of a fluorogenic real-time reverse transcription-polymerase chain reaction method for the specific detection of all known serotypes of porcine teschoviruses.

Performance of a real-time reverse-transcription polymerase chain reaction method for the rapid, simple and reliable detection of porcine teschovirus (PTV) was assessed. The method was based on the use of a set of oligonucleotides consisting of two specific primers and a fluorogenic TaqMan-MGB probe. Reverse transcription and PCR reactions were performed sequentially in one step. As a result the whole procedure was simple and rapid, taking less than 3h for completion. The method reacted in a dose-dependent manner with prototype strains for the eleven known PTV serotypes (PTV1-11), with higher analytical sensitivity than other gel-based RT-PCR methods described, which were performed in parallel to allow for a comparison. The assay did not cross-react with other related viruses or porcine viruses tested. The diagnostic performance of the method was analyzed using a panel of field samples consisting of pig fecal and pig slurry samples. As a conclusion, this technique is adequate and convenient for porcine teschovirus detection, both for diagnosis as well as in environmental investigations.

DNA immunization of pigs with foot-and-mouth disease virus minigenes: from partial protection to disease exacerbation.

Despite several attempts to design new vaccines, there are as of yet no available alternatives to the conventional FMDV vaccines. Here, we present the divergent results obtained in pigs after immunization with two experimental DNA vaccines encoding one B and two T cell FMDV epitopes, either expressed alone (pCMV-BTT) or fused to a strong signal peptide (pCMV-spBTT). While all pigs vaccinated with pCMV-spBTT showed both a delay in the disease onset and reduced severity of signs and lesions after FMDV challenge, pigs immunized with pCMV-BTT showed an exacerbation of the disease and most of the pigs remained viremic at 10 days post-infection, the end-point of the experiment, thus opening concerns about FMDV-suboptimal immunization. Interestingly, only one of the four pigs vaccinated with pCMV-spBTT showed neutralizing antibodies before challenge, demonstrating that partial protection against FMDV could be afforded in the absence of preexisting neutralizing antibodies.

A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.

In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.

Experimental infection of European red deer (Cervus elaphus) with bluetongue virus serotypes 1 and 8.

Bluetongue (BT) is a climate change-related emerging infectious disease in Europe. Outbreaks of serotypes 1, 2, 4, 6, 8, 9, 11, and 16 are challenging Central and Western Europe since 1998. Measures to control or eradicate bluetongue virus (BTV) from Europe have been implemented, including movement restrictions and vaccination of domestic BTV-susceptible ruminants. However, these measures are difficult to apply in wild free-ranging hosts of the virus, like red deer (Cervus elaphus), which could play a role in the still unclear epidemiology of BT in Europe. We show for the first time that BTV RNA can be detected in European red deer blood for long periods, comparable to those of domestic ruminants, after experimental infection with BTV-1 and BTV-8. BTV RNA was detected in experimentally infected red deer blood up to the end of the study (98-112 dpi). BTV-specific antibodies were found in serum both by enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VNT) from 8 to 12 dpi to the end of the study, peaking at 17-28 dpi. Our results indicate that red deer can be infected with BTV and maintain BTV RNA for long periods, remaining essentially asymptomatic. Thus, unvaccinated red deer populations have the potential to be a BT reservoir in Europe, and could threaten the success of the European BTV control strategy. Therefore, wild and farmed red deer should be taken into account for BTV surveillance, and movement restrictions and vaccination schemes applied to domestic animals should be adapted to include farmed or translocated red deer.

DNA vaccines expressing B and T cell epitopes can protect mice from FMDV infection in the absence of specific humoral responses.

Despite foot-and-mouth disease virus (FMDV) being responsible for one of the most devastating animal diseases, little is known about the cellular immune mechanisms involved in protection against this virus. In this work we have studied the potential of DNA vaccines based on viral minigenes corresponding to three major B and T-cell FMDV epitopes (isolate C-S8c1) originally identified in natural hosts. The BTT epitopes [VP1 (133-156)-3A (11-40)-VP4 (20-34)] were cloned into the plasmid pCMV, either alone or fused to ubiquitin, the lysosomal targeting signal from LIMPII, a soluble version of CTLA4 or a signal peptide from the human prion protein, to analyze the effect of processing through different antigenic presentation pathways on the immunogenicity of the FMDV epitopes. As a first step in the analysis of modulation exerted by these target signals, a FMDV infection inhibition assay in Swiss outbred mice was developed and used to analyze the protection conferred by the different BTT-expressing plasmids. Only one of the 37 mice immunized with minigene-bearing plasmids developed specific neutralizing antibodies prior to FMDV challenge. As expected, this single mouse that had been immunized with the BTT tandem epitopes fused to a signal peptide (pCMV-spBTT) was protected against FMDV infection. Interestingly, nine more of the animals immunized with BTT-expressing plasmids did not show viremia at 48 h post-infection (pi), even in the absence of anti-FMDV antibodies prior to challenge. The highest protection (50%, six out of 12 mice) was observed with the plasmid expressing BTT alone, indicating that the targeting strategies used did not result in an improvement of the protection conferred by BTT epitopes. Interestingly, peptide specific CD4+ T-cells were detected for some of the BTT-protected mice. Thus, a DNA vaccine based on single FMDV B and T cell epitopes can protect mice, in the absence of specific antibodies at the time of challenge. Further work must be done to elucidate the mechanisms involved in protection and to determine the protective potential of these vaccines in natural FMDV hosts.