Deletion Mutants of the Attenuated Recombinant ASF Virus, BA71ΔCD2, Show Decreased Vaccine Efficacy.

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71∆CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding ß-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71∆CD2 as a template: the single mutant BA71∆CD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71∆CD2f, BA71∆CD2DP96R and BA71∆CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71∆CD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.

Transposon-associated polymorphisms of stress-responsive gene promoters in selected accessions of Arabidopsis thaliana.

Genetic diversity caused by transposable element movement can play an important role in plant adaptation to local environments. Regarding genes, transposon-induced alleles were mostly related to gene bodies and a few of them to promoter regions. In this study, promoter regions of 9 stress-related genes were searched for transposable element insertions in 12 natural accessions of Arabidopsis thaliana. The promoter screening was performed via PCR amplification with primers designed to flank transposable element insertions in the promoter regions of the reference accession Col-0. Transposable element-associated insertion/deletion (indel) polymorphisms were identified in 7 of the 12 promoter loci across studied accessions that can be developed further as molecular markers. The transposable element absence in the promoter regions of orthologous genes in A. lyrata indicated that the insertion of these transposable elements in A. thaliana lineage had occurred after its divergence from A. lyrata. Sequence analysis of the promoter regions of CML41 (Calmodulin-like protein 41) and CHAP (chaperone protein dnaJ-related) confirmed the indel polymorphic sites in four accessions - Col-0, Wassilewskija, Shahdara, and Pirin. The observed indel polymorphism of the CHAP promoter region was associated with specific gene expression profiles in the different accessions grown at a normal and elevated temperature in a plant growth chamber. The collected data can be a starting point for gene expression profiling studies under conditions resembling the natural habitats of accessions.

Molecular cloning and characterization of cDNAs of the superoxide dismutase gene family in the resurrection plant Haberlea rhodopensis.

Resurrection plants can tolerate almost complete water loss in their vegetative parts. The superoxide dismutases (SODs) are essential enzymes of defense against the oxidative damage caused by water stress. Here, we cloned and characterized cDNAs of the SOD gene family in the resurrection plant Haberlea rhodopensis. Seven full-length cDNAs, and their partial genomic clones, were obtained by combination of degenerate PCR, RT-PCR and RACE. The derived amino acid sequences exhibited a very high degree of similarity to cytosolic Cu,Zn-SODs (HrCSD2, HrCSD3), chloroplastic Cu,Zn-SODs (HrCSD5), other Cu,Zn-SODs (HrCSD4), Mn-SODs (HrMSD) and Fe-SODs (HrFSD). One cDNA turned out to be a pseudogene (HrCSD1). All identified SOD genes were found expressed at transcriptional level--the HrCSD2, HrCSD5, HrMSD and HrFSD were constitutively expressed in all organs, while the HrCSD3 and HrCSD4 were organ-specific. The transcripts of the housekeeping SOD genes were detected at significant levels even in air-dry leaves. The multigene SOD family of H. rhodopensis is the first studied SOD family amongst resurrection plant species. Our finding of well expressed SOD transcripts in fully dehydrated leaves correlates with retention of SOD activity, and with the ability of H. rhodopensis to revive upon rehydration. Because of the endemic relict nature of that species, our findings may help to further elucidate the evolutionary relationships among different SOD isoforms from distinct plant species.