Search

Background and Aim: Adeno-associated virus serotype 2 (AAV2) represents a promising basis for developing a virus-vector vaccine against African swine fever (ASF). This study aimed to create genetic constructs based on AAV2 to deliver the immunodominant genes of ASF virus (ASFV) and to evaluate their functionality in vitro . The efficiency and specificity of transgene expression, as well as their non-toxicity in cells of target animals, were evaluated., Materials and Methods: Bioinformatics analysis methods were used to identify the immunodominant genes of ASFV. The target genes B646L , E183L , CP204L , and CP530R were identified and subsequently cloned into the pAAV-MCS vector. Assembly of recombinant AAV2 (rAAV2) was performed by cotransfection of AAV293 cells with the following plasmids: pAAV-MCS with the gene of interest, envelope, and packaging. Quantitative polymerase chain reaction was used to determine the AAV2 titer. The functionality of the constructs was evaluated in HEK293 and SPEV cells by determining the presence of mature proteins in the cell lysate and the expression levels of messenger RNA. The specificity of the target proteins in cell lysates was confirmed by Western blotting., Results: The proposed AAV2 assembly protocol makes it possible to achieve a concentration of mature viral particles of at least 280 billion/mL of virus-containing material. The rAAV2 could effectively transduce host SPEV cells. The expression of both cistrons was detectable during the transduction of cells; therefore, the combined expression of immunogens in the cells of target animals should be possible using this method., Conclusion: This study demonstrated the potential of using genetic constructs based on AAV2 for the delivery of ASFV genes in vitro ., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Ravilov, et al.)

Background: The occurrence of a congenital heart defect has long been thought to have a multifactorial basis, but the evidence is indirect. Complex trait analysis could provide a more nuanced understanding of congenital heart disease., Methods and Results: We assessed the role of genetic and environmental factors on the incidence of ventricular septal defects (VSDs) caused by a heterozygous Nkx2-5 knockout mutation. We phenotyped >3100 hearts from a second-generation intercross of the inbred mouse strains C57BL/6 and FVB/N. Genetic linkage analysis mapped loci with lod scores of 5 to 7 on chromosomes 6, 8, and 10 that influence the susceptibility to membranous VSDs in Nkx2-5(+/-) animals. The chromosome 6 locus overlaps one for muscular VSD susceptibility. Multiple logistic regression analysis for environmental variables revealed that maternal age is correlated with the risk of membranous and muscular VSD in Nkx2-5(+/-) but not wild-type animals. The maternal age effect is unrelated to aneuploidy or a genetic polymorphism in the affected individuals. The risk of a VSD is not only complex but dynamic. Whereas the effect of genetic modifiers on risk remains constant, the effect of maternal aging increases over time., Conclusions: Enumerable factors contribute to the presentation of a congenital heart defect. The factors that modify rather than cause congenital heart disease substantially affect risk in predisposed individuals. Their characterization in a mouse model offers the potential to narrow the search space in human studies and to develop alternative strategies for prevention.