Although smallpox has been eradicated globally, the potential use of the smallpox virus in bioterrorism indicates the importance of stockpiling smallpox vaccines. Considering the advantages of microneedle-based vaccination over conventional needle injections, in this study, we examined the feasibility of microneedle-based smallpox vaccination as an alternative approach for stockpiling smallpox vaccines. We prepared polylactic acid (PLA) microneedle array patches by micromolding and loaded a second-generation smallpox vaccine on the microneedle tips via dip coating. We evaluated the effect of excipients and drying conditions on vaccine stability in vitro and examined immune responses in female BALB/c mice by measuring neutralizing antibodies and interferon (IFN)-γ-secreting cells. Approximately 40% of the virus titer was reduced during the vaccine-coating process, with or without excipients. At −20 °C, the smallpox vaccine coated on the microneedles was stable up to 6 months. Compared to natural evaporation, vacuum drying was more efficient in improving the smallpox vaccine stability. Microneedle-based vaccination of the mice elicited neutralizing antibodies beginning 3 weeks after immunization; the levels were maintained for 12 weeks. It significantly increased IFN-γ-secreting cells 12 weeks after priming, indicating the induction of cellular immune responses. The smallpox-vaccine-coated microneedles could serve as an alternative delivery system for vaccination and stockpiling.

This study applied a molecular-based method to detect parainfluenza virus 5 (PIV5) collected from 2016 to 2018 in nine provinces of Republic of Korea. We demonstrated that PIV5 was detectable in both serum and pooled organs at an average positive rate of 1.78% (99/5566). Among these, the complete genome sequence of 15,246 nucleotides was obtained for 12 field strains. Three out of the 12 strains had the lowest genetic identity (96.20–96.68%) among the 21 porcine PIV5 genomes collected in Germany, China, India, and Republic of Korea from 1998 to 2017. By analyzing a large collection of complete genome sequences of the structural protein-coding F and HN genes, this study proposed a classification of PIV5 into two lineages, 1 and 2, and identified that group 2.2.2 within sub-lineage 2.2 was substantially divergent. The evolution of two structural protein-coding genes was largely under purifying selection. A few codons (6/9 for the F gene, 7/8 for the HN gene) had elevated dN/dS values, which were loaded on internal branches and were predicted to be related to beneficial trait(s) of the virus.