Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen–free chickens were immunised and their humoral and cellular immune responses were analysed. The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4⁺/CD8⁺ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4⁺/CD8⁺ ratio and the increase in IL-4 did not promote anti-HA antibodies. Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

In 2007 Nevill, Venter, Meiswinkel & Nevill demonstrated that the pupae of five Culicoides species belonging to the Imicola complex of the subgenus Avaritia could readily be differentiated from one another using various morphological characters. Three of the described species, Culicoides bolitinos Meiswinkel 1989; Culicoides loxodontis Meiswinkel 1992 and Culicoides sp. # 107 (= C. kwagga, Meiswinkel, unpublished thesis 1995), were reared from the dung of large herbivores, which included buffaloes, elephants, white and black rhinoceroses and zebras. However, during that study a further two Avaritia species, neither of which belonged to the Imicola complex, were reared from dung and these are the subject of the present study. For the past 20 years the adults of these two new closely related species have been known as Culicoides sp. # 54 pale form (p.f.) Meiswinkel and Culicoides sp. # 54 dark form (d.f.) Meiswinkel. The taxonomic description and formal naming of the adults of these two species has yet to be done. The present description and comparison of their pupae show that they are two clearly distinct species; that there is no group of morphological characters that can be used to differentiate these two species from the previously described five species of the Imicola complex; and finally that there was no difference between the pupae of C. sp. # 54 d.f. nor C. sp. # 54 p.f. reared from the dung of different host animals.