Cyprinid herpesvirus 2 (CyHV-2) is a virus that causes mass mortality in economically important Carassius spp. However, there have been no comprehensive studies into host susceptibility or permissivity with respect to developmental stage, and the major portal of viral entry into the host is still unclear. To help bridge these knowledge gaps, we developed the first ever recombinant strain of CyHV-2 expressing bioluminescent and fluorescent reporter genes. Infection of Carassius auratus hosts with this recombinant by immersion facilitated the exploitation of various in vivo imaging techniques to establish the spatiotemporal aspects of CyHV-2 replication at larval, juvenile, and adult developmental stages. While less susceptible than later developmental stages, larvae were most permissive to CyHV-2 replication, leading to rapid systemic infection and high mortality. Permissivity to CyHV-2 decreased with advancing development, with adults being the least permissive and, thus, also exhibiting the least mortality. Across all developmental stages, the skin was the most susceptible and permissive organ to infection at the earliest sampling points post-infection, indicating that it represents the major portal of entry into these hosts. Collectively these findings provide important fundamental insights into CyHV-2 pathogenesis and epidemiology in Carassius auratus with high relevance to other related economically important virus-host models.

Schizochytrium limacinum SR21, a kind of eukaryotic heterotrophic organism rich in unsaturated fatty acids, is an emerging microbial alternative to fish oil. The dietary inclusion of 15% SR21 was optimal for the growth performance of zebrafish. Previous studies demonstrated that fructose-1,6-bisphosphate aldolase (FBA) of Edwardsiella tarda is a valuable broad-spectrum antigen against various pathogens in aquaculture (e.g., Aeromonas hydrophila, Vibro anguillarum, Vibro harveyi, Vibro alginolyticus). We pioneered the development of stable S. limacinum SR21 transformants expressing the antigen protein FBA, exploring their potential as a novel oral vaccine for the aquaculture industry. The model animal zebrafish (Danio rerio) and ornamental fish koi carp (Cyprinus carpio koi) were harnessed to assess the immunoprotective effect, respectively. According to the quantitative expression analysis, zebrafish fed with recombinant Schizochytrium expressing FBA exhibited specific immune responses in the intestine. The expression levels of MHC-I and MHC-II, involved in cell-mediated adaptive immune responses, were significantly upregulated on the 14th and 28th days post-immunization. Additionally, the expression of highly specialized antibody genes IgZ1 and IgZ2 in mucosal immunity were significantly triggered on the 14th day post-immunization. Feeding koi carp with recombinant S. limacinum SR21-FBA increased the production of myeloperoxidase and FBA-specific antibodies in the sera. Furthermore, the sera of koi fed with recombinant S. limacinum SR21-FBA exhibited significant bactericidal activities against pathogen E. tarda. Thus, S. limacinum SR21 is a natural and highly promising oral vaccine carrier that not only provides essential nutrients as a functional feed ingredient but also offers specific immune protection to aquatic animals. This dual application is vital for promoting the sustainable development of the aquaculture industry.

Background/Objectives: Anguillid herpesvirus 1 (AngHV-1) (recently renamed Cyvirus anguillidallo 1) is the etiologic agent of a lethal disease that affects several eel species. It is thought to be one of the main infectious agents causing a population decline in wild eels and economic loss within the eel aquaculture sector. To date, no vaccines are available against AngHV-1. Recently, we developed a safe and efficacious live attenuated recombinant vaccine against Cyprinid herpesvirus 3 (CyHV-3). This CyHV-3 recombinant vaccine encodes a deletion of ORF57. Orthologues of CyHV-3 ORF57 exist in Cyprinid herpesvirus 2 (CyHV-2, ORF57) and AngHV-1 (ORF35). Methods: In the present study, using recombinant strains and bioluminescent in vivo imaging, we investigated the effect of AngHV-1 ORF35 deletion on virus replication in vitro, virulence in vivo, and the potential of an AngHV-1 ORF35-deleted recombinant as a vaccine candidate for the mass vaccination of eels by immersion. With this goal in mind, we produced ORF35-deleted recombinants using two parental strains: a UK strain and a recombinant derived from the former strain by insertion of a Luciferase–GFP reporter cassette into a non-coding intergenic region. Results: Analyses of ORF35-deleted recombinants led to the following observations: (i) AngHV-1 ORF35 is not essential for viral growth in cell culture, and its deletion does not affect the production of extracellular virions despite reducing the size of viral plaque. (ii) In contrast to what has been observed for CyHV-3 ORF57 and CyHV-2 ORF57, in vivo bioluminescent analyses revealed that AngHV-1 ORF35 is an essential virulence factor and that its deletion led to abortive infection in vivo. (iii) Inoculation of the AngHV-1 ORF35-deleted recombinant by immersion induced a protective immune response against a wild-type challenge. This protection was shown to be dose-dependent and to rely on the infectivity of AngHV-1 ORF35-deleted virions. Conclusions: This study suggests that the AngHV-1 ORF35 protein has singular properties compared to its orthologues encoded by CyHV-2 and CyHV-3. It also supports the potential of AngHV-1 ORF35-deleted recombinants for the mass vaccination of eels by immersion.