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Background African horse sickness (AHS) is a severe, noncontagious disease of equines caused by the African horse sickness virus (AHSV). The virus has nine serotypes and is transmitted by the Culicoides midge. AHS is endemic in South Africa and other sub-Saharan African countries. Currently, the disease is managed using a live attenuated vaccine manufactured by Onderstepoort Biological Products (OBP). Although this vaccine has been in use for decades, it has several drawbacks, including the possibility of reversion to virulence, and it does not allow for the differentiation of infected horses from vaccinated horses (DIVA). Previously, our group developed recombinant AHSV serotype 4 and 5 virus-like particle (VLP) vaccine candidates in plants that elicited an immune response in guinea pigs and horses. In this research, we aimed to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) using an AHSV-VP6 antigen expressed in plants, allowing for the differentiation of horses infected with the virus from those vaccinated with VLP vaccine candidates. For this DIVA ELISA, we utilized a robust, cost-effective, and easily scalable manufacturing process that employs transient expression of VP6 in Nicotiana benthamiana . Results AHSV-VP6 sequences from all nine serotypes were aligned to obtain a consensus sequence, which was then used to design the VP6 gene. The VP6 gene was successfully expressed in Nicotiana benthamiana plants via Agrobacterium -mediated infiltration. The VP6 protein was extracted from infiltrated leaves and purified. A purified yield of approximately 7.7 mg of recombinant VP6/kg fresh weight leaf material was obtained. The VP6 protein was also expressed in E. coli , yielding a purified product of 9.4 mg/L. Preliminary data revealed that AHSV-VP6 antigen expressed in both plants and E. coli could be used to differentiate between sera from infected horses and those vaccinated with the candidate AHSV4 and AHSV5 VLP vaccines. The plant-produced VP6 could detect more anti-VP6 antibodies than the E. coli- produced VP6. Conclusions In this study, we expressed the AHSV-VP6 protein in plants, which enabled the differentiation of infected AHSV horse sera from those of horses vaccinated with the candidate VLP vaccines. To our knowledge, this is the first evidence of AHSV-VP6 expression in plants and the first demonstration of its diagnostic ability.

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery.

Objective Rotavirus is known for its pathogenicity in children under the age of five, causing severe gastroenteritis with concerning mortality and morbidity rates in endemic regions. The complex, three-layered capsid of this pathogen is a substantial field of study, and deepening our understanding, particularly of double-layered particles (DLPs), is crucial for advancing various rotavirology areas, such as vaccine development, pathogenesis, host-virus interactions, and diagnostic research. In this pilot study, we designed and evaluated three immunostimulation regimens to generate the highest level of anti-VP6 antibody in vivo for a range of downstream applications, which may require these antibodies as a starting point for further investigation. Results This study indicated that immune stimulation can vary based on the combination of antigens and the order in which they are introduced into the host. According to our findings, a heterologous regimen composed of recombinant VP6 protein with complete Freund’s adjuvant as the priming shot and rotavirus particles with incomplete Freund’s adjuvant as the booster shot could elicit an antibody response at least two-fold stronger than the other homologous regimens applied in the present study. This preliminary observation suggests a high-yielding yet cost-effective protocol to access abundant anti-VP6 polyclonal antibodies as a base for different DLP-relying research.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402 ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock’s health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

Indirect immunofluorescence techniques targeting the rotavirus (RV) protein VP6 are used to differentiate RV species. The ICTV recognizes RV species A to E and two tentative species, F and G. A potential new RV species, ADRV-N, has been described. Phylogenetic trees and pairwise identity frequency graphs were constructed with more than 400 available VP6 sequences and seven newly determined VP6 sequences of RVD strains. All RV species were separated into distinct phylogenetic clusters. An amino acid sequence cutoff value of 53% firmly permitted differentiation of RV species, and ADRV-N was tentatively assigned to a novel RV species H (RVH).

Rotavirus (RV) structural proteins VP4 and VP7, located on the surface of viral particles, elicit neutralizing antibodies (Abs) and are therefore considered to be important components of RV vaccines. However, despite inducing neutralizing Abs, limits of cross-neutralizing activity and lack of full correlation with protection limit the usefulness of these proteins as protective agents against RV disease. VP6 protein, which forms the middle layer of RV particles, is discussed as an alternative vaccine candidate since it can induce cross-protective immune responses against different RV strains although the Ab raised is not neutralizing. This report reviews different functions of VP6 that can lead to considering it as an alternative vaccine against RV disease.

Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of \"living\" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.

The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.

Rotavirus (RV) is the leading cause of viral gastroenteritis in neonates and VP6 protein has been discussed as a potential candidate vaccine. CRISPR/Cas9 was the latest generation of gene editing tools that can mediate the site-specific knock-in of exogenous genes, providing strong support for the expression of recombinant proteins. Here, seeking to design a rotavirus vaccine that would be suitable for both mammary-gland-based production and milk-based administration, rabbit β-casein (CSN2) locus was chosen as the target site to integrate the VP6 gene. The efficiency of inducing mutations in different target sites of rabbit CSN2 locus was analyzed and g4 site seems to be the best one to generate mutations (g4 72.76 ± 0.32% vs gl 30.14 ± 1.93%, g2 38.53 ± 0.75%, g3 52.26 ± 1.16%, P < 0.05). We further compared the knock-in efficiency through cytoplasmic injection of two group mixtures (containing 100 ng/µL Cas9 mRNA or Cas9 protein, 20 ng/µL sgRNA4, and 100 ng/µL donor vector) in rabbit zygotes, though the Cas9 mRNA group induced an HDR efficiency as high as 20.0% ± 2.6% than Cas9 protein group (10.3% ± 3.1%), 37.5% of the knock-in events were partial integration in the target site, when Cas9 protein used in the CRISPR/Cas9 system, all of the positive blastocysts showed completely integrated, results showed that the use of Cas9 protein is better than Cas9 mRNA to integrate the correct exogenous gene into the target site. Moreover, the transgenic rabbit that harbored correct integration of VP6 gene was obtained using Cas9 protein group and was used to produce an experimental milk-based rotavirus vaccine. Our research provides a novel strategy to produce rotavirus subunit vaccine and make a foundation for building broader milk-based vaccine protection against other pathogens.

Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines.