Porcine Rotavirus (PoRV) is one of the key pathogens causing viral diarrhea in newborn and young piglets, and its infection can lead to severe gastrointestinal dysfunction in the host, with clinical manifestations, including dehydration, diarrhea, and even death, resulting in huge economic losses to the global pig industry. The VP4 protein is one of the key structural proteins on the surface of PoRV virions. The VP4 protein is cleaved by trypsin to generate two functional subunits, such as VP8 and VP5, which play a core role in the initial stage of viral infection of host cells, mediating the adsorption of the virus to host cell receptors and the subsequent membrane penetration process. Meanwhile, the VP4 protein is also an important target antigen that elicits the host immune response. However, current research on specific monoclonal antibodies (mAbs) against the PoRV VP4 protein is relatively scarce, which limits the development of related diagnostic methods and new vaccines.

This study aimed to prepare specific monoclonal antibodies (mAbs) against the VP4 protein of Porcine Rotavirus (PoRV), and on this basis, comprehensive biological characterization of these mAbs were analyzed, including reactivity, antigenic epitope types (conformational or linear), and subtype identification, and key neutralizing activities were evaluated too. The aim was to precisely identify the antigenic epitope regions with important functional significance on the VP4 protein, thereby providing support for the accurate diagnosis of PoRV infection and the design of novel vaccines.

BALB/c mice were immunized with purified VP4*P23 recombinant protein, and hybridoma cell lines were screened using splenocyte fusion technology. The reactivity of monoclonal antibodies was identified by Western blot, indirect immunofluorescence assay (IFA), and immunoperoxidase monolayer assay (IPMA). The conformational sensitivity of monoclonal antibodies was evaluated by indirect ELISA. VP5 truncated proteins were constructed to determine the antigenic epitope regions, and the neutralizing ability of VP5 monoclonal antibodies was assessed by in vitro neutralization assay.

A total of 26 hybridoma cell lines stably secreting antibodies were successfully obtained. The antibody subtypes included multiple types such as IgG1, IgG2a, IgG2b and IgM, among which the light chain type was mainly κ chain. Among the obtained 26 mAbs, 15 of them were confirmed to specifically react with natural PoRV virions (detected by IFA and IPMA). Indirect ELISA detection showed that mAb11, 14, 15 and 23 were conformation-insensitive mAbs, while mAb16, 17, 18, 19, 21, 24, 25 and 26 were conformation-sensitive mAbs. It was worth noting that the reactivity of three mAbs (mAb1, 2 and 22) was enhanced after antigen denaturation. Western blot analysis further focused on mAbs recognizing linear epitopes (mAb11, 14, 15), and the results showed that they could all specifically recognize the linear epitope within the amino acid region of about 300-360 on the VP5 protein, possibly targeting the same antigenic epitope. However, in vitro neutralization assay evaluation showed that these three mAbs had no neutralizing effect on PoRV strains.

Multiple mAbs targeting the PoRV VP4 protein were successfully prepared, among which 15 mAbs exhibited the ability to bind to natural viruses. Through systematic characterization, not only the subtype distribution and light chain type of mAbs were clarified, but more importantly, the antigen recognition characteristics of mAbs were deeply analyzed: conformation-sensitive and conformation-insensitive mAbs were successfully distinguished, and the linear antigenic epitope regions recognized by mAb11, 14, and 15 were precisely located. This study provided key antibody resources for the optimization of PoRV diagnostic reagents and the development of subunit vaccines, and laid a foundation for in-depth research on the immunological functions and antiviral mechanisms of the VP4 protein.

Rotavirus (RV) is one of the main causes of acute viral gastroenteritis in young children and young animals worldwide, and it is of great significance in public health. Porcine rotavirus disease, caused by Porcine Rotavirus (PoRV), is an acute intestinal infectious disease that often results in gastrointestinal dysfunction in piglets, leading to severe symptoms, such as vomiting, diarrhea, and dehydration. Outbreaks of PoRV can result in significant economic losses in the pig industry. At present, there is no specific drug treatment for PoRV infection, so vaccination is the most economical way to control the infection. However, PoRV genotypes are various and easily mutable, and cross-protection between different genotypes is poor. Therefore, it is urgent to strengthen the epidemiological surveillance and pathogenic mechanism research of PoRV to explore new prevention and control strategies.

The prokaryotic system of Escherichia coli was employed to express the non-structural proteins (NSP), such as NSP2, NSP4, and NSP5 of PoRV. Subsequently, rabbits were immunized to produce polyclonal antibodies (pAbs) specific to these proteins, which could offer novel insights to the detection and prevention of PoRV.

The NSP2, NSP4, and NSP5 genes of PoRV were codon-optimized and cloned into the pCold-sumo vector. The positive recombinant plasmids, with correct sequencing, were transformed into E. coli BL21 (DE3), and the recombinant proteins were obtained with IPTG induction. Protein expression was identified using SDS-PAGE and Western blot assay. The recombinant proteins were then purified and quantified using affinity chromatography. New Zealand white rabbits were immunized with NSP2, NSP4, and NSP5 recombinant proteins by a subcutaneous multi-point injection method to prepare pAbs. The titers of pAbs were determined using indirect ELISA technology. The reactivity of pAbs with PoRV was verified by indirect immunofluorescence (IFA) and Western blot assay, and their applications in PoRV infection were also explored.

SDS-PAGE analysis indicated that the recombinant proteins of NSP2, NSP4, and NSP5 were expressed well in a soluble form. Indirect ELISA analysis showed that the titers of pAbs against the three proteins reached 1∶81 000, indicating good immunogenicity of the expressed proteins. The results from IFA and Western blot assay demonstrated that the prepared pAbs could specifically react with the dominant prevalent genotypes of PoRV, but had no reaction with other common diarrhea pathogens. Western blot assay results also showed that the pAbs could be used for dynamic expression analysis of NSPs during PoRV infection and validating transfection of the three recombinant eukaryotic plasmids.

Here, the NSP2, NSP4 and NSP5 of PoRV were successfully expressed in E. coli, and their pAbs with high titer and good specificity were obtained, which laid the foundation for the study of PoRV pathogenesis and the development of prevention and control strategies.