Development of murine monoclonal antibodies for porcine rotavirus group B and C and use in immunoassays

Porcine rotavirus (PRV) is a non-enveloped icosahedral virus belonging to the genus Rotavirus and family Rotaviridae. Rotavirus genome has 11 segments which encode 6 nonstructure proteins (NSP1,2,3,4,5,6) and 6 structure proteins (VP1,2,3,4,6,7). Virion has three layers. The outer most layer comprise of VP4 and VP7 which are necessary for virus entry. The middle layer is VP6 protein, the most abundant and conserve structure protein. Base on the antigenicity and sequence of VP6, rotaviruses are divided into 8 serogroups (A-H). Serogroup A, B, C, E and H have been implicated in swine enteric disease. To date, isolates and genomic information of PRV group B and C are limited due to substantial difficulties in virus isolation. As a consequence, not many laboratory tools are available for virus detection and research. The goal of this study is producing monoclonal antibodies (mABs) against PRV B and C that can be used in diagnostics as well as research.

Molecular cloning and recombinant protein technology were used to produce mABs specific for PRV B and C. Full-length of PRV B was achieved by using full-length amplification of cDNA method. Whole VP6 of PRV B and C were amplified by RT-PCR and then cloned and expressed in a baculovirus system using Bac-to-Bac cloning and expression kit. The VP6 proteins of PRV were purified in native conditions using a Ni-NTA Purification System. The proteins then were used to produce mABs. Mouse immunization and care, fusion, and hybridoma production and maintenance were done at the Iowa State University Hybridoma Facility. VP6-based ELISA and IFA using PRV C-infected cells or Sf9 cells expressing VP6 of PRV B were used to screen hybridomas. Antibody-producing hybridomas were selected and cloned for further evaluation. The mABs then were verified and characterized by Western blot, isotyping, IFA, Immunohistochemistry (IHC).

A 1269 nucleotide- long PRV B VP6 was achieved and used to design primers to amplify full-length VP6 of PRV B. 45-kDa VP6 proteins of PRV B and C were successfully expressed and purified.A total of 8 mABs each for PRV B and C were initially obtained based on reactivity with VP6 on ELISA. Among those, mAB 10B1, 10F7 and 10B5, 11F3 were selected for PRV B and C, respectively, as they were positive by ELISA, IFA and Western blot. Furthermore, these MAbs could be used in IHC to specifically detect PRV B or C in intestinal tissues from pigs with enteritis.

Murine monoclonal antibodies specific for VP6 of PRV B and C were successfully developed. The availability of these antibodies would enhance the pathogenesis study of various serogroups of rotaviruses in addition to its utility in diagnostic investigations.

Disclosure of Interest: None Declared.