Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins

Abstract

AbstractRotaviruses, segmented dsRNA viruses of the Reoviridae family, are a primary cause of acute gastroenteritis in young children. In countries where rotavirus vaccines are widely used, norovirus (NoV) has emerged as the major cause of acute gastroenteritis. Towards the goal of creating a combined rotavirus-NoV vaccine, we explored the possibility of generating recombinant rotaviruses (rRVs) expressing all or portions of NoV GII.4 VP1 capsid proteins. This was accomplished by replacing the segment 7 NSP3 ORF with a cassette encoding sequentially NSP3, a 2A stop-restart translation element, and all or portions (P, P2) of NoV VP1. In addition to successfully recovering SA11 rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the RIX4414 segment 7 RNA. Immunoblot assay confirmed that rRVs expressed NoV capsid proteins as independent products. Moreover, VP1 expressed by rRVs underwent dimerization and was recognized by conformational-dependent anti-VP1 antibodies. Serially passaged rRVs that expressed the NoV P and P2 were genetically stable, retaining sequences up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.5 kb VP1 sequence were less stable and gave rise to virus populations with segment 7 RNAs lacking VP1 coding sequences. Together, these studies suggest that it may be possible to develop combined rotavirus-NoV vaccines using modified segment 7 RNA to express NoV P or P2. In contrast, development of potential rotavirus-NoV vaccines expressing NoV VP1 will need additional efforts to improve genetic stability.ImportanceRotavirus (RV) and norovirus (NoV) are the two most important causes of viral acute gastroenteritis (AGE) in young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of live attenuated RV vaccines, similar highly effective NoV vaccines are not available. To pursue the development of a combined RV-NoV vaccine, we examined the potential usefulness of RV as an expression vector of all or portions of the NoV capsid protein VP1. Our results showed that by replacing the NSP3 open reading frame in RV genome segment 7 RNA with a coding cassette for NSP3, a 2A stop-restart translation element, and VP1, recombinant RVs can be generated that express NoV capsid proteins as separate products. These findings raise the possibility of developing a new generation of RV-based combination vaccines that can provide protection against a second enteric pathogen, such as the NoV.