Molecular cloning and host range analysis of three cytomegaloviruses from <i>Mastomys natalensis</i>

Herpesvirus-based vectors are attractive for use as conventional or transmissible vaccines against emerging zoonoses in inaccessible animal populations. In both cases, cytomegaloviruses (CMVs) as members of the subfamily <i>Betaherpesvirinae</i> are particularly suitable for vaccine development as they are highly specific for their natural host species, infect a large proportion of their host population, and cause mild infections in healthy individuals. The Natal multimammate mouse (<i>Mastomys natalensis</i>) is the natural reservoir of Lassa virus, which causes deadly hemorrhagic fever in humans. <i>M. natalensis</i> was recently reported to harbor at least three different cytomegaloviruses (MnatCMV1, MnatCMV2, and MnatCMV3). Herein, we report the molecular cloning of three complete MnatCMV genomes in a yeast and bacterial artificial chromosome (YAC-BAC) hybrid vector. Purified viral genomes were cloned in yeast by single-step transformation-associated recombination (STAR cloning) and subsequently transferred to <i>Escherichia coli</i> for further genetic manipulation. The integrity of the complete cloned viral genomes was verified by sequencing, and the replication fitness of viruses reconstituted from these clones was analyzed by replication kinetics in <i>M. natalensis</i> fibroblasts and kidney epithelial cells. We also found that neither parental nor cloned MnatCMVs replicated in mouse and rat fibroblasts, nor did they show sustained replication in baby hamster kidney cells, consistent with the expected narrow host range for these viruses. We further demonstrated that an exogenous sequence can be inserted by BAC-based mutagenesis between open reading frames M25 and m25.1 of MnatCMV2 without affecting replication fitness <i>in vitro</i>, identifying this site as potentially suitable for the insertion of vaccine target antigen genes.IMPORTANCECytomegaloviruses (CMVs) recently discovered in the Natal multimammate mouse (<i>Mastomys natalensis</i>) are widespread within the <i>M. natalensis</i> population. Since these rodents also serve as natural hosts of the human pathogen Lassa virus (LASV), we investigated the potential suitability of <i>M. natalensis</i> CMVs (MnatCMVs) as vaccine vectors. We describe the cloning of three different MnatCMV genomes as bacterial artificial chromosomes (BACs). The replicative capacity and species specificity of these BAC-derived MnatCMVs were analyzed in multiple cell types. We also identified a transgene insertion site within one of the MnatCMV genomes suitable for the incorporation of vaccine target antigens. Together, this study provides a foundation for the development of MnatCMVs as transmissible MnatCMV-based LASV vaccines to reduce LASV prevalence in hard-to-reach <i>M. natalensis</i> populations and, thereby, zoonotic transmission to humans.