Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis)

Abstract

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Keywords: Mastomys natalensis; Mastomys natalensis cytomegalovirus; cytomegalovirus; genome sequence; herpesvirus; muromegalovirus.

Fig. 1.

Sequence alignment of a 178 bp region of the DNA polymerase gene in MnatCMV primary isolates. The alignment is presented in three contiguous sections separated by grey horizontal lines. The sequences are grouped with the MnatCMV references (names in bold font), and differences are also highlighted in bold font. Dataset Mnat36 represented MnatCMV1 and MnatCMV3, dataset Mnat29 represented MnatCMV1, MnatCMV2 and MnatCMV3, and dataset Mnat2 represented two MnatCMV2 strains (MnatCMV2A and MnatCMV2B). No isolate represented MnatCMV4.

Fig. 2.

Map of the MnatCMV1 genome (GenBank accession OP429138.1). The terminal direct repeats are shown in a thicker format. The ORFs comprising protein-coding regions are indicated by coloured arrows grouped according to the key, with corresponding gene names below. Introns connecting protein-coding regions are shown as narrow white bars. The colours of protein-coding regions indicate genes that are conserved in the family Orthoherpesviridae (core genes) or only in the subfamilies Betaherpesvirinae and Gammaherpesvirinae (betagamma genes). The remaining coding regions (non-core genes) include six families of related genes (the lectin, US22, UL25, DURP, GPCR and m145 families). M72 is shown as a member of the DURP family and is also a core gene. The alternative right genome end that would extend the standard genome by about 2 % is indicated by the vertical black bar between genes a3 and a4.

Fig. 3.

Map of the MnatCMV2 genome (GenBank accession OP429139.1). See the Fig. 2 legend for details.

Fig. 4.

Map of the MnatCMV3 genome (GenBank accession OP429140.1). See the Fig. 2 legend for details. The genome lacks an alternative right end.

Fig. 5.

Maximum-likelihood phylogenetic tree showing relationships among MnatCMVs and related viruses in the subfamily Betaherpesvirinae. The names of the genera in which the viruses group are shown on the right. Fractional bootstrap values are shown at the nodes. Bar, 0.1 amino acid substitutions per site.