Identifying the genetic basis of viral spillover using Lassa virus as a test case

The rate at which zoonotic viruses spill over into the human population varies significantly over space and time. Remarkably, we do not yet know how much of this variation is attributable to genetic variation within viral populations. This gap in understanding arises because we lack methods of genetic analysis that can be easily applied to zoonotic viruses, where the number of available viral sequences is often limited, and opportunistic sampling introduces significant population stratification. Here, we explore the feasibility of using patterns of shared ancestry to correct for population stratification, enabling genome-wide association methods to identify genetic substitutions associated with spillover into the human population. Using a combination of phylogenetically structured simulations and Lassa virus sequences collected from humans and rodents in Sierra Leone, we demonstrate that existing methods do not fully correct for stratification, leading to elevated error rates. We also demonstrate, however, that the Type I error rate can be substantially reduced by confining the analysis to a less-stratified region of the phylogeny, even in an already-small dataset. Using this method, we detect two candidate single-nucleotide polymorphisms associated with spillover in the Lassa virus polymerase gene and provide generalized recommendations for the collection and analysis of zoonotic viruses

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

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.</jats:p