Human Cytomegalovirus Genomes Sequenced Directly from Clinical Material: Variation, Multiple-Strain Infection, Recombination and Mutation

Abstract

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination and natural mutation. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analysed, in the process facilitating the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating diversity, (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination, (iii) mutants with non-functional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae, and (iv) intrahost diversity in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology and pathogenesis of HCMV.

Multiple-Strain Infections of Human Cytomegalovirus with High Genomic Diversity are Common In Breast Milk from HIV-Positive Women in Zambia

“…In a recent study, we highlighted the importance of monitoring the quality of datasets produced directly from clinical material by target enrichment and high-throughput sequencing [24]. We implemented this here by assembling the datasets against the reference strain Merlin genome (GenBank accession AY446894), noting the number of matching HCMV reads ( Supplementary Table 1 line 19), and deriving two parameters: (1) the percentage of matching HCMV reads in the dataset and (2) the percentage of the reference genome represented ( Supplementary Table 1 lines 20-21).…”

“…UL73 and UL74 genotypes [7, 12] were investigated in 243 different HCMV strains [24]. MEGA 6.06 [25] was used to generate Muscle-derived amino acid sequence alignments and phylogenetic trees based on the Jones-Taylor-Thornton model and discrete gamma distribution with five categories.…”

“…Bowtie2 [26] was used to remove reads mapping to the Genome Reference Consortium Human Reference 38 sequence, and also quality-checked and trimmed to create purged datasets ( Supplementary Table 1 row 14). Dataset quality parameters were ( Supplementary Table 1 rows 19-23) [24] on the basis of thresholds described in Results.…”

“…Long motifs identifying common mutations in three genes (RL5A, UL111A and US9) were also counted ( Supplementary Table 1 rows 167-174). The derivation of the long motifs as described [24], were used on the basis of thresholds described in Results ( Supplementary Table 1 row 17).…”

“…High-throughput sequencing studies at the whole-genome level have started to facilitate a broader view of HCMV diversity, but most have involved isolating the virus in cell culture, which is prone to strain loss or mutation, or have depended on direct sequencing of PCR amplicons generated from clinical samples [11, 14, 16, 21]. Recent studies have avoided these limitations by using target enrichment to enable direct sequencing of strains present in clinical samples, most of which originated from patients in developed countries with congenital or transplantation-associated infections [11, 22-24]. We have used this approach to examine HCMV strain diversity in a developing country by analysing breast milk from women in Zambia, who constitute an HIV endemic population in sub-Saharan Africa, where we have previously demonstrated the negative developmental effects of early infection of infants with HCMV, particularly alongside HIV exposure [1, 3].…”