As bone remodelling requires the immigration, proliferation and differentiation of osteoblasts at the fusion site, high dosages of intrawound vancomycin might interfere with regenerative processes and increase the risk of non-union. To allow an appropriate balance of infection risk and the risk of non-union, the minimal local concentration required should be determined by controlled in vivo studies.

Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Mixed human cytomegalovirus (HCMV) strain infections are frequent in lung transplant recipients (LTRs). To date, the influence of the donor (D) and recipient (R) HCMV-serostatus on intra-host HCMV strain composition and viral population dynamics after transplantation is only poorly understood. Here, we investigated ten pre-transplant lungs from HCMV-seropositive donors, and 163 sequential HCMV-DNA positive plasma and bronchoalveolar lavage samples from 50 LTRs with multiviremic episodes post-transplantation. The study cohort included D+R+ (38%), D+R- (36%), and D-R+ (26%) patients. All samples were subjected to quantitative genotyping by short amplicon deep sequencing, and 24 thereof were additionally PacBio long-read sequenced for genotype linkages. We find that D+R+ patients show a significantly elevated intra-host strain diversity compared to D+R- and D-R+ patients (P=0.0089). Both D+ patient groups display significantly higher viral population dynamics than D- patients (P=0.0061). Five out of ten pre-transplant donor lungs were HCMV-DNA positive, whereof in three multiple HCMV strains were detected, indicating that multi-strain transmission via lung transplantation is likely. Using long reads, we show that intra-host haplotypes can share distinctly linked genotypes, which limits overall intra-host diversity in mixed infections. Together, our findings demonstrate donor-derived strains as a main source for increased HCMV strain diversity and dynamics post-transplantation. These results foster strategies to mitigate the potential transmission of the donor strain reservoir with the allograft such as ex vivo delivery of HCMV-selective immunotoxins prior to transplantation to reduce latent HCMV.

No abstract

Mixed human cytomegalovirus (HCMV) strain infections are frequent in lung transplant recipients (LTRs). To date, the influence of the donor (D) and recipient (R) HCMV-serostatus on intra-host HCMV strain composition and replication dynamics after transplantation is only poorly understood.Here, we investigated ten pre-transplant lungs from HCMV-seropositive donors, and 163 sequential HCMV-DNA positive plasma and bronchoalveolar lavage samples from 50 LTRs with multiviremic episodes post-transplantation. The study cohort included D+R+ (38%), D+R− (36%), and D−R+ (26%) patients. All samples were subjected to quantitative genotyping by short amplicon deep sequencing, and 24 thereof were additionally PacBio long-read sequenced for genotype linkages.We find that D+R+ patients show a significantly elevated intra-host strain diversity compared to D+R− and D−R+ patients (P=0.0089). Both D+ patient groups display significantly higher replication dynamics than D− patients (P=0.0061). Five out of ten pre-transplant donor lungs were HCMV-DNA positive, whereof in three multiple HCMV strains were detected, indicating that multi-strain transmission via lung transplantation is likely. Using long reads, we show that intra-host haplotypes can share distinctly linked genotypes, which limits overall intra-host diversity in mixed infections. Together, our findings demonstrate donor-derived strains as a main source for increased HCMV strain diversity and dynamics post-transplantation, while a relatively limited number of intra-host strains may facilitate rapid adaptation to changing environments in the host. These results foster targeted strategies to mitigate the potential transmission of the donor strain reservoir with the allograft.