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Effect of low-passage number on dengue consensus genomes and intra-host variant frequencies.

ABSTRACT: Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low-passage number increases viral load, but few studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence and the intra-host virus population frequencies. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T. splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 500×, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNVs with frequencies ≥5 % were often preserved between the samples, and that the number of iSNV positions, and sample diversity, at this frequency cutoff did not differ significantly between the sample pairs (clinical sample and isolate) in either DENV1 or DENV2 data. Our results show that low-passage DENV isolate consensus genomes are largely representative of their direct sample parental viruses, and that low-passage isolates often mirror high frequency within-host variants from direct samples.

SUBMITTER: Fung CK

PROVIDER: S-EPMC8515859 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

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Effect of low-passage number on dengue consensus genomes and intra-host variant frequencies.

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