Search

Hepatitis B Virus (HBV) genome sequencing can be used to provide more complete genetic information at the population and individual level to shed light on the limitations of current interventions, and inform new strategies for elimination. HBV sequencing is challenging due to the partially dsDNA genome, high diversity, low viral loads and presence of large amounts of host genetic material in clinical samples. Here we describe the design and use of a pan-genotypic panel of 74 HBV specific capture-probes and nuclease treatment in improving sequencing efficiency. We processed 20 plasma samples (viral loads 1.98 to 4.07 log10, genotypes A-E) and three positive controls (human total brain RNA and bacteriophage lambda DNA) in triplicate to compare DNAse vs. RNAse vs. no nuclease treatment. We prepared libraries using the Takara Bio SMARTer Stranded Total RNA-Seq Kit v3, split the library in two, enriching half with the custom-designed probe panel and xGen Hybridization and Wash Kit (IDT), the other half was not enriched. Both libraries were sequenced on the NovaSeq6000 platform with 2×150nt paired-end reads. Capture resulted in a 47,970 fold increase in the number of reads mapped to the HBV genome in the “no nuclease” arm (243 HBV reads per million reads sequenced in the capture pool vs. 5×10−3reads per million in the no-capture pool). Out of 20 samples, only 1 without capture generated HBV reads (viral load 3.89 log10IU/ml) vs. 19 samples with capture. HBV sequence yield was increased in the capture arm and resulted in 2.30 log10(95% confidence interval 1.99 - 2.48 log10) increase in HBV reads (per million reads sequenced) per log10increase in viral load. The proportion of HBV reads increased a median of 12 fold with RNAse treatment. We developed a targeted pan-genotypic sequencing method using a custom panel of biotinylated oligos that increases the sequencing efficacy of HBV. This method will allow us to gain a better insight into HBV diversity.

BACKGROUNDRespiratory syncytial virus (RSV) is the leading cause of hospitalisation associated with acute respiratory infection in infants and young children, with substantial disease burden globally. The impact of additional respiratory pathogens on RSV disease severity is not completely understood.OBJECTIVESThe objective of this study was to explore the associations between RSV disease severity and the presence of other respiratory pathogens.METHODSNasopharyngeal swabs were prospectively collected from two infant cohorts: a prospective longitudinal birth cohort study and an infant cross-sectional study recruiting infants RESULTSRSV was detected in 433 infants. Nearly one in four of the infants (24%) harboured at least one additional non-RSV respiratory virus, with human rhinovirus being the most frequently detected (15% of the infants), followed by seasonal coronaviruses (4%). In this cohort, RSV-infected infants harbouring any other virus tended to be older (median age: 4.3 vs. 3.7 months) and were more likely to require intensive care and mechanical ventilation than those who did not.Moraxella, Streptococcus, andHaemophilusspecies were the most frequently identified target bacteria, together found in 392 (91%) of the 433 infants (S. pneumoniaein 51% of the infants andH. influenzaein 38%). The strongest contributors to severity of presentation were younger age and the co-detection ofHaemophilusspecies alongside RSV. Across all age groups in both cohorts, detection ofHaemophilusspecies was associated with higher overall severity, as captured by ReSVinet scores, and specifically with increased rates of hospitalisation and respiratory distress. In contrast, presence ofMoraxellaspecies was associated with lower ReSVinet scores and reduced need for intensive care and mechanical ventilation. Infants with and withoutStreptococcusspecies (orS. pneumoniaein particular) had similar clinical outcomes. No specific RSV strain was associated with co-detection of other pathogens.CONCLUSIONOur findings provide strong evidence for associations between RSV disease severity and the presence of additional respiratory viruses and bacteria. The associations, while not indicating causation, are of potential clinical relevance. Awareness of coexisting microorganisms could inform therapeutic and preventive measures to improve the management and outcome of RSV-infected infants.