Influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (RSV) shedding.

RSV is the most important viral cause of pneumonia and bronchiolitis in children worldwide and has been associated with significant disease burden. With the renewed interest in RSV vaccines, we provide realistic estimates on duration, and influencing factors on RSV shedding which are required to better understand the impact of vaccination on the virus transmission dynamics. The data arise from a prospective study of 47 households (493 individuals) in rural Kenya, followed through a 6-month period of an RSV seasonal outbreak. Deep nasopharyngeal swabs were collected twice each week from all household members, irrespective of symptoms, and tested for RSV by multiplex PCR. The RSV G gene was sequenced. A total of 205 RSV infection episodes were detected in 179 individuals from 40 different households. The infection data were interval censored and assuming a random event time between observations, the average duration of virus shedding was 11·2 (95% confidence interval 10·1-12·3) days. The shedding durations were longer than previous estimates (3·9-7·4 days) based on immunofluorescence antigen detection or viral culture, and were shown to be strongly associated with age, severity of infection, and revealed potential interaction with other respiratory viruses. These findings are key to our understanding of the spread of this important virus and are relevant in the design of control programmes

A Bayesian Approach to Analyse Genetic Variation within RNA Viral Populations

The development of modern and affordable sequencing technologies has allowed the study of viral populations to an unprecedented depth. This is of particular interest for the study of within-host RNA viral populations, where variation due to error-prone polymerases can lead to immune escape, antiviral resistance and adaptation to new host species. Methods to sequence RNA virus genomes include reverse transcription (RT) and polymerase chain reaction (PCR). RT-PCR is a molecular biology technique widely used to amplify DNA from an RNA template. The method itself relies on the in vitro synthesis of copy DNA from RNA followed by multiple cycles of DNA amplification. However, this method introduces artefactual errors that can act as confounding factors when the sequence data are analysed. Although there are a growing number of published studies exploring the intra- and inter-host evolutionary dynamics of RNA viruses, the complexity of the methods used to generate sequences makes it difficult to produce probabilistic statements about the likely sources of observed sequence variants. This complexity is further compounded as both the depth of sequencing and the length of the genome segment of interest increase. Here we develop a Bayesian method to characterise and differentiate between likely structures for the background viral population. This approach can then be used to identify nucleotide sites that show evidence of change in the within-host viral population structure, either over time or relative to a reference sequence (e.g. an inoculum or another source of infection), or both, without having to build complex evolutionary models. Identification of these sites can help to inform the design of more focussed experiments using molecular biology tools, such as site-directed mutagenesis, to assess the function of specific amino acids. We illustrate the method by applying to datasets from experimental transmission of equine influenza, and a pre-clinical vaccine trial for HIV-1

Evaluation of Protective Efficacy of Respiratory Syncytial Virus Vaccine against A and B Subgroup Human Isolates in Korea

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates (‘KR/A/09-8’ belonging to HRSV-A and ‘KR/B/10-12’ to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses

This work received support from the Biotechnology and Biological Sciences Research Council (grant BB/M010996/1) and Marine Scotland Science. The authors thank Dr Elena Fringuelli (Agri-Food and Biosciences Institute, Belfast) for providing some of the SAV material used.Peer reviewedPublisher PD

Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing

Human respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies

Evaluating the performance of tools used to call minority variants from whole genome short-read data.

Background: High-throughput whole genome sequencing facilitates investigation of minority virus sub-populations from virus positive samples. Minority variants are useful in understanding within and between host diversity, population dynamics and can potentially assist in elucidating person-person transmission pathways. Several minority variant callers have been developed to describe low frequency sub-populations from whole genome sequence data. These callers differ based on bioinformatics and statistical methods used to discriminate sequencing errors from low-frequency variants. Methods: We evaluated the diagnostic performance and concordance between published minority variant callers used in identifying minority variants from whole-genome sequence data from virus samples. We used the ART-Illumina read simulation tool to generate three artificial short-read datasets of varying coverage and error profiles from an RSV reference genome. The datasets were spiked with nucleotide variants at predetermined positions and frequencies. Variants were called using FreeBayes, LoFreq, Vardict, and VarScan2. The variant callers’ agreement in identifying known variants was quantified using two measures; concordance accuracy and the inter-caller concordance. Results: The variant callers reported differences in identifying minority variants from the datasets. Concordance accuracy and inter-caller concordance were positively correlated with sample coverage. FreeBayes identified the majority of variants although it was characterised by variable sensitivity and precision in addition to a high false positive rate relative to the other minority variant callers and which varied with sample coverage. LoFreq was the most conservative caller. Conclusions: We conducted a performance and concordance evaluation of four minority variant calling tools used to identify and quantify low frequency variants. Inconsistency in the quality of sequenced samples impacts on sensitivity and accuracy of minority variant callers. Our study suggests that combining at least three tools when identifying minority variants is useful in filtering errors when calling low frequency variants

Genomic analysis of respiratory syncytial virus infections in households and utility in inferring who infects the infant

Infants (under 1-year-old) are at most risk of life threatening respiratory syncytial virus (RSV) disease. RSV epidemiological data alone has been insufficient in defining who acquires infection from whom (WAIFW) within households. We investigated RSV genomic variation within and between infected individuals and assessed its potential utility in tracking transmission in households. Over an entire single RSV season in coastal Kenya, nasal swabs were collected from members of 20 households every 3-4 days regardless of symptom status and screened for RSV nucleic acid. Next generation sequencing was used to generate >90% RSV full-length genomes for 51.1% of positive samples (191/374). Single nucleotide polymorphisms (SNPs) observed during household infection outbreaks ranged from 0-21 (median: 3) while SNPs observed during single-host infection episodes ranged from 0-17 (median: 1). Using the viral genomic data alone there was insufficient resolution to fully reconstruct within-household transmission chains. For households with clear index cases, the most likely source of infant infection was via a toddler (aged 1 to <3 years-old) or school-aged (aged 6 to <12 years-old) co-occupant. However, for best resolution of WAIFW within households, we suggest an integrated analysis of RSV genomic and epidemiological data