Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak

Abstract

BackgroundHigh genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.Methods and Principal FindingsWe deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.Conclusions and SignificanceDespite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.

High temporal and spatial diversity in marine RNA viruses implies that they have an important role in mortality and structuring plankton communities

“…Read abundance of OTUs can be considered semi-quantitative and good for comparisons of richness and diversity among samples (but not for absolute counts of genes) (Amend et al, 2010 ; Pinto and Raskin, 2012 ; Ibarbalz et al, 2014 ). Moreover, by using control sequences obtained by cloning and Sanger sequencing alongside pyrosequenced libraries containing the same sequence (Supplemental Methods, Figure S2 ) we verified that amplicon deep sequencing and our sequence processing methodology recovered accurate environmental viral sequences and non-inflated estimates of richness like in studies for bacterial amplicons (Sogin et al, 2006 ; Huse et al, 2008 ; Kirchman et al, 2010 ; Caporaso et al, 2011 ) and clinical viral studies (Romano et al, 2013 ; Watson et al, 2013 ).…”

Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data

“…We processed the data with simple thresholding and repeated our ANOVA analyses. If we remove minor allele frequencies using a threshold of ≤ 1 %, which is commonly done [ 24 ], we lose most of the minor alleles in our data and practically all significant effects in ANOVA . If we use a threshold of ≤ 0.05 %, the reported 454-platform substitution error rate [ 38 ], we retain more alleles and some significant effects, but still lose power to detect interactions at gene and genome scales.…”

“…The development of algorithms to differentiate between errors and actual genetic variants and/or to perform error correction in NGS data is an active area of research. These algorithms commonly utilize error thresholds [ 24 ] or Poisson/Binomial error distributions [ 18 , 19 , 25 , 26 ] which can be site-specific. Thresholds or error distribution parameters are fixed in a variety of ways; e.g.…”

“…Thresholds or error distribution parameters are fixed in a variety of ways; e.g. using values from existing literature [ 24 ], values derived from quality scores [ 19 , 26 ], or values estimated by computing errors from sequences of cloned samples obtained under conditions matching those of the samples under consideration [ 18 , 25 ].…”

Complete Genome Sequence of an Atypical Dengue Virus Serotype 2 Lineage Isolated in Brazil

“…The virus was PCR amplified using a previously described protocol ( 2 ) with three subsets of primers that covered the complete polyprotein. Fragments were sequenced using the Ion Torrent platform (Life Technologies).…”