Divergent picobirnaviruses in human feces.
ABSTRACT The near-complete genomes of two picobirnaviruses (PBVs) in diarrheal stool samples, human picobirnaviruses D and E (HuPBV-D and -E), were genetically characterized. Their RNA-dependent RNA polymerase (RdRp) protein sequences had <66% identities to known PBVs. Due to a single nucleotide insertion, the open reading frame 2 (ORF2) in segment 1 of HuPBV-D was interrupted by a stop codon. A small stem-loop structure overlying the stop codon may result in translational readthrough into the rest of ORF2.
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ABSTRACT: Next-generation sequencing (NGS) approaches rapidly produce millions to billions of short reads, which allow pathogen detection and discovery in human clinical, animal and environmental samples. A major limitation of sequence homology-based identification for highly divergent microorganisms is the short length of reads generated by most highly parallel sequencing technologies. Short reads require a high level of sequence similarities to annotated genes to confidently predict gene function or homology. Such recognition of highly divergent homologues can be improved by reference-free (de novo) assembly of short overlapping sequence reads into larger contigs. We describe an ensemble strategy that integrates the sequential use of various de Bruijn graph and overlap-layout-consensus assemblers with a novel partitioned sub-assembly approach. We also proposed new quality metrics that are suitable for evaluating metagenome de novo assembly. We demonstrate that this new ensemble strategy tested using in silico spike-in, clinical and environmental NGS datasets achieved significantly better contigs than current approaches. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 01/2015; · 8.81 Impact Factor
Divergent Picobirnaviruses in Human Feces
Terry Fei Fan Ng,a,bEverardo Vega,cNikola O. Kondov,aChristopher Markey,aXutao Deng,a,bNicole Gregoricus,cJan Vinjé,c
Blood Systems Research Institute, San Francisco, California, USAa; Department of Laboratory Medicine, University of California at San Francisco, San Francisco, California,
USAb; National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia, USAc
Received 10 April 2014 Accepted 1 May 2014 Published 15 May 2014
Citation Ng TFF, Vega E, Kondov NO, Markey C, Deng X, Gregoricus N, Vinjé J, Delwart E. 2014. Divergent picobirnaviruses in human feces. Genome Announc. 2(3):e00415-14.
Copyright © 2014 Ng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Address correspondence to Terry Fei Fan Ng, firstname.lastname@example.org, or Eric Delwart, email@example.com.
first identified in rats in 1988 (1) and partly sequenced first from
rabbits in 1999 and then from humans in 2000 (2, 3). The first
ple (4). PBVs have been reported in fecal and respiratory samples
in humans, other mammals, reptiles, and birds (5–9). The patho-
ment 1 sequences have been reported and only one fully charac-
terized human PBV genome sequence has been deposited in
GenBank. Most PBV sequences in GenBank include only a short
conserved region of the RNA-dependent RNA polymerase
(RdRp) gene in segment 2 (6).
A total of 62 fecal samples from 15 outbreaks of unexplained
diarrheal disease in humans with a typical viral gastroenteritis
epidemiology (10) were analyzed by viral metagenomics. The
pathogens (norovirus GI, GII, and GIV, sapovirus, astrovirus, ro-
tavirus, adenovirus, and enterovirus). Unbiased deep sequencing
using an Illumina Miseq platform was performed on enriched
viral particles according to previously described protocols (11,
12). BLASTx was used to identify viral sequences based on trans-
lated protein sequence similarity to virus sequences in GenBank.
nucleic acid reextraction, reverse transcription-PCR (RT-PCR),
and Sanger sequencing. Near-complete genomes from these
two human picobirnavirus were assembled. Both viruses
shared ?66% identities with other PBVs in the RdRP proteins,
including each other, reflecting highly distinct PBV species. To
distinguish these sequences from the three existing human pi-
cobirnavirus segment 1 sequences (complete [GenBank acces-
sion no. AB186897] and partial [GU968923 and AF246941]),
we named these human picobirnavirus D strain CDC23
(HuPBV-D-CDC23) and human picobirnavirus E strain
genome consisting of two double-stranded RNA segments
For HuPBV-E, segment 1 was partially sequenced (2,056
bases), encoding a nearly complete capsid protein, whereas seg-
ment 2 of HuPBV-E was completely sequenced (1,717 bases). A
near-complete genome was obtained for HuPBV-D, with 2,509
2,525 and 1,745 bases of the reference human PBV genome
(NC_007026). Segment 1 normally contains 3 open reading
stop codon in the middle of ORF2. We confirmed this insertion
and premature ORF2 stop codon using both Illumina (30? cov-
erage) and Sanger sequencing (4? coverage). A small stem-loop
structure (5-bp stem and 9-nucleotide loop) was predicted to
overlie the stop codon, indicating possible ribosomal frameshift-
ing and translational readthrough.
With the use of RT-PCR with specific primers, only one
patient from each diarrhea outbreak (among 2 and 3 tested
individuals, respectively) tested positive for HuPBV-D and -E,
suggesting a lack of direct association with diarrhea. A lack of
association between PBV detection and diarrhea has been pre-
viously reported (3, 5), although given the wide genetic diver-
sity of PBVs, it remains possible that certain genotypes are
pathogenic in susceptible populations such as immunodefi-
cient individuals (13).
Nucleotide sequence accession numbers. The genome se-
quences of HuPBV-D and -E have been submitted to GenBank
under the accession numbers KJ663813 to KJ663816.
We thank Sarah Ives for proofreading the manuscript.
This work was supported by NHLBI grant R01 HL105770 (to E.D.)
and the Blood Systems Research Institute.
The findings and conclusions in this article are those of the authors
and do not necessarily represent the views of the Centers for Disease
Control and Prevention.
Genome AnnouncementsMay/June 2014 Volume 2 Issue 3 e00415-14genomea.asm.org 1
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