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 Announcements May/June 2014 Volume 2 Issue 3 e00415-14genomea.asm.org 1
REFERENCES Download full-text
1. Pereira HG, Flewett TH, Candeias JA, Barth OM. 1988. A virus with a
bisegmented double-stranded RNA genome in rat (Oryzomys nigripes)
intestines. J. Gen. Virol. 69:2749–2754. http://dx.doi.org/10.1099/0022
2. Green J, Gallimore CI, Clewley JP, Brown DW. 1999. Genomic charac-
terization of the large segment of a rabbit picobirnavirus and comparison
with the atypical picobirnavirus of Cryptosporidium parvum. Arch. Virol.
3. Rosen BI, Fang ZY, Glass RI, Monroe SS. 2000. Cloning of human
picobirnavirus genomic segments and development of an RT-PCR de-
tection assay. Virology 277:316–329. http://dx.doi.org/10.1006/
4. Wakuda M, Pongsuwanna Y, Taniguchi K. 2005. Complete nucleotide
ods 126:165–169. http://dx.doi.org/10.1016/j.jviromet.2005.02.010.
5. van Leeuwen M, Williams MM, Koraka P, Simon JH, Smits SL, Oster-
haus AD. 2010. Human picobirnaviruses identified by molecular screen-
ing of diarrhea samples. J. Clin. Microbiol. 48:1787–1794. http://
6. Woo PC, Lau SK, Bai R, Teng JL, Lee P, Martelli P, Hui SW, Yuen KY.
birnavirus, discovered in California sea lions. J. Virol. 86:6377–6378.
7. Browning GF, Chalmers RM, Snodgrass DR, Batt RM, Hart CA, Or-
marod SE, Leadon D, Stoneham SJ, Rossdale PD. 1991. The prevalence
of enteric pathogens in diarrhoeic thoroughbred foals in Britain and Ire-
land. Equine Vet. J. 23:405–409. http://dx.doi.org/10.1111/j.2042
8. Pereira HG, Fialho AM, Flewett TH, Teixeira JM, Andrade ZP. 1988.
Novel viruses in human faeces. Lancet ii:103–104.
9. Masachessi G, Martínez LC, Giordano MO, Barril PA, Isa BM, Ferreyra
L, Villareal D, Carello M, Asis C, Nates SV. 2007. Picobirnavirus (PBV)
natural hosts in captivity and virus excretion pattern in infected animals.
Arch. Virol. 152:989–998. http://dx.doi.org/10.1007/s00705-006-0900-2.
10. Kaplan JE, Gary GW, Baron RC, Singh N, Schonberger LB, Feldman
R, Greenberg HB. 1982. Epidemiology of Norwalk gastroenteritis and
the role of Norwalk virus in outbreaks of acute nonbacterial gastroen-
teritis. Ann. Intern. Med. 96:756–761. http://dx.doi.org/10.7326/0003
11. Ng TF, Kondov NO, Hayashimoto N, Uchida R, Cha Y, Beyer AI,
Wong W, Pesavento PA, Suemizu H, Muench MO, Delwart E. 2013.
Identification of an astrovirus commonly infecting laboratory mice in
the US and Japan. PLoS One 8:e66937. http://dx.doi.org/10.1371/
12. Ng TF, Marine R, Wang C, Simmonds P, Kapusinszky B, Bodhidatta L,
Oderinde BS, Wommack KE, Delwart E. 2012. High variety of known
and new RNA and DNA viruses of diverse origins in untreated sewage. J.
Virol. 86:12161–12175. http://dx.doi.org/10.1128/JVI.00869-12.
13. Grohmann GS, Glass RI, Pereira HG, Monroe SS, Hightower AW,
Weber R, Bryan RT. 1993. Enteric viruses and diarrhea in HIV-infected
patients. Enteric Opportunistic Infections Working Group. N. Engl. J.
Ng et al.
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