Identification of New CRF51_01B in Singapore Using
Full Genome Analysis of Three HIV Type 1 Isolates
Oon Tek Ng,1,2Lindsay M. Eyzaguirre,3Jean K. Carr,4Kuan Kiat Chew,2Li Lin,2Arlene Chua,2
Yee Sin Leo,2Andrew D. Redd,5Thomas C. Quinn,1,5and Oliver Laeyendecker1,5
A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form,
which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of
these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B re-
combinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and
shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has
been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1
transmission in Singapore.
recombination is coinfection of a single cell, and by implica-
tion the human host, with genetic material originating from at
least two HIV-1 viruses belonging to different strains. In
Thailand and Malaysia, emergence of distinct CRF01_AE/B
circulating recombinant forms (CRFs) was documented
mainly among injection drug users.1–4
was performed to determine if there was any cross-border
transmission of the newly identified CRFs.5Unexpectedly, a
novel recombinant form, with subtype B in protease and gp41
and CRF01_AE in gp120, was identified in 13 (11.9%) of 109
From January 2011 to March 2011, three patients from the
prior study at the Singapore Communicable Disease Centre
outpatient clinic, identified as infected with the novel re-
combinant, were contacted to participate in this follow-up
and transmission risk factor data were obtained from clinical
chart review. The National Healthcare Group ethics com-
mittee approved this study.
After obtaining written, informed consent, peripheral ve-
nous blood was drawn for peripheral blood mononuclear cell
(PBMC) isolation using Ficoll-Paque Plus (GE Healthcare,
Waukesha, WI). PBMC DNA was extracted using the Qiagen
he emergence of HIV-1 recombinant forms is a mar-
ker of ongoing, active transmission.1A prerequisite for
DNA Mini Kit (Qiagen, Valencia, CA) and used for near full-
length proviral amplification using a hot start nested PCR
method. As previously described, approximately 9kb of the
for the first-round amplification and primers F2NST/UNIN-
EF7 for the second-round amplification.6,7Amplified prod-
uctsweresequenced usingtheABI 3130automated sequencer
(Applied Biosystems Inc, Foster City, CA). Sequences were
analyzed, edited, and assembled with Sequencher 4.6 (Gene
Codes Corporation, Ann Arbor, MI). Phylogenetic analysis
was performed using MacGDE and MEGA3.8–10Reference
sequences were downloaded from the Los Alamos HIV Se-
quence Database.11Recombination breakpoints were deter-
mined using SimPlot v.3.5.1, RIP, and jpHMM.12–14
All three patients were Singapore citizens of Chinese eth-
nicity (Table 1). The ages of the patients were 23, 36, and 48
years. The transmission risk factor reported was homosexual
in two and bisexual in one. Two of the three patients reported
sexual exposures only in Singapore. Information on the lo-
cation of sexual exposure for one of the patients was un-
available. There was no obvious epidemiologic link between
the study participants.
Using phylogenetic analysis, near full-length sequences of
the three patients formed a monophyletic branch supported
by a bootstrap value of 100%, separate from global subtypes
and known CRFs in Southeast Asia (Fig. 1). SimPlot analysis
1Johns Hopkins School of Medicine, Johns Hopkins Medical Institution, Baltimore, Maryland.
2Department of Infectious Disease, Tan Tock Seng Hospital, Singapore.
3Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland.
4St James School of Medicine, The Q_ uarter, Anguilla.
5Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National
Institutes of Health, Baltimore, Maryland.
AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 28, Number 5, 2012
ª Mary Ann Liebert, Inc.
revealed that the breakpoints corresponded to HXB2 nucleo-
tide positions 4300, 5200, 5800, and 7600 (Fig. 2). The break-
point were identical in all three isolates. Similar results were
obtained using RIP and jpHMM. Phylogenetic analysis of the
subgenomic fragments confirmed the four breakpoints iden-
tified (Fig. 3). The genomic fragments 796 to 4300, 4301 to
5200, 5201 to 5800, 5801 to 7600, and 7601 to 8701 branched
with reference subtype B, CRF01_AE, subtype B, CRF01_AE,
and subtype B, respectively.
In genomic regions I, III, and V, the Singapore strains form
a cluster distinct from the reference subtype B strains, sup-
ported by strong bootstrap values. A potential reason for this
would be that the subtype B progenitor of the CRF51_01B
recombinant had diverged significantly from the reference B
strains used. However, full-length subtype B sequences from
Singapore to test this hypothesis are not available. Another
potential reason could be the presence of CRF01_AE se-
quences withinthegenomic fragmentsas non-Bsegments less
than 100bp would potentially be undetected as a separate
recombinant fragment. As the new strains were phylogenet-
ically more similar to ‘‘western’’ B than B-prime reference
sequences, "western" B sequences were used for the genomic
region trees (data not shown).
This study confirmed the presence of a novel circulating
recombinant form, named CRF51_01B, in Singapore.15The
breakpoints identified in 11SG.HM021, 11SG.HM062, and
11SG.HM091 are identical among the three samples but were
distinct from the four CRF01_AE/B CRFs described to date in
Thailand and Malaysia (Fig. 1). As CRF51_01B has only been
described in Singapore and accounted for 11.9% of HIV-1
infections in our previous molecular epidemiology survey, it
is likely that CRF51_01B was generated within Singapore and
is currently established as a circulating strain. In our prior
study, this CRF had a higher prevalence among homosexuals
and bisexuals compared to subtype B and CRF01_AE.5Con-
tinual monitoring of the molecular epidemiology of HIV-1 in
Singapore will provide vital insight concerning local and
Table 1. Demographic Characteristics
for the Three Study Patients
mony tree representing the
relationship of the three near
Singapore with reference se-
quences. The scale denotes the
number of informative site
mutations. Bootstrap analysis
was performed with 100 rep-
licates. The taxon name of the
study sequences represents the
year of sample collection (11)
followed by country (SG) and
unique study identifier (e.g.,
HM021). The bootstrap value
at the node of the newly se-
quenced strains is shown.
528 NG ET AL.
A Singapore National Medical Research Training Fellow-
ship grant provided salary support for O.T. Ng. We ac-
service at the Communicable Disease Centre, Singapore, who
made this study possible. Additional support was provided
by the Division of Intramural Research, NIAID, NIH. We
thank Dr. Mark I.C. Chen, Mr. Ridzwan Abdullah, and Ms.
Tan Pei Ling for help with patient recruitment and sample
collection. O.T.N. and L.M.E. contributed equally to this
work. The sequences analyzed in this study have been de-
posited in GenBank under accession numbers JN029801,
JN029802, and JN029803.
the three Singapore isolates.
The subtype reference strains
used were Subtype B, HXB2
(black), CRF01_AE, CM240
(light gray), and subtype C,
length, based on HXB2, was
used following end trimming.
The bootscan window was
350bp with a step of 50bp.
The recombinant gene mo-
saic structure is shown at the
bottom of the figure with
sponding to the reference
Bootscan analysis of
for genome segments of the
methods described in Fig. 1,
performed based on recombi-
nation breakpoints shown in
Fig. 2. The genome segments
correspond to the following
HXB2 positions: (I) nts 796 to
4300; (II) nts 4301 to 5200; (III)
nts 5201 to 5800; (IV) nts 5801
to 7600; and (V) nts 7601 to
8701. The bootstrap value at
the node of the newly se-
quenced strains is shown.
NEW CRF51_01B IN SINGAPORE529
Author Disclosure Statement Download full-text
No competing financial interests exist.
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Address correspondence to:
Johns Hopkins University School of Medicine
Rangos Building, Room 527
855 North Wolfe Street
Baltimore, Maryland 21205
530 NG ET AL.