Article Figures & Data
- FIG 1
Gel electrophoresis of PCR products. (A) Cyclovirus-like sequence PCR; (B) inverse PCR of cyclovirus. Lanes 1 and 8, 100-bp and 1-kb ladders, respectively; lanes 2 to 6, 9, and 10, patient samples; lanes 7 and 11, negative controls. Arrows indicate products of expected sizes. The thin black line indicates the spliced margin where the two unrelated lanes between lanes 10 and 11 were removed from the original gel picture.
- FIG 2
(A)Predicted genome organization of CyCV-VN; (B) stem-loop structure of CyCV-VN, with nonamer sequence in red.
- FIG 3
Reconstructed phylogeny trees of cycloviruses. (A) Tree based on complete genome sequences of 7 strains of CyCV-VN (red), 16 cycloviruses, 12 circoviruses, and a gyrovirus as an outlier. (B) Tree based on partial amino acid sequences of Rep proteins of 28 reported CyCV species and 27 CyCV-VN strains, indicated by the compressed branch. Trees were built by means of neighbor joining (MEGA 4.1); bootstrap tests of the reconstructed trees were done with 1,000 replicates. Sequence accession numbers are in brackets. CV, circovirus.
- TABLE 1
Oligonucleotide sequences of primers and a probe used in this study
Primers Oligonucleotide sequence (5′–3′) Source CyCV31_53F GAGCGCACATTGAAAGAGCTAAA Newly designed CyCV178-153R TCTCCTCCTTCAATGACAGAAACAAC Newly designed CyCV65-96Probe* FAM-CGADAATAAGGMATACTGCTCTAAAGSTGGCG-BHQ1 Newly designed CVF1 GGIAYICCICAYYTICARGG 3 CVR1 AWCCAICCRTARAARTCRTC 3 CVF2 GGIAYICCI CAYYTICARGGITT 3 CVR2 TGYTGYTCRTAICCRTCCCACCA 3 CyCV31_53F-rc TTTAGCTCTTTCAATGTGCGCTC Newly designed CyCV178-153R-rc GTTGTTTCTGTCATTGAAGGAGGAGA Newly designed CyCV_FL1 ACTTATTTCTAATTCATATTGCCGGGTA Newly designed CyCV_FL2 TCAGCGTCCAGCAGAATCTAC Newly designed CyCV_FL3 AAGCTCATCGTATTTGATCCATCC Newly designed CyCV_FL4 CCTCACTGAGCTATGTAAATTTGCT Newly designed - TABLE 2
Characteristics and clinical outcome of patients with CNS infections and positive for CyCV-VN by PCR
Group
and
patient
(n)aOther
confirmed
diagnosisbSample
dateNo. (%) of males
in group or sex
of patientc,dAge
(yr)dLength of
hospital
stay
(days)dIllness
dayNo. of patients withd: CSF white
blood cell
count% of
lympho-
cytes
in CSFCSF/blood
glucose
ratioOut-
comefSequelae
at
dischargeFever Convul-
sionsNeck
stiff-
nessLimb
weak-
nessHead-
acheVom-
itingGCS
scoreeGroup 1
(12)9
(75)19.5
(<1–50)8
(2–41)5
(2–20)11
(92)4
(33)7
(58)2
(18)6
(86)8
(89)40
(1–464)1 None Jan. 5, 2004 F <1 7 5 Y N N Y NA NA 2 (BCS) 1 0.88 1 None 2 None Jun. 8, 2004 M <1 8 7 Y N N N NA NA 3 (BCS) 3 0.53 1 None 3 None Feb. 7, 2004 M 12 8 3 Y N N N NA NA 6 26 90 0.96 1 None 4 None Oct. 18, 1999 M 27 5 7 Y N Y NA NA Y 15 54 68 0.55 1 None 5 None Jun. 12, 2008 M 31 2 2 Y Y N N Y Y 15 10 0.54 3 Unknown 6 None Nov. 25, 2009 F <2 9 2 Y Y N N NA Y 5 (BCS) 10 60 0.56 1 Unknown 7 None Nov. 29, 2008 M 20 41 20 Y Y Y Y Y Y 7 160 30 0.73 1 Unknown 8 None Sep. 4, 2009 M 15 15 9 Y N Y N Y Y 15 78 20 0.62 1 Unknown 9 None Sep. 7, 2009 F 50 15 7 Y N Y N Y Y 15 105 22 0.44 1 Unknown 10 None Nov. 18, 2009 M 19 5 5 Y Y Y N Y N 11 14 36 0.55 1 Unknown 11 None Mar. 31, 2008 M 43 3 5 N N Y N Y Y 9 78 20 0.57 3 Unknown 12 None Apr. 16, 2008 M 24 17 4 Y N Y N N Y 15 464 20 0.51 1 Unknown Group 2
(4)3
(75)14
(2–19)14
(4–38)5.5
(2–7)2
(50)3
(100)2
(50)1
(25)2
(100)2
(100)50 (0–110) 1 JEV Jan. 30 2004 M 2 4 2 N N N N NA NA 13 0 0.98 1 None 2 JEV Aug. 12 2004 M 11 5 5 Y N Y N NA NA 11 22 65 0.81 1 None 3 DENV Oct. 24 2008 M 19 38 7 N NA Y Y Y Y 9 79 21 0.41 1 Unknown 4 JEV May 05 2008 F 17 23 6 Y N N N Y Y 15 110 25 0.71 1 Unknown Group 3
(7)6
(85)5
(<1–61)19
(1–34)2
(1–3)6
(85)2
(28)5
(71)1
(14)4
(100)4
(57)1,439
(2–9,160)1 S. suis Aug. 31, 2008 M 29 21 1 N N Y N Y Y 15 3,500 0.04 1 Unknown 2 S. pneumoniae Nov. 17, 2008 M 25 16 2 Y N Y N Y Y 15 5,513 20 0.23 1 Unknown 3 H. influenzae Jan. 20, 2009 M <1 19 1 Y N Y N NA N 5 (BCS) 9,160 9 0.36 1 Unknown 4 S. pneumoniae Jan. 27, 2009 M 5 1 1 Y Y N Y Y Y 8 30 11 0.18 2 5 S. suis Mar. 16, 2009 F 61 34 3 Y N Y N NA N 11 1,439 28 0.19 1 Unknown 6 S. pneumoniae Apr. 17, 2009 M 2 4 2 Y Y N N NA N 0 (BCS) 2 0.65 1 Unknown 7 S. pneumoniae Dec. 23, 2009 M 3 20 2 Y N Y N y Y 11 80 33 0.95 1 Unknown Group 4
(5)1
(20)32
(29–68)4
(2–10)5
(3–7)4
(80)0 4
(80)0 NA NA 296
(100–950)1187 M. tuberculosis Aug. 4, 2009 F 30 4 7 Y N Y N NA NA 13 296 NA 0.18 3 Unknown 1847 None Feb. 16, 2009 M 32 10 3 Y N Y N NA NA 15 100 20 0.13 1 Unknown 1854 None Jul. 24, 2009 F 29 2 3 Y N N N NA NA 15 603 20 0.34 1 Unknown 2117 M. tuberculosis Feb. 9, 2010 F 55 NA 7 N Unknown Y N NA NA 7 950 60 NA 3 Unknown 2460 None Nov. 13, 2008 F 68 NA 5 Y N Y N NA NA 15 296 NA 0.72 3 Unknown ↵a Group 1, patients with CNS infections of unknown etiology; group 2, patients with laboratory-confirmed CNS infections; group 3, bacterial meningitis patients; group 4, PCR confirmed/clinically suspected tuberculosis meningitis patients. Names of provinces from which the patients were originated are intentionally not shown; patients 5 and 6 were the index cases in whose samples the CyCV-like sequence was detected by VIDISCA-454.
↵b For M. tuberculosis, denominators may vary. JEV, Japanese encephalitis virus; DENV, dengue virus.
↵c M, male; F, female.
↵d Data are numbers (percentages) of patients; continuous variables are presented as medians (ranges). NA, not available; Y, yes; N, no.
↵e Scores are on the Glasgow coma scale (GCS) unless the Blantyre coma scale (BCS) is indicated.
↵f 1, recovery; 2, death; 3, unknown.
- TABLE 3
Nonamer sequences of CyCV-VN and 15 different CyCV species and lengths of the stems
CyCV species Nonamer sequencea Loop length VN TAATACTAT 11 PK 5006 TAATACTAT 13 PK 5034 TAATACTAT 13 PK 5222 TAATACTAT 13 PK 5510 TAATACTAT 12 Chimp 11 TAATACTAT 13 NG12 TAATACTAT 13 NG 14 TAATACTAT 11 TN 18 TAATACTAT 12 NG chicken 8 TAATACTAA 12 PK goat 11 TAATACTAT 12 PK goat 21 TAATACTAG 13 TB bat USA TAATACTAT 12 Bat GF-4c TAATACTAT 11 NG 13 TAGTATTAC 9 DfCyCV-A1 TAATACTAT 13 FWCasCyV-GS140 TAGTATTAC 11 ↵a Underlined nucleotides are those that differ from the consensus sequence.
- TABLE 4
Degree of sequence identities between CyCV-VN and other cyclovirusesa
CyCV species % identity of: Host Complete genome (nt level) Cap protein (aa level) Rep protein (aa level) USAbat-TB/2009 46 22 51 Bats BatGF-4c 45 22 50 Bats Chimp12 43 24 46 Chimpanzees NG12 44 26 51 Humans NG13 40 14 43 Humans NG14 44 22 50 Humans NGchicken15/2009 45 23 49 Chickens PK5006 46 22 48 Humans PK5034 44 23 51 Humans PK5222 44 30 49 Humans PK5510 46 28 51 Humans PKgoat11/2009 44 25 49 Sheep PKgoat21/2009 67 50 70 Sheep TN18 70 48 74 Humans DfCyCV-A1 50 30 47 Dragonflies FWCasCyV-GS140 45 23 33 Cockroaches NG23 NA NA 50 Humans Chimp13 NA NA 51 Chimpanzees PK5192 NA NA 40 Humans PKgoat24 NA NA 30 Sheep Pkbeef25 NA NA 76 Bovines TN12 NA NA 46 Humans TN9 NA NA 46 Humans TN26 NA NA 49 Humans NG6 NA NA 46 Humans NG15 NA NA 46 Humans Chimp73 NA NA 45 Chimpanzees TN6 NA NA 46 Humans Chimp53 NA NA 48 Chimpanzees ↵a NA, not available. Sequence accession numbers are shown in Fig. 3.
- TABLE 5
Samples for PCR screening of CyCV-VN
Samplea Disease/syndrome No. Collection period Hospitalb Hospital location CSFc CNS 642 1999-2009 —a Southern and central Vietnam CSFd Noninfectious condition 122 1997-2011 Hospital for Tropical Diseases and Cho Ray Hospital Ho Chi Minh City, southern Vietnam Bloode Unknown 90 2009-2010 Hue Central Hospital Hue City, central Vietnam Human feces Healthy children 188 2011 NA Southern Vietnam Animal fecesf NA 65 2011 NA Dong Thap Province, southern Vietnam ↵a Prior to PCR screening, nucleic acid was isolated from clinical samples with use of the easyMAG (bioMérieux, Marcy l’Étoile, France) or the MagNA pure96 system (Roche), following the manufacturer’s instructions.
↵b The hospitals from which the noninfectious CSF and blood samples were collected are referral hospitals for southern and central provinces in Vietnam. NA, not applicable.
↵c From children and adult patients enrolled in the BMD, PVE, or 01SS study (see Materials and Methods for study details).
↵d Indications for lumbar puncture included headache/migraine (n = 17), trauma (n = 16), postoperative CSF leakage (n = 2), tumors (n = 13), benign intracranial hypertension (n = 1), epilepsy/convulsion (n = 15), encephalopathy (n = 5), external ventricular draining (n = 4), hemorrhage/stroke (n = 29), Guillain-Barré syndrome (n = 3), cranial nerve palsies (n = 6), and others (n = 11). The patients included both children and adults [age median (range): 36 (1 to 91) years; 67% male], and they were from 26 provinces in central or southern Vietnam.
↵e Anonymized residual blood samples from an influenza sero-surveillance study.
↵f Including samples collected from individual animals and boot swabs—from pigs (n = 20), chickens (n = 12), and ducks (n = 33).
Supplemental Material
Figure S1
Nucleotide sequence alignment showing sequence identity between primers/probe of CyCV-VN PCR used and CyCV-VN and CyCV-20 (including CyCV-TN8, -TN15, -TN16, -TN18, -TN22, and -TN25) sequences. Degenerate nucleotides: D = A, G, or T; M = A or C; S = G or C. Download Figure S1, TIF file, 1.2 MB.
Copyright © 2013 Tan et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
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Supplementary Data
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