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Detection of human bocavirus from children and adults with acute respiratory tract illness in Guangzhou, southern China
© Liu et al; licensee BioMed Central Ltd. 2011
Received: 27 February 2011
Accepted: 14 December 2011
Published: 14 December 2011
Human bocavirus (HBoV) is a newly discovered parvovirus associated with acute respiratory tract illness (ARTI) and gastrointestinal illness. Our study is the first to analyze the characteristics of HBoV-positive samples from ARTI patients with a wide age distribution from Guangzhou, southern China.
Throat swabs (n=2811) were collected and analyzed from children and adults with ARTI over a 13-month period. The HBoV complete genome from a 60 year-old female patient isolate was also determined.
HBoV DNA was detected in 65/2811 (2.3%) samples, of which 61/1797 were from children (<18 years old) and 4/1014 from adults (≥18 years old). Seasonal peaks of 4.8% and 7.7% were detected in May and June, respectively. 28 of 65 (43.1%) HBoV-positive samples were co-detected with 11/16 other potential pathogens. Mycoplasma pneumoniae had the highest frequency of 16.9% (11/65). Upper and lower respiratory tract illness were common symptoms, with 19/65 (29.2%) patients diagnosed with pneumonia by chest radiography. All four adult patients had systemic influenza-like symptoms. Phylogenetic analysis of the complete genome revealed a close relationship with other HBoVs, and a more distant relationship with HBoV2 and HBoV3.
HBoV was detected from children and adults with ARTI from Guangzhou, southern China. Elderly people were also susceptive to HBoV. A single lineage of HBoV was detected among a wide age distribution of patients with ARTI.
Respiratory tract infection etiology is complex and diverse, and new pathogens are continuously being reported. Over the past few years, several novel respiratory viruses including human metapneumovirus (hMPV) , severe acute respiratory syndrome (SARS) coronavirus , human coronavirus NL63 (HCoV-NL63) [3, 4], and coronavirus HKU1 (HCoV-HKU1) [5–7] have been identified.
In 2005, Allander et al.  reported a previously undescribed human parvovirus, human bocavirus (HBoV) that belongs to the genus Bocavirus, in respiratory secretions of children with respiratory tract disease in Sweden. HBoV is a single-stranded deoxyribonucleic acid (DNA) virus with a small genome size of approximately 5.3 kilo-bases (kb), which has three open reading frames (ORF) encoding two non-structural proteins NS1 and NP1, and the two structural proteins VP1 and VP2. VP1 and VP2 are located within the same ORF but have different initiator codon positions .
Subsequently, HBoV was reported in respiratory samples from different countries and regions worldwide [9–14], where HBoV was detected in 1.5%-8.3% of respiratory samples from individuals with acute respiratory tract illness (ARTI), especially young children and infants. The virus was also found in stool samples from patients with gastrointestinal illness [15–22]. These reports suggest that HBoV might be associated with upper and lower respiratory disease and gastrointestinal illness throughout the world. In 2009, two viruses closely related to HBoV, named HBoV2  and HBoV3 , were found in stool samples, and suggested HBoV diversity.
HBoV infection has recently attracted increasing attention all over the world. However, the incidence and clinical presentation of this infection varies widely, and often involves co-infection with other potential pathogens [9–22]. Such characteristics have led to debate over the role of HBoV as a true pathogen. Therefore, additional evidence and studies are needed throughout the world to gain a better understanding of this virus. In this study, 2811 respiratory samples were collected from patients (with an age range of 9 days to 84 years) with ARTI in Guangzhou, southern China, from November 2009 to November 2010 to analyze the characteristics of HBoV-positive patients.
Samples in this study were taken as part of standard care. The First Affiliated Hospital of Guangzhou Medical University Ethics Committee approved the experimental design and patient involvement in this study.
Respiratory samples collection
Throat swab samples (n = 2811) were collected from patients with ARTI (presented at least two of the following symptoms: cough, pharyngeal discomfort, rhinobyon, snivel, sneeze, dyspnea) at three hospitals in Guangzhou, southern China between November 2009 and November 2010. Patients' ages ranged from nine days to 84 years, and included 1797 children (<18-years-old) and 1014 adults (≥18-years-old). Clinical characteristics of the patients were recorded for further analysis.
Real-time polymerase chain reaction (PCR) for HBoV detection
DNA from respiratory samples was extracted using a QIAamp DNA Mini Kit (Qiagen), in accordance with the manufacturer's protocol. Taqman real-time PCR primers and probe were designed based on the conserved region of the NP1 gene. Sequences were as follows: forward primer, 5'- GAG AGA GGC TCG GGC TCA TA-3' (2545-2564 nt); reverse primer, 5'- TCG AAG CAG TGC AAG ACG AT-3' (2592-2611 nt); and probe, 5'-FAM- CAT CAG GAA CAC CCA ATC AGC CAC C-BHQ1-3' (2566-2590 nt). Primers and the probe were synthesized by TaKaRa. Premix Ex Taq (Perfect Real Time) real-time PCR reaction buffer was also purchased from TaKaRa. Amplification was conducted using 10 pmol of primers, 3 pmol of probe and 5 μl DNA in a final volume of 25 μl. Cycling conditions included an initial incubation at 94°C for 2 min, followed by 40 cycles of 94°C for 10 sec and 55°C for 35 sec (ABI-7500 real-time PCR instrument,Applied Biosystems). The amplified NP1 gene target sequence (2545-2611 nt) was inserted into the pMD18-T vector (TaKaRa) and used as a positive control for quantification analysis. Sensitivity of the PCR assay was calculated to be 10 copies of plasmid DNA using positive control plasmid diluted gradients.
Detection of common respiratory pathogens in HBoV-positive samples
HBoV DNA positive samples were tested for 16 other potential pathogens, including influenza A virus, influenza B virus, parainfluenza virus (1, 2, 3, 4), respiratory syncytial virus, adenovirus, enterovirus, human metapneumovirus, human coronavirus (229E, OC43, NL63, HKU1), Mycoplasma pneumoniae, and Chlamydia pneumoniae by Taqman real-time PCR, in accordance with the manufacturer's protocol (Guangzhou HuYanSuo Medical Technology Co., Ltd).
Complete genome sequencing
Primer sets for HBoV genome sequencing
Amplification position (bases)
The HBoV complete genome (GU338055) was aligned to other HBoV genomes available in the GenBank database using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of 18 complete genomes of HBoV, HBoV2 and HBoV3 from different countries and regions, including USA, Sweden, Thailand, Japan, Australia, China, Hong Kong, and Taiwan was conducted using Molecular Evolutionary Genetics Analysis Version 4.0 (MEGA 4.0, http://www.megasoftware.net/). Phylogenetic trees were inferred from VP1/VP2 (3056-5071 nt), NS1 (253-2172 nt), NP1 gene (2410-3069 nt) and complete genome data (1-5299 nt) using the neighbor-joining method, and bootstrap values were calculated from 1000 replicates.
For comparison of categorical data, χ2 test and Fisher's exact test where appropriate. All tests were two-tailed and p < 0.05 was considered statistical significant.
Detection of HBoV from patients with ARTI
The 65 positive patients were aged between 54 days and 70 years, comprising 83.1% (54/65) that were ≤ 3 years-old, 10.8% (7/65) that were 4-17 years-old, and 6.2% (4/65) adult patients. Four HBoV-positive adults were 19, 22, 60 and 70 years-old, respectively.
Seasonal distribution of HBoV
Co-detection with common respiratory pathogens
Co-pathogens of HBoV-positive patients
Patient No. (%)
Respiratory syncytial virus
Parainfluenza virus type 3
Influenza A virus
Human coronavirus OC43
Human coronavirus HKU1
Influenza B virus
Parainfluenza virus type 1
The clinical characteristics of HBoV-positive patients
Clinical characteristics of HBoV-positive patients
Abnormal pulmonary breathing sound c
Pharyngeal discomfort d
Lung markings increasing
Abnormal pulmonary breathing sounds and dyspnea were detected in 35 (53.9%) and 30 (46.2%) of 65 patients, respectively (Table 3). Nineteen (29.2%) were diagnosed as pneumonia by chest radiography, and 13/19 (68.4%) pneumonia patients were co-detected with one or two other pathogens, and a statistic difference was found for the symptom of "pneumonia" (p = 0.008) between the two groups "Single HBoV" and "Co-pathogens". Two of seven patients with systemic influenza-like symptom samples were co-detected with influenza A virus-enterovirus (triple pathogens) and influenza B virus (dual pathogens), respectively, while the remaining five patients were detected with single HBoV. All four adult patients (≥18 years old) presented with systemic influenza-like symptoms; three had only HBoV detection and the other 19 year-old female patient was co-detected with influenza B virus.
Complete genome sequencing and phylogenetic analysis
HBoV is a novel parvovirus first described in 2005 by Allander and colleagues . Since that time, it has been associated with upper and lower-respiratory tract disease and gastrointestinal illness throughout the world. However, most studies were focused on young children and infants [9–14], and only a few papers have described the characteristics of HBoV infection in adult patients [16, 25, 26]. Our study successfully analyzed the characteristics of HBoV-positive samples from ARTI-infected patients with a wide age distribution from Guangzhou, southern China for the first reported time. Similar to previous study , the detection rate in pediatric patients (<18 years old) was significantly higher than that in adults (≥18 years old) (p < 0.001), and most HBoV-positive patients were ≤3 years old. HBoV was detected in four adult patients, including 60 and 70 year-old patients. This suggested that older people were also susceptive to HBoV infection, although at much lower positive rates. Four adult patients also presented with systemic influenza-like symptoms, which might suggest that HBoV infection in adults is a more complex and serious problem than in children.
While seasonal peaks of HBoV infection vary among different counties and regions because of climate and other factors, many previous studies suggest a higher detection rate in winter [27, 28]. In our study, a higher frequency of HBoV was observed between May and June during the 13-month testing period (Figure 2). This result was similar to the report of Choi and colleagues, in which a seasonal peak was observed between May and June .
Characteristics of HBoV-positive patients in our study were also similar to previous reports [9–14]. The male:female ratio in the HBoV-positive patients did not differ significantly from the HBoV-negative patients. In all clinical characteristics, seven major symptoms (cough, fever (≥38°C), abnormal pulmonary breath sound, dyspnea, expectoration, snivel and pneumonia) had the highest ratio (ratio > 10%) (Table 3) and were common as URTI and LRTI.
As previous studies [13, 29–34], co-detection with other potential pathogens was common in HBoV-positive patients. In this work, 43.1% (28/65) patients were co-detected with other 16 potential pathogens, and there would be higher ratio if human rhinovirus were concerned . Furthermore, not only co-pathogens but also single HBoV groups had a high ratio of major symptoms (Table 3), which might suggest HBoV is an important pathogen in URTI and LRTI. A statistic difference was found for the symptom of "pneumonia" between the two groups "Single HBoV" and "Co-pathogens" (Table 3), which might further suggest HBoV is an important pathogen. Further studies were required to determine whether HBoV played a causative role in these co-infections or acted as an exacerbation factor.
Before 2009, little variation was found in the surface protein of HBoV (VP1-VP2), and some researchers predicted that HBoV infection might only occur after the subsequently development of life-long immunity via the neutralization of the target antibody . However, novel types of HBoV2 and HBoV3 were described successively in 2009 [23, 24], suggested diversity within this group of viruses. Furthermore, we successfully sequenced the complete HBoV genome (GU338055) for an isolate from an adult patient and phylogenetic analysis revealed the existence of three lineages: group I, HBoV; group II, HBoV2; group III, HBoV3, and GU338055 was located in group I.
The frequency and clinical presentations of HBoV infection vary widely [9–14, 16, 25, 26] and are typically associated with other pathogens [13, 29–34]. Such characteristics have subsequently led many scientists to question the role of HBoV as a potential pathogen. This lack of information is largely because of the inability of HBoV to grow in standard cell lines . To confirm the effects of HBoV, more studies are required throughout the world, focusing on various aspects of this infection, including epidemiology, serology, molecular biology, in vitro culture and animal models.
HBoV was detected from children and adults with ARTI from Guangzhou, southern China, and the features were described in this study. HBoV was confirmed in elderly patients (60 and 70 years old), suggesting that older people were also susceptive to HBoV. All four adult patients with HBoV positive in this study presented systemic influenza-like symptoms, which potentially suggest that HBoV infection in adults may develop more serious symptoms than those in children. Phylogenetic analysis suggested that HBoV-GU338055 from an elderly patient is in a single lineage with other HBoVs.
The State Major Infectious Disease Research Program (China Central Government, 2009ZX10004-109) provided financial support for this work.
- van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RAM, Osterhaus ADME: A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med. 2001, 7: 719-724.View ArticlePubMedGoogle Scholar
- Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, Lim W, Nicholls J, Yee WKS, Yan WW, Cheung MT, Cheng VCC, Chan KH, Tsang DNC, Yung RWH, Ng TK, Yuen KY: Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. 2003, 361: 1319-1325.View ArticlePubMedGoogle Scholar
- Fouchier RA, Hartwig NG, Bestebroer TM, Niemeyer B, de Jong JC, Simon JH, Osterhaus ADME: A previously undescribed coronavirus associated with respiratory disease in humans. Proc Natl Acad Sci USA. 2004, 101: 6212-6216.View ArticlePubMedPubMed CentralGoogle Scholar
- van der Hoek L, Pyrc K, Jebbink MF, Vertheim-Oost W, Berkhout RJM, Wolthers KC, van Dillen PMEW, Kaandorp J, Spaargaren J, Berkhout B: Identification of a new human coronavirus. Nat Med. 2004, 10: 368-373.View ArticlePubMedGoogle Scholar
- Woo PCY, Lau SKP, Tsoi HW, Huang Y, Poon RW, Chu CM, Lee RA, Luk WK, Wong GK, Wong BH, Cheng VC, Tang BS, Wu AK, Yung RW, Chen H, Guan Y, Chan KH, Yuen KY: Clinical and molecular epidemiological features of coronavirus HKU1-associated community-acquired pneumonia. J Infect Dis. 2005, 192: 1898-1907.View ArticlePubMedGoogle Scholar
- Woo PCY, Lau SKP, Chu CM, Chan KH, Tsoi HW, Huang Y, Wong BHL, Poon RWS, Cai JJ, Luk WK, Poon LLM, Wong SSY, Guan Y, Peiris GSM, Yuen KY: Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J Virol. 2005, 79: 884-895.View ArticlePubMedPubMed CentralGoogle Scholar
- Lau SKP, Woo PCY, Chu CM, Yip CCY, Tse H, Tsoi HW, Cheng VCC, Lee P, Tang BSF, Cheung CHY, Lee RA, So LY, Lau YL, Chan KH, Yuen KY: Coronavirus HKU1 and other coronavirus infections in Hong Kong. J Clin Microbiol. 2006, 44: 2063-2071.View ArticlePubMedPubMed CentralGoogle Scholar
- Allander T, Tammi MT, Eriksson M, Bjerkner A, Lindell AT, Andersson B: Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA. 2005, 102 (36): 12891-12896.View ArticlePubMedPubMed CentralGoogle Scholar
- Kesebir D, Vazquez M, Weibel C, Shapiro ED, Ferguson D, Landry ML, Kahn JS: Human bocavirus infection in young children in the United States: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus. J Infect Dis. 2006, 194: 1276-1282.View ArticlePubMedGoogle Scholar
- Fry AM, Lu X, Chittaganpitch M, Peret T, Fischer J, Dowell S, Anderson LJ, Erdman D, Olsen SJ: Human bocavirus: a novel parvovirus pidemiologically associated with pneumonia requiring hospitalization in Thailand. J Infect Dis. 2007, 195: 1038-1045.View ArticlePubMedGoogle Scholar
- Maggi F, Andreoli E, Pifferi M, Meschi S, Rocchi J, Bendinelli M: Human bocavirus in Italian patients with respiratory diseases. J Clin Virol. 2007, 38: 321-325.View ArticlePubMedGoogle Scholar
- Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay LM: Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol. 2006, 78: 1232-1240.View ArticlePubMedGoogle Scholar
- Arnold JC, Singh KK, Spector SA, Sawyer MH: Human bocavirus: prevalence and clinical spectrum at a children's hospital. Clin Infect Dis. 2006, 43: 283-288.View ArticlePubMedGoogle Scholar
- Qu XW, Duan ZJ, Qi ZY, Xie ZP, Gao HC, Liu WP, Huang CP, Peng FW, Zheng LS, Hou YD: Human bocavirus infection, People's Republic of China. Emerg Infect Dis. 2007, 13 (1): 165-168.View ArticlePubMedGoogle Scholar
- Chieochansin T, Thongmee C, Vimolket L, Theamboonlers A, Poovorawan Y: Human bocavirus infection in children with acute gastroenteritis and healthy controls. Jpn J Infect Dis. 2008, 61 (6): 479-481.PubMedGoogle Scholar
- Lau SK, Yip CC, Que TL, Lee RA, Au-yeung RKH, Zhou B, So LY, Lau YL, Chan KH, Woo PCY, Yuen KY: Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong. J Infect Dis. 2007, 196 (7): 986-993.View ArticlePubMedGoogle Scholar
- Lee JI, Chung JY, Han TH, Song MO, Hwang ES: Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis. 2007, 196 (7): 994-997.View ArticlePubMedGoogle Scholar
- Albuquerque MC, Rocha LN, Benati FJ, Soares CC, Maranhão AG, Ramírez ML, Erdman D, Santos N: Human bocavirus infection in children with gastroenteritis, Brazil. Emerg Infect Dis. 2007, 13 (11): 1756-1758.View ArticlePubMedPubMed CentralGoogle Scholar
- Cheng WX, Jin Y, Duan ZJ, Xu ZQ, Qi HM, Zhang Q, Yu JM, Zhu L, Jin M, Liu N, Cui SX, Li HY, Fang ZY: Human bocavirus in children hospitalized for acute gastroenteritis: a case-control study. Clin Infect Dis. 2008, 47 (2): 161-167.View ArticlePubMedGoogle Scholar
- Yu JM, Li DD, Xu ZQ, Cheng WX, Zhang Q, Li HY, Cui SX, Jin M, Yang SH, Fang ZY, Duan ZJ: Human bocavirus infection in children hospitalized with acute gastroenteritis in China. J Clin Virol. 2008, 42 (3): 280-285.View ArticlePubMedGoogle Scholar
- Campe H, Hartberger C, Sing A: Role of human bocavirus infections in outbreaks of gastroenteritis. J Clin Virol. 2008, 43 (3): 340-342.View ArticlePubMedGoogle Scholar
- Schildgen O, Muller A, Simon A: Human bocavirus and gastroenteritis. Emerg Infect Dis. 2007, 13 (10): 1620-1621.View ArticlePubMedPubMed CentralGoogle Scholar
- Kapoor A, Slikas E, Simmonds P, Chieochansin T, Naeem A, Shaukat S, Alam MM, Sharif S, Angez M, Zaidi S, Delwart E: A newly identified bocavirus species in human stool. J Infect Dis. 2009, 199 (2): 196-200.View ArticlePubMedPubMed CentralGoogle Scholar
- Arthur JL, Higgins GD, Davidson GP, Givney RC, Ratcliff RM: A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog. 2009, 5 (4): e1000391-View ArticlePubMedPubMed CentralGoogle Scholar
- Bastien N, Brandt K, Dust K, Ward D, Li Y: Human bocavirus infection, Canada. Emerg Infect Dis. 2006, 12: 848-850.View ArticlePubMedPubMed CentralGoogle Scholar
- Kupfer B, Vehreschild J, Cornely O, Kaiser R, Plum G, Viazov S, Franzen C, Tillmann RL, Simon A, Müller A, Schildgen O: Severe pneumonia and human bocavirus in adult. Emerg Infect Dis. 2006, 12: 1614-1616.View ArticlePubMedPubMed CentralGoogle Scholar
- Chow BD, Huang YT, Esper FP: Evidence of human bocavirus circulating in children and adults, Cleveland, Ohio. J Clin Virol. 2008, 43 (3): 302-306.View ArticlePubMedGoogle Scholar
- Bastien N, Chui N, Robinson JL, Lee BE, Dust K, Hart L, Li Y: Detection of human bocavirus in Canadian children in a 1-year study. J Clin Microbiol. 2007, 45 (2): 610-613.View ArticlePubMedGoogle Scholar
- Choi EH, Lee HJ, Kim SJ, Eun BW, Kim NH, Lee JA, Lee JH, Song EK, Kim SH, Park JY, Sung JY: The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children, 2000-2005. Clin Infect Dis. 2006, 43 (5): 585-592.View ArticlePubMedGoogle Scholar
- Sloots TP, McErlean P, Speicher DJ, Arden KE, Nissen MD, Mackay IM: Evidence of human coronavirus HKU1 and human bocavirus in Australian children. J Clin Virol. 2006, 35: 99-102.View ArticlePubMedGoogle Scholar
- Regamey N, Frey U, Deffernez C, Latzin P, Kaiser L: Isolation of human bocavirus from Swiss infants with respiratory infections. Swiss Paediatric Respiratory Research Group. Pediatr Infect Dis J. 2007, 26: 177-179.View ArticlePubMedGoogle Scholar
- Kaplan NM, Dove W, Abu-Zeid AF, Shamoon HE, Abd-Eldayem SA, Hart CA: Human bocavirus infection among children, Jordan. Emerg Infect Dis. 2006, 12: 1418-1420.View ArticlePubMedPubMed CentralGoogle Scholar
- Foulongne V, Olejnik Y, Perez V, Elaerts S, Rodiere M, Segondy M: Human bocavirus in French children. Emerg Infect Dis. 2006, 12: 1251-1253.View ArticlePubMedPubMed CentralGoogle Scholar
- Weissbrich B, Neske F, Schubert J, Tollmann F, Blath K, Blessing K, Kreth HW: Frequent detection of bocavirus DNA in German children with respiratory tract infections. BMC Infect Dis. 2006, 6: 109-View ArticlePubMedPubMed CentralGoogle Scholar
- Chow BD, Esper FP: The human bocaviurses: a review and discussion of their role in infection. Clin Lab Med. 2009, 29 (4): 695-713.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/345/prepub
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