Detection of human bocavirus from children and adults with acute respiratory tract illness in Guangzhou, southern China

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.

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.

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).

The complete genome of HBoV from a 60-year-old female patient isolate was sequenced and analyzed. Sequencing primer sets were designed according to HBoV genome sequences available in the GenBank database (Table 1). DNA template (2 μl) was added to the Pfu DNA polymerase reaction mixture (Fermentas) in a final volume of 25 μl and PCR amplified. Products were purified after 1% agarose gel electrophoresis using a QIAquick Gel Extraction Kit (Qiagen). The purified DNA was then sequenced (ABI Genetic Analyzer 3130XL) and assembled using DNASTAR-SeqMan software (DNASTAR, http://​www.​dnastar.​com/​t-products-lasergene.​aspx). PCR amplification and sequencing were conducted at least twice to ensure sequence accuracy.

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.

HBoV DNA positive samples were detected in 65/2811 patients with a total positive rate of 2.3%. In children, positive rates were 4.1% (28/687) for 0-1 year-olds, > 4.8% (16/335) for 1-2 year-olds, 4.4% (10/225) for >2-3 year-olds, 1.7% (2/121) for >3-4 year-olds, 1.3% (1/80) for >4-5 year-olds, and 1.1% (4/349) for >5-17 year-olds (Figure 1), with a total of 3.4% (61/1797). The positive rate of adults (≥18 year-old) was 0.4% (4/1014) (Figure 1).

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.

During our 13-month study period, more than 100 samples were collected for detection each month, and the ratio of ≤3 year-old patients ranged from 20.2% to 66.5%. Low positive rates of 0.3% (1/369) (single HBoV: 0, co-pathogen: 0.3%) were detected in March 2010 and 0.7% (1/135) (single HBoV: 0.7%, co-pathogen: 0) in October 2010, while high positive rates of 4.8% (8/166) (single HBoV: 3.0%, co-pathogens: 1.8%) were found in May 2010 and 7.7% (11/143) (single HBoV: 1.4%, co-pathogens: 6.3%) in June 2010 (Figure 2). The positive rates of other seven months fluctuated around the total rate of 2.3% (65/2811) (Figure 2).

HBoV DNA positive samples were co-detected with 11 of 16 upper referred pathogens in 28/65 (43.1%) of the patients. Mycoplasma pneumoniae had the highest frequency of 16.9% (11/65), followed by RSV with 7.7% (5/65) (Table 2).

The male:female ratio was 42:23 in the HBoV-positive patients which did not differ significantly from the HBoV-negative patients (p = 0.40). The clinical characteristics of the patients are listed in Table 3. Most patients presented with symptoms of upper respiratory tract illness (URTI), including cough (93.8%) and expectoration (40.0%); 44 (67.7%) patients presented with symptoms of fever (≥38°C); 41 (63.1%) patients had lower respiratory tract illness symptoms (LRTI) and 19 (29.2%) patients were diagnosed as pneumonia by chest radiography; 10 (15.4%) patients had gastrointestinal symptoms; seven (10.8%) patients had systemic influenza-like symptoms (chilly, swirl, headache, myodynia or debilitation); and one (1.5%) patient had herpetic angina.

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 HBoV genome sequences for isolates from a 60 year-old patient were sequenced and submitted to the GeneBank (Accession Number:GU338055). The full length of the genome was 5299 bases and the distribution of A/G/C/T was 32.4%/20.4%/21.8%/25.4%, respectively. Compared to the other HBoV genomes available in the GeneBank database, GU330588 showed a 98% similarity with the HBoV previously described by Allander et al. [8]. Phylogenetic trees were inferred from VP1/VP2, NS1, and NP1 gene data, in addition to the complete genome sequence (Figure 3). GU330588 was similar to other HBoVs, although it displayed obviously sequence variations from HBoV2 and HBoV3. Three HBoV lineages were illustrated in all four phylogenetic trees (Figure 3).