Novel Human Bocavirus in Children with Acute Respiratory Tract Infection

Human bocavirus (HBoV) and HBoV2, two human bocavirus species, were found in 18 and 10 of 235 nasopharyngeal aspirates, respectively, from children hospitalized with acute respiratory tract infection. Our results suggest that, like HBoV, HBoV2 is distributed worldwide and may be associated with respiratory and enteric diseases.


The Study
NPA samples were collected from 235 children hospitalized with ARTI at the First Hospital of Lanzhou University, Gansu Province, China during December 2007-November 2008. All patients were <15 years of age, and informed consent was obtained from their parents. Demographic data and clinical fi ndings were recorded. The study protocol was approved by the hospital ethics committee.
In total, 260 viruses were identifi ed in 196 (83.4%) of the 235 children. Using nested PCR, we found 21 positive specimens; further nucleotide sequence analysis showed that 10 (4.3%) were HBoV2 and 11 were HBoV (Figure, panel A). All 11 HBoV strains detected by using HBoV2 nested-PCR were included in the 18 HBoV-positive patients as determined by PCR using primers 188F and 542R. Of the 10 HBoV2-positive patients, 7 (70%) were co-infected with other respiratory viruses, including 4 patients with RSV. Of the 18 HBoV-positive patients, 12 (66.7%) displayed co-infections. There were no statistically signifi cant differences in the HBoV2 and HBoV detection (p = 0.119 by χ 2 test) and co-infection (p = 1.000 by Fisher exact test) rates.
HBoV infections were detected in every month except August, with peaks in December (3 cases) and January (4 cases). The main diagnoses of the 3 patients with HBoV2 monoinfection were acute asthmatic bronchopneumonia, bronchopneumonia, and acute upper respiratory tract infection in 1 patient each. For the 6 patients with HBoV monoinfection, the main diagnoses were acute asthmatic bronchopneumonia (4 cases) and bronchopneumonia (2 cases). The clinical signs and symptoms of HBoV2 and HBoV positive patients included cough, fever, sputum production, crack- les, wheezing, rhinorrhea, cyanosis, vomiting, and diarrhea (Table 1). For patients with HBoV2 monoinfection, the median hospital stay was 11.3 days (range 4-23 days), and 2 had underlying illnesses (idiopathic pulmonary hemosiderosis and iron defi ciency anemia). The chest radiograph of 1 patient showed upper middle zone air-space shadows.

Conclusions
Using nested PCR and sequencing, we identifi ed HBoV2 infections in 10 (4.3%) of 235 NPAs from children hospitalized with ARTI. Most of the patients were <3 years old. In HBoV2-positive patients, co-infection was high (70%), with RSV being the most common co-pathogen. Primers SN1 and SN2 were designed to detect the NP1 gene in the 10 HBoV2-positive patients. However, only 4 gave positive results, which occurred because of the low PCR sensitivity with this pair of primers and because the NP1 gene of HBoV2 is divergent, as described (6). Furthermore, as previous studies (6,12) pointed out, potential recombination upstream from the NP1 gene may explain the lower detection. Phylogenetic analysis showed that the NS-1 region of the HBoV2 strain (LZ480 and LZ578) clustered closely with that of the HBoV2 PK-2255 strain (FJ170279), and the NP-1 region clustered closely with that of the HBoV2 W153 strain (EU082213), suggesting potential recombination in the HBoV2 strains ( Figure). In addition, 11 HBoV sequences were amplifi ed by using nested-PCR for HBoV2. In the future, HBoV2-specifi c primers should be designed to investigate the prevalence of HBoV2 and its potential association with disease.
We found no difference in the clinical symptoms or length of hospital stay between the groups with HBoV2 and HBoV monoinfection, as well as between the groups with HBoV2 monoinfection and HBoV2 co-infection (Table 1). Statistical analysis indicated that HBoV2 and HBoV coinfection obviously did not correlate with disease severity (data not shown). Two of 3 patients with HBoV2 monoinfection had diarrhea with no vomiting (Table 1), and only 1 of 10 patients who were HBoV2 positive vomited. Further investigation is needed to exclude oral or inhaled gastric viruses as possible sources of NPA-associated HBoV2. Phylogenetic analysis showed a high degree of similarity between HBoV2 sequences found in China and those in other areas (Figure). Our results suggest that like HBoV, HBoV2 is distributed worldwide and may be associated with respiratory and enteric diseases. Additional studies are needed to confi rm the association between human bocavirus species (HBoV2 and HBoV) and respiratory tract infections or other diseases.