key: cord-0728020-oggmck8r authors: Wang, Kai; Wang, Wei; Yan, Huajie; Ren, Peijun; Zhang, Jing; Shen, Jun; Deubel, Vincent title: Correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection date: 2010-02-28 journal: Journal of Clinical Virology DOI: 10.1016/j.jcv.2009.11.015 sha: 3248603667ebc528d651336bff45e0b4cd208ae2 doc_id: 728020 cord_uid: oggmck8r Abstract Background Human bocavirus (HBoV), a recently discovered virus, is prevalent among children with respiratory tract infection throughout the world. Co-infection was frequently found in HBoV-positive patients. Thus, whether HBoV is responsible for the respiratory disease is still arguable. Objectives A comprehensive study was carried out to integrate clinical and virological prevalence in HBoV-positive outpatient children, and to determine genetic and serologic characteristics of HBoV in Shanghai, China. Study design Nasal/throat swabs and sera were obtained over a 2-year period from 817 children with respiratory tract infection to examine the presence of HBoV and its co-infection. The seroepidemiology of HBoV was studied by ELISA and Western blot against the capsid protein VP2-based fragment. Persistence of HBoV was also analyzed in 12 pairs of return-visit cases. Results HBoV was identified in 96 samples (11.8%). The co-infection rate with other respiratory viruses was 51%. IgM was detected in 55.7% of HBoV RT-PCR-positive patients, and in 72.7% of those who had high viral genome load. In addition, persistent viral DNA positivity was detected in 10 of 12 HBoV-positive cases tested, an average of 14 days later, and one child was still HBoV-positive after 31 days. Conclusion HBoV was found frequently in children with respiratory tract symptoms associated with other respiratory viruses, and persisted in the respiratory tract and in serum and urine. The presence of IgM was significantly more prevalent in viremic patients and those diagnosed with high load of HBoV DNA in nasal/throat swabs. In 2005, Human bocavirus (HBoV), a novel member in the genus Parvovirus, family Parvoviridae, was first identified in children with lower respiratory tract infection (LRTI). 1 Since then, the detection of HBoV in acute respiratory illness was reported worldwide, with prevalence rates between 1.5% and 19%. [2] [3] [4] Among parvoviruses, HBoV has the closest amino acid identity with bovine parvovirus (42%) and canine minute virus (43%). HBoV is a single-stranded DNA virus with a genome of around 5 kb, encoding 4 genes (NS1, NP1, VP1, and VP2). The majority of genetic variations occurred in the capsid VP1/VP2 genes, allowing a classification into two genotypes, ST1 and ST2. 1 HBoV was detected in various samples, including nasopharyngeal aspirate, 1 serum, 5,6 feces, 7, 8 and urine. 9 HBoV detection was significantly higher in patients with symptoms of respiratory tract or gastroenteritis than in asymptomatic individuals. 3, 5, [10] [11] [12] In addition, higher HBoV prevalence was observed in children aged from 6 months to 2 years, compared to older individuals. 2, 6, [13] [14] [15] Co-infection with other viruses was commonly observed in HBoVpositive patients with respiratory tract symptoms. 5, 6, [15] [16] [17] [18] Thus, whether HBoV is a major cause of respiratory disease remains questionable. Recently, several studies on the seroepidemiology of HBoV were reported by immunofluorescence assay, 19 Western blot, 20 or ELISA. [21] [22] [23] [24] [25] Anti-HBoV IgG was detected in a large fraction of the population, especially in older children and adults, while IgM was found predominantly in HBoV-positive patients. Only few studies have undertaken a comprehensive analysis of clinical, virological, and serological aspects in HBoV infection. 24, 25 The present study aimed at better characterizing the virological and serological profiles of HBoV in children with acute respiratory infection (ARI) in samples collected during a 2-year period in one hospital in Shanghai, China. Co-infecting respiratory viruses were analyzed, and serological diagnosis using a highly antigenic fragment of VP2 was developed. In addition, persistent HBoV shedding was assessed in nasal, throat, serum and urine specimens. -time PCR NP-1 5 -AGAGGCTCGGGCTCATATCA-3 5 -CACTTGGTCTGAGGTCTTCGAA-3 81 26 Probe 5 FAM-AGGACCACCCAATCARCCACCTATCGTCT-BHQ1 3 NS1 for conventional RT-PCR NS-1 5 -TATGGCCAAGGCAATCGTCCAAG-3 5 -GCCGCGTGAACATGAGAAACAG-3 245 13 VP1 for sequence VP1 5 -CCACTAGTATGCCTCCAATTAAGAGACAGC-3 5 -CCTCTAGATTACAACACTTTATTGATGTTTGT-3 2016 VP2 VP2-P1 5 -CCCAAGCTTGCATGTCTGACACTGACATTCAA-3 5 -CCGCTCGAGTTAATCACAAAAGATGTGAACGC-3 480 VP2 VP2-P2 5 -CCCAAGCTTGCATG CAAATACTTTCAAATGGTGCT-3 5 -CCGCTCGAGTTATGTGCTTCCGTTTTGTCTTAT-3 435 VP2 VP2-P3 5 -CCCAAGCTTGCATGCTAATGTTTAATCCAAAAGTC-3 5 -CCGCTCGAGTTA TCTGGTGATAGGAAATCTGTC-3 465 VP2 VP2-P4 5 -CCCAAGCTTGCATGCTATACAAAGGAAACCAAACC-3 5 -CCGCTCGAGTTACAACACTTTATTGATGTTTG-3 474 From October 2006 to September 2008, 817 samples were collected from children aged between 6 months and 9 years, who were diagnosed with an ARI with accompanying fever. All specimens were collected at the Shanghai Nanxiang Hospital, with informed consent from the children's parents. Standardized protocols accepted by the ethical committee of the hospital and reviewed by the medical committee of Institut Pasteur in Paris were used to record the patients' clinical symptoms and medical history, and for specimen collection. Nasal and throat swab samples were collected and stored in viral transport medium, and blood samples were collected in tubes containing 3.2% sodium citrate. The doctor who was involved in the study and did the sampling remained the same throughout the study. All samples were aliquoted at arrival in the laboratory and stored at −80 • C. Twelve patients who were diagnosed positive for HBoV were recalled between 8 and 31 days after the initial visit, and nasal swabs, throat swabs, blood, as well as urine samples were collected. Total RNA and DNA were extracted from 140 l of each specimen by QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. Nucleic acids were then eluted in 50 l RNase-free water and stored at −80 • C until use. The primers and the probe used to detect HBoV were targeted to the NP1 gene, as published previously (Table 1) . 26 The real-time PCR assays were performed using Takara's Premix Ex Taq TM Perfect Real Time (Takara Biotechnology, Dalian, China) in 20 l reaction mixture, containing 1 l of extracted sample, 0.2 M each of the forward and reverse primers, and 0.2 M of probe. PCR amplification included a step at 95 • C for 10 s followed by 40 cycles of 5 s at 95 • C and 30 s at 60 • C. The reactions were run and analyzed in an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA). The standard curve was obtained using serial dilutions of pskHBoV containing the whole genome of HBoV ST2 genotype (a kind gift from Dr Jianming Qiu, University of Missouri-Columbia, USA). The housekeeping gene GAPDH was detected as the internal control to monitor the quality of the specimens and to normalize HBoV DNA in clinical samples for comparative analysis. HBoV-positive specimens were also examined with conventional RT-PCR, using the One-Step RT-PCR kit (QIAGEN) that targets the NS1 gene, as described previously. 13 Multiplex RT-PCR, described previously, was used to diagnose other respiratory viruses in HBoV-positive samples, 27 The entire VP1 gene was amplified with primer pairs (Table 1) The phylogenetic tree was drawn by the Neighbor-joining method with bootstraps of 1000 replications. The recombinant bacmid pFastBac1 containing HBoV VP2 gene (kindly provided by Dr Jianming Qiu) was transfected into Spodoptera frugiperda Sf21 cells to recover the recombinant baculovirus. High Five cells were infected with the virus at a multiplicity of infection of five plaque-forming units/cell and cells were harvested 3 days later by centrifugation. The cell pellet was resuspended in 25 mM NaHCO 3 at a density of 2 × 10 7 cells/ml and left on ice for 30 min until the cells were completely lysed. Cell debris was removed by centrifugation at 13,000 rpm. The proteins were further precipitated in 20% ammonium sulfate, and resuspended in PBS. Four regions of VP2 were amplified by PCR from the pskHBoV clone. The forward and reverse primers (Table 1) contained Hind III and Xho I restriction sites, respectively. After digestion with the restriction enzymes, the amplicons were cloned into pET30a containing 6xHis tag at the 5 terminal. The recombinant proteins were expressed in E. coli BL21(DE3) host strains by induction with IPTG for 3 h. Cells were collected by centrifugation and the pellets were resuspended in lysis buffer and lysed by sonication on ice. The recombinant proteins were further purified by Ni-NTA agarose (Invitrogen) under denaturing conditions, following the manufacturer's instructions. The protein needed for ELISA was further dialyzed into PBS. Fifteen micrograms of each protein were loaded and separated on a 12.5% SDS-PAGE gel, and then transferred onto nitrocellulose membranes. After blocking for 2 h in 5% nonfat dry milk in TBS-T (0.5% Tween-20 in TBS buffer) blocking buffer, the membranes were incubated for 1 h in patient serum samples diluted in blocking buffer (dilution 1:50 for IgM test, 1:100 for IgG test). After incubation, the membranes were washed four times in TBS-T, and incubated in anti-human IgG or IgM conjugated with horseradish peroxidase (HRP) (Bethy Laboratories, Montgomery, TX, USA). After 1 h, the membranes were washed four times in TBS-T buffer and 3,3 -diaminobenzidine and H 2 O 2 were added to reveal the peroxidase activity. ELISA 96-well plates (Nunc TM , Roskilde, Denmark) were coated with 200 ng/well of purified proteins in coating buffer (0.05 M carbonate/bicarbonate, pH 9.6) overnight at 4 • C. After washing with PBS-T (0.05% Tween-20), the wells were saturated in 5% dry milk in PBS-T at 37 • C for 2 h. Serum samples diluted in 1% dry milk in PBS-T were added and incubated for 1 h at 37 • C. After washing, HRPconjugated anti-human IgG (1:10,000 diluted in dry milk in PBS-T) was added for 1 h at 37 • C. Plates were washed five times with PBS-T, and OPD (o-Phenylenediamine Dihydrochloride) (Sigma, St. Louis, MO, USA) was used as a substrate. After 20 min of incubation, the absorbance at OD 450 was read. All samples were measured in triplicate, and the average value was calculated for each serum sample. A purified heptapeptide fragment of SARS-HCoV spike protein produced in E. coli was used as a negative antigen (the corresponding expression plasmid pet30a-SARS spike was a kind gift of Dr. YongJin Wang, East Normal University, Shanghai, China). The threshold for positivity was calculated as twice the mean OD 450 value obtained when sera were tested against the SARS spike protein fragment. The statistical analysis was performed via SAS6.12 software (SAS institute Inc., Cary, NC, USA). The p value was measured by twosided analysis, and a p value less than 0.05 was considered to be statistically significant. Comparisons between different variations were evaluated by chi-squared or Fisher's test. During a 2-year period, a total of 817 pairs of nose and throat swabs were obtained from pediatric patients (male: 531, female: 286) with an age range from 6 months to 9 years. To detect HBoV, we first used conventional RT-PCR and then real-time PCR, targeting conserved NS1 and NP1 gene sequences, 13, 25 respectively. Samples with discrepant results between negative RT-PCR and positive real-time PCR were retested by increasing the amount Table 2 Multiple viral infections in HBoV-positive samples. Three virus infection (6 cases Table 2) . The HBoV loads detected in nose and throat swabs ranged from <500 to 10 8 DNA copies/ml, and those in serum ranged from <500 to 10 5 copies/ml. Thirteen (59.1%) of 22 patients showing high copies (>10 4 copies/ml) of HBoV in throat swabs were also positive in their serum, whereas only 8 (14.0%) out of 57 patients showing low copies of HBoV were positive in their serum (p = 0.00012). Forty patients (62.5%) among 64 with a low level of HBoV copies in their throat were co-infected with another virus, whereas only nine (28.1%) from 32 with high copies of HBoV were co-infected (p = 0.0022). HBoV was detected in 17.1% of children younger than 1 year, 14.3% in 1-2-year-old children, 8.2% in 2-4-year-olds, and 11.2% in older children. The gender ratio of HBoV-positive patients was 55% male and 45% female. The main clinical symptoms in HBoV-positive patients involved fever (100%), cough (74.7%), and wheezing (20.9%). No specific symptom was prevalent in the HBoV-positive patients ( Table 3 ). The number of HBoV RT-PCR-positive patients with bronchitis, however, was low (5.4%) when compared to the high prevalence of this disease (Table 3) . The VP1 gene of HBoV from 17 samples containing enough DNA/RNA material was amplified and sequenced. The nucleotide identity among different strains was over 99.3%. All strains tested in this study clustered with European, Asian and American strains into genotype ST2 (Fig. 1) . The VP2 capsid protein and four fragments of VP2 (designated P1 to P4) were first tested in a panel of serum samples by Western blots to detect HBoV-specific IgG and IgM antibodies. Eight of the samples contained high HBoV load, six contained low viral load, and six contained no HBoV. VP2 and its P3 fragment were the most immunoreactive with both IgG and IgM, and the P1 fragment was reactive mostly with IgM (Fig. 2) . Therefore, the 79 available serum samples from HBoV RT-PCR-positive patients were tested in Western blots against VP2 protein and P1 and P3 polypeptides (Table 4 ). The sera of patients were tested in ELISA using a purified P3 antigenic fragment derived from the VP2 protein, and SARS-HCoV (HR2 spike protein fragment) as a negative control. At a serum dilution of 1:100, the mean OD 450 value for negative controls using the SARS-HCoV spike HR2 fragment was 0.165. The assay was further confirmed by Western blots and the ELISA values were considered either negative (OD 450 < 0.33 corresponding to twice the mean value of the negative control), equivocal (OD 450 = 0.33-0.42), or positive (OD 450 > 0.42). In all, 713 serum samples were available to test HBoV-specific IgG antibodies. 314 samples (44.0%) were seropositive, and 95 (13.3%) were equivocal. The number of IgG-positive individuals increased with their age, with a statistical significance for age distribution (p < 0.0001) (Fig. 3) . Fewer children younger than 2 years old had anti-HBoV IgG (41.2%) than those older than 4 years old (67.93%) (p < 0.0001). In addition, 17 out of 29 sera showing an OD 450 value above 0.85 were from children older than 4 years. Twelve HBoV-positive patients were called back around 2 weeks after the first sampling. The mean interval of specimen collection was 16 days (days . None of these children showed any symptoms of ARI, and obviously recovered from their previous infection. However, HBoV could still be detected in nose and throat swabs, serum or urine (Table 5 ) from 10 children with a viral load ranging from less than 10 2 to 10 4 copies/ml. In all four specimens, the virus loads were lower or similar in the second samples compared to the first samples, and two samples were found to be free of HBoV. No urine samples were collected during the first sampling, and three urine specimens were positive for HBoV in return-visit samples. In one case, virus genes were still detected in nose/throat swabs as well as in urine collected 31 days apart. Seven serum samples showed a seroconversion for IgG and three for IgM. All but two samples contained IgM in return samples. An anti-HBoV IgG antibody response was detected in all but two patients showing low virus titer during the second visit. Co-infecting viruses found in four children at the first visit were absent at the second visit but HRV or ADV were newly detected in three children showing low levels of persistent HBoV (Table 5 ). In this study, the prevalence rate of HBoV-positive nonhospitalized infants with ARI was as high as 11.8%, and infants less than 2-year-old were more frequently positive than older ones. This is similar to that reported by other studies, [14] [15] [16] All the HBoV strains clustered in the ST2 genotype, as previously identified in Table 4 Antibody responses in sera (via Western blot) compared with viral loads in HBoV-positive samples. mainland China and Hong Kong in children with LRTI. 28, 29 The limited number of specimens studied, however, does not preclude the possible presence of the ST1 genotype of HBoV. Interestingly, higher HBoV positivity and load were observed in throat swabs (n = 96) than in nasal swabs (n = 78), suggesting an infection preferentially of the lower respiratory tract, as indicated by previous studies on bronchoalveolar lavage (Fig. 4) . 11, 30, 31 In addition, HBoV was detected in sera of infected children, as previously observed with other parvoviruses. 1 Consistent with other reports, samples with high copies of HBoV detected in nasal and throat swabs were more frequently positive in sera, compared with those with low copies, 5, 6, 20, 24 suggesting that viremia often occurs during acute infection. A high multi-infection rate was also detected in HBoV-infected patients (51.0%), with RSV and HRV having the highest prevalence. The high ratio of these two co-infecting viruses may reflect their prevalence in children visiting the Pediatrics Department of Shanghai Nanxiang Hospital with ARI (Wang and Deubel, unpublished data). In addition, the percentage of HBoV-positive patients with co-infecting viruses was 47.6%, suggesting a correlation between the presence of HBoV and that of other respiratory viruses (p < 0.0001). Patients with lower copies of HBoV were more frequently associated with co-infection, as previously reported. 32, 33 Conversely, data published in a recent paper 25 reported a higher co-infection rate in patients with high HBoV load. It was probably Previous studies reported that the majority of epitopes recognized by antibodies in HBoV-infected patients were located in the VP2 protein. 20 In this study, we divided VP2 into 4 fragments (P1-P4) and tested their antigenic reactivity by Western blot. Sixteen of 20 sera contained IgG/IgM antibodies that reacted with VP2 and its P3 fragment, and four did not show reactivity with VP2 and any of the four fragments. Strong immunoreactivity with IgG and IgM was observed for fragment P3, consistent with results with the whole VP2 protein. However, preparation of large amounts of purified P3 antigen in E. coli is easier and cheaper than that of VP2, which requires a more tedious preparation in the baculovirus system. Therefore, the purified P3 fragment of VP2 represents an alternative antigen for IgG detection in ELISA to test for acute or recent infection. It remains to be determined, however, whether this polypeptide is still recognized by long lasting antibodies to prevent false negative result for past HBoV infection. 20, 24, 33 The number of IgG-positive cases increased with the age of the children, suggesting that infection of HBoV likely occurred during childhood. The data validate other studies that showed a high number of IgGpositive serum samples in adults. 23, 24 Since some previous studies reported the absence or only few cases of anti-HBoV IgM in virusnegative samples from children, and rare positive results in healthy adults, 24, 25 only sera of patients confirmed with HBoV infection were tested by Western blot for IgM. Anti-HBoV IgM antibodies were likely detected more often in patients viremic for HBoV or with high copies of HBoV in nasal and throat swabs, as previously observed. 20, 24, 25 In 11 patients with viremia, high copy number in swabs, and infected only by HBoV, 10 (90.9%) were IgM-positive, which is consistent with previous studies. 20, 25 Patients infected singly with HBoV had comparatively more anti-HBoV IgM and IgG than multi-infection ones (p = 0.0119) ( Table 4 ). In contrast, in 57 patients with low viral load in swab samples, the amount of IgG or IgM remained at a low level (data not shown). Persistent HBoV shedding was reported in a few patients with symptoms lasting for several weeks, 9, 25 and in immunocompromised children. 4, 27, 34, 35 In this study, 12 patients were called for a second visit within an interval of 8-31 days (Table 5) . No fever or symptoms of the respiratory tract were noted in these patients. Low copies of HBoV were still found in nasal and throat swabs, serum, and urine, indicating that infection of HBoV may last for a long period of time at a low level, and in the presence of anti-HBoV antibodies (Table 5 ). This situation was also observed in parvovirus B19 infection with systemic and long-term persistent infection, high IgG reactivity and activated CD8+ T cell responses. 36 All ten patients with persistent PCR positivity of HBoV were found to be free of respiratory disease, but three were diagnosed with a secondary infection with ADV or HRV, suggesting that the persistence of HBoV was of less clinical significance, but may have played a role in promoting co-infection. Altogether, our study suggests that acute infection of HBoV causes systemic infection, induces immune responses, and may play a crucial role in respiratory disease of children, and is often associated with co-infection. Further studies are needed to understand the mechanism of the interactions between the host immune response, HBoV infection and prolonged virus shedding. Cloning of a human parvovirus by molecular screening of respiratory tract samples Human bocavirus Clinical and epidemiological aspects of human bocavirus infection Detection of human bocavirus in respiratory, fecal, and blood samples by realtime PCR Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand Human bocavirus and acute wheezing in children Real-time PCR for diagnosis of human bocavirus infections and phylogenetic analysis Human bocavirus, a respiratory and enteric virus High incidence of human bocavirus infection in children in Spain Human bocavirus infection in young children in the United States: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus Human bocavirus in Italian patients with respiratory diseases Human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection Evidence of human coronavirus HKU1 and human bocavirus in Australian children Detection of human bocavirus in Japanese children with lower respiratory tract infections Human bocavirus in Iranian children with acute respiratory infections Epidemiological profile and clinical associations of human bocavirus and other human parvoviruses The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children The incidence of human bocavirus infection among children admitted to hospital in Singapore Seroepidemiology of human bocavirus in Hokkaido prefecture Serodiagnosis of human bocavirus infection ELISAs using human bocavirus VP2 virus-like particles for detection of antibodies against HBoV Seroepidemiology of human bocavirus defined using recombinant virus-like particles CD4+ T helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults Humoral immune response against human bocavirus VP2 virus-like particles Clinical assessment and improved diagnosis of bocavirusinduced wheezing in children Realtime PCR assays for detection of bocavirus in human specimens Detection of human bocavirus in hospitalised children Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong Human bocavirus infection in children hospitalized with acute gastroenteritis in China Respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults High prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard PCR protocol and a novel real-time PCR protocol Human Bocavirus quantitative DNA detection in French children hospitalized for acute bronchiolitis Human bocavirus commonly involved in multiple viral airway infections Advances in the biology, diagnosis and host-pathogen interactions of parvovirus B19 Human bocavirus in children with acute lymphoblastic leukemia Prolonged activation of virus-specific CD8+ T cells after acute B19 infection We thank Dr Jianming Qiu, University of Missouri-Columbia, USA, for sharing the plasmid pskHBoV and bacmid pFastBac1 containing HBoV genes, and Dr Yongjin Wang, East China University, Shanghai, China, for providing the plasmid pet30a-SARS spike containing the SARS-HCoV heptapeptide HR2 gene. This work was supported by the Li Ka-Shing Foundation (RESPARI project), the French Agency for Development (SISEA project), Chinese Academy of Sciences visiting professorship for senior international scientists, and the National Science and Technology Major Project 2009ZX10004-105 (the establishment of pathogen and immunoresponse detection platforms for respiratory and central nervous system viral infections). WW is a recipient of a fellowship from the AREVA Foundation. The authors declare no conflict of interest.