Newly recognized bocaviruses (HBoV, HBoV2) in children and adults with gastrointestinal illness in the United States

Journal of Clinical Virology 47 (2010) 143–147

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Newly recognized bocaviruses (HBoV, HBoV2) in children and adults with gastrointestinal illness in the United States Brian D.W. Chow a,b , Zhen Ou c , Frank P. Esper a,c,∗ a Department of Pediatrics, University Hospitals – Case Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106, United States b Department of Internal Medicine, University Hospitals – Case Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106, United States c Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, United States

a r t i c l e

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Article history: Received 23 September 2009 Received in revised form 25 November 2009 Accepted 27 November 2009 Keywords: Bocavirus HBoV Viral gastroenteritis Gastrointestinal disease Gastroenteritis

a b s t r a c t Background: The human bocavirus (HBoV) is a newly recognized parvovirus associated with respiratory and gastrointestinal disease. Recently, two new members of the parvovirus family have been recognized, HBoV2 and HBoV3. Objectives: Here we investigate stool and respiratory samples for the presence of HBoV, HBoV2 and HBoV3. Study design: Stool samples collected from 12/1/2007 to 3/31/2008 were screened by PCR for the presence of HBoV, HBoV2, and HBoV3. Extracted DNA from respiratory specimens archived between 10/17/2005 and 3/29/2006 were screened by PCR for HBoV2 and HBoV3. Medical records for all bocavirus positive patients were reviewed. Results: Of 479 stool samples screened, 328 (68.5%) were from adults, and 151 (31.5%) were from children. Sixteen (3.4%) patients were positive for the presence of a bocavirus, including 10 (2.1%) HBoV and 6 (1.3%) HBoV2. No HBoV3 was detected in stool samples. Frequency of HBoV and HBoV2 in stool samples from children was 3.3% and 0.7%, and from adults was 1.5% and 1.5% respectively. Clinical findings in patients with HBoV and HBoV2 in stool include diarrhea (50% and 83.3%), abdominal pain (40%, 33.3%), and cough (10%, 50%). Of 868 respiratory samples screened, none were positive for either HBoV2 or HBoV3. Conclusions: The newly recognized parvovirus HBoV2 circulates in the United States. Patients with bocaviruses in stool have evidence of gastrointestinal illness. HBoV2 was not detected in respiratory samples. HBoV3 was not detected in either stool or respiratory samples. © 2009 Elsevier B.V. All rights reserved.

1. Background Gastroenteritis is a major cause of morbidity and mortality, with acute gastroenteritis causing 3 million deaths annually worldwide.1 Many viruses are associated with acute gastroenteritis, including rotaviruses, astroviruses, adenoviruses, and calciviruses.2–5 Despite sensitive molecular techniques, most cases of gastroenteritis remain without an identifiable cause, suggesting the presence of unrecognized pathogens.6–8 Parvoviruses have been recognized as a cause of gastrointestinal illness in numerous animal species. Diarrheal illness has been associated with parvovirus species in dogs and cats, and hepatitis in geese and hamsters.9 Prior to the description of human bocaviruses,

∗ Corresponding author at: Department of Pediatrics, Division of Pediatric Infectious Diseases, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106, United States. Tel.: +1 216 844 3645; fax: +1 216 844 8362. E-mail address: [email protected] (F.P. Esper). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.11.030

the only recognized human parvoviruses were parvovirus B19,9 PARV410 and PARV5.11 Since its discovery in 2005, the human bocavirus (HBoV) has been associated with upper and lower respiratory tract illness.12–18 HBoV is now implicated in gastrointestinal illness with 0.8–9.1% of stool samples screening positive for HBoV DNA.19–25 Recently, two viruses related to HBoV have been recognized, provisionally named human bocavirus 2 (HBoV2) and human bocavirus 3 (HBoV3). Human bocavirus 2 was discovered in stool samples from patients with acute flaccid paralysis in Pakistan.26 Soon thereafter, researchers in Australia identified HBoV2 as well as HBoV3.27 In genomic analysis, HBoV2 has between 67% and 80% nucleotide homology compared to HBoV.26 Analysis of the HBoV3 genome shows close homology to HBoV in the nonstructural protein encoding regions NS1 and NP-1 (87% nucleotide similarity), but is more similar to HBoV2 in the structural protein encoding regions VP1/VP2 (77% nucleotide similarity). This suggests that HBoV3 may have arisen from a recombination event between HBoV and HBoV2.26 The epidemiology and clinical man-

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Fig. 1. Human bocavirus primer locations. Diagrammatic representation of a typical bocavirus genome and primer sets used in screening and analysis. Locations based on the primer locations of the following isolates: HBoV (GenBank accession number NC 007455), HBoV2 (NC 012042), HBoV3 (NC 012564) and Bocavirus Consensus (Displayed primer position based on NC 007455; GC clamp underlined). Approximate primer locations are marked with black arrows on genomic map.

ifestations associated with these viruses are only now being realized.28 2. Objectives To investigate the presence of HBoV, HBoV2, and HBoV3 in patients with gastrointestinal illness and to determine if HBoV2 and HBoV3 have involvement with respiratory tract disease. 3. Study design 3.1.1. Gastrointestinal sample collection From December 1, 2007 to March 31, 2008, stool samples were collected from the core laboratory at University Hospitals – Case Medical Center of Cleveland, OH. We obtained all clinical specimens from children and adults that screened negative for Clostridium difficile A and B toxin by enzyme immunoassay (Meridian Bioscience, Cincinnati, OH). For each month within the study period, random samplings of stool specimens were selected for bocavirus screening. Other than age, no selection criteria were used. 3.1.2. Respiratory sample collection Archived DNA originating from pediatric and adult respiratory samples previously analyzed for HBoV15 were selected for HBoV2 and HBoV3 screening. DNA originated from patients presenting from October 17, 2005 to March 29, 2006 and have been stored at −20 ◦ C. All clinical specimens had negative results for RSV, parainfluenza viruses (1–3), influenza A and B, and adenovirus by direct immunofluorescence assay (DFA).

used.29 Bocavirus consensus primers were constructed targeting a conserved region of the NP-1 gene identified through ClustalW multiple sequence alignment of human and animal parvovirus sequences (Bioedit, Version 7.0.5.330 ). A GC clamp was applied to the consensus reverse primer to ensure compatibility of annealing temperature. Each sample was separately screened for the presence of HBoV, HBoV2 or HBoV3 by PCR using Platinum Taq Polymerase (Invitrogen, Carlsbad, CA) according to the manufacturer’s specification. Amplification conditions for all reactions were as follows: 95 ◦ C for 3 min; followed by 40 cycles of 94 ◦ C for 1 min, 55 ◦ C for 1 min (for bocavirus consensus primer reaction, annealing was set at 50 ◦ C), and 72 ◦ C for 30 s; and completed with a final extension cycle of 72 ◦ C for 10 min. Each set of polymerase chain reactions contained appropriate positive and negative controls. An amplicon of the HBoV3 NS1 region was generously provided by Jane Arthur and Rodney Ratcliff for use in this study. Sequence confirmation was performed on ABI Prism 3730 DNA Analyzer automated sequencers at the Genomics Core Facility, Case Western Reserve University School of Medicine. All isolates positive for bocaviruses were screened for the presence of common gastrointestinal viruses including adenovirus, rotavirus, and noroviruses by RT-PCR using primer sets and reaction conditions as described.31–33 3.1.4. Clinical data Medical records of all bocavirus positive patients were reviewed. Demographic data, history of illness, physical exam and laboratory studies were recorded on a standard collection form. Collection of specimens and clinical data was approved by the University Hospitals – Case Medical Center Human Investigation Committee and is compliant with the Health Insurance Portability and Accountability Act regulations. 3.1.5. Phylogenetic analysis Cleveland isolates of HBoV and HBoV2 were amplified using a bocavirus consensus primer set and sequenced. The region of analysis corresponded to a 307 base pair region of the NP1 gene spanning nucleotides 2593–2900 of the HBoV genome (Fig. 1). The phylogenetic analysis included representative samples of Cleveland isolates in addition to GenBank sequence data from original bocavirus prototype strains [HBoV, NC 007455; HBoV2, NC 012042; HBoV3, NC 012564] and animal parvoviruses [Bovine Parvovirus, DQ335247; Canine Minute Virus, NC 004442]. ClustalW alignment was generated using Bioedit 7.0.5.3 alignment software.30 One hundred bootstrap data sets were created using the PHYLIP program SEQBOOT. Phylogenetic analyses were constructed using the PHYLIP program DNAML, with the default transition to transversion ratio of 2.0 and 1 jumble.34 4. Results

3.1.3. DNA extraction and polymerase chain reaction (PCR) Nucleic acid from each stool specimen was extracted with the MagMAXTM -96 Total Nucleic Acid Isolation Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol using 0.3–0.5 g of stool. After disruption and centrifugation, 175 ␮l of supernatant were used for nucleic acid extraction. Primers used for HBoV screening originated from published reports15 (Fig. 1). Primers used for screening of HBoV2 and HBoV3 were constructed using GenBank sequences provided by the original reports.26,27 The sensitivity and specificity of bocavirus primer sets are unknown. Recent analysis suggest detection rates of HBoV by PCR is similar despite genes targeted or primer sets

Four hundred seventy-nine stool samples were collected between December 2007 and March 2008 corresponding to the peak prevalence of HBoV in several North American studies.15,35 Of these, 151 were from children (<18 years of age), and 328 from adults (≥18 years of age). Sixteen samples (3.4%) contained one of the bocavirus species; 6 from children, and 10 from adults (4.0% and 3.0% respectively) (Table 1). Ten (2.1%) stool samples screened positive for HBoV; 5 from children and 5 from adults (3.3% and 1.5% respectively). Six (1.3%) stool samples contained HBoV2; 1 from a child, and 5 from adults (0.7% and 1.5% respectively). No stool sample screened positive for multiple bocaviruses. HBoV3 was not detected in any stool samples.

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Table 1 Occurrence of HBoV, HBoV2, and HBoV3 in stool samples from adults and children. Pediatric, n = 151

Adult, n = 328

Total, n = 479

HBoV HBoV2 HBoV3

4 (2.6%) 1 (0.7%) 0 (0%)

5 (1.5%) 5 (1.5%) 0 (0%)

9 (1.9%) 6 (1.3%) 0 (0%)

Total

5 (3.3%)

10 (3.0%)

15 (3.1%)

The highest proportion of positive samples occurred in January, where 9 of 121 (7.4%) samples screened were positive for one of the human bocavirus species (Fig. 2). Of these, 6 (5.0%) were positive for HBoV and 3 (2.5%) were positive for HBoV2. No stool samples were positive for any bocavirus in March. Clinical characteristics of patients with HBoV and HBoV2 positive stool samples are outlined in Table 2. One child with HBoV was coinfected with adenovirus and was excluded from clinical analysis. Patients with HBoV in stool tended to be younger compared to HBoV2 (median age 23.4 yrs vs. 53.2 yrs). The median age of adults was similar (HBoV: 67.4 yrs; HBoV2: 66.8 yrs), as were the median ages for children (HBoV: 12 mo; HBoV2: 8 mo). Comorbid conditions were present at similar frequency in patients identified with HBoV and HBoV2. All patients with HBoV2 positive stool sample had gastrointestinal findings, compared to 66.7% of patients with HBoV. Gastrointestinal symptoms in patients with HBoV or HBoV2 include diarrhea (HBoV: 55.5%; HBoV2: 83.3%), nausea or emesis (HBoV: 22.2%; HBoV2: 33.3%) and abdominal pain, tenderness, or distention (HBoV: 44.4%, HBoV2: 33.3%). There were no substantial differences in the severity of gastrointestinal symptoms between children and adults. Many patients with HBoV and HBoV2 positive stool samples had respiratory findings. These include cough (HBoV: 11.1%; HBoV2:

Fig. 2. HBoV and HBoV2 positive stool samples by month. Monthly totals for each virus are tabulated below.

50%), tachypnea (HBoV: 33.3%) and hypoxia (HBoV: 33.3%; HBoV2: 33.3%). To determine whether HBoV2 or HBoV3 is involved with respiratory disease, we screened 868 archived respiratory samples. In this collection, 34 (3.9%) were positive for HBoV in our previous investigation.15 None screened positive for HBoV2 or HBoV3. Characteristics of patients with HBoV positive respiratory samples are reported elsewhere.15 There were similar rates of hospital admission in both groups (HBoV 66.7%; HBoV2 66.7%). The median hospital stay was slightly longer in patients with HBoV2 (19 days vs. 15 days). In both groups, children had a shorter median hospital stay. One patient was found

Table 2 Clinical characteristics of children and adults with HBoV and HBoV2 positive stool samples* . HBoV

HBoV2

Pediatric patients, n = 1 Adult patients, n = 5 All patients, n = 9

Pediatric patients, n = 1

Adult patients, n = 5 All patients, n = 6

Demographics Median age Male sex

12 mo 2 (50%)

67.4 yrs 4 (80%)

23.4 yrs 6 (86.7%)

8 mo 0 (0%)

66.8 yrs 3 (60%)

53.2 yrs 3 (50%)

Comorbidities Pulmonary diseasesa Cardiac diseasesb Immunodeficiencyc Any comorbidity

0 (0%) 1 (25%) 0 (0%) 1 (25%)

2 (40%) 3 (60%) 4 (80%) 4 (80%)

2 (22.2%) 4 (44.4%) 4 (44.4%) 5 (55.5%)

0 (0%) 0 (0%) 0 (0%) 0 (0%)

2 (40%) 1 (20%) 2 (40%) 3 (60%)

3 (50%) 1 (16.7%) 2 (33.3%) 3 (50%)

Gastrointestinal findings Feverd Diarrhea Nausea/emesis Abdominal complaintse Hypoactive bowel sounds Any gastrointestinal finding

1 (25%) 3 (75%) 2 (50%) 1 (25%) 1 (25%) 3 (75%)

1 (20%) 2 (40%) 0 (0%) 3 (60%) 1 (20%) 3 (60%)

2 (22.2%) 5 (55.5%) 2 (22.2%) 4 (44.4%) 2 (22.2%) 6 (66.7%)

1 (100%) 0 (0%) 1 (100%) 0 (0%) 0 (0%) 1 (100%)

2 (40%) 5 (100%) 1 (20%) 2 (40%) 0 (0%) 5 (100%)

3 (50%) 5 (83.3%) 2 (33.3%) 2 (33.3%) 0 (0%) 6 (100%)

Respiratory findings Cough Tachypnea Hypoxiaf Any respiratory finding

0 (0%) 2 (50%) 1 (25%) 2 (50%)

1 (20%) 1 (20%) 2 (40%) 4 (80%)

1(11.1%) 3 (33.3%) 3 (33.3%) 6 (66.6%)

1 (100%) 0 (0%) 0 (0%) 1 (100%)

2 (40%) 0 (0%) 2 (40%) 2 (40%)

3 (50%) 0 (0%) 2 (33.3%) 4(66.7%)

Disposition Admitted to hospital Median hospital days

2 (50%) 11.5

4 (80%) 15.5

6 (66.7%) 15

1 (100%) 16

3 (60%) 17.5

4 (66.7%) 19

* a b c d e f

Pediatric patients are <18 years of age, adult patients are ≥18 years of age. Includes chronic obstructive pulmonary disease and history of tracheostomy. Includes atrial fibrillation, coronary artery disease, mitral regurgitation, congestive heart failure, ventricular septal defect, and total anomalous pulmonary venous return. Includes diabetes, solid organ transplant (kidney), and malignancy. Clinical history of fever or documented temperature >38.0 ◦ C. Includes pain, tenderness, and distention. Pulse oximetry <92%.

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Fig. 3. Phylogenetic analysis of human and animal bocaviruses. Sequences of the bocavirus NP-1 gene from representative pediatric and adult isolates originating from Cleveland using a 307 pb region of the bocavirus NP-1 region. Additional strains of HBoV [NC 007455], HBoV2 [NC 012042], HBoV3 [NC 012564] and the animal parvoviruses: Canine Minute Virus [NC 004442] and Bovine Parvovirus [DQ335247] are included. Bootstrap values are displayed at major branch points.

to have HBoV2 in separate stool samples obtained 5 days apart during his hospitalization. 4.1. Phylogenetic analysis Phylogenetic analysis confirmed that strains of HBoV and HBoV2 which circulate in Cleveland, OH are similar to strains described worldwide. HBoV and HBoV3 are more closely related in the NP-1 region used in the analysis. There was no difference between bocavirus strains circulating in children and adults (Fig. 3). 5. Discussion We demonstrate the identification of HBoV and HBoV2 genomic DNA from stool samples of children and adults with gastroenteritis. This is the first report of HBoV2 infecting patients in the United States, suggesting that HBoV2 has a worldwide distribution. Numerous reports have recently detailed HBoV in patients with gastrointestinal illness.19,20,22,23,27,36 Recently, Arthur et al. performed a case–control study on acute gastroenteritis, examining stool specimens for potential pathogens, including HBoV, HBoV2 and HBoV3.27 They identified human bocaviruses as the second most common viral agents behind rotavirus (37.1%). In their study, a statistically significant association with acute gastroenteritis was detected with HBoV2, but not HBoV. HBoV3 was not present in enough samples to draw a definitive conclusion. However the authors caution that long term post-infectious shedding may account for detection in asymptomatic groups. Quantitative analysis finds the viral load of HBoV in stool is substantially lower than in respiratory tract specimens and may impact the sensitivity of detection.36

Similar to studies of respiratory illness, investigations of gastrointestinal illness find HBoV is commonly co-detected with recognized enteric pathogens, with coinfection rates ranging from 21% to 77.6%.21,24 Coinfection with multiple viral pathogens is not uncommon in studies of acute gastroenteritis.2 However, coinfection was not prominent in our study with only 10% of HBoV samples having an identified copathogen and none with HBoV2. Still, not all copathogens were investigated. Increased screening for other enteric pathogens may demonstrate a higher coinfection rate. HBoV is well described in association with respiratory disease.12,15,35 A recent report from China finds children with HBoV2 identified in stool samples often presented with cough, fever and wheezing.28 The absence of HBoV2 in respiratory samples in our study suggests this virus is not directly involved in respiratory tract illness and may predominantly be a gastrointestinal pathogen. Further investigations are needed to determine if HBoV2 may be associated with clinical manifestations outside the gastrointestinal tract. We were unable to detect the presence of HBoV3 in either gastrointestinal or respiratory samples. Possible explanations include primer mismatch secondary to genetic variation of local HBoV3 isolates, low circulation of the virus during the study period, and low frequency of occurrence. Further studies using larger sample sizes and year round sampling to detect HBoV3 are warranted. This study has several limitations, the most important being the lack of an asymptomatic control group. The presence of HBoV genetic sequences in stool samples suggests these viruses may play a role in enteric disease. However causality cannot be addressed through our study’s design. In addition, by selecting specimens from a tertiary care hospital, a bias towards finding individuals with more severe disease may occur. Investigations as to whether human bocaviruses play a role in mild disease should be undertaken. While the highest proportion of positive samples occurred in January, the study period limits our ability to determine seasonality. Further investigations which include the summer and fall seasons are warranted. Despite these shortcomings, our data suggests these viruses likely play a role in human gastroenteritis. In summary, we find that HBoV and HBoV2 are circulating in fecal samples from children and adults with gastrointestinal disease in Cleveland, OH. While HBoV is well described in association with respiratory disease, there is no evidence of HBoV2 in respiratory samples. HBoV3 was not detected in either stool or respiratory samples. This study aids our understanding as to the role these viruses play in gastrointestinal illness. Conflict of interest statement None of the authors report conflicts of interest. Acknowledgements We are indebted the staff of the Core Laboratory at University Hospitals Case Medical Center for their assistance and support. We appreciate the generosity of Jane Arthur and Rodney Ratcliff. References 1. King CK, Glass R, Bresee JS, Duggan C. Managing acute gastroenteritis among children: oral rehydration, maintenance, and nutritional therapy. MMWR Recomm Rep 2003;52(November(RR-16)):1–16. 2. Lyman WH, Walsh JF, Kotch JB, Weber DJ, Gunn E, Vinje J. Prospective study of etiologic agents of acute gastroenteritis outbreaks in child care centers. J Pediatr 2009;154(February(2)):253–7. 3. Vabret A, Dina J, Gouarin S, Petitjean J, Corbet S, Freymuth F. Detection of the new human coronavirus HKU1: a report of 6 cases. Clin Infect Dis 2006;42(March (5)):634–9.

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