Human Bocavirus infection, Canada.

Human Bocavirus was detected in 18 (1.5%) of 1,209 respiratory specimens collected in 2003 and 2004 in Canada. The main symptoms of affected patients were cough (78%), fever (67%), and sore throat (44%). Nine patients were hospitalized; of these, 8 (89%) were <5 years of age.

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A new parvovlrus, human Bocavirus (HBoV), was recently identified in Sweden (1). The virus was identified in clinical specimens from infants and children with respiratory tract illness. Phylogenetic analyses of the complete genome of HBoV showed that that the virus is most closely related to canine minute virus and bovine parvovirus, which are members of the genus Bocavirus, family Parvoviridae (1). To date, the only parvovirus known to be pathogenic in humans is B 19, which is responsible for Fifth disease in children (2). The role of HBoV in respiratory tract illnesses is unknown. We retrospectively investigated HBoV in Canadian patients with acute respiratory infection (ARI) in 2003 and 2004 to assess the impact of HBoV infections on respiratory tract illnesses and identify the signs and symptoms of this illness.

The Study

A total of 1,209 specimens from patients with ARI from January 2003 to December 2004 were tested for HBoV. The specimens originated from the Saskatchewan provincial public health laboratories. Specimen types analyzed included throat swabs, nasopharyngeal swabs, nasopharyngeal aspirates, and auger suctions. All specimens were negative for influenza viruses A and B; parainfluenza viruses 1, 2, and 3; adenovirus; and respiratory syncytial virus (RSV) by direct or indirect fluorescence assays or virus isolation and for human metapneumovirus (HMPV) by reverse transcription-polymerase chain reaction. Specimens were collected from all age groups: 290 (24%) from those <5 years of age, 59 (5%) from those 6-10 years of age, 90 (7.4%) from those 11-15 years of age, 86 (7.1%) from those 16-20 years of age, 358 (29.6%) from those 21-50 years of age, and 324 (27%) from those >50 years of age, The age of the patients was unknown for 2 (0.2%) specimens.

HBoV was detected by polymerase chain reaction (PCR) using primers specific for 2 different regions of the genome. The screening primers 188F (2281-5'-GACCTCTGTAAGTACTATTAC-3'-2301) and 542R (2634-5'CTCTGTGTTGACTGAATACAG-3'-2614), reported by Allander et al. (1), were based on the sequence of the putative noncapsid protein 1 (NP-1) gene. The second set of primers, VP1/VP2F (4492-5'-GCAAACCCATCACTCTCAATGC-3'-4513) and VP1/VP2R (4895-5'-GCTCTCTCCTCCCAGTGACAT-3'-4875), was used for confirmation and was based on the published HBoV putative VP1/VP2 gene sequences (DQ000495) (1). Viral DNA was extracted from 285 [micro]L of original samples with a BioRobot MDx and the QiAamp Virus BioRobot MDX kit (Qiagen, Valencia, CA, USA). We used 5 [micro]L of DNA in a volume of 50 [micro]L containing 20 pmol of each primer. The thermocycler conditions were 95[degrees]C for 15 min for activation of HotStartTaq DNA polymerase (Qiagen); 35 cycles of 94[degrees]C for 1 min, 54[degrees]C for 1 min, and 72[degrees]C for 2 min; and extension at 72[degrees]C for 10 min. Nucleotide sequences of NP1 gene amplicons were determined with an ABI 377 Sequencer and a fluorescent dye terminator kit (Applied Biosystems, Foster City, CA, USA). DNA sequences were assembled and analyzed with SEQMAN, EDITSEQ, and MEGALIGN programs in Lasergene (DNASTAR, Madison, WI, USA). To avoid cross-contamination, specimen processing, DNA extraction, amplification, and analyses were conducted in different rooms. For DNA extraction and PCR procedures, we included 12 negative controls per 96-well plate.

A total of 18 (1.5%) of the 1,209 specimens tested were positive for HBoV by PCR. HBoV activity was found throughout the year with no apparent seasonal prevalence (Table 1). The sex distribution of patients was 61% (11) male and 39% (7) female (Table 2). Patients with HBoV ranged in age from 10 months to 60 years (median 11.5 years), and no significant difference in infection rates was observed between age groups.

The main clinical symptoms were cough (78%), fever (67%), and sore throat (44%) (Table 2). Other clinical symptoms included flulike symptoms (28%), headache (22%), nausea (17%), and myalgia (11%). Five patients had rhinitis, 1 had pneumonia, and 1 had bronchiolitis. One patient had rhinitis, bronchiolitis, and pneumonia, and 1 patient had rhinitis and pneumonia. Nine (50%) HBoV patients were hospitalized; 8 (89%) were [less than or equal to]5 years of age, and 1 was between 21 and 50 years of age. The incidence of lower respiratory tract infection was lower in outpatients: 1 with bronchiolitis and no pneumonia (Table 2). Although the infection rates were similar in all age groups, a significant increase in hospitalization rates was seen in those [less than or equal to]5 years of age compared with those >6 years of age (8/8 vs. 1/10, p = 0.001) (Table 2). All patients with pneumonia (3/3) and half of those with bronchiolitis (1/2) were also in this age group.

Nucleotide sequences were determined for nucleotides 2342-2581 that encode the NP-1 gene of HBoV (GenBank accession nos. DQ267760-DQ267775). No differences in nucleic acid sequences were found between different Canadian isolates. These isolates were also identical to 2 Swedish isolates (ST1 and ST2, GenBank accession nos. DQ000495-DQ000496) (1).

Conclusions

Although a causal relationship still needs to be demonstrated by including a control group of healthy persons, detection of HBoV in respiratory tract specimens from patients with undiagnosed ARI suggests that this virus may be associated with respiratory illness. This finding supports those of Allander et al. with regard to the association of HBoV with respiratory disease (1). It also demonstrates that HBoV was present in Canada in 2003 and 2004, which suggests that it may be circulating worldwide. Since this study used only samples from ARI patients who tested negative for influenza viruses A and B, parainfluenza viruses 1-3, adenovirus, RSV, and HMPV, dual infection cannot be excluded. In addition, whether HBoV is present asymptomatically in humans cannot be excluded because samples from healthy persons were not tested.

Allander et al. reported HBoV only in infants and children, which was probably the result of testing fewer specimens from adults patients (1). Most respiratory viruses show a seasonal distribution with peak activity in winter. Human parvovirus B 19, the only parvovirus that is pathogenic in humans, is also seasonal, with peak occurrences in spring and summer (3). In contrast, no seasonal prevalence was observed for HBoV infection; the virus was found throughout the year. The lack of seasonality observed for HBoV may have been caused by the low prevalence in this study. Thus, additional year-round studies are needed to better understand the epidemiology of HBoV. Most (89%) hospitalizations were in persons [less than or equal to]5 years of age, which suggests that HBoV may cause more severe respiratory illness in infants and children, similar to disease caused by RSV (4,5), HMPV (6, 7), human coronavirus NL63 (8-14), and human coronavirus 229E (15). More comprehensive studies with data on prevalence, risk factors, and use of health services are needed to determine the role of HBoV in ARI and its effect on the healthcare system.

This study was supported by the Public Health Agency of Canada.

Dr Bastien is a scientist at the National Microbiology Laboratory of the Public Health Agency of Canada in Winnipeg. Her research interests include the diagnosis and pathogenesis of respiratory viruses.

References

(1.) Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A. 2005;102: 12891-6.

(2.) Bloom MT, Young NS, 2001. Parvoviruses. In: Knipes DM, Howley PM, editors. Fields virology. Philadelphia: Lippincott Williams and Wilkins; 2001. p.2361-80.

(3.) Heegaard ED, Brown KE. Human parvovirus B19. Clin Microbiol Rev. 2002;15:485-505.

(4.) Collins PL, Chanock RW, Murphy BR. Respiratory syncytial virus. In: Knipe DL, HoMey PM, editors. Fields virology. Philadelphia: Lippincott Williams and Wilkins; 2001. p. 1443-86.

(5.) Glezen WE Greenberg SB, Atmar RL, Piedra PA, Couch RB. Impact of respiratory virus infections on persons with chronic underlying conditions. JAMA. 2000;283:499-505.

(6.) Bastien N, Ward D, van Caeseele P, Brandt K, Lee SH, McNabb G, et al. Human metapneumovirus infection in the Canadian population. J Clin Microbiol. 2003;41:4642-6.

(7.) Esper F, Martinello RA, Boucher D, Weibel C, Ferguson D, Landry ML, et al. A 1-year experience with human metapneumovirus in children aged <5 years. J Infect Dis. 2004;189:1388-96.

(8.) Arden KE, Nissen MD, Sloots TP, Mackay IM. New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia. J Med Virol. 2005;75:455-62.

(9.) Bastien N, Anderson K, Hart L, van Caeseele P, Brandt K, Milley D, et al. Human coronavirus NL63 infection in Canada. J Infect Dis. 2005;191:503-6.

(10.) Chiu SS, Chan KH, Chu KW, Kwan SW, Guan Y, Poon LL, et al. Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong, China. Clin Infect Dis. 2005;40:1721-9.

(11.) Ebihara T, Endo R, Ma X, Ishiguro N, Kikuta H. Detection of human coronavirus NL63 in young children with bronchiolitis. J Med Virol. 2005;75:463-5.

(12.) El Sahly HM, Atmar RL, Glezen WP, Greenberg SB. Spectrum of clinical illness in hospitalized patients with "common cold" virus infections. Clin Infect Dis. 2000;31:96-100.

(13.) McIntosh K, Chao RK, Krause HE, Wasil R, Mocega HE, Mufson MA. Coronavirus infection in acute lower respiratory tract disease of infants. J Infect Dis. 1974;130:502-7.

(14.) Van der Hoek L, Pyrc K, Jebbink MF, Vermeulen-Oost W, Berkhout RJ, Wolthers KC, et al. Identification of a new human coronavirus. Nat Med. 2004;10:368-73.

(15.) Pene F, Merlat A, Vabret A, Rozenberg F, Buzyn A, Dreyfus F, et al. Coronavirus 229E-related pneumonia in immunocompromised patients. Clin Infect Dis. 2003;37:929-32.

Nathalie Bastien,* Ken Brandt, [dagger] Kerry Dust,* Diane Ward, * and Yan Li *

* National Science Center for Human and Animal Health, Winnipeg, Manitoba, Canada; and [dagger] Saskatchewan Health, Regina, Saskatchewan, Canada

Address for correspondence: Yan Li, National Microbiology Laboratory, Canadian Science Center for Human and Animal Health, 1015 Arlington St, Winnipeg, Manitoba R3E 3R2, Canada; email: yan_li@phac-aspc. ge.ca

Table 1. Distribution of human Bocavirus--positive specimens by
month, Canada

Date    No. positive/no. tested

2003
  Jan            1/48
  Feb            2/51
  Mar            0/44
  Apr            1/49
  May            0/48
  Jun            4/54
  Jul            1/53
  Aug            0/46
  Sep            0/49
  Oct            0/51
  Nov            1/50
  Dec            1/50
2004
  Jan            0/50
  Feb            1/51
  Mar            0/48
  Apr            0/50
  May            0/50
  Jun            0/50
  Jul            1/50
  Aug            0/66
  Sep            1/52
  Oct            0/49
  Nov            3/50
  Dec            1/50

Table 2. Data from medical files of patients infected with human
Bocavirus, Canada *

                                      Patient
Specimen no.   Date collected   Sex   status     Age

883             Jan 17, 2003     M       O      23 y
947             Feb 5, 2003      M       H      9 mo
963             Feb 28, 2003     F       O      11 y
1029            Apr 10, 2003     F       O      16 y
1122            Jul 8, 2003      F       H       1 y
1166            Jun 10, 2003     M       O      17 y
1178            Jun 16, 2003     M       H      28 y
1179            Jun 16, 2003     F       H       3 y
1181            Jun 18, 2003     M       H       1 y
1368            Nov 1, 2003      F       O      60 y
1431            Dec 16, 2003     F       O      41 y
1545            Feb 24, 2004     M       H      10 mo
1776            Jul 12, 2004     M       H       2 y
1871            Sep 3, 2004      M       H      11 mo
1979            Nov 8, 2004      F       O      12 y
2013            Nov 28, 2004     M       H      9 mo
2016            Nov 25, 2004     F       O      14 y
2021            Dec 1, 2004      M       O      37 y

Specimen no.                          Symptoms

883                                 Fever, cough
947                   Fever, cough, nausea, rhinitis, pneumonia
963                      Fever, cough, sore throat, rhinitis
1029                            Sore throat, headache
1122                                Fever, cough
1166                            Sore throat, rhinitis
1178                                    Fever
1179                           Fever, cough, rhinitis
1181                           Fever, cough, rhinitis
1368             Cough, flulike symptoms, myalgia, headache, nausea
1431             Cough, sore throat, flulike symptoms, bronchiolitis
1545                           Fever, cough, pneumonia
1776                                    Fever
1871                  Cough, rhinitis, bronchiolitis, pneumonia
1979            Fever, cough, sore throat, flulike symptoms, headache
2013                                    Cough
2016                 Fever, cough, sore throat, flulike symptoms
2021            Fever, cough, sore throat, flulike symptoms, headache

* O, outpatient; H, hospitalized.