ISOLATION OF HUMAN BOCAVIRUS FROM SWISS INFANTS WITH RESPIRATORY INFECTIONS

Abstract

Human bocavirus (HBoV) is a novel agent associated with respiratory symptoms in adults and children. We studied prospectively the first acute respiratory infection in a birth cohort of healthy neonates in Switzerland. HBoV was identified in 5 (4.5%) of 112 infants as young as 3 months of age. In 4 of the 5 infants, HBoV was associated with other respiratory viruses. We conclude that HBoV circulates in the community and is acquired early in life.

Human bocavirus (HBoV), a newly recognized parvovirus, has been identified in respiratory samples from children with lower respiratory tract disease in Sweden.¹ Subsequently, HBoV has been reported in stored respiratory samples from Australian, Japanese, Canadian and French children, suggesting that the virus may be circulating worldwide.^2–5 All previous studies reporting HBoV have been retrospective and hospital-based and did not target a predefined population. So our understanding of the symptoms associated with HBoV and of its epidemiologic pattern is limited. Infants are particularly prone to respiratory viral infections and are an important population to study the timing and the potential impact of new respiratory viruses. No study has assessed the potential impact of bocavirus infections in a prospective manner in this population.

To assess the role of HBoV in respiratory tract infection in otherwise healthy infants living in the community, we analyzed the first acute respiratory infection with cough or wheeze in a prospective birth cohort of healthy Swiss neonates between April 1999 and December 2004.⁶

MATERIALS AND METHODS

Neonates enrolled in the study were followed for any disease and its treatment by weekly telephonic standardized interviews focusing on respiratory symptoms using a previously described symptom score for standardized assessment of respiratory morbidity.⁷ An acute respiratory infection (ARI) was defined as more than 2 days with fever >38°C, acute rhinitis, otitis or pharyngitis together with cough or wheeze. A nasopharyngeal swab was collected at the first ARI as well as 3 weeks later and immediately stored at −80°C. Specimens were thawed and analyzed with specific reverse transcriptase–polymerase chain reaction assays for HBoV and for all other respiratory viruses that commonly infect humans, namely rhinovirus, coronavirus OC43, E229, NL63 and HKU1, parainfluenza 1, 2 and 3, respiratory syncytial virus (RSV) A and B, influenza A and B, adenovirus, human metapneumovirus and enterovirus.⁸ Polymerase chain reaction and detection of HBoV were performed using extraction procedures as previously described.⁸ Polymerase chain reaction was performed using 5 μL of extracted DNA, Taqman Universal Mastermix containing Rox passive reference (Applied Biosystems, PE Europe B.V., Rotkrenz, Switzerland) with an ABI Prism 7′900 HT (Applied Biosystems) under the following conditions: 2 minutes at 50°C, 10 minutes at 95°C and 55 cycles of 15 seconds at 95°C and 1 minute at 60°C. Selected primers and probe were directed to gene NS1. Primer forward 5′-CAC TGG CAG ACA ACT CAT CAC A-3′ and primer reverse 5′-GAT ATG AGC CCG AGC CTC TCT-3′ were used at 900 nM and 600 nM, respectively, and probe 5′-AGC AGG AGC CGC AGC CCG A-3′ at 200 nM. As positive control and to validate the analytic sensitivity of our polymerase chain reaction assay, we used a plasmid containing the sequence of HBoV isolate ST2 (DQ000496) produced and provided by Dr. Tobias Allander from the Karolinska Institute (Karolinska University Hospital, Stockholm, Sweden). The limit of detection of the polymerase chain reaction assay was estimated at 8 copies/5 μL of reaction mixture. The Ethics Committee of the University of Berne approved the study and written informed parental consent was obtained for all infants.

RESULTS

We analyzed the first ARI in 112 infants. HBoV was identified in 5 (4.5%) cases at the onset of respiratory symptoms (Table 1). HBoV infections occurred at a median age of 9 months (range, 3.4–11.3 months) with predominance during the winter months.

TABLE 1: Clinical Features of the Infants at the First Acute Respiratory Infection With Recovery of Human Bocavirus in Nasal Samples

Dual viral respiratory infections were documented in 3 cases: once with adenovirus, once with rhinovirus and once with RSV. In a fourth case, 3 respiratory viruses were recovered: HBoV, rhinovirus and coronavirus NL63. In one infant, HBoV was the only virus identified. All but one infant had not had upper respiratory tract symptoms in the 3 weeks preceding the onset of the ARI. In addition to cough, all infants had an acute rhinitis and 2 had fever. No infant had wheeze. All ARI were mild and did not require a visit to a physician nor the use of antibiotic or inhalation therapy, except the one in whom RSV was recovered in addition to HBoV. No infant with HBoV infection was hospitalized. Median duration of symptoms was 1 week (range, 1–3 weeks). HBoV was still detected in the follow-up sample 3 weeks after the acute episode in one infant (viral load in the follow-up sample comparable to the initial sample), whereas the infection had cleared in the other 4. In this infant presenting with a triple viral infection, HBoV only was detected in the follow-up sample.

DISCUSSION

We report for the first time HBoV infection in previously healthy infants living in the community, suggesting that the virus circulates in the community in an endemic fashion. In this prospective study, we show that bocavirus is temporally associated with acute respiratory symptoms in Swiss infants, thus confirming the worldwide distribution of HBoV and its likely association with respiratory disease.^1–5

The 4.5% incidence of HBoV in ARI observed in our study compares with that reported previously (from 1.5% to 5.7%) in children and adults with respiratory disease.^1–5 In accordance to previous studies,^1,3 we found a predominance of HBoV infections during the winter months, although others have observed a distribution of HBoV throughout the year.⁵

We found an association of HBoV with predominantly mild respiratory symptoms in infants as young as 3 months of age. This suggests that HBoV is acquired early in life and is not associated with severe disease in most infants. One case with HBoV infection had a severe respiratory disease course with need of medical advice, but in this case, RSV was concomitantly recovered, raising the possibility that the severity of symptoms in this case was mainly caused by RSV and not HBoV. Our findings are in accordance to those of Foulongne et al,⁴ who reported a relatively mild respiratory disease course in children <5 years of age infected with HBoV. In their study, dyspnea, cough and fever were the most common signs observed, and all 9 children recovered and were discharged from hospital within 6 days. Previous studies have also reported more severe airway disease associated with HBoV infections in hospitalized children, mostly wheezy bronchitis, pneumonia and bronchiolitis.^1–3,5

The presence of one or more other respiratory viruses in 4 of 5 cases in our study is puzzling. This rate of coinfections is high compared with other studies in which dual respiratory infections were usually found in fewer than 20% of cases.^9,10 It also contrasts with previous studies that have shown HBoV as the only infectious agent at the time of acute respiratory symptoms in most cases, although Sloots et al reported coinfections with other viruses in 56% of all HBoV-infected cases.^1–5 We used a panel of sensitive reverse transcriptase–polymerase chain reaction assays targeting a large number of respiratory viruses, which might have increased the detection rate of coinfections. On the other hand, these findings complicate our understanding of the pathophysiological role of HBoV because symptoms may have been caused by the other viruses recovered. Other DNA viruses like SEN virus, TT virus and hepatitis G virus have been identified as possible causes of human disease (non-A-to-E hepatitis), but their pathogenic role is still unclear.¹¹ A causal relationship between HBoV and respiratory symptoms thus needs to be formally established in prospective studies including appropriate control groups.

To date, HBoV has not been considered to be a cause of latent infection, but the biology of this DNA virus from the Parvoviridae family has not yet been described in detail and its potential ability to cause prolonged infections needs to be elucidated. In our study, HBoV could not be detected in respiratory samples 3 weeks after the onset of the ARI in all but one infant with HBoV infection, which suggests that the virus is rapidly cleared in most of the cases.

ACKNOWLEDGMENTS

The authors thank Christine Becher, Helen Gehr, Monika Graf, Gisela Wirz and Dr. David Baldwin for their help in obtaining the data presented. The authors also thank the patients and families that agreed to take part in the study.

Funding for this study provided by Swiss National Foundation Grants No. 32-68025.02 and 3200B0-10167/1 to U.F.