Oliver Schildgen

Human Bocavirus: Passenger or Pathogen in Acute Respiratory Tract Infections?

SUMMARY Human bocavirus (HBoV) is a newly identified virus tentatively assigned to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. HBoV was first described in 2005 and has since been detected in respiratory tract secretions worldwide. Herein we review the literature on HBoV and discuss the biology and potential clinical impact of this virus. Most studies have been PCR based and performed on patients with acute respiratory symptoms, from whom HBoV was detected in 2 to 19% of the samples. HBoV-positive samples have been derived mainly from infants and young children. HBoV DNA has also been detected in the blood of patients with respiratory tract infection and in fecal samples of patients with diarrhea with or without concomitant respiratory symptoms. A characteristic feature of HBoV studies is the high frequency of coinciding detections, or codetections, with other viruses. Available data nevertheless indicate a statistical association between HBoV and acute respiratory tract disease. We present a model incorporating these somewhat contradictory findings and suggest that primary HBoV infection causes respiratory tract symptoms which can be followed by prolonged low-level virus shedding in the respiratory tract. Detection of the virus in this phase will be facilitated by other infections, either simply via increased sample cell count or via reactivation of HBoV, leading to an increased detection frequency of HBoV during other virus infections. We conclude that the majority of available HBoV studies are limited by the sole use of PCR diagnostics on respiratory tract secretions, addressing virus prevalence but not disease association. The ability to detect primary infection through the development of improved diagnostic methods will be of great importance for future studies seeking to assign a role for HBoV in causing respiratory illnesses.

Human Bocavirus Can Be Cultured in Differentiated Human Airway Epithelial Cells

ABSTRACT In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.

Frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction.

Background: Respiratory viruses are the main cause of acute respiratory tract infection (ARI) in children. Real-time polymerase chain reaction (PCR) technology is highly practicable for the rapid detection of viral pathogens. The simultaneous detection of a broad spectrum of viruses enables the diagnosis and evaluation of viral coinfection in ARI. Methods: A 1-step real-time PCR was developed for the detection of 12 respiratory viruses (10 RNA and 2 DNA viruses) in clinical samples. Clinical samples from 254 children admitted to the Departments of Pediatrics with ARI during a 10-month period were tested. Results: Respiratory syncytial virus (RSV) was the most frequently detected pathogen in 112 samples (44.1%), followed by human bocavirus (hBoV) in 49 (19.3%), and rhinovirus in 17 samples (6.7%). Viral coinfection was detected in 41 (16.1%) samples with RSV and hBoV being the most dominating combination (27 cases, 10.6%). Viral coinfection was found in 10 cases (17%) of children with bronchitis (n = 58) and in 7 cases (23%) of bronchiolitis (n = 30). In patients with pneumonia (n = 51), 17 cases (33%) were positive for 2 or more viral pathogens. Conclusions: Simultaneous testing of respiratory viruses by real-time PCR is a suitable tool for the detection of viral coinfections. In children hospitalized because of respiratory infection viral coinfection is frequently detected with RSV and hBoV being a common combination.

Human Metapneumovirus: Lessons Learned over the First Decade

SUMMARY It has been 10 years since human metapneumovirus (HMPV) was identified as a causative agent of respiratory illness in humans. Since then, numerous studies have contributed to a substantial body of knowledge on many aspects of HMPV. This review summarizes our current knowledge on HMPV, HMPV disease pathogenesis, and disease intervention strategies and identifies a number of areas with key questions to be addressed in the future.

Hospitalized children with respiratory syncytial virus infection and neuromuscular impairment face an increased risk of a complicated course

Background: Respiratory syncytial virus (RSV) infection is an important cause of viral respiratory tract infection in children. In contrast to other confirmed risk factors that predispose to a higher morbidity and mortality, the particular risk of a preexisting neuromuscular impairment (NMI) in hospitalized children with RSV infection has not been prospectively studied in a multicenter trial. Methods: The DMS RSV Paed database was designed for the prospective multicenter documentation and analysis of all clinically relevant aspects of the management of inpatients with RSV infection. Patients with clinically relevant NMI were identified according to the specific comments of the attending physicians and compared with those without NMI. Results: This study covers 6 consecutive seasons; the surveillance took place in 14 pediatric hospitals in Germany from 1999 to 2005. In total, 1568 RSV infections were prospectively documented in 1541 pediatric patients. Of these, 73 (4.7%) patients displayed a clinically relevant NMI; 41 (56%) NMI patients had at least 1 additional risk factor for a severe course of the infection (multiple risk factors in some patients; prematurity in 30, congenital heart disease in 19, chronic lung disease 6 and immunodeficiency in 8). Median age at diagnosis was higher in NMI patients (14 vs. 5 months); NMI patients had a greater risk of seizures (15.1% vs. 1.6%), and a higher proportion in the NMI group had to be mechanically ventilated (9.6% vs. 1.9%). Eventually, the attributable mortality was significantly higher in the NMI group (5.5% vs. 0.2%; P < 0.001 for all). Multivariate logistic regression confirmed that NMI was independently associated with pediatric intensive care unit (PICU) admission (OR, 4.94; 95% CI, 2.69–8.94; P < 0.001] and mechanical ventilation (OR, 3.85; 95% CI, 1.28–10.22; P = 0.017). Conclusion: This is the first prospective multicenter study confirming the hypothesis that children with clinically relevant NMI face an increased risk for severe RSV-disease. It seems reasonable to include NMI as a cofactor into the decision algorithm of passive immunization.

Norovirus outbreak in a pediatric oncology unit

Objective. Norovirus (NV) is an etiologic agent of outstanding importance that can cause severe epidemic gastroenteritis in day-care centers, schools, nursing homes, and hospitals. Therefore NV requires foremost attention as a pathogen responsible for epidemics of gastroenteritis in immunocompromised inpatients. In this study, a NV outbreak in a pediatric oncology unit is described and the consequences for this high-risk population are discussed. Material and methods. Stool and vomitus samples from 11 patients were tested for NV and other relevant viruses during the outbreak by reverse transcriptase-polymerase chain reaction (RT-PCR) and/or enzyme-linked immunosorbent assay (ELISA) (whenever an appropriate ELISA was available). Norwalk virus PCR amplifications were sequenced and phylogenetic analysis was performed.Results. The index patient and the chain of infection were identified. Follow-up investigation surprisingly demonstrated viral shedding for a maximum of 140 days (median 23 days). Three patients experienced severe or life-threatening symptoms, probably related to NV infection.Conclusions. In the event of an outbreak of gastroenteritis (involving two or more symptomatic patients) in a pediatric oncology unit, the search for NV in stool or vomitus specimens should be initiated in good time. As long as the data are limited regarding whether a detectable viral antigen or RNA in stools represents an infectious virus, patients have to be isolated as long as the diagnostic assays remain positive. During the acute phase of the illness, health-care workers should wear masks in addition to practicing meticulous hand hygiene with a disinfectant of proven activity against NV. Pediatric oncology patients must be closely monitored during follow-up investigations as they may shed the virus for months. There is some evidence from the outbreak described here that those patients face a greater risk of severe NV-related complications.

Human Metapneumovirus RNA in Encephalitis Patient

We describe a fatal case of encephalitis that might be correlated with primary human metapneumovirus (HMPV) encephalitis. Postmortem HMPV RNA was detected in brain and lung tissue samples from the patient. Furthermore, HMPV RNA was found in culture fluids from cells coincubated with lung tissue.

Successful therapy of hepatitis B with tenofovir in HIV-infected patients failing previous adefovir and lamivudine treatment

Three HIV-infected patients with chronic hepatitis B (genotype A) were switched to adefovir therapy after unsuccessful lamivudine treatment. Surprisingly, adefovir therapy failed, although none of the virus isolates displayed mutations known to be associated with adefovir resistance (A181V, N236T). In two isolates we identified hepatitis B virus DNA polymerase mutation L217R, in one case we found multiple frameshifts in the same region. In all cases adefovir was replaced by tenofovir, resulting in a significant drop in the viral load.

Prospective study of Human Bocavirus (HBoV) infection in a pediatric university hospital in Germany 2005/2006

Abstract Background Human Bocavirus (HBoV), a new species of the genus parvovirus newly detected in 2005, seems to be a worldwide distributed pathogen among children with respiratory tract infection (prevalence 2%–18%). Recently published retrospective studies and one prospective birth cohort study suggest that HBoV-primary infection occurs in infants. Methods Prospective single center study over one winter season (November 2005–May 2006) with hospitalized children without age restriction using PCR-based diagnostic methods. Results HBoV DNA was detected in 11 (2.8%) of 389 nasopharyngeal aspirates from symptomatic hospitalized children (median age 9.0 months; range: 3–17 months). RSV, HMPV, HCoV, and Influenza B were detected in 13.9% (n =54), 5.1% (n =20), 2.6% (n =10), and 1.8% (n =7), respectively. There was no influenza A DNA detected in any of the specimens. The clinical diagnoses were acute wheezing (bronchitis) in four patients, radiologically confirmed pneumonia in six patients (55%) and croup syndrome in one patient. In five to six patients with pneumonia, HBoV was the only pathogen detected. While no patient had to be mechanically ventilated, 73% needed oxygen supplementation. In four (36.4%) patients at least one other viral pathogen was found (plus RSV n =3; 27.3%; Norovirus n =1; 9.1%). Conclusion HBoV causes severe respiratory tract infections in infants and young children. Its role as a copathogen and many other open questions has to be defined in further prospective studies.

Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples

Parvoviruses are single stranded DNA viruses that replicate in a so called “rolling-hairpin” mechanism, a variant of the rolling circle replication known for bacteriophages like ϕX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus.