Cloning of a human parvovirus by molecular screening of respiratory tract samples

Abstract

The identification of new virus species is a key issue for the study of infectious disease but is technically very difficult. We developed a system for large-scale molecular virus screening of clinical samples based on host DNA depletion, random PCR amplification, large-scale sequencing, and bioinformatics. The technology was applied to pooled human respiratory tract samples. The first experiments detected seven human virus species without the use of any specific reagent. Among the detected viruses were one coronavirus and one parvovirus, both of which were at that time uncharacterized. The parvovirus, provisionally named human bocavirus, was in a retrospective clinical study detected in 17 additional patients and associated with lower respiratory tract infections in children. The molecular virus screening procedure provides a general culture-independent solution to the problem of detecting unknown virus species in single or pooled samples. We suggest that a systematic exploration of the viruses that infect humans, ''the human virome,'' can be initiated.bioinformatics ͉ nucleotide sequencing ͉ respiratory tract infection ͉ virus V irus infections impose an enormous disease burden on humanity, but our knowledge of the viruses that infect humans is still incomplete. Because of the elusive nature of viruses, most studies are limited to the investigation of already known viruses, whereas the discovery of an unknown virus and production of the first diagnostic reagent is very difficult and remains a rare occurrence. The majority of viruses known today were first identified by animal experiments or virus replication in tissue culture. We know that there are viruses that cannot be replicated in the laboratory, and some of these have been identified by molecular methods (1-4). No generally applicable method for the identification of such viruses has been available, and, in fact, a very large number of unidentified human viruses may exist (2). Thus, numerous acute and chronic diseases with unknown etiology may be caused by unidentified viruses, and the systematic search for unknown viruses is an urgent scientific task (2).Lower respiratory tract infection (LRTI) is a leading cause for hospitalization of infants and young children and accounts for Ϸ250,000 hospitalizations a year in the United States alone (5). The most important viral agent in this group of patients is respiratory syncytial virus (RSV). Other important agents are influenza viruses, parainfluenza viruses, adenoviruses, rhinoviruses, coronaviruses, and human metapneumovirus (6-8). Human metapneumovirus was cultured and characterized in 2001, and, more recently, a renewed interest in coronaviruses after the severe acute respiratory syndrome epidemic has led to the characterization of two coronavirus species associated with LRTI (9-11). In comprehensive studies of the etiology of LRTI, no etiologic agent has been found in 12-39% of cases, which suggests that additional unknown agents may be involved in the etiology of LRTI (6-8).The identification of viruses...

The impact of respiratory viruses on lung health after preterm birth

“…Human bocavirus (hBoV) was first identified in 2005 from the nasopharyngeal aspirate of patients with unresolved respiratory infection [130]. HBoV has also been associated with acute respiratory illnesses, predominantly among young children [131–133], and has been suggested as a potential cause of acute respiratory illnesses and recurrent wheezing episodes across several studies of preterm infants and children [134–136].…”

Human bocavirus 1 and 3 infection in children with acute gastroenteritis in Brazil

“…107(6): 800-804, September 2012 Human bocaviruses (HBoVs) were first detected in a pool of respiratory aspirates obtained from children presenting with respiratory illness (Allander et al 2005) and, more recently, in faecal samples from children presenting with fever and diarrhoea with or without associated respiratory symptoms (Albuquerque et al 2007). …”

“…Since its discovery, the HBoV-1 detection rate has varied from 2-19% in patients with upper or lower respiratory disease (Allander et al 2005, Lau et al 2007, Monteny et al 2007, Tozer et al 2009). HBoV-2, HBoV-3 and HBoV-4 have mainly been detected in stool samples, with HBoV-2 and possibly HBoV-3 being associated with gastroenteritis , Jartti et al 2012.…”

“…The second ORF encodes for two major structural proteins (VP1 and VP2) that comprise the viral capsid. The third ORF encodes for a non-structural nuclear phosphoprotein (NP1) (Allander et al 2005, Schildgen et al 2008, Jartti et al 2012.…”

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

“…For some agents, such as influenza, monitoring can be managed by virus isolation in selected laboratories, but for many unculturable agents, a DNA-sequence-based monitoring system is recommended. A principal approach for such a system has been described [Allander et al, 2005]. If such a monitoring system is systematically applied, monitoring based on virus isolation may be unnecessary.…”