Bocavirus: Molecular diagnosis by Qualitative Real time PCR (rt-PCR)
Human Bocavirus (HBoV) is a Parvovirus identified in 2005 in samples of swabs and nasopharyngeal washes of children with respiratory infections without known etiology in 3.1% of cases, so it was proposed as a causative pathogen of diseases of the respiratory tract. Subsequently, three additional subtypes were found in stool samples called HBoV2, HBoV3 and HBoV4 to differentiate from the subtype found in the respiratory system, HBoV1. The genotypes of HBoV belong to the family Parvoviridae, subfamily Parvovirinae, genus Parvovirus, which infect only vertebrates.
Parvoviruses are small, icosahedral, non-enveloped viruses, 18-26 nm in diameter, with a 5 kb linear, positive or negative single-stranded DNA, and 32-52 nt terminal sequence that participates in replication, with a sequence very similar to that of BPV (Bovine Parvovirus) and CMV (Canine Minute virus). The assigned name of Bocavirus comes from the combination of "Bovine Parvovirus" (BPV) and "Canine Minute virus" (CMV) due to the genomic similarities between these viruses. Its grouping has been proposed in two species (clusters): Human Bocavirus 1 (to include HBoV1 and HBoV3) and Human Bocavirus 2 (to include HBoV2 and HboV4).
Parvoviruses associated with human infections are Parvovirus B19 included in the genus Erythroparvovirus, the non-pathogenic virus associated with Adenovirus (Dependoparvovirus) and the recently discovered Parvovirus 4 (PARV4) and 5 (PARV5) included in the new genus Tetraparvovirus or genus Hokovirus, not yet related to any infectious process.
Among human Parvoviruses, B19V is known to be the agent of infectious erythema (fifth disease), also related to other affectations in children (transient arthritis) and adults (nonimmune Hydrops fetalis), several autoimmune diseases, spontaneous abortions and arthropathies. Despite the phylogenetic relationship between B19V and HBoV, they appear to be very divergent. B19P shows tropism by bone marrow and persists in cardiac tissues, while HBoV persists in lymphatic tissue and in tissues of chronic sinusitis.
The Bocaviruses have been found in individuals of all ages, although mainly in children from 6 to 24 months affected by respiratory symptoms. However, it has not been possible to confirm its pathogenic nature because Koch´s postulates could not be reproduced since it lacks an experimental model and there are difficulties to obtain its in vitro replication in cell culture.
The fact that it was found in the respiratory system and in feces along with other potential pathogens suggested that it could be a passenger without involvement instead of a true pathogen. In addition, it has been found in peripheral blood, saliva, feces, urine, environmental samples, including river and wastewater.
The pathogenesis of HBoV is poorly characterized, mainly due to the lack of specific cell lines for its culture, and of susceptible experimental animals. The only susceptible cells are HAE-ALI pseudostratified cells, derived from primary human bronchial epithelial cells.
The virus penetrates by respiratory route and by blood or by direct ingestion would reach the gastrointestinal tract. HBoV1 has been found in both the respiratory and gastrointestinal tracts. Several studies have shown the association between HBoV1 and the upper and lower respiratory tract. The most frequent clinical manifestations include cough, fever, rhinorrhea, asthmatic exacerbations, bronchiolitis, and pneumonia.
HBoV1 has also been found in samples of adult patients with gastrointestinal manifestations of nausea, vomiting and diarrhea. However, the viral load of HBoV1 in stool samples from pediatric patients with acute gastroenteritis is lower than in respiratory samples. HBoV2 is more frequent in stool samples, and possibly together with HBoV3, would be associated with gastroenteritis.
Unique HBoV infections are rare, whereas double coinfections are frequent associated with other viruses such as human Rhinovirus, Adenovirus, Norovirus or Rotavirus. In the respiratory tract, the existence of coinfection has been reported in 83% of respiratory samples, in particular associated with respiratory syncytial virus (89.5%).
HBoV1 can be detected in samples of nasopharyngeal aspirate in immunocompetent subjects up to 6 months after infection. This fact would facilitate its detection along with other viruses that at the same time could facilitate the reactivation of the latent virus during superinfection. The virus has little tropism in the human organism and has been found in lymphatic tissue.
Relevant clinical infections requiring hospitalization are associated with coinfections with up to 6 different pathogens in the same subject. It has been pointed out that when there is a high viral load (> 104 copies/mL), it is statistically associated with more serious clinical manifestations and more long-lasting hospitalization, although this fact has not been confirmed by others.
The main method to detect human Bocavirus infections (HBoV) in respiratory or intestinal samples has been conventional PCR, followed by nested PCR and real-time PCR (rt-PCR).
PCR allows detection in nasopharyngeal aspirates, bronchoalveolar lavage, stool, serum and urine, using the amplification of NP1, NS1 and/or VP1/2 genes. The NP1 and NS1 regions are more conserved than VP1/2, so they are the preferred targets. Real time PCR (rt-PCR) has the advantage over conventional PCR of being more specific and rapid with a sensitivity of 100% and a specificity of 94% and 93% respectively for tests with NP1 and VP1.
Also, serological methods have been developed to detect specific antibodies in serum samples such as western-blot, immunofluorescence and ELISA for IgG and IgM, including antibody avidity tests to differentiate antibodies from primary or secondary infections. However, considering that Bocaviruses are very prevalent and can establish persistent infections, the interpretation of the results is just as complicated as the molecular detection tests (PCR).
Tests carried out in IVAMI:
- Molecular detection by real-time qualitative PCR (RT-PCR) for NP1 gene.
- In respiratory infections: nasopharyngeal aspirate, bronchoalveolar lavage or similar.
- In gastrointestinal infections: stool sample.
Conservation and sending of the sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: more than 2 days.
Cost of the test:
- Real-time PCR (Real Time PCR): Consult to firstname.lastname@example.org.