Detection and Molecular Epidemiology of Human Bocavirus in Children with Acute Gastroenteritis from Brazil

Human Bocavirus (HBoV) is a recently discovered virus and was first detected in the nasopharyngeal aspirate samples and after in stool samples, suggesting that HBoV may be a causative agent for human enteric infections. Due to absence of treatment options, there is a need to understand the disease-causing mechanism of these viruses. The aim of this was to demonstrate the prevalence of HBoV from children less than 10 years with acute gastroenteritis in Brazil, during November 2011 to November 2012. Stool samples from hospitalized children ≤ 10 years who presented symptoms of acute gastroenteritis were analyzed for the presence of HBoV DNA by nested-PCR. HBoV- positivity was detected in 24.0% (54/225) of samples. Two peaks of HBoV detection were observed, during November 2011 and July to September 2012. Co-infections between HBoV and rotavirus A were identified in 50.0% (27/54) of specimens. Phylogenetic analysis identified the presence of HBoV-1 (94.8%), HBoV-2 (2.6%) and HBoV-3 (2.6%) species, with only minor variations among them. Further investigations are necessary to improve the knowledge on the role of HBoV in gastrointestinal infections.


INTRODUCTION
Globally, diarrhoeal disease in 2015 were responsible for 499 000 deaths of children aged < 5 years (1). Several pathogens are associated with acute gastroenteritis (AGE), however ~40% of cases remain of unknown etiology (2). Human Bocavirus (HBoV) is a recently discovered virus and was first detected in the nasopharyngeal aspirate samples and after in stool samples, suggesting that HBoV may be a causative agent for human enteric infections (3)(4)(5).
HBoV belongs to Parvoviridae family, Parvovirinae subfamily, Bocaparvovirus genus (6). This parvovirus is a small single stranded DNA virus with a diameter of 18-26 nanometers and contains a non-enveloped icosahedral capsid (7). The genome of HBoV consists of three ORFs that encode two non structural proteins (NS1 and NP1) and two structural proteins (VP1 and VP2) (7,8). Based on genetic variability of VP1 region, HBoV are divided into 4 species: HBoV1 -HBoV4. Several groups have detected HBoV1 in respiratory tract infections, while HBoV2, 3 and 4 were reported in fecal samples (9,10).
The role of HBoV in respiratory disease and acute gastroenteritis remains unclear. It is believed that HBoV can persist for a longer period in respiratory tract mucosa, reaches the bloodstream or ingestion and migrates to gastrointestinal tract, where it may generate a new infection or be excreted in an asymptomatic way (11).
However, there are increasing reports on HBoV detection in fecal specimens from patients with acute diarrhoea, particularly in children (12)(13)(14).
HBoV have been detected mostly in respiratory tract secretions and stool, however the viruses have been found in serum and cerebrospinal fluid causing viremia (15,16). Likewise, several studies have reported HBoV in sewage and river water (17)(18)(19).
Since the first HBoV detection, several studies reported its association with respiratory and gastrointestinal infections, mostly in young children (12,20,21).
Nevertheless, the significance of this virus as a causative agent in human infections remains unknown, due to high prevalence of co-infection with other viruses in symptomatic patients as well as to frequent detection of HBoV in asymptomatic individuals. A recent systematic review suggests that even though HBoVs are often considered bystanders in acute gastroenteritis course, HBoV-2 infection may increase the risk for this disease (5,10,22).
Due to absence of treatment options, there is a need to understand the disease-causing mechanism of these viruses. (10) have reported that HBoV prevalence was similar between subjects with or not gastrointestinal symptoms. HBoV has also been found to co-infect humans with other enteric viruses. A study conducted in Pakistan has related a frequency of 98% involved co-infection between HBoV and rotavirus from children with AGE (24). In Brazil in an investigation that also enrolled diarrheic children have described HBoV coinfection with norovirus, adenovirus and rotavirus in 19%, 3% and 3% of patients, respectively (12).
The aim of this was to demonstrate the prevalence of HBoV from children less than 10 years with AGE in Brazil, from November 2011 to November 2012, to report co-infection with rotavirus A (RVA) and to describe HBoV species.

HBoV Screening and DNA Extraction
Viral genome was extracted from 10% fecal suspensions using guanidinium isothiocyanate-silica method (25). Posteriorly, samples were subjected to nested-PCR targeting the partial region of VP1 gene. The first-round of the nested-PCR was carried out using the AK-VP-F1 and AK-VP-R1 primers set, and the second round using the AK-VP-F2 and AK-VP-R2, as described previously (26).

Nucleotide Sequencing and Phylogenetic Analysis
Sequencing of the PCR amplicons HBoV strains were performed using the same primers as those used in the second-round of the nested-PCR and carried out with a Big Dye Terminator cycle sequencing kit v 3.1 (Applied Biosystems, Foster City, CA). Electrophoresis was performed on the ABI Prism 3130xl automatic sequencer (Applied Biosystems) and the sequences obtained were aligned and edited using the BioEdit Sequence Alignment Editor program (v. 7.0.5.2). Neighbor-joining method was used to perform the phylogenetic analysis, in which distance was calculated from aligned sequences (27). Dendrograms were constructed using MEGA program v.5.0.1, and bootstrap analysis was performed using 2,000 replicas. Partial nucleotide sequences from this study were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov), under the following access number: MH003642-MH003679.

Statistical Analysis
HBoV frequencies and genotypes description were calculated using Microsoft Excel software. Comparisons of HBoV infection rates in distinct groups were performed using chi-square test ( 2 ) through BioEstat 5.0 with statistical significance established of P values <0.05 (28).

DISCUSSION
This study reports HBoV detection among pediatric patients with acute gastroenteritis from Brazil, is demonstrated the prevalence of HBoV and describe its dominant species. HBoV was detected in 24.0% of patients. Presents findings were higher than most of the previously reported in Brazil, including a survey with HIVseropositive children hospitalized with acute diarrhoea, in which HBoV overall positivity rate was 14% (4,(29)(30)(31).
HBoV and RVA co-infection is usually related. In the current study, this condition was observed in 50% of samples tested, a number comparable to the obtained for HBoV and diverse gastroenteritis viruses in an investigation performed in West China, between 2012 and 2013, in which the overall rate of co-detection was equivalent to 43,3% (23). In Pakistani children, 98% of HBoV-positive samples also tested positive to RVA, a higher number than observed in the present results. It is important to notice that RVA vaccine is not included in vaccination program in Pakistan, and this may be an important contributing factor to the high prevalence of RVA among infants from that population (24). On the other hand, in Brazil, where the immunization program includes RVA vaccine, HBoV and RVA association was also found, but the major co-infection rate was noted between HBoV and noroviruses (12).
An investigation performed at the same period (year of 2012) in Bahia state, Northeast Brazil, revealed, however, that 70% of HBoV-positive sequenced samples were correspondent to specie 2 (subtype 2A), while only 30% were related to HBoV-1 (12). In Southeast Brazil, a preview study has shown that 20.8% of a diarrheic group was characterized as HBoV-2, whereas 1.2% corresponded to HBoV-1 (29).
There is a current lack of data regarding to HBoV infection in Northern