RESEARCH ARTICLE Soares et al., Journal of Medical Microbiology 2019;68:1233–1239 DOI 10.1099/jmm.0.001026

Molecular epidemiology of in children with acute gastroenteritis from North Region of Brazil

Luana S. Soares*, Ana Beatriz F. Lima, Kamilla C. Pantoja, Patrícia S. Lobo, Jonas F. Cruz, Sylvia F. S. Guerra, Delana A. M. Bezerra, Renato S. Bandeira and Joana D. P. Mascarenhas

Abstract Purpose. Human bocavirus (HBoV) is a DNA that is mostly associated with respiratory infections. However, because it has been found in stool samples, it has been suggested that it may be a causative agent for human enteric conditions. This under- pins the continuous search for HBoVs, especially after the introduction of the rotavirus vaccine due to acute gastroenteritis cases related to emergent , as HBoVs are more likely to be found in this post-vaccine scenario. Therefore, the aim of this study is to demonstrate the prevalence of HBoV in children aged less than 10 years with acute gastroenteritis in Brazil from November 2011 to November 2012. Methodology. Stool samples from hospitalized children ≤10 years old who presented symptoms of acute gastroenteritis were analysed for the presence of rotavirus A (RVA) by an enzyme-linked immunosorbent assay (ELISA), and for HBoV DNA by nested PCR. Results. HBoV positivity was detected in 24.0 % (54/225) of samples. Two peaks of HBoV detection were observed in Novem- ber 2011 and from 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. Conclusion. Our findings provide evidence for the circulation of most HBoV genotypes (except HBoV-4) in the North Region of Brazil at a considerable rate and further investigations are necessary to improve our knowledge in the context of HBoV infec- tions and their role in gastrointestinal diseases.

INTRODUCTION However, subsequent studies revealed its presence in stool as Acute gastroenteritis (AGE) is a global public health problem a potential cause of diarrhoea worldwide [3–5]. that is responsible for >500 000 deaths per year worldwide HBoV belongs to the family , subfamily Parvo- and represents the third largest cause of childhood mortality, virinae, genus Bocaparvovirus [6]. This parvovirus is a small especially among children in low-income and lower-middle- single-stranded DNA virus with a diameter of 18–26 nm income countries [1]. Although several agents (bacterial, and contains a non-enveloped icosahedral capsid [7]. The parasitic, viral) are currently associated with this condi- genome of HBoV consists of three open reading frames tion, the aetiology of approximately 40 % of cases remains (ORFs) that encode two nonstructural proteins (NS1 and unknown [2]. This fact encourages the performance of inves- NP1) and two structural proteins (VP1 and VP2) [7, 8]. tigations aiming to identify potential causes for AGE. Human Based on the genetic variability of the VP1 region, HBoV bocavirus (HBoV) is a viral agent that was first detected in is divided into four species: HBoV-1 to HBoV-4. Usually, nasopharyngeal aspirate samples from children and because HBoV-1 is most associated with respiratory tract infec- of this it was primarily associated with respiratory infections. tions, whereas HBoV-2, 3 and 4 are most likely related to

Received 16 November 2018; Accepted 07 June 2019; Published 19 June 2019 Author affiliations: 1Secretaria de Vigilância em Saúde, Instituto Evandro Chagas, Seção de Virologia, Ananindeua, PA, Brazil. *Correspondence: Luana S. Soares, luanasoares@​ iec.​ gov.​ br​ Keywords: human bocavirus; children; acute gastroenteritis; Brazil. Abbreviations: AGE, Acute gastroenteritis; ELISA, Enzyme-linked immunosorbent assay; HBoV, Human Bocavirus; RVA, Rotavirus A. The GenBank accession numbers for the VP1 sequences of HBoV are MH003642–MH003679.

001026 © 2019 The Authors

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gastroenteric infections, because of their reported presence Detection of RVA in faecal samples [9, 10]. Selected faecal samples were screened for the presence The role of HBoV in respiratory disease and acute gastroen- of RVA using a commercially available enzyme-linked teritis remains unclear because of its high rate of co-infection immunosorbent (ELISA) assay according to the manufac- with other viruses in symptomatic patients, as well as its turer’s instructions (Ridascreen kit, R-Biopharm, Darmstadt, frequent detection in asymptomatic individuals [5, 10]. It is Germany). believed that after a long period of persistence in respiratory tract mucosa, HBoV reaches the bloodstream and migrates HBoV screening and DNA extraction to the gastrointestinal tract, where it may either generate a Total DNA was extracted from faecal suspensions prepared at new infection or be excreted in an asymptomatic way [11]. 10 % (v/v) in buffered Tris-Ca++ 0.01 M using the guanidinium A recent systematic review suggested that even though HBoVs isothiocyanate–silica method [18]. Posteriorly, samples were are often considered to be bystanders in the course of acute subjected to a nested PCR targeting a partial region of the gastroenteritis, HBoV-2 infection may enhance the risk for HBoV VP1 gene. The first round of the nested PCR was this disease [12]. However, there are increasing reports on carried out using the AK-VP-F1 and AK-VP-R1 primer set, single detection of HBoV in faecal specimens from patients and the second round was carried out using the AK-VP-F2 with AGE, particularly among children under 5 years of age and AK-VP-R2 set, as described previously [19]. Fig. 2 shows [13–15]. the amplified area of the HBoV genome that was used for PCR and gene sequencing. This situation underpins the continuous search for HBoVs, especially after the introduction of rotavirus vaccine, which occurred in Brazil and several other countries in 2006. The Nucleotide sequencing and phylogenetic analysis significant reduction of rotavirus diarrhoea episodes since Sequencing of the PCR amplicons from HBoV strains was then is notable, but AGE cases that are related to emergent performed using the same primers as those used in the nested viruses, such as HBoV and calicivirus, are more likely to be PCR, and was carried out with a Big Dye Terminator cycle found in this post-vaccine scenario [16, 17]. Alongside this, sequencing kit (v 3.1; Applied Biosystems, Foster City, CA, the lack of treatment options available for HBoV infections USA). Electrophoresis was performed on the ABI Prism highlights the need to understand the disease-causing mecha- 3130xl automatic sequencer (Applied Biosystems) and the nisms and circulation patterns of these viruses. obtained sequences were aligned and edited using the BioEdit Sequence Alignment Editor program (v 7.0.5.2). The neigh- Therefore, the current investigation aimed to demonstrate the bour-joining method was used in the phylogenetic analysis, frequency of HBoV among children with AGE aged <10 years in which distance was calculated from aligned sequences [20]. in North Region of Brazil, report HBoV co-infection with Dendrograms were constructed using mega v 5.0.1, and boot- rotavirus A (RVA) and describe the HBoV species circulating strap analysis was performed using 2000 replications. Partial in this region. nucleotide sequences from this study were deposited in the GenBank database (http://www.​ncbi.​nlm.​nih.​gov) under the METHODS access numbers MH003642–MH003679. Clinical specimens Statistical analysis The faecal samples analysed in this study were derived from a HBoV frequencies and genotypes description were calcu- Brazilian public health surveillance system that receives faecal lated using Microsoft Excel software. Comparisons of HBoV specimens collected from hospitalized patients presenting infection rates in distinct groups were performed using the with AGE symptoms at several hospitals located throughout chi-square test (χ2) in BioEstat 5.0 software with statistical North Region to monitor the circulation of different RVA significance established for P-values <0.05 [21]. genotypes. From November 2011 to November 2012, a total of 541 faecal samples were transported to Evandro Chagas Institute, a Brazilian Ministry of Health National Reference Laboratory. In this study, acute diarrhoea was defined as the RESULTS presence of three or more liquid and semi-liquid stools within From November 2011 to November 2012, HBoV was detected a 24 h period for up to 14 days. An aliquot of each sample was by nested PCR in 24.0 % (54/225, range 0–50 %) of samples stored at –20 °C until processing. from hospitalized children ≤10 years old. Fig. 3 shows that there were two peaks in the monthly frequency of HBoV From the total of 541 samples, a smaller number were selected detection during which the HBoV detection rates were over for inclusion in the present study according to the following 30 % (November 2011 and July to September 2012). criteria: patient age (<10 years old) and availability of material (enough for HBoV detection procedures). After this prelimi- These samples were also tested for RVA, the most impor- nary screening, specimens were randomly chosen for testing, tant viral agent associated with acute gastroenteritis. The with a 95 % confidence level and a confidence interval of 5 %. RVA detection rate was equivalent to 40.9 % (92/225) and The study’s methodology is represented in Fig. 1. co-infection between HBoV and RVA was present in 50.0 %

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Fig. 1. Study methodology.

(27/54) of samples, with a median age of 17 months (data children aged 7–24 months. With regard to gender, 59.2 % not shown). (32/54) of infected individuals were male. No statistically significant differences (P>0.05) were seen when comparing The general clinical and epidemiological characteristics of age groups and gender. HBoV-positive cases are summarized in Table 1. The median age of HBoV-positive children was 16 months, and a higher Phylogenetic analysis of a partial region from VP1 genes was HBoV positivity rate (66.6%, 36/54) was observed among performed in 38 (70.3 %) of the HBoV-positive samples that

Fig. 2. Amplified area analysed in PCR and VP1 gene sequencing of the HBoV genome.

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Fig. 3. Temporal distribution of HBoV in Brazil, 2011–2012.

had good DNA quality. Based on this analysis, it was observed HBoV-2 specimens showed nt and aa similarities ranging that HBoV-1 was found in 94.8 % of the samples (n=36), while from 93.3–98.6% and 78.6–95.6 %, respectively. With regard the remaining isolates involved the HBoV-2 and HBoV-3 to the to HBoV-3 strains, the nt and aa similarities were genotypes (2.6 % each), respectively. 97.1–98.2% and 91–95.5 %, respectively (Fig. 4). All HBoV-1 strains shared 97–100% and 93.1–100 % nucleo- tide (nt) and amino acid (aa) similarities among themselves, DISCUSSION respectively. When compared with other isolates, the nt and This study reports HBoV detection among paediatric patients aa similarities ranged from 95.8–100% and 93.2–100 %, with acute gastroenteritis from Brazil, demonstrating the respectively. When compared with the prototype strains, the frequency of HBoV and describing its dominant species. HBoV was detected in 24.0 % of patients. The detection rates Table 1. Baseline characteristics of HBoV-positive Cases in Brazil, in this study were higher than those in most of the previous 2011–2012 reports from Brazil, including a survey involving HIV-sero- positive children hospitalized with acute diarrhoea, in which HBoV-positive/tested (%) the overall positivity rate for HBoV was 14 % [4, 22, 23]. Age group (months) HBoV infection and RVA infection are frequently related. 0–6 11/50 (22.0) In the present study, co-infection was observed in 50 % of

7–24 36/126 (28.6) samples tested, a comparable figure to that obtained for HBoV and diverse gastroenteritis viruses in an investiga- 25–60 6/45 (14.2) tion performed in western China between 2012 and 2013, >61 1/4 (25.0) in which the overall rate of co-detection was equivalent to 43.3 % [24]. In Pakistani children, 98 % of HBoV-positive Clinical characteristics samples also tested positive for RVA, a higher percentage than Fever 24/110 (21.8) was observed in our results. It is important to note that RVA vaccine is not included in the vaccination programme in Paki- Vomiting 28/141 (17.0) stan, and this may be an important contributing factor to the Gender high prevalence of RVA among infants from that population [25]. On the other hand, in Brazil, where the immunization Male 32/134 (23.8) programme includes RVA vaccine, an association between Female 22/91 (24.1) HBoV and RVA was also found, but the major co-infection Brazilian state rate was noted between HBoV and noroviruses [13].

Acre 13/47 (27.6) As the co-infection data were limited to RVA in the present investigation, since it was the only viral agent tested other Amazonas 38/133 (28.5) than HBoV, it was not possible to determine the relationship Pará 2/40 (5.0) of HBoV with other acute gastroenteritis causative viruses. Another limitation was the absence of sufficient clinical data Roraima 1/5 (20.0) regarding HBoV mono- and co-infections. Hence, it was not

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Fig. 4. Phylogenetic analysis of the VP1 protein of HBoV Brazilian strains. The level of bootstrap support is indicated at each node (values <70 % were omitted) based on neighbour-joining analysis of 2000 replications. The HBoV strains analysed in this study are in bold and are marked with a circle (•) (HBoV-1, red; HBoV-2, blue; HBoV-3, green).

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possible to establish if there were significant differences in Acknowledgements The authors would like to acknowledge the staff of the State Central disease severity between these groups. Laboratories (LACENs) and the technical assistance given by the labo- ratory personnel at the Virology Section of the Evandro Chagas Insti- Despite the fact that HBoV−2, -3 and -4 are most associated tute. The authors are also thankful to the children/mothers who agreed with AGE symptoms, while HBoV-1 is mostly associated to participate in this study as volunteers and permitted the analysis of with respiratory tract diseases [5, 10], we identified a higher their relevant biological material. presence of HBoV-1 among HBoV-positive stool samples Author contributions compared to the other species. Similar results were observed A. B. F. L., K. C. P. and J. F. C. performed the experiments. P. S. L., S. F. S. G., in Chile, where HBoV-1 was found in 14.1 % of the HBoV- D. A. M. B. and R. S. B. carried out the molecular and the phylogenetic analyses. L. S. S. and A. B. F. L. analysed and interpreted the data and positive faecal specimens between 1985 and 2010, and in other drafted the manuscript. L. S. S. and J. D. P. M. provided critical review of countries such as India, Thailand, Pakistan, the Republic of the manuscript. All authors read and approved the final manuscript. Korea and Australia [15, 25–29]. Conflicts of interest An investigation performed over the same period (2012) in The authors declare that there are no conflicts of interest. Bahia state, Northeast Brazil, revealed, however, that 70 % of Ethical statement HBoV-positive sequenced samples corresponded to species This study was approved by the Evandro Chagas Institute’s Human Research Ethics Committee, protocol number 414.389, in accordance 2 (subtype 2A), while only 30 % corresponded to HBoV-1 with the National Health Council’s Resolution 466/2012. The authors [13]. In Southeast Brazil, a preview study has shown that ensured that all procedures contributing to this work complied with the 20.8 % of isolates from a diarrhoeic group were characterized ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as HBoV-2, whereas 1.2 % corresponded to HBoV-1 [30]. as revised in 2008.

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