Background: Human Bocavirus (HBoV) infection is of worldwide distribution. There is increasing evidence

that HBoV is pathogenic for the human gastroenteric tract. However, less data are available on the role of HBoV

in gastroenteritis. The present study was aimed to determine the prevalence of HBoV in children with gastroenteritis.

Methods: Real-time PCR TaqMan was used to screen 200 stool specimens that had been referred to the virology

laboratory for HBoV evaluation. All of samples were collected on viral transport media.

Results: Of the 200 stool samples analyzed, 16 (8%) were positive for HBoV. Human Bocavirus positive samples

from patients aged between 1 to 5 years with acute gastroenteritis infection suggest a minor role of HBoV

in gastroenteritis (p=0.0001).

Conclusion: The study showed a high prevalence of human Bocavirus in young children with acute gastroenteritis

diseases in Iran, suggesting that HBoV play a role in the pathogenesis of gastroenteritis.

Keywords: Gastroenteritis, Child, Human Bocavirus, Real-time PCR

Abedi Kiasari, Bahman

Fazlalipour, Mehdi

Mollaie, Hamidreza

Monavari, Seyed Hamidreza

Noorbakhsh, Samileh

Simorgh Research Repository

   Original Article                     http://mjiri.tums.ac.ir Medical Journal of the Islamic Republic of Iran, Vol. 27, No. 3, Aug 2013,  pp. 127-131 _______________________________________________________________________________________________________ 1. (Corresponding author)  PhD, Associate professor, Department of Virology and Anti-Microbial Resistance Research Cen-ter, Tehran University of Medical Sciences, Hemmat Highway, Tehran, Iran. hrmonavari@yahoo.com 2. Professor of Pediatric Infectious Diseases, Research Center of Pediatric Infectious Diseases, Tehran University of Medical Sciences, Tehran, Iran. samileh_noorbakhsh @yahoo.com 3. PhD student, Department of Virology, Tehran University of Medical Sciences, Tehran, Iran. hamid2008kmu@gmail.com 4. PhD student, Department of Virology, Tehran University of Medical Sciences, Tehran, Iran. 5. Assistant professor, Human Viral Vaccine Department, Razi Vaccine & Serum Research Institute, Karaj, Iran.  Human Bocavirus in Iranian children with acute gastroenteritis  Seyed Hamidreza Monavari1, Samileh Noorbakhsh2, Hamidreza Mollaie3   Mehdi Fazlalipour4, Bahman Abedi Kiasari5  Department of Virology and Anti-Microbial Resistance Research Center, Tehran University of Medical  Sciences, Tehran, Iran.  Received: 21 Jan 2013        Revised: 23 Feb 2013        Accepted: 25 Feb 2013    __________________________________________________________________________________________ Abstract    Background: Human Bocavirus (HBoV) infection is of worldwide distribution. There is increasing evidence that HBoV is pathogenic for the human gastroenteric tract. However, less data are available on the role of HBoV in gastroenteritis. The present study was aimed to determine the prevalence of HBoV in children with gastroen-teritis.   Methods: Real-time PCR TaqMan was used to screen 200 stool specimens that had been referred to the virol-ogy laboratory for HBoV evaluation. All of samples were collected on viral transport media.  Results: Of the 200 stool samples analyzed, 16 (8%) were positive for HBoV. Human Bocavirus positive sam-ples from patients aged between 1 to 5 years with acute gastroenteritis infection suggest a minor role of HBoV in gastroenteritis (p=0.0001).   Conclusion: The study showed a high prevalence of human Bocavirus in young children with acute gastroen-teritis diseases in Iran, suggesting that HBoV play a role in the pathogenesis of gastroenteritis.     Keywords: Gastroenteritis, Child, Human Bocavirus, Real-time PCR.  ________________________________________________________________________________________   Introduction  The human Bocavirus (HBoV), a member of the Parvoviridae family, has been cloned from nasopharyngeal aspirates of children with acute respiratory infection (ARI) in Sweden (1). Phylogenetic analyses of the complete genome of HBoV revealed that the virus is most closely related to canine minute virus and bovine parvovirus, and thus, it was called “human Bocavirus” (bo for bovine and ca for canine) (2, 3).  Hu-man Bocavirus has been detected in urine, fecal, and blood samples of children with gastrointestinal tract infections (4). Acute gastroenteritis (AGE) known by vomiting, diarrhea, and dehydration, is one of the most common causes of morbidity and mortality in infants and children (5). Many viruses such as Norovirus, Rota-virus, Astrovirus, Sapovirus, and Adenovi-rus have been known to associate with the-se diseases and recently, human Bocavirus has been considered as agent associated with diarrhea in humans (6). The associa-tion of HBoV with gastroenteritis is sus-pected since the relation between bovine parvovirus (BPV) enteritis in cattle and gastroenteritis symptoms were found(7). Initial studies showed that HBoV is a common cause of acute upper and lower respiratory tract infection in children. It has been detected in 1.5%–11.3% of individu-als with ARI in Europe, United States, Canada, Asia, South Africa, and Australia (8,9).  Human Bocavirus (HBoV) in acute gastroenteritis 128  MJIRI, Vol. 27, No. 3, Summer, Aug 2013, pp. 129-131  http://mjiri.tums.ac.ir  The aim of this study was to determine the prevalence of Bocavirus in fecal speci-mens collected from gastroenteritis cases in the Hazrat-e-Rasool Akram Hospital of Tehran, Iran and to evaluate its contribu-tion as a cause of acute gastroenteritis dis-eases.   Methods This cross-sectional study has been done in the Hazrat-e-Rasool Akram Hospital af-filiated to Tehran University of Medical Sciences from November 2010 to Novem-ber 2011. It has been approved by the Eth-ics Committee of the university. Stool samples (n = 200) were collected from pa-tients with acute gastroenteritis, presented at least with two of the following symp-toms: vomiting, diarrhea, abdominal pain, cough, exanthematic disease or skin rash. Moreover, 200 fecal samples were collect-ed from controls without acute gastroen-teritis within the same age group.  The samples were diluted 1:10 in PBS (Pe-ripheral Blood Smear) and then were vortexed and centrifuged. Supernatants were collected and were frozen at -70˚C until use. DNA was extracted with High Pure Viral Nucleic Acid kit (Roche Diagnostics GmbH, Mannheim, Germany). Extracted DNA stored at -20°C until use. Real-time PCR as-say (Rotor Gene 6000; Corbett Research, Australia) with Hydrolyses TaqMan probe was utilized to detect HBoV DNA. The am-plification reaction mixture was contained 5 μL of extracted sample, 10 μL TaqMan uni-versal PCR master mix (PE Applied Biosystems), 0.1 μL bovine serum albumin (20 mg/ml), 300 nmol/liter of each primer (Boca-forward, 5'-AGAGGCTCGGGCTC ATATCA-3'; Boca-reverse, 5'-CACTTGG TCTGAGGTCTTCGAA-3'), and 150 nmol/ liter of the Boca probe (5'-FAM- AGGACCACCCAATCARCCACCTATCGTCT-TAMRA-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine). Amplifica-tion was performed under standard amplifi-cation conditions with the following set-tings: 95°C for 10 min; 42 cycles of 95°C for 15 seconds and 60°C for 1 min (10). HBoV positive samples were confirmed in a second run including dilutions of the samples. Also the HBoV negative samples were retested and confirmed. The medical histories of the children were re-evaluated 5 to 7 days after enrollment and until the resolution of their illness by means of interviews and clinical examina-tions by trained investigators using stand-ardized questionnaires. During this evalua-tion, information was also obtained regard-ing acute illnesses and related morbidity in their households. All the data were verified from medical records.  Statistical analysis: The data were ana-lyzed using SPSS v.13 (SPSS, Chicago, IL). P-value of <0.05 was considered statis-tically significant. Parametric data were compared by analysis of variance (ANO-VA). Skewed distributed or nonparametric data were analyzed using the Kruskal-Wallis test. The categorical data were ana-lyzed by means of contingency analysis and the chi-square or Fisher’s Exact test.  Results Patients Characteristics: Patients’ ages ranged from 2 to 3.5 years old, and includ-ed 144 (72%) male. Mean age was 3.08 ± 1.13 years (mean ± standard deviation) Clinical characteristics of the patients were recorded for further analysis. Twenty (10%) had evidence of acute respiratory tract infection with cough, 140 (70%) had vomiting, 92 (46%) had fever without any source, and 116 (58%) had exanthematic disease or skin rash; 152 (76%) had ab-dominal pain. Among the studied cases, 16 (8%) had evidence of chronic underlying diseases, 4 (2%) had chronic asthma, 8 (4%) had chronic kidney insufficiency, and 4 (2%) had chronic diarrhea.   Human Bocavirus Prevalence: HBoV DNA positive samples were detected in 16 patients (from a total of 200) with a total positive rate of 8%. In the control group none of samples were positive for HBoV  S.H. Monavari, et al.  129  MJIRI, Vol. 27, No. 3, Summer, Aug 2013, pp. 129-131   http://mjiri.tums.ac.irDNA. HBoV DNA positive samples were also tested for 5 other potential pathogens, including: Rota virus, Norwalk, Adenovi-rus, Escherichia coli and helicobacter pylo-ri by TaqMan real-time PCR, (Guangzhou HuYanSuo Medical Technology Co., Ltd). Samples were all negative for these patho-gens.  Discussion Viral gastroenteritis is one of the most common illnesses in humans worldwide. However, different viral agents have caused the disease and the clinical presen-tation of patients with acute gastroenteritis symptoms is not generally indicative of a specific pathogen (11, 12). Acute gastroen-teritis is one of the causes of morbidity and mortality during childhood in developing countries. Human Bocavirus has been rec-ognized as an important cause of severe gastroenteritis in childhood and has been detected in hospitalized children (10, 13).  The present work is a prospective study to find one of the etiologies of community-acquired gastroenteritis in symptomatic small children in Iran. This study demon-strated the presence of HBoV genomic DNA in stool samples from a group of children with acute gastroenteritis. The study is in line with previous studies in China, Australia, USA, and Brazil (14,15). The frequency of HBoV in the present study (8%) was higher than the frequency reported in the previous studies (Canada 1.5%, Sweden 3.1%, Australia 5.6%, and Japan 5.7%, Germany 10.3% and Korea 11.3%) (23). An increasing evidence sug-gests a relationship between HBoV and the disease. High titers of DNA in some sam-ples suggest that the virus replicates in the human gut and none of the HBoV-positive patients showed the respiratory symptoms. The low HBoV-DNA titers in other stool samples could be explained by swallowed virus from the respiratory tract; but HBoV cannot be ruled out as a cause for single gastroenteritis cases. Control groups were used to support evidence demonstrating an association between HBoV infection and gastroenteritis (16,17). HBoV is an im-portant agent for outbreaks of gastroenteri-tis in day care facilities for children (18, 19).  In a study done in Hong Kong, diarrhea was found in 11% out of 79 patients with respiratory symptoms. In that study, human Bocavirus was identified is fecal samples of 25 children with gastroenteritis. Of these children, 16% had blood in the stool, 8% had mucus in the stool, 32% had vomiting, and 68% had fever. The following respira-tory findings were found in these children with gastroenteritis: coryza (56%), acute bronchitis (16%), and pneumonia (12%). Co-pathogens were identified in 56 % of the children: rotavirus (36%), Salmonella spp. (8%), Campylobacter spp. (4%), Staphylococcus aureus (4%), and Clostrid-ium difficile (4%) (20-22). In a study from Korea, viral agents were found in 44.4% of the stool specimens (n=962) from children with acute gastroenteritis. The viral agents included rotavirus (25.7%), Norovirus (13.7%), adenovirus (3.0%), Astrovirus (1.1%), and human Bocavirus (0.8%). In another study from Brazil, stool samples of 2 % of 705 cases with diarrhea were PCR positive for human Bocavirus. In studies of respiratory illness associated with human Bocavirus infection, diarrhea has been re-ported in 9-38% of patients.  Previous studies have been utilized con-ventional nested PCR assays with agarose gel electrophoresis. To improve the diag-nostic methods for HBoV detection, a real-time PCR assay was applied in this study. This assay was sensitive, specific, and reli-able for HBoV DNA amplification. Obvi-ously, this real-time PCR is simple and less time consuming. It is truly a cost-effective laboratory method to detect different virus-es (24, 25). The designed primers used in this novel real-time PCR were designed based on virus sequences circulating re-cently and are appropriate for screening Bocavirus in clinical specimens from in-fants and children with acute gastroenteritis to detect recent strains circulating in pa-tients (26).   Human Bocavirus (HBoV) in acute gastroenteritis 130  MJIRI, Vol. 27, No. 3, Summer, Aug 2013, pp. 129-131  http://mjiri.tums.ac.ir Conclusion The study showed a high prevalence of human Bocavirus in young children with acute gastroenteritis in Iran, suggesting that HBoV plays an important role in the path-ogenesis of gastroenteritis.  This was a small study, and these obser-vations must be confirmed in a larger sam-ple of patients with more analysis works.  Acknowledgments  This investigation has been funded by Tehran University of Medical Sciences.   References  1. Tattersall P, Fauquet M, Mayo J, Maniloff U, Desselberger A. Virus taxonomy: classification and nomenclature of viruses. 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Human Bocavirus in Iranian children with acute gastroenteritis

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Human Bocavirus in Iranian children with acute gastroenteritis

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