Human Bocavirus Prevalence In Children With Acute Gastroenteritis From Rural Communities In The Northen Region Of South Africa

BACKGROUND Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality in young children worldwide. Human Bocavirus (HBoV) is an emerging virus globally associated with diarrhea. The aim of this study was to demonstrate the prevalence of HBoV genotypes in children (≤5 years) from rural communities in South Africa (SA) suffering from AGE. MATERIAL AND METHOD A total of 141 fecal samples of children ≤5 years with acute gastroenteritis (AGE) were collected from rural Primary Health Care facilities in the Vhembe district of SA between June 2017 and July 2018. Clinical symptoms and demographic data were also recorded. A total of 102 (72%) were outpatients and 39 (28%) were hospitalized patients. Human Bocavirus (HBoV) genotypes were determined using Real-Time Multiplex PCR. DNA extracts of positive samples were confirmed by conventional PCR targeting the NS1 gene. Co-infection with other enteric viruses were determined in HBoV positive samples using Real-Time PCR. RESULTS HBoV was detected in 8 (5.7%) children with AGE. Children were in the age group between 1-24 months. HBoV1 and HBoV3 genotypes were each detected in 3 (37.5%) stool samples and HBoV2 in 2 (25%) stool samples. Co-infection with other enteric viruses included Rotavirus (37.5%); Adenovirus (37.5%); Norovirus (25%) and Astrovirus (12.5%). CONCLUSION HBoV infections could be seen as a potential emerging diarrheal pathogen in South Africa. Further studies are required to understand the role of HBoV infections in children and adults with acute gastroenteritis. Author summary Acute gastroenteritis (AGE) is recognized as a major cause for mortality in children ≤5 years of age in Africa and other developing countries. Viruses known to be involved in AGE includes Rotavirus, Norovirus, Astrovirus and Adenovirus and have been reported globally. Recently the Human Bocavirus (HBoV) have been reported in numerous studies globally as a potential cause of diarrhea. In this study, the prevalence and genetic diversity of human Bocavirus in children with AGE from rural communities in Limpopo, South Africa were investigated. In total, 141 stool samples from children ≤ 5 years with AGE were assessed for the presence of HBoV using Real-Time PCR. HBoV were detected in 8 (5.7%) patients and included 3 positive samples for HBoV1 and HBoV3 respectively and 2 positive for HBoV2. No HBoV4 were detected. Among the 8 positive HBoV samples, co-infection with other enteric viruses were found in 7 (87.5%) samples, while mono infection with HBoV alone was detected in 1 (12.5%) patient. HBoV mixed infection with Rotavirus (3/8; 37.5%); Adenovirus (3/8; 37.5%); Norovirus (2/8; 25%) and Astrovirus (1/8; 12.5%) were observed in this study. This study reported for the first time on the prevalence of human Bocavirus in children with AGE from rural communities in South Africa.


Author summary
Acute gastroenteritis (AGE) is recognized as a major cause for mortality in children ≤5 years of age in Africa and other developing countries. Viruses known to be involved in AGE includes Rotavirus, Norovirus, Astrovirus and Adenovirus and have been reported globally.
Recently the Human Bocavirus (HBoV) have been reported in numerous studies globally as a potential cause of diarrhea. In this study, the prevalence and genetic diversity of human This study reported for the first time on the prevalence of human Bocavirus in children with AGE from rural communities in South Africa.

Introduction
Acute gastroenteritis (AGE) is recognized as one of major causes of mortality in children ≤5 years of age in Africa and other developing countries (1, 2). AGE can be caused by several viral pathogens including Human Bocavirus (HBoV), which is an emerging viral agent reported as a potential cause of diarrhea, especially in young children (3,4).
HBoV is a member of the Parvoviriadae family, Parvovirinae subfamily and the genus HBoV investigations in South Africa (SA) have only reported on the detection of HBoV genotypes in children with respiratory tract infections (7)(8)(9). There is no published work on the detection of this virus from stools in children with AGE or from children living in rural communities with poor water and sanitation infrastructure in South Africa. This study aimed to determine the prevalence and genetic diversity of HBoV in children with AGE from rural communities in Limpopo, South Africa.

Detection and genotyping of HBoV Isolates
General characteristics of HBoV positive patients is summarized in Table 2. The prevalence of HBoV genotypes 1 to 4 in stool samples of children with AGE was determined using Real-Time PCR. In total, 8 (5.7%) samples were found positive for Human Bocavirus (Table 2). HBoV1 was detected in 3 female patients with a median age of ± 9 months, HBoV2 was detected in 2 patients of which 1 was male and 1 female with a median age of ± 9.5 months, HBoV3 was detected in 3 patients (37%) of which 2 were female and 1 was male with a median age of ± 19 months ( Table 2).
HBoV4 was not detected in any of the 141 patients. Five (62%) of the positive cases were from clinics and 3 (37%) of the positive cases were from hospitals ( Table 2).
In Real-Time PCR C T levels are used as a surrogate measurement of viral load in combination with standards of known quantities (10). In this study, HBoV1 was detected in 3 patients with a median C T value of ± 23.8, and HBoV2 in 2 patients with a median C T value of ± 36, whereas HBoV3 was detected in 3 patients with a median C T value of ± 27.7 (Table 2). Genotypes were determined through Sanger DNA sequencing and confirmed by comparison with reference genotypes available in the NCBI GenBank using BLAST tool. HBoV Sequences from the current study were  (Table 2).

Discussion
Several studies have reported Human Bocavirus in children with respiratory tract infections in South Africa (7)(8)(9). However, no data is available on the prevalence of HBoV in children with AGE from rural communities suggesting that cases from these rural areas are most likely to be under investigated and underreported (11). This study, for the first time investigated the prevalence of HBoV in children with AGE from rural communities in South Africa.
Altogether, a total of 141 stool samples from children with AGE were assessed for the presence of HBoV. The prevalence of 5.7% for HBoV in this study was comparable to other studies which have investigated HBoV in children with diarrhea worldwide (12)(13)(14). Several of these studies have indicated that HBoV prevalence in patients with AGE ranges between 0.8% to 42% (12,13). The high detection of HBoV in children from the clinics (outpatient) in the current study suggested that HBoV could be associated with both severe and asymptomatic diarrheal cases (15). Results from this study further suggests that HBoV could be seen as an emerging viral pathogen in the rural communities of South Africa. Although HBoV is considered a potential cause of diarrhea, available evidence supporting the causative role of the virus in acute gastroenteritis is inconclusive (16). The presence of HBoV in asymptomatic individuals raises questions regarding the role of the virus in gastrointestinal infections as a pathogen or just a bystander (16). This is due to limited available studies investigating the pathogenesis of HBoV due to the lack of cell culture system or animal model (17)(18)(19).
Reports worldwide have indicated that young children are prone to HBoV infections (20). In this study HBoV was detected in children between 1-24 months of age (≤24 months) (  (15) have reported that the transmission of HBoV was through ingestion of contaminated food/water (e.g. via flies, inadequate sanitation facilities, inadequate sewage and water treatment systems, and the cleaning food with contaminated water), direct contact with infected faces (faecaloral-route), person-to-person contact and poor personal hygiene. In this study, the patients came from rural communities with no or inadequate water and sanitation infrastructure and poor hygiene practices (21). From the 8 positive cases of HBoV in this study, 7 (87%) used tap water as a source of water and only 1 (12%) used spring water as a source of water at home. Rural communities in South Africa usually collect water in storage tanks for both domestic and sanitation use due to scarcity of water in rural areas, this results in the contamination of water through faecal-oral-route transmission (21). Waterborne viruses represent a major health risk to population worldwide (22). There is currently limited data worldwide exploring the circulation of HBoV from environmental samples. Some studies have shown the prevalence of Human Bocavirus in river water (22,23) and wastewater samples (24)(25)(26). Even though the role of HBoV in gastrointestinal infections still remains to be not fully understood, the risk of infection via contaminated water should be taken into consideration since many rural communities still face challenges of poor sanitation and hygiene practices (11). All 8 HBoV positive cases used a pit latrine at home. The use of a pit latrine plays a role in the transmission of pathogens since the facility lack hand washing facility and usually has flies moving in and out between the pit latrine facility and house (21).
Only, HBoV1, HBoV2 and HBoV3 were detected in this study. HBoV1 and HBoV3 were detected in 3 (37%) stool samples each, while HBoV2 was detected in 2 (25%) stool samples. Some studies have indicated that HBoV2, HBoV3 and HBoV4 are highly associated with gastroenteritis (27,28). The widespread distribution of HBoV1 and HBoV3 in comparison to HBoV2 could presumably be due to differences in pathogenesis that may influence their transmission route and ability to establish persistence (29). A study in Thailand (30) also only detected HBoV1, HBoV2 and HBoV3. Likewise, studies from Finland and Pakistan also detected genotypes HBoV1 to 3 (31,32). In this study we were unable to detect HBoV4. This was also the case in several other recent studies (6,31,33,34), which makes the role of HBoV4 genotype unclear.
HBoV co-infection with other enteric viruses have been reported worldwide. In this study, HBoV was co-detected with other enteric viruses that are involved in acute gastroenteritis including Adenovirus F which was detected in 3/8 (37.5%) samples, and Norovirus were the most predominant co-infections (13,30,36,37).
A study in China (33), suggested that differences in prevalence of certain HBoV genotypes might be due to regional differences in viral epidemiology. Some studies have suggested that HBoV have a seasonal peak during the spring months, while other studies suggested the winter months, the geographic location and seasonality (38,39) plays an important role in HBoV prevalence. In this study, samples were collected over a period of twelve months. However, samples were not available for collection in all the months. Therefore, overall HBoV prevalence was inconclusive concerning seasonal patterns.
Limitations of the study included the absence of a control group (asymptomatic cases) and mild cases of gastroenteritis. Another limitation was the small number of stool

Genotype analysis
All positive samples from Real-Time PCR assay were subjected to subsequent PCR targeting the NS1-nonstructural protein gene using the primers listed in Table 1.
The TopTaq Master Mix Kit (Qiagen) was used for the amplification of all positive HBoV samples following the manufactures instructions. Amplification reactions were performed in a volume of 50µl containing 25µl master mix, 5µl each of primer F and R, 15µl Free-RNase water and 5µl DNA. Amplification condition for HBoV1 were initial denaturation for 10 min at 94°C, 35 cycles of amplification (94°C for 1 min, 54°C for 1 min, and 72°C for 2 min) and the expected product size was 354 bp. HBoV2/4 amplification was initiated with a denaturation step of 15 min at 95ºC followed by 40 cycles of 94ºC for 15 sec, 53ºC for 30 sec, and 72ºC for 1 min, the expected product size was 454 bp for HBoV2/4. Human Bocavirus 2 and 4 used the same antisense primers devised by Kapoor et al. (18), which was unable to differentiate between HBoV2 and 4 genotypes. Therefore, genotypes were further confirmed through sequencing. The amplification of HBoV3 consisted of 1 step of 95ºC for 15 min followed by 45 cycles of 94ºC for 20 sec, 52ºC for 20 sec, and 72ºC for 40 sec, followed by an extension step at 72ºC for 10 min, the expected product size for HBoV3 was 440 bp (41).
All PCR products were visualized on 2% agarose gel electrophoresis and ethidium bromide staining. All positive samples were sequenced, the Sanger DNA sequencing was performed on the ABI 3500XL Genetic Analyzer POP7TM (Thermo-Scientific).
The nucleotide sequences were compared with those of the reference strains available in the NCBI GenBank using BLAST tool available at http://www.ncbi.nlm.nih.gov/blast then analyzed for their respective genotypes.

Co-infection viruses detected
Co-infection with other enteric viruses were determined using a CFX96 (Bio-Rad) realtime PCR. The Allplex gastrointestinal panel virus assays (Seegene Technologies Inc., California, USA) was used to determine the co-detected enteric viruses following the manufactures instructions.

Conflict of interest
The authors declare no conflict of interest.