Study of Cosavirus, Salivirus, and Bufavirus viruses in children with acute gastroenteritis
Beni-Suef University Journal of Basic and Applied Sciences volume 12, Article number: 89 (2023)
Acute gastroenteritis (AGE) in children represents a health problem. Besides common enteric viruses such as rotavirus and adenovirus, new viruses such as cosavirus, salivirus, and bufavirus may be associated with AGE. The objective of the study was to detect cosavirus, salivirus, and bufavirus viruses in children below 5 years with acute gastroenteritis by the use of real-time polymerase chain reaction (PCR) besides detection of rotavirus and adenovirus by enzyme-linked immunosorbent assay (ELISA).
The study included 150 children ≤ 5 years with community-acquired diarrhea. Stool samples from children were subjected for the detection of rotavirus and adenovirus antigens by ELISA and for detection of buvavirus, salivirus, and cosavirus by real-time PCR.
The commonest virus detected in the stool samples of children with AGE was rotavirus 31.3% followed by adenovirus 24%. Among the new viruses studied, salivirus was detected in six samples (4.0%), buvavirus was detected in four samples (2.7%), and cosavirus was detected in two samples (1.3%). The mixed rotavirus detection with studied viruses was 23.4% for adenovirus, 4.3% for calicivirus, and 2.1% for bocavirus, and none of the detected cosavirus was associated with rotavirus. In the studied children, at least one of the new viruses was detected in ten children (6.7%). Buvavirus, salivirus, and cosavirus were detected as a single virus (0.7%) in the children with acute gastroenteritis and buvavirus was detected with cosavirus without other viruses in one sample (0.7%).
The study reports the occurrence of buvavirus, cosavirus, and salivirus in the pediatric patients with community-acquired acute gastroenteritis. There was a high prevalence of rotavirus and adenovirus antigens in those patients with low positivity for buvavirus, cosavirus, and salivirus viruses. There is a need for a large cohort study to study the prevalence of buvavirus, cosavirus, and salivirus in pediatrics with acute gastroenteritis and to validate their association with the disease.
Acute gastroenteritis (AGE) in children remains a major health problem worldwide. It has high morbidity and mortality rates in addition to the financial burden of the disease . The infectious organisms' responsible for AGE include mainly viruses like rotaviruses noroviruses, astroviruses, and human adenoviruses . There is a continuous discovery of new viruses that may be implicated as the etiology of acute gastroenteritis since about 40% of AGE is of unknown etiology [3,4,5].
Studies have reported the emergence of newly discovered viruses cosavirus (CosV), salivirus (SalV), and bufavirus (BuV) that may be implicated in acute gastroenteritis in children . The CosaV and salivirus are RNA viruses, while bufavirus is a DNA virus.
The CosV is an RNA virus with single RNA strand of around 7.6 Kb organized in a typical picornavirus genome. There are around six species of the virus from A to F; the A species had 24 t genotypes, and D species had five different genotypes .
The salivirus is an RNA virus with a single RNA strand genome of around 7.1Kb that is picornavirus genome typically. SalV had one genotype with 2 clusters . Human salivirus has been reported to be associated with AGE from different geographical locations and in sewage from studies in Spain and Hong-Kong .
The third virus, bufavirus a single stranded DNA virus, was associated with AGE from children in Burkina Faso, from which it gets its name , and from other countries [11,12,13,14]. It is a DNA virus that is around 4.9 Kb, that encodes viral structural protein (VP2) and nonstructural protein 1 (NS1). There are three types of the genotypes (BuV1, 2, and 3) ; however, due to the diversity of the capsid gene, there may be other undiscovered genotypes .
The link of these viruses to AGE as an etiology is controversy and there is a need to detect these viruses in the AGE in children to establish their role. There are various reports about the detection of CosV, SalV, and bufavirus in children with AGE in different geographical regions [7, 10]. However, there are no reports about the detection of these viruses in children from Egypt.
Therefore, the objective of the study is to detect the viruses cosavirus, salivirus, and bufavirus in pediatric patients below 5 years with acute gastroenteritis by using the real-time polymerase chain reaction (real-time PCR) besides rotavirus and adenovirus detection by enzyme-linked immunosorbent assay (ELISA).
This report was a cross-sectional study which included 150 children ≤ 5 years complaining of community-acquired diarrhea. The included children were recruited from outpatient clinics, Egypt, from March 2022 to December 2022. The size of the sample depended upon the time nonprobability sampling consecutive that depended on the time of study in which enrolled pediatric patients with diarrhea with the inclusion criteria will be presented to the clinicians during the time of the study .
The inclusion criteria were children ≤ 5 years with diarrhea during the past 72 h. The children with diarrhea due to systemic conditions, drug intake, or associated with hepatic or renal disorders or with isolated bacterial pathogens or parasites were excluded from the study.
The children were subjected to medical examination and the condition severity classification was assessed by medical history taking from the parents of each child and recorded by the use of the scale of Vesikari . The Vesikari scale depends upon 20 points calculation regarding the number of diarrhea and its severity with the presence of vomiting, fever, and dehydration. Vesikari score is reported as mild if below 7, moderate from 7 to 10, and severe if ≥ 11.
2.1 Stool samples
The obtained stool sample from each child was taken in clean container and transferred within thirty minutes to the laboratory.
3 Stool examination and culture
Stool samples were examined for the presence of r parasites by the method of previously described . The microbiological specific culture was used for exclusion of common bacterial pathogens like Aeromonas species, E. coli O157:H7, Pleisiomonas shigelloides, Salmonella, and Shigella. Microbiological culture was performed by culture on agar of sheep's blood, plate of MacConkey, plate of sorbitol-MacConkey and Selenite F broth (BBL; Becton Dickinson) was used for Salmonella–Shigella with pre-inoculation. All culture plates were incubated at 35 °C for 24 h .
4 Stool examination for rotavirus and human adenovirus antigen
Detection of rotavirus will be performed by commercially available enzyme-linked sorbent assay (ELISA)-RIDASCREEN® kit (R-Biopharm, Germany) and detection of adenovirus was performed by ELISA by RIDASCREEN® Adenovirus (R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany). The kits were used according to the manufacturer instructions.
5 Real-time PCR for CosV, SalV, and bufavirus
5.1 Nucleic acid extraction (DNA–RNA) from stool samples
Nucleic acid was extracted from stool sample, by the use of 400 μL of 12.5% stool suspension in phosphate buffer solution and the RNA, DNA extraction kits (Promega, Madison, WI, USA) were used for the relevant virus according to the manufacturer’s instructions. The DNA and RNA were eluted in the final volume of 50 μL and kept frozen at − 20°C till other amplification procedures.
6 Reverse transcription for cosavirus and salivirus
The reverse transcriptase reactions for cosavirus and salivirus were carried on the 5 μL of the extracted RNA by the use of a Promega kit for reverse transcription by the same manufacturer’s instructions. The reaction for the reverse transcription was performed at 42°C for 60 min, followed by 70 °C for 15 min.
7 Amplification by real-time PCR
Ready-to-use amplification kit with primers and probe will be used to ready the primers and probes commercial kits: PP-BioMole073 for salivirus, PP-BioMole074 for cosavirus, and PP-BioMole077 for bufavirus (BioMole, Turin, Italy). The amplification was performed by the following sequences: heating for 2 min at 50 °C, 2 min at 95 °C, 45 cycles of 15 s at 95 °C, and 1 min at 60 °C on the 7500 ABI real-time instrument (Life-Technologies).
7.1 Statistical analysis
The SPPS22 was used to analyze the data. The qualitative data were expressed as number and percentages. The comparison between qualitative data was done by the use of Chi-square test and P was significant if it was below 0.05. The numerical data were mentioned as mean and standard deviation (mean ± SD).
The study included 150 children with acute gastroenteritis, 96 (64%) males and 54 females (36%), with mean age ± SD 43.8 ± 13.02 months. The presenting symptoms were watery diarrhea 79(52.7%), fever 65 (43.3%), and vomiting 64 (42.7%). The Vesikari score was mainly mild (46%) and moderate (45.3%), Table 1.
The commonest virus detected in the stool samples of children with AGE was rotavirus 31.3% followed by adenovirus 24%. Among the new viruses studied, salivirus was detected in six samples (4.0%), buvavirus was detected in four samples (2.7%), and cosavirus was detected in two samples (1.3%), Table 2. The mixed rotavirus detection with studied viruses was 23.4% for adenovirus, 4.3% for calicivirus, and 2.1% for bocavirus, and none of the detected cosavirus was associated with rotavirus, Table 3.
In the studied children, at least one of the new viruses was detected in ten children (6.7%). Buvavirus, salivirus, and cosavirus were detected as a single virus (0.7%) in the children with acute gastroenteritis and buvavirus was detected with cosavirus without other viruses in one sample (0.7%), data not shown.
In the present study, the severe Vesikari score was defined in 8.7% of the children. Previous study reported similar severity of AGE in children . The presenting symptoms were watery diarrhea 79(52.7%), fever 65 (43.3%), and vomiting 64 (42.7%). A previous study of the AGE in children below 5 years and vomiting in. reported fever and vomiting as a common symptom in children with AGE . The vomiting is a leading cause for the severity of AGE and failure of oral rehydration therapy.
The common etiology of AGE in children in less developed countries is viral pathogens . The present study shows high prevalence of rotavirus (31.3%) followed by adenovirus (24%). The mixed rotavirus detection with studied viruses was 23.4% for adenovirus. The prevalence of adenovirus as an etiology for AGE ranged from 1% up to 23% in different geographical locations [24,25,26,27,28,29]. The range of rotavirus detection in AGE was from 16.7% up to 73.7% [30,31,32,33].
In a previous study from Egypt, a higher prevalence rate was reported for rotavirus (58%) and a lower rate for adenovirus (6.7%) and mixed rotavirus and adenovirus was 8% in children admitted to hospital . The difference in the presence of rotavirus and adenovirus may be attributed to the age of the included children and the type of acute gastroenteritis, as the included children in the present study were children 5 years with community-acquired diarrhea. However, there is a need to introduce the rotavirus vaccine in the national program the vaccination of children in Egypt.
In the studied children, at least one of the new viruses was detected in ten children (6.7%). Among the new viruses studied, salivirus was detected in six samples (4.0%), buvavirus was detected in four samples (2.7%), and cosavirus was detected in two samples (1.3%). A previous study reported detection rates of these new viruses in 11 patients (5.4%) with CosV 2 (1.0%), SalV 7 (3.5%), and 2 BuV (1.0%) .
The ranges of detection rates of cosavirus in diarrheal children in previous studies ranged from 1% up to 3.6% in children with diarrhea (1%) [35, 36]. The detection rates of BuV 0% to 4.0% in patients of all ages [[10, 12, 37,38,39]. SalV had a prevalence ranges from 0.1 up to 8.8% in patients with gastroenteritis [4, 14, 40,41,42,43,44,45,46,47,48]. The discrepancy of the detection rates of these virus may be attributed to the difference in the patients age in various studies, whether children or adults.
In the present report, mixed presence of buvavirus was detected with cosavirus without other viruses in one sample (0.7%) and mixed with rotavirus was present in 4.3% of the stool samples for salivirus, and in the stool samples 2.1% for bufavirus. The mixture of enteric rotavirus with the bufavirus and salivirus may indicate environmental contamination of soiled water as a possible source of infection . There were various studies that indicated the wide distribution of salivirus in different environmental samples [29,30,31,32]. This finding may indicate a fear-oral transmission of the virus. There is a need for larger children studies from multiple geographical regions in Egypt to assess the association of these viruses with AGE in children.
The study shows the presence of buvavirus, cosavirus, and salivirus in the pediatric patients with community-acquired acute gastroenteritis. There was a high presence of rotavirus and adenovirus antigens in those patients with low positivity for buvavirus, cosavirus, and salivirus viruses. There is a need for a large cohort study to study the prevalence buvavirus, cosavirus, and salivirus in children with acute gastroenteritis and to validate their association with the disease.
Availability of data and materials
Enzyme-linked immunosorbent assay
Nonstructural protein 1
Polymerase chain reaction
Viral structural protein
Eckardt AJ, Baumgart DC (2011) Viral gastroenteritis in adults. Recent Patents Anti Infect Drug Discov 6:54–63. https://doi.org/10.2174/157489111794407877
Arashkia A, Bahrami F, Farsi M, Nejati B, Jalilvand S, Nateghian A et al (2019) Molecular analysis of human adenoviruses in hospitalized children < 5 years old with acute gastroenteritis in Tehran, Iran. J Med Virol 91:1930–1936. https://doi.org/10.1002/jmv.25539
Smits SL, Osterhaus AD, Koopmans MP (2016) Newly identified viruses in human gastroenteritis: pathogens or not? Pediatr Infect Dis J 35:104–107. https://doi.org/10.1097/INF.0000000000000950
Daprà V, Galliano I, Montanari P, Zaniol E, Calvi C, Alliaudi C et al (2021) Bufavirus, Cosavirus, and Salivirus in diarrheal Italian infants. Intervirology 64:165–168. https://doi.org/10.1159/000514384
Menage L, Yodmeeklin A, Khamrin P, Kumthip K, Maneekarn N (2017) Prevalence of human cosavirus and saffold virus with an emergence of saffold virus genotype 6 in patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2014–2016. Infect Genet Evol 53:1–6. https://doi.org/10.1016/j.meegid.2017.05.005
Ayouni S, Estienney M, Hammami S, Neji Guediche M, Pothier P, Aouni M et al (2016) Cosavirus, salivirus and bufavirus in diarrheal Tunisian infants. PLoS ONE 11:e0162255. https://doi.org/10.1371/journal.pone.0162255
Kapusinszky B, Phan TG, Kapoor A, Delwart E (2012) Genetic diversity of the genus Cosavirus in the family Picornaviridae: a new species, recombination, and 26 new genotypes. PLoS ONE 7(5):e36685. https://doi.org/10.1371/journal.pone.0036685
Boros A, Raab M, Karoly E, Karai A, Katai A, Bolba N et al (2016) A cluster of salivirus A1 (Picornaviridae) infections in newborn babies with acute gastroenteritis in a neonatal hospital unit in Hungary. Arch Virol 161(6):1671–1677. https://doi.org/10.1007/s00705-016-2824-9
Holtz LR, Finkbeiner SR, Zhao G, Kirkwood CD, Girones R, Pipas JM et al (2009) Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread. Virol J 6:86. https://doi.org/10.1186/1743-422X-6-86
Phan TG, Vo NP, Bonkoungou IJ, Kapoor A, Barro N, O’Ryan M et al (2012) Acute diarrhea in West African children: diverse enteric viruses and a novel parvovirus genus. J Virol 86(20):11024–11030. https://doi.org/10.1128/JVI.01427-12
Altay A, Yahiro T, Bozdayi G, Matsumoto T, Sahin F, Ozkan S et al (2015) Bufavirus genotype 3 in Turkish children with severe diarrhoea. Clin Microbiol Infect. https://doi.org/10.1016/j.cmi.2015.06.006
Chieochansin T, Vutithanachot V, Theamboonlers A, Poovorawan Y (2015) Bufavirus in fecal specimens of patients with and without diarrhea in Thailand. Arch Virol 160(7):1781–1784. https://doi.org/10.1007/s00705-015-2441-z
Huang DD, Wang W, Lu QB, Zhao J, Guo CT, Wang HY et al (2015) Identification of Bufavirus-1 and Bufavirus-3 in feces of patients with acute diarrhea, China. Sci Rep 5:13272. https://doi.org/10.1038/srep13272
Smits SL, Schapendonk CM, van Beek J, Vennema H, Schurch AC, Schipper D et al (2014) New viruses in idiopathic human diarrhea cases, the Netherlands. Emerg Infect Dis 20(7):1218–1222. https://doi.org/10.3201/eid2007.140190[
Yahiro T, Wangchuk S, Tshering K, Bandhari P, Zangmo S, Dorji T et al (2014) Novel human bufavirus genotype 3 in children with severe diarrhea, Bhutan. Emerg Infect Dis 20(6):1037–1039. https://doi.org/10.3201/eid2006.131430
Martínez-Mesa J, González-Chica DA, Duquia RP, Bonamigo RR, Bastos JL (2016) Sampling: how to select participants in my research study? An Bras Dermatol 91(3):326–330. https://doi.org/10.1590/abd1806-4841.20165254
Schnadower D, Tarr PI, Gorelick MH, O’Connell K, Roskind CG, Powell EC, Rao J, Bhatt S, Freedman SB (2013) Validation of the modified Vesikari score in children with gastroenteritis in 5 US emergency departments. J Pediatr Gastroenterol Nutr 57(4):514–519. https://doi.org/10.1097/MPG.0b013e31829ae5a3
Humphries RM, Linscott AJ (2015) Practical guidance for clinical microbiology laboratories: diagnosis of bacterial gastroenteritis. Clin Microbiol Rev 28(1):3–31
Moore NE, Wang J, Hewitt J, Croucher D, Williamson DA, Paine S, Yen S, Greening GE, Hall RJ (2015) Metagenomic analysis of viruses in feces from unsolved outbreaks of gastroenteritis in humans. J Clin Microbiol 53:15–21. https://doi.org/10.1128/JCM.02029-14
Ramani S, Kang G (2009) Viruses causing childhood diarrhoea in the developing world. Curr Opin Infect Dis 22:477–482. https://doi.org/10.1097/QCO.0b013e328330662f
Jones S (2003) A clinical pathway for pediatric gastroenteritis. Gastroenterol Nurs 26:7–18. https://doi.org/10.1097/00001610-200301000-00003
Weghorst AAH, Bonvanie IJ, Holtman GA et al (2022) Course of uncomplicated acute gastroenteritis in children presenting to out-of-hours primary care. BMC Prim Care 23:125. https://doi.org/10.1186/s12875-022-01739-2
Seo H, Duan Q, Zhang W (2020) Vaccines against gastroenteritis, current progress and challenges. Gut Microbes 11:1486–1517. https://doi.org/10.1080/19490976.2020.1770666
Oh DY, Gaedicke G, Schreier E (2003) Viral agents of acute gastroenteritis in German children: prevalence and molecular diversity. J Med Virol 71:82–93. https://doi.org/10.1002/jmv.10449
Ouyang Y, Ma H, Jin M, Wang X, Wang J, Xu L, Lin S, Shen Z, Chen Z, Qiu Z et al (2012) Etiology and epidemiology of viral diarrhea in children under the age of five hospitalized in Tianjin, China. Arch Virol 157:881–887. https://doi.org/10.1007/s00705-012-1235-9
Cunliffe NA, Booth JA, Elliot C, Lowe SJ, Sopwith W, Kitchin N, Nakagomi O, Nakagomi T, Hart CA, Regan M (2010) Healthcare-associated viral gastroenteritis among children in a large pediatric hospital, United Kingdom. Emerg Infect Dis 16:55. https://doi.org/10.3201/eid1601.090401
Meqdam MM, Thwiny IR (2007) Prevalence of group a rotavirus, enteric adenovirus, norovirus and astrovirus infections among children with acute gastroenteritis in Al-Qassim, Saudi Arabia. Pak J Med Sci 23:551
Aminu M, Ahmad A, Umoh J, De Beer M, Esona M, Steele A (2007) Adenovirus infection in children with diarrhea disease in Northwestern Nigeria. Ann Afr Med 6:168–173. https://doi.org/10.4103/1596-3519.55702
Olesen B, Neimann J, Bottiger B, Ethelberg S, Schiellerup P, Jensen C, Helms M, Scheutz F, Olsen KEP, Krogfelt K et al (2005) Etiology of diarrhea in young children in Denmark: a case-control study. J Clin Microbiol 43:3636–3641. https://doi.org/10.1128/JCM.43.8.3636-3641.2005
Bates PR, Bailey AS, Wood DJ, Morris DJ, Couriel JM (1993) Comparative epidemiology of rotavirus, subgenus F (types 40 and 41) adenovirus and astrovirus gastroenteritis in children. J Med Virol 39(3):224–228
Ozsari T, Bora G, Kaya B, Yakut K (2016) The prevalence of rotavirus and adenovirus in the childhood gastroenteritis. Jundishapur J Microbiol 9(6):e34867
Allayeh AK, El Baz RM, Saeed NM, Osman ME (2018) Detection and genotyping of viral gastroenteritis in hospitalized children below five years old in Cairo, Egypt. Arch Pediatr Infect Dis 6(3):e6288
Tekin A (2010) The frequency of rotavirus and enteric adenovirus in children with acute gastroenteritis in Mardin. J Clin Exp Invest 1(1):41–45
Montasser KA, Youssef MI, Ghandour AA, Kamal M (2022) Infection with adenovirus, rotavirus, and coinfection among hospitalized children with gastroenteritis in an Egyptian university hospital. J Med Virol 94(10):4950–4958. https://doi.org/10.1002/jmv.27935
Dai XQ, Hua XG, Shan TL, Delwart E, Zhao W (2010) Human cosavirus infections in children in China. J Clin Virol 48(3):228–229
Stocker A, Souza BF, Ribeiro TC, Netto EM, Araujo LO, Correa JI et al (2012) Cosavirus infection in persons with and without gastroenteritis, Brazil. Emerg Infect Dis 18(4):656–659
Oka T, Katayama K, Hansman GS, Kageyama T, Ogawa S, Wu FT et al (2006) Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction. J Med Virol 78(10):1347–1353
Altay A, Yahiro T, Bozdayi G, Matsumoto T, Sahin F, Ozkan S et al (2015) Bufavirus genotype 3 in Turkish children with severe diarrhoea. Clin Microbiol Infect 21:965-e1
Huang DD, Wang W, Lu QB, Zhao J, Guo CT, Wang HY et al (2015) Identification of Bufavirus-1 and Bufavirus-3 in Feces of patients with acute diarrhea. China Sci Rep 5:13272
Allard A, Albinsson B, Wadell G (2001) Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis. J Clin Microbiol 39(2):498–505
Yamashita T, Sugiyama M, Tsuzuki H, Sakae K, Suzuki Y, Miyazaki Y (2000) Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the Picornavirus family associated with gastroenteritis in humans. J Clin Microbiol 38(8):2955–2961
Ayouni S, Estienney M, Sdiri-Loulizi K, Ambert-Balay K, de Rougemont A, Aho S et al (2015) Relationship between GII.3 norovirus infections and blood group antigens in young children in Tunisia. Clin Microbiol Infect 21(9):874 e1–8
Ayouni S, Sdiri-Loulizi K, de Rougemont A, Estienney M, Ambert-Balay K, Aho S et al (2015) Rotavirus P infections in persons with secretor and nonsecretor phenotypes, Tunisia. Emerg Infect Dis 21(11):2055–2058
Yu JM, Ao YY, Liu N, Li LL, Duan ZJ (2015) Salivirus in children and its association with childhood acute gastroenteritis: a paired case-control study. PLoS ONE 10(7):e0130977
Reuter G, Pankovics P, Boros Á (2017) Saliviruses—the first knowledge about a newly discovered human picornavirus. Rev Med Virol 27(1):e1904
Haramoto E, Otagiri M (2013) Prevalence and genetic diversity of klassevirus in wastewater in Japan. Food Environ Virol 5(1):46–51
Kitajima M, Iker BC, Rachmadi AT, Haramoto E, Gerba CP (2014) Quantification and genetic analysis of salivirus/klassevirus in wastewater in Arizona, USA. Food Environ Virol 6(3):213–216
Badru S, Khamrin P, Kumthip K, Yodmeeklin A, Surajinda S, Supadej K et al (2018) Molecular detection and genetic characterization of Salivirus in environmental water in Thailand. Infect Genet Evol 65:352–356
Ethics approval and consent to participate
The study was approved by the Ethical Committee of Mansoura Faculty of Medicine, Egypt (R.22.12.1987). The study was performed according to the Declaration of Helsinki. Informed written consent was obtained from each child's parent.
Consent for publication
There are no competing interests for any of the authors.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Salem, O.A.M., El Sayed Zaki, M., Elsayed, A.G. et al. Study of Cosavirus, Salivirus, and Bufavirus viruses in children with acute gastroenteritis. Beni-Suef Univ J Basic Appl Sci 12, 89 (2023). https://doi.org/10.1186/s43088-023-00429-x