Molecular detection of enteric viruses in the stool samples of children without diarrhea in Bangladesh

Journal Pre-proof Molecular detection of enteric viruses in the stool samples of children without diarrhea in Bangladesh

Shoko Okitsu, Pattara Khamrin, Sayaka Takanashi, Aksara Thongprachum, Sheikh Ariful Hoque, Haruko Takeuchi, Md Alfazal Khan, S.M. Tafsir Hasan, Tsutomu Iwata, Hiroyuki Shimizu, Masamine Jimba, Satoshi Hayakawa, Niwat Maneekarn, Hiroshi Ushijima PII:

S1567-1348(19)30281-3

DOI:

https://doi.org/10.1016/j.meegid.2019.104055

Reference:

MEEGID 104055

To appear in:

Infection, Genetics and Evolution

Received date:

25 May 2019

Revised date:

25 September 2019

Accepted date:

29 September 2019

Please cite this article as: S. Okitsu, P. Khamrin, S. Takanashi, et al., Molecular detection of enteric viruses in the stool samples of children without diarrhea in Bangladesh, Infection, Genetics and Evolution(2018), https://doi.org/10.1016/j.meegid.2019.104055

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© 2018 Published by Elsevier.

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Molecular detection of enteric viruses in the stool samples of children without diarrhea in Bangladesh Shoko Okitsu1, 2, Pattara Khamrin3, 4, Sayaka Takanashi2, Aksara Thongprachum5, Sheikh Ariful Hoque6, Haruko Takeuchi7, Md Alfazal Khan8, S. M. Tafsir Hasan8, Tsutomu Iwata9, Hiroyuki Shimizu10, Masamine Jimba7, Satoshi Hayakawa1, Niwat Maneekarn3, 4,

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Hiroshi Ushijima1, 2

Division of Microbiology, Department of Pathology and Microbiology, Nihon

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University School of Medicine, Japan

Department of Developmental Medical Sciences, Graduate School of Medicine, The

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University of Tokyo, Japan

Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,

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Thailand

Center of Excellence in Emerging and Re-emerging Diarrheal Viruses, Chiang Mai

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University, Chiang Mai, Thailand

Faculty of Public Health, Chiang Mai University, Chiang Mai, Thailand

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Cell and Tissue Culture Laboratory, Centre for Advanced Research in Sciences,

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University of Dhaka, Dhaka, Bangladesh Department of Community and Global Health, Graduate School of Medicine, The

University of Tokyo, Japan 8

Nutrition and Clinical Services Division, International Centre for Diarrhoeal Disease

Research, Bangladesh, Dhaka, Bangladesh 9

The Graduate School, Tokyo Kasei University, Japan

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Department of Virology II, National Institute of Infectious Diseases, Japan

Correspondence: Shoko Okitsu Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi, Tokyo 173-8610, Japan 1

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Tel: +81-3-3972-8111 ext: 2263 Fax: +81-3-3972-9560

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E-mail: [email protected]

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Abstract A number of molecular epidemiological studies reported the detection of enteric viruses in asymptomatic children. The role of these viruses in an asymptomatic infection remains unclear. This study investigated the enteric viruses in the stool samples collected from children without diarrhea. Stool samples were collected during June to October, 2016, from 227 children who lived in Matlab, Bangladesh. Seventeen enteric viruses,

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including rotavirus A, B, and C (RVA, RVB, and RVC), norovirus GI (NoV GI), norovirus GII (NoV GII), sapovirus (SaV), adenovirus (AdV), human astrovirus (HAstV),

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Aichivirus (AiV), human parechovirus (HPeV), enterovirus (EV), human bocavirus

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(HBoV), Saffold virus (SAFV), human cosavirus (HCoSV), bufavirus (BufV), salivirus

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(SalV), and rosavirus (RoV), were investigated by RT-PCR method. One hundred and eighty-two (80.2%; 182/227) samples were positive for some of these viruses, and 19.8%

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(45/227) were negative. Among the positive samples, 46.7% (85/182) were a single

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infection, and 53.3% (97/182) were coinfection with multiple viruses. The HCoSV was the most prevalent virus (41.4%), followed by EV (32.2%), NoV GII (25.6%), HPeV

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(8.8%), RVA (6.2%), AdV (5.7%), AiV (5.3%), SAFV (4.4%), and SaV (2.6%). Each of NoV GI, HAstV, HBoV, and BufV was detected at 0.4%. However, RVB, RVC, SalV, and RoV were not detected in this study. Phylogenetic analysis showed that diverse HCoSV species and genotypes were circulating in Bangladesh, and four strains of species A are proposed to be new genotypes. The data indicated that non-diarrheal Bangladeshi children were asymptomatically infected with wide varieties of enteric viruses.

Key words: Asymptomatic infection; Bangladesh; Coinfection; Enteric virus; Human cosavirus

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1. Introduction Despite decrease of child deaths due to diarrhea worldwide, it is still the second most leading cause of global child mortality. Diarrhea caused 8.6% (527,000 children) of death in 2000-2015 (Liu et al., 2016). In developing countries, diarrheal diseases are one of the most important problems for child health, meanwhile, they are common diseases and remain to cause serious economic burden in developed countries. Viral

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infection causes diarrheal diseases, and the most common virus is still rotavirus A (RVA), although rotavirus vaccination had been already introduced in more than 60

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countries worldwide (Tate et al., 2016). In addition, many other viruses are known to

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cause diarrheal diseases, such as noroviruses (NoV GI and GII), adenovirus (AdV),

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human astrovirus (HAstV), sapovirus (SaV), and various viruses in the family Picornaviridae (Thongprachum et al., 2016; 2017a).

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The previous studies reported asymptomatic infection of enteric viruses in the

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children. Kapusinszky et al. (2012a) described that various enteric viruses were detected in the stool of two healthy infants. Using metagenomics, 127 different viruses were

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detected in the sample pools of Ethiopian children (Altan et al., 2018). Norovirus and rotavirus have also been reported to associate with asymptomatic infection and persistent infection in relation to the host genetic factors, such as Histo Blood Group Antigen status (Colston et al., 2019; Piedade et al., 2019). Diarrhea-associated AdV is mainly F species (AdV40 and 41), however, other species of AdVs are also found in the diarrheal specimens, and the pathogenesis remains unknown. HAstV infection is associated with diarrhea, however, asymptomatic infection of HAstV has also been reported (Cortez et al., 2017; Olortegui et al., 2018).

On the other hand, these

asymptomatic enteric viral infections were described to be a risk of related outbreaks of acute gastroenteritis or foodborne diarrhea (Wang et al., 2018). Some viruses recently 4

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identified, especially the viruses in the family Picornaviridae, were found in asymptomatic population (Tapparel et al., 2013). Recently, components of the gut virome of mammalian hosts and their ability to modulate the responses of the hosts during homeostasis and disease have been described (Neil and Cadwell, 2018). However, the roles of enteric viruses in transient and asymptomatic infections are unclear.

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In Bangladesh, a pediatric mortality rate due to diarrhea was 6.4% (https://www.who.int/immunization/monitoring_surveillance/burden/estimates/rotavirus

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/en/) in 2013, and RVA was the most important pathogen that caused 32.8% of death in

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children with the age of less than 5 years old. Other viruses such as NoV GI and NoV

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GII, AdV, HAstV, SaV, and viruses in the family Picornaviridae were also reported to be associated with diarrhea in the country (Dey et al., 2007a; 2007b; 2009; Haque et al.,

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2018; Hoque et al., 2019; Mitui et al., 2014; Nelson et al., 2018; Olortegui et al., 2018;

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Pham et al., 2007). The association of enteric viruses with diarrhea has been established and most of data has been reported in only symptomatic individuals.

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This study investigated the enteric viruses in the stool samples collected from children without diarrhea in the rural area of Bangladesh in 2016.

2. Materials and Methods 2.1 Stool samples Stool samples were collected from 227 children (73 ± 4 months of age: 66 to 81 months of age) without diarrhea, who were formerly enrolled in the study investigating the association between asthma and parasitic infection at Matlab Hospital which is a 120-bed hospital and provides maternal and child healthcare service in Matlab, Bangladesh, from June to October, 2016 (Takeuchi et al., 2019). In this study site, the 5

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rotavirus vaccination was introduced and the vaccine coverage was demonstrated more than 70% (Schwartz et al., 2019; Zaman et al., 2017). The stool samples were stored at 30°C until use.

2.2 Ethical clearance The study was conducted with the approval by the Ethical Committees of the

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University of Tokyo (No. 11018), Japan, and icddr,b (#PR-15054), Bangladesh. Written informed consent was obtained from the legal guardians of all the subjects in the

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original study (Takeuchi et al., 2019).

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2.3 Viral genome extraction and reverse transcription The viral genome was extracted by using QIAamp Viral RNA mini kit (QIAgen,

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Hilden, Germany). The reverse transcription (RT) was performed with Superscript

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Japan).

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reverse transcriptase III (Invitrogen, Carlsbad, CA) with random primer (Takara, Shiga,

2.4 Detection of enteric viruses Seventeen viruses were detected by the monoplex and/or multiplex RT-PCR assays, using the primer sets as reported previously (Thongprachum et al., 2018; 2017a). The enteric viruses investigated in this study were RVA, rotavirus B and C (RVB and RVC), AdV, HAstV, NoV GI and NoV GII, SaV, Aichivirus (AiV), human parechovirus (HPeV), enterovirus (EV), human bocavirus (HBoV), Saffold virus (SAFV), human cosavirus (HCoSV), bufavirus (BufV), salivirus (SalV), and rosavirus (RoV). Additionally, nested-PCR assay was conducted for the detection of NoV GII, HBoV, HCoSV, SAFV, BufV, and SalV. The presence of enteric viruses was determined by 6

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electrophoresis of PCR products and visualized under the LED blue–light transilluminator.

2.5 Determination of the VP1 and partial 3D sequences of HCoSV strains The nucleotide sequences of the VP1 region and partial 3D region of HCoSV strains were amplified by using the primer sets reported previously (Kapusinszky et al.,

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2012b; Okitsu et al., 2014). The VP1 and 3D amplicons were sequenced and analyzed in

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comparison with the reference strains available in the GenBank database.

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2.6 DNA sequencing and phylogenetic analysis

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The PCR products of RVA, AdV, HAstV, NoV GI, NoV GII, SaV and HCoSV were sequenced for identification of species and genotypes of the viruses. The DNA

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sequencing was performed by using the BigDye terminator Cycle Sequencing Kit

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(Perkin Elmer-Applied Biosystems. Inc., Foster City, CA, USA). The genotypes of NoV GII were identified by the Novovirus Typing Tool Version 2.0 server

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(https://www.rivm.nl/mpf/typingtool/norovirus/). The nucleotide sequences were analyzed together with the reference strains deposited in the GenBank database. Multiple sequence alignments were conducted by the Clustal X program and phylogenetic trees were constructed according to the Maximum Likelihood method using MEGA version 7 software (Kumar et al., 2016). The nucleotide sequences of the HCoSV strains were deposited in the GenBank database under the accession numbers LC480465 to LC480513, LC481376 to LC481382, and LC496474 to 496480.

3. Results 3.1 Detection of enteric viruses 7

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One hundred and eighty-two (80.2%) samples were positive for some of the enteric viruses. The HCoSV was the most prevalent virus (41.4%), followed by EV (32.2%), NoV GII (25.6%), HPeV (8.8%), RVA (6.2%), AdV (5.7%), AiV (5.3%), SAFV (4.4%), and SaV (2.6%). Each of NoV GI, HAstV, HBoV, and BufV was detected at 0.4%. However, RVB, RVC, SalV, and RoV were not detected in this study (Table 1). Among the enteric viruses detected, 46.7% (85/182) were a single infection, and other 53.3 %

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(97/182) were multiple infections, including double, triple, quadruple and quintuple infections (Table 2). However, only 19.8% (45/227) of the stool samples were negative

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for any of enteric viruses investigated.

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3.2 Distribution of enteric virus genotypes and species The genotypes and species of enteric viruses detected in this study had been

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identified as shown in Table 3. The distributions of RVA, NoV GI, NoV GII, HAstV and

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SaV genotypes are presented in Table 3a. For the RVA, four genotypes, G1, G2, G3, and G8 were detected, and the G3 genotype was most prevalent at 42.9%. For NoV, only

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one GI.9 genotype of NoV GI was detected while eight genotypes of NoV GII, including GII.2, GII.3, GII.4, GII.6, GII.9, GII.15, GII.17, and GII.21 were detected, and the most prevalent genotype was GII.2 at 70.7%, following by GII.4, GII.3, and GII.6. For HAstV, only one HAstV of MLB2 type was detected. For SaV, four genotypes of GI, including GI.3, GI.4, GI.5, GI.7, and one genotype of GII (GII.1) were detected. The distributions of AdV and HCoSV species are presented in Table 3b. For AdV, five species of AdV, including species A, B, D, E, and F were detected, with species D being the most prevalent at 61.5%, followed by species A, B, E, and F. For HCoSV, four species of HCoSV, A, B, C, and D were detected, and A and D were the most prevalent species at 45.7% and 43.6%, respectively, and 2 samples (2.1%) of 8

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HCoSV detected in this study were unidentified species.

3.3 Phylogenetic analysis of HCoSV strains Phylogenetic tree of the nucleotide sequences of 5’UTR of 49 representative strains of HCoSV detected in this study was constructed and shown in Fig. 1. Four species of HCoSV, including species A, B, D, and C were identified (Fig. 1). Among HCoSV

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species A (HCoSV-A), 7 strains (105-629/BGD/2016, 212-1455/BGD/2016, 128808/BGD/2016, 224-1526/BGD/2016, 140-944/BGD/2016, 155-1050/BGD/2016, and

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82-500/BGD/2016) formed distinctive clusters separated from other HCoSV-A

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reference strains and therefore their genotypes could not be identified by 5’-UTR

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nucleotide sequence analysis in this phylogenetic tree. The VP1 gene of these strains were further amplified and sequenced in order to identify their genotypes. The

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nucleotide sequences were then converted to deduced amino acid sequences and

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compared with those of the HCoSV reference strains. The nucleotide and amino acid sequences identities of VP1 region of these HCoSV-A strains as compared to different

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genotypes of HCoSV-A reference strains are shown in Table 4. The HCoSV-A strains 128-808 and 224-1526 showed 99% nucleotide and amino acid sequences identities to each other, and showed 90% nucleotide, 97% and 96% amino acid sequence identities with A23, respectively, suggesting that the 128-808 and 224-1526 strains belonged to HCoSV genotype A23. The 155-1050 strain showed 80% nucleotide and 89% amino acid sequence similarities with that of A21 reference strain, suggesting the 155-1050 strain belonged to the HCoSV genotype A21. The other four HCoSV strains (105-629, 212-1455, 140-944, and 82-500) showed the nucleotide and amino acid sequence identities below the cutoff value of the same genotype using the genetic distance criteria of less than 75 % and 88% nucleotide and amino acid sequence identities, respectively 9

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(Kapusinszky et al., 2012b; Oberste et al., 1999). Therefore, the genotypes of these four strains could not be identified by analysis of nucleotide and amino acid sequence alignments with the reference strains. In order to identify the genotypes of these seven HCoSV-A strains, the VP1 nucleotide and deduced amino acid sequences of these strains were used to construct the phylogenetic tree together with different genotypes of the reference strains as shown in

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Fig. 2. The 128-808 and 224-1526 strains formed a monophyletic cluster with A23 genotype strain (AF198775-NG213-2/2007/NGA-A23), confirming that the 128-808

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and 224-1256 strains belong to A23 genotype. However, from the phylogenetic tree, the

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105-629, 212-1455, 140-944 and 82-500 strains formed distinct clusters separated from

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known genotypes. This observation was in line with the data of nucleotide and amino acid sequence alignment analyses described above. Therefore, the genotypes of these

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strains could not be identified and were proposed as novel genotypes.

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The full-genome sequences of strains detected in this study (the 82-500, 105-629, 128-808, 140-944, 155-1050, 212-1455, and 224-1526 strains) were attempted to

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determine by the primer walking method, however, it was not succeeded. To study the possibilities of recombination of four strains, the nucleotide sequences of partial 3D region (704bp) were determined. Phylogenetic analyses of the nucleotide sequences of the 3D region indicated that these four HCoSV strains belonged to species A, and formed clusters separated from other known genotypes within species A (Fig.3).

4. Discussion Enteric viruses are a diverse group of viruses that cause acute gastroenteritis worldwide and may be detected in both symptomatic and asymptomatic persons (Aiemjoy et al., 2019; Altan et al., 2018; Ayukekbong et al., 2011; Fan et al., 2019; Harb 10

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et al., 2019; Kapusinszky et al., 2012a; Moyo et al., 2017; Ouedraogo et al., 2016; Siqueira et al., 2018; Yinda et al., 2019). The recent studies conducted by the viral metagenomics analysis reported the detection of numerous enteric viruses, which were mainly from the families Picornaviridae and Caliciviridae (Altan et al., 2018; Siqueira et al., 2018). Our study investigated for seventeen enteric viruses in the stool samples of non-diarrheal children in Bangladesh, and then thirteen enteric viruses were detected.

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More than 80% of the samples at least one enteric virus was detected, and more than half were multiple virus infections. The HCoSV was detected as the most prevalent

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viruses, followed by EV, and NoV GII, and the other enteric viruses, HPeV, RVA, AdV,

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AiV, SAFV, SaV, NoV GI, HAstV, HBoV, and BufV were detected with less prevalent.

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The results of this study highlight an extremely high prevalence (approximately 80%) and high diversity (13 different kinds) of enteric viruses detected in Bangladeshi

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children without any symptom of diarrhea. In addition, the results are comparable with

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those reported from other developing countries in Africa and South America (Altan et al., 2018; Ouedraogo et al., 2016; Siqueira et al., 2018; Yinda et al., 2019). The enteric

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viruses are transmitted by the fecal-oral-route, and an important feature of these viruses is the silent shedding of these viruses from asymptomatic persons which may facilitate the transmission and spreading of these viruses to susceptible persons and also into the environment and eventually contamination in the sources for drinking water. The findings show that the risk of infection by enteric viruses is higher in developing countries compared to developed countries probably due to suboptimal sanitation and hygienic conditions, particularly low quality of drinking water in rural areas (Emch, 1999; Okoh et al., 2010). The HCoSV was originally detected in the feces of South Asian children with nonpolio acute flaccid paralysis (Kapoor et al., 2008). Later, it was reported worldwide, 11

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including Asia, Australia, Africa, Europe, North and South America with low prevalence (Blinkova et al., 2009; Dai et al., 2010; Holtz et al., 2008; Khamrin et al., 2012; Khamrin and Maneekarn, 2014; Okitsu et al., 2014; Rovida et al., 2013; Stocker et al., 2012). However, in some countries such as Afghanistan, Pakistan, North India, Tunisia, and Bolivia, the HCoSV was reported with high prevalence, ranging from 22.5% to 52.9% (Kapoor et al., 2008; Maan et al., 2013; Nix et al., 2013; Rezig et al., 2015). The

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present study also reported high prevalence of HCoSV (41.4%) in the rural area of Bangladesh which is one of the South Asian countries where the HCoSV was reported

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with high prevalence, suggesting a wide spread of HCoSV in this geographical area.

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Although the HCoSV was detected with higher prevalence in patients with diarrhea than

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in healthy persons (Dai et al., 2010; Oude Munnink et al., 2014), it was also reported previously as well as shown in the present study with high prevalence in healthy

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asymptomatic persons (Kapoor et al., 2008; Nix et al., 2013; Stocker et al., 2012). The

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role of HCoSV in association with diarrhea is unclear and awaits for future elucidation. Based on the criteria for classification of HCoSV, the VP1 nucleotide or amino acid

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sequence identities of less than 75% or 88%, respectively, is defined as a new genotype/serotype (Kapusinszky et al., 2012b; Oberste et al., 1999). The nucleotide and amino acid sequence identities of four strains of HCoSV-A (105-629, 212-1455, 140944, and 82-500) detected in this study were compared to those of known genotypes. The identities were below the cutoff value of the same genotypes, suggesting that these strains might be the new genotype in the species A of HCoSV. For genotyping in the species A, distribution of pairwise distance between the VP1 regions of strains in this study and the reference strains available in the GenBank database were analyzed, however, frequencies of nucleotide and amino acid distances could not clearly form clusters. At present HCoSV studies are very limited and only 33 complete VP1 12

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nucleotide sequences are available in the Genbank database that makes analysis unsuccessful. On the other hand, recombination event plays an important role for evolution of picornaviruses (Lukashev, 2010). The previous studies reported two highly similar recombinant strains of species D and E in Nigeria and Brazil (Kapusinszky et al., 2012; da Cost et al., 2018). However, the inter- and intra-species recombinations of species A

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have never been previously reported and needed to be further investigated. In addition, the possibility of four strains of HCoSV detected in this study (82-500, 105-629, 140-

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944, and 212-1455) and proposed as a new genotype could be the recombinant strains is

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also needed to be elucidated.

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Although it is well-established that the RVA causes gastroenteritis in infants and young children, the detection of RVA in healthy asymptomatic children is not

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uncommon (Abiodun et al., 1985; Barron-Romero et al., 1985; Phillips et al., 2010).

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Most recently, asymptomatic RVA infection in post-vaccinated children has been reported and the RVA genotypes are homologous to those causing gastroenteritis in

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unvaccinated children of the same community (Gunawan et al., 2019). The children included in this study were the RVA vaccinated children under the routine immunization program in Matlab (Schwartz et al., 2019), Bangladesh. The prevalence of asymptomatic RVA infection observed in our study was 6.2% which was much lower than those of the general population of children with acute gastroenteritis in Bangladesh at 24.0 – 32.0% (Haque et al., 2018; Rahman et al., 2007). The RVA genotypes, G1, G2, G3, and G8 detected in this study in asymptomatic persons who live in Matlab, Bangladesh, are closely corresponded to those reported in the sewage water in Dhaka city, Bangladesh (Hoque et al., 2019) suggesting that the sewage water could be the potential source of RVA infection in human in Bangladesh. 13

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The prevalence of NoV detected in the present study in asymptomatic children in Matlab, Bangladesh was 25.6% which is approximately two times higher than those reported recently in Mirzapur, another rural city of Bangladesh (Hossain et al., 2018). The NoV GII.2 was reported to emerge in Asia in 2016, particularly in China (Ao et al., 2017) and in Japan (Thongprachum et al., 2017b). In the present study, the stool specimens were collected in 2016 and the NoV GII.2 was found to be the most

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predominant genotype at 70.7% (Table 3a) rather than the NoV GII.4 that has been to be the most predominant worldwide. The findings confirm the emergence of NoV GII.2 in

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Asia in 2016 and became the most predominant genotype to replace the NoV GII.4.

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The limitation of this study is a small sample size which was collected in a short

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time period, and some of the subjects were more than 5 years of age.

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5. Conclusion

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Thirteen enteric viruses including RVA, AdV, HAstV, NoV GI, NoV GII, SaV, AiV, HPeV, HBoV, EV, SAFV, HCoSV, and BufV, were detected in stool samples collected

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from non-diarrheal children who lived in the rural area of Bangladesh. About 80% of stool samples were detected at least one type of enteric viruses and more than half of the positive samples were co-infected with multiple enteric viruses.

Acknowledgements This research was supported by Grants-in-Aid for Scientific Research under Japan Society for the Promotion of Science (JSPS) grant numbers 26257507 and 16H05360, and by Japan Agency for Medical Research and Development (AMED) under grant number JP18fk0108004.

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Zaman, K., Sack, D.A., Neuzil, K.M., Yunus, M., Moulton, L.H., Sugimoto, J.D., Fleming, J.A., Hossain, I., Arifeen, S.E., Azim, T., Rahman, M., Lewis, K.D.C., Feller, A.J., Qadri, F., Halloran, M.E., Cravioto, A., Victor, J.C., 2017. Effectiveness of a live oral human rotavirus vaccine after programmatic introduction in Bangladesh: A cluster-randomized trial. PLoS Med.

14, e1002282. https://doi.org/10.1371/journal.pmed.1002282

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Table 1.

Detection of enteric viruses in non-diarrheal children in

Bangladesh Numbers

Detection rates

RVA

14

6.2%

RVB

0

0.0%

RVC

0

0.0%

AdV

13

5.7%

HAstV

1

NoV GI

1

NoV GII

58

SaV

6

Pr

e-

pr

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f

Virus

AiV

0.4% 25.6% 2.6%

12

5.3%

20

8.8%

al

HPeV

0.4%

1

0.4%

73

32.2%

SAFV

10

4.4%

HCoSV

94

41.4%

BufV

1

0.4%

SalV

0

0.0%

RoV

0

0.0%

rn

HBoV

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EV

RVA: rotavirus A, RVB: rotavirus B, RVC: rotavirus C, AdV: adenovirus, HAstV: human astrovirus, NoV GI: norovirus genogroup I, NoV GII: norovirus genogroup II, SaV: sapovirus, AiV; Aichi virus, HPeV: human parechovirus, HBoV: human bocavirus,

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EV: enterovirus, SAFV: Saffold virus, HCoSV: human cosavirus, BufV: bufavirus, SalV: salivirus, RoV: rosavirus

Table 2.

Single and multiple infections of enteric viruses in non-diarrheal

children in Bangladesh

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Single infection (n=85, 37.4%,) Number of samples

Detection rate (%)

RVA

4

1.8%

AdV

6

e-

NoV GII

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SAFV

rn

EV

al

AiV

HCoSV

2.6%

17

7.5%

2

0.9%

1

0.4%

6

2.6%

17

7.5%

3

1.3%

29

12.8%

Pr

SaV

HPeV

pr

Diarrheal viruses

Double infection (n=76, 33.5%) Diarrheal viruses

Number of samples

Detection rate (%)

RVA+NoV GII

1

0.4%

RVA+EV

1

0.4%

RVA+HCoSV

5

2.2%

AdV + EV

3

1.3%

AdV + HCoSV

2

0.9%

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1

0.4%

NoV GI + NoV GII

1

0.4%

NoV GII + AiV

2

0.9%

NoV GII + HPeV

2

0.9%

NoV GII + EV

10

4.4%

NoV GII + HCoSV

13

5.7%

SaV + EV

1

SaV + HCoSV

1

AiV + HPeV

1

AiV + EV

3

e-

pr

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f

HAstV + HPeV

1

Pr

AiV + HCoSV AiV + SAFV

0.4% 0.4% 0.4% 1.3% 0.4%

1

0.4%

1

0.4%

3

1.3%

1

0.4%

EV + SAFV

2

0.9%

EV + HCoSV

18

7.9%

SAFV + HCoSV

1

0.4%

HCoSV + BufV

1

0.4%

al

HPeV + SAFV

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HBoV + HCoSV

rn

HPeV + HCoSV

Triple infection (n=18, 7.9%) Diarrheal viruses

Number of samples

Detection rate (%)

RVA + AiV + EV

1

0.4%

RVA + SaV + HCoSV

1

0.4%

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2

0.9%

NoV GII + HPeV+ HCoSV

2

0.9%

NoV GII + EV + HCoSV

7

3.1%

SaV + AiV + EV

1

0.4%

AiV + EV + HCoSV

1

0.4%

HPeV + EV + HCoSV

1

0.4%

EV + SAFV + HCoSV

2

0.9%

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f

AdV + EV + HCoSV

pr

Quadruple infection (n=2, 0.9%) 2

0.9%

Pr

e-

NoV GII + HPeV + EV + HCoSV

Quintuple infection (n=1, 0.4% ) 1

0.4%

rn

al

RVA + NoV GII + HPeV + EV + HCoSV

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Table 3a. Distributions of RVA, NoV GI, NoV GII, HAstV and SaV genotypes RVA

NoV GI

NoV GII

HAstV

Genoty

Numb

Genoty

Numb

Genoty

Numb

pe

er

pe

er

pe

er

G1

4

GI.9

1

GII.2

41

SaV

Numb

Genoty

Numb

er

pe

er

1

GI.3

1

Type

MLB 2 G2

2

GII.3

4

GI.4

1

G3

6

GII.4

5

GI.5

1

G8

2

GII.6

3

GI.7

2

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Total

14

Total

1

GII.9

1

GII.15

1

GII.17

2

GII.21

1

Total

58

Total

1

GII.1

1

Total

6

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Table 3b. Distributions of AdV and HCoSV species

HCoSV

2

B

1

D

8

E

1 1

rn

F

A

43

B

2

C

6

D

41

Unidentified

2

13

Total

94

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Total

Number

e-

A

Species

Pr

Number

al

Species

pr

AdV

Table 4. Identities of nucleotide and amino acid sequences of VP1 region of the HCoSV strains detected in this study and reference strains Identities of nucleotide sequences S

1 2

1 2 1

1 8

t A A

A 2 2

A

A A

r 1 2

A A A 1 8 4

2

2 1

a 9 0

A A A 2 7 8 5

3 -

-

3

A A 2 6 9

4

A A 0 1 4 A A A A A A A 5 1 2 5 2 0 1 1 1 1 1 1 2 5 3

5

A 1

8 1

28

1

0 2 -

-

1 2 4 5 6 7 1 -

6 1 9

1

-

8 5

i

B

2

Journal Pre-proof

n

0 5

2 4 4

0 0

n

8 2

9 5 4

5 0

a

6

5

0

m e /

oo

f

g e

pr

n

e-

o

Pr

t y

al

p

rn

e

6 5 6 5 5 5 5 5 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 4

A

I

Identities of amino acid sequences

D

%%%%% % %%%%%%%%%%%% % %%%%%%%%% %%%

9

A

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6 6 0 8 6 6 8 6 6 8 0 7 7 6 6 6 8 5 5 5 8 8 4 7 7 9 6 3 6 4 9

1

6

5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 5 I

2

7

0

%

8 7 0 8 8 8 8 7 6 9 8 7 4 6 8 5 7 6 5 7 7 6 9 6 9 9 8 9 5 2 D %%%% % %%%%%%%%%%%% % %%%%%%%%% %%%

5 5

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 5 5 5 5 4 I

A

6 2

7 7 9 9 7 9 9 7 8 8 5 6 5 7 5 6 5 8 9 9 6 7 7 8 8 8 7 8 9 D

2

%%

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29

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The name of HCoSV strains detected in this study and the strains that showed highest nucleotide and amino acid sequence identities are shown in bold face. Reference strains: A1: 0553; A2: 6344; A3: 6572; A5: NG6; A6: TN06-S231; A7: NP11; A8: NG18; A9: NP7; A10: NG11; A11: NP1; A12: NG14; A13: NP6; A14: NG376; A15:

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NG23; A16: NP10; A17: TN06-E34; A18: NG15; A19: PK6187; A20: NG263; A21: NG295-2; A22: NG295-1; A23: NG213-2; A24: NP8-1; A25: NG2; HCoSVB1: 2263.

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The VP1 sequence of A4 strain was not registered in the GenBank database.

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Figure legends Figure 1. Phylogenetic analysis of 5’-UTR nucleotide sequences of HCoSV strains (173 nt). A Kimura-2 parameter model was used for the Maximum Likelihood method. Bootstrap values greater than 70% are shown. A scale bar indicates the number of nucleotide substitution per site. The HCoSV strains detected in this study are indicated with filled circle.

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Figure 2. Phylogenetic analysis of the VP1 (a) nucleotide (834nt) and (b) amino acid (277aa) sequences of HCoSV strains. (a) A Kimura-2 parameter model was used for the

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Maximum Likelihood method. The 2263/PAK in the species B1 (FJ438907) was used as

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outgroup. (b) A Jones-Taylor-Thornton model was used for the Maximum Likelihood

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method. The 2263/PAK in the species B1 (ACL15190) was used as outgroup. Bootstrap value greater than 70% are shown. A scale bar indicates the number of amino acid

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substitution per site. The HCoSV strains detected in this study are indicated with filled

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circle. The established reference strains of A genotypes are indicated in boldface. Figure 3. Phylogenetic analysis of the nucleotide sequences of partial 3D region (621

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nt) of HCoSV strains. A Kimura-2 parameter model was used for the Maximum Likelihood method. Bootstrap values greater than 70% are shown. A scale bar indicates the number of nucleotide substitution per site. Four HCoSV strains detected in this study are indicated with filled circle. The 2263/PAK in the species B1 (FJ438907) was used as outgroup.

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Highlights ●Investigation of stool samples collected from healthy children without diarrhea revealed that 80.2% of the samples contained enteric viruses ●Enteric viruses including RVA, AdV, HAstV, NoVGI, NoVGII, SaV, AiV, HPeV, HBoV, EV, SAFV, HCoSV, and BufV were detected

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●Both single and multiple types of virus infections were observed in these children

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Conflict of Interest

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The authors have no conflict of interest to declare regarding this study.

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Figure 1

Figure 2A

Figure 2B

Figure 3