Genetic recombination and diversity of sapovirus in pediatric patients with acute gastroenteritis in Thailand, 2010–2018

Background Human sapovirus (SaV) is an etiologic agent of acute gastroenteritis (AGE) in all age groups worldwide. Genetic recombination of SaV has been reported from many countries. So far, none of SaV recombinant strain has been reported from Thailand. This study examined the genetic recombination and genotype diversity of SaV in children hospitalized with AGE in Chiang Mai, Thailand. Methods Stool samples were collected from children suffering from diarrhea who admitted to the hospitals in Chiang Mai, Thailand between 2010 and 2018. SaV was detected by RT-PCR and the polymerase and capsid gene sequences were analysed. Results From a total of 3,057 samples tested, 50 (1.6%) were positive for SaV. Among positive samples, SaV genotype GI.1 was the most predominant genotype (40%; 20/50), followed by GII.1 and GII.5 (each of 16%; 8/50), GI.2 (14%; 7/50), GIV.1 (4%; 2/50), and GI.5 (2%; 1/50). In addition, 4 SaV recombinant strains of GII.1/GII.4 were identified in this study (8%; 4/50). Conclusions The data revealed the genetic diversity of SaV circulating in children with AGE in Chiang Mai, Thailand during 2010 to 2018 and the intragenogroup SaV recombinant strains were reported for the first time in Thailand.


Sample collection
Stool samples were collected from children who admitted to hospitals with AGE during the period 2010 to 2018 from five major hospitals in Chiang Mai province, northern Thailand, including Maharaj Nakorn Chiang Mai Hospital, Sriphat Medical Center, Nakornping Hospital, Sanpatong Hospital, and Rajavej Chiang Mai Hospital. The age of patients enrolled in this study ranged from neonate to 15 years old. Acute gastroenteritis was defined by watery diarrhea with three or more stool episodes per day for less than 14 days (Green, 2013). All specimens were stored at −20 • C until investigation. This work was conducted under the approval of the Research Ethics Committee of the Faculty of Medicine, Chiang Mai University (MIC-2557-02710). The written informed consent form was obtained from parents before samples were collected from their children

Detection of SaV by RT-PCR
Viral nucleic acid was extracted from 200 µl of the supernatant of a 10% stool suspension prepared in phosphate-buffered saline (pH 7.4) using the Geneaid Viral Nucleic Acid Extraction Kit II (Geneaid, Taiwan) according to the manufacturer's protocol. The viral RNA was reverse transcribed to cDNA using random hexamer primers and RevertAid TM reverse transcriptase (https://www.thermofisher.com/, USA) according to the manufacturer's instruction. SaV was screened by conventional PCR method using GoTaq DNA polymerase (Promega, USA) with primers SLV5731 and SLV5749 (Table 1)  targeting capsid region (VP1 gene) as described previously (Yan et al., 2003). The PCR cycling condition was as follows: initial denature at 94 • C for 3 min, 35 cycles step of denature at 94 • C for 1 min, anneal at 58 • C for 1 min, and extend at 72 • C for 1 min, followed by final extension at 72 • C for 10 min. Amplicon size of 434 bp was separated on 1.5% agarose gel electrophoresis and stained with nucleic acid staining solution (RedSafe, INtRON Biotechonology, South Korea) before subjecting to visualize under ultraviolet transilluminator. In addition to SaV screening, the same set of stool specimens were also tested for several other diarrhea-causing viruses, including rotavirus, norovirus, astrovirus, adenovirus, enterovirus, parechovirus, and Aichivirus using the protocol described previously (Khamrin et al., 2011).

Sequence analysis and genotype identification
A Gel/PCR DNA Fragment Extraction Kit (Geneaid, Taiwan) was used to purify amplicons of SaV-capsid gene according to the manufacturer's protocol. All purified PCR products were direct sequenced using Applied Biosystems BigDye R Terminator Cycle Sequencing Kit v3.1 (Life Technologies, USA) with forward and reverse primers SLV5731 and SLV5749, and analyzed by using Applied Biosystems 3100 Genetic Analyzer (Life Technologies, USA). Nucleotide sequences of partial capsid gene were analyzed manually using BioEdit and Clustal X softwares. Identification of virus genotype was initially determined by using the BLAST Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Human Calicivirus Typing Tool (https://norovirus.ng.philab.cdc.gov/) and confirmed by phylogenetic analysis using MEGA7 software package (Kumar, Stecher & Tamura, 2016). The tree was statistically supported by bootstrapping with 1,000 replicates.

Recombination analysis
To examine genetic recombination of SaV strains detected in this study, the polymerase (RdRp) region was amplified by using primers shown in  , 1997;Harada et al., 2013;Honma et al., 2001;Martinez et al., 2002). Phylogenetic tree of the partial capsid gene sequences was constructed by using the Maximum Likelihood method based on the Kimura 2-parameter model. Phylogenetic tree of the partial RdRp region was inferred by using the Maximum Likelihood method based on the General Time Reversible model. To predict the putative recombination point for SaV recombinant strains, nucleotide sequences of the polymerase and capsid genes, spanning the RdRp-VP1 junction region (positions 5078-5265 according to SaV genome of accession no. X86560), were analyzed. The possible recombination point of SaV strain was determined by using SimPlot software v.3.5.1 (Lole et al., 1999). In addition, the confidence interval for recombination between the query strain and parent strains was examined by the Recombination Detection Program v.4.71 (RDP4) complemented with the Max-Chi test to confirm the significant events (p < 0.01) (Martin et al., 2015).

Nucleotide accession number
Nucleotide sequences of the polymerase and capsid regions of SaV strains detected in this study were deposited in the GenBank database under the accession numbers MN245671 to MN245579 and MN253492 to MN253541 for the polymerase and capsid gene sequences, respectively.

Prevalence of SaV
A total of 3,057 stool samples collected between 2010 and 2018 were screened for the presence of SaV. The average of SaV detection rate over the study period of nine years was 1.6% (50 out of 3057) (Table 1). From 2010 to 2015, SaV was detected at low prevalence (0-1.8%). After that, the prevalence of SaV infection increased year by year to 2.2%, 2.5%, and 2.8% in 2016, 2017, and 2018, respectively. There was no specific seasonal pattern of SaV detection observed in this study. The age of SaV-infected patients varied from 8 months up to 11 years (Table 2). Among 50 SaV positive cases, 26 (52%) were male and 24 (48%) were female. All SaV positive samples were also tested for the presence of other enteric viral pathogens. The results showed that majority of cases were single infection with SaV (74%, 37 out of 50) whereas the rest of cases (26%, 13 out of 50) were co-infected with other enteric viruses including rotavirus, norovirus, adenovirus, parechovirus, enterovirus, or astrovirus (Table 3).

Recombination analysis
In the SaV genome, recombination event typically occurs between the polymerase (RdRp) and capsid (VP1) genes. To investigate the genetic recombination of SaV strains detected in this study, we further amplified the polymerase gene of the viruses. The partial RdRp gene sequence (745 nucleotides) was successfully obtained from 49 of 50 SaV-positive samples. Phylogenetic tree of the partial RpRp region of 49 SaV strains was constructed (Fig. 2) and the results showed that all strains, except for four GII.4 strains (CMH-S057-17, CMH-S050-17, CMH-N021-13, and CMH-N145-12), were clustered into the same genotypes assigned by the capsid gene sequence as shown previously in Fig. 1. Based in the capsid gene sequence (Fig. 1), CMH-S057-17, CMH-S050-17, CMH-N021-13, and CMH-N145-12 were assigned as the GII.4 genotype whereas based on the RdRp sequence they clustured together with SaV GII.1 reference strains and shared 91.8-95.0% nucleotide sequence identities. The data suggested that these four SaV strains were the SaV GII.

SaV infection in different age groups of patients
Among SaV positive cases, the highest detection rate was seen in patients with the age of 2 to <3 years (40.5%), followed by 1 to <2 years (35.7%), less than 1 year (9.5%), 3 to <4 years (7.1%), 4 to <5 years (4.8%), and more than 5 years (2.4%). In addition, this study identified 6 different genotypes and one recombinant pattern of SaV. Distribution of SaV genotypes detected in different age groups of patients is shown in Fig. 4. It was found that GI.1 genotype was detected in all age groups except for patients with more than 5 years of age. The GII.5 was also identified in patients with age groups of less than 4 years. Interestingly, recombinant SaV GII.1/GII.4 was detected in patients between 1 and 2 years of age.

DISCUSSION
Enteric caliciviruses including noroviruses and sapoviruses are the major causes of AGE of human in all age group worldwide (Khamrin et al., 2017;Lu et al., 2014;Neo et al., 2017;Sala et al., 2014). The detection rate of SaV infection in many countries around the world have been reported with a range from 0.2 to 39% in children and 2.2 to 15.6% in all age groups (Magwalivha et al., 2018;Oka et al., 2015;Varela et al., 2019). In Thailand, the prevalence of SaV was detected between 0% and 15.0% (Kumthip & Khamrin, 2018 et al., 2017;Supadej et al., 2019). However, when compared to the studies conducted in different geographical regions, the prevalence of SaV detected in this study was lower than those reported from Japan (4.8%), Philippines (7%), USA (5.4%), Italy (6%), Denmark (8.8%), Finland (9.3%), Spain (15.6%), and Nicaragua (17%) (Biscaro et al., 2018;Bucardo et al., 2014;Chhabra et al., 2013;Oka et al., 2015;Thongprachum et al., 2015;Varela et al., 2019). The variation of SaV prevalence in different studies could be explained, at least in part, by the difference in study locations, the detection methods, and the emergence of new epidemic strains. It was noticed that our study as well as other studies that had similar SaV prevalence used single CMH-S004-13 CMH-N131-13 CMH-S174-18 CMH-ST169-18 CMH-S023-17 MG012400 GI.  round PCR as a screening method while other studies that reported higher SaV-positive rate performed real-time PCR for the screening process. Among different genogroups of SaV, the SaV GI (GI.1 and GI.2) and GII (GII.1 and GII.2) are the most predominant genogroups circulating worldwide while other genotypes are rarely detected in some particular countries (Diez-Valcarce et al., 2018;Magwalivha et al., 2018). Similar to other reports, SaV GI.1, GI.2, and GII.1 were the most common genotypes detected in our study. Nonetheless, there was no GII.2 strain observed in the present study. The SaV GII.5 is not often detected. The occurrence of GII.5 in human stool samples was reported in some particular areas such as Guatemala, Peru, South Africa, and USA (Diez-Valcarce et al., 2018;Liu et al., 2016;Murray et al., 2016). In addition to these countries, it should be noted that a high proportion of GII.5 strain (16%, 8 out of 50) was observed in our study. Interestingly, 7 out of 8 SaV GII.5-infected patients were from the same hospital (Sanpatong hospital) and 5 of them were detected in the same year of 2018, suggesting that this particular genotype is circulating in a particular location.
RNA recombination plays a key role in virus evolution and leads to its pathogenicity and virus diversity (Worobey & Holmes, 1999). At least, two types of recombination events of SaV including intergenogroup and intragenogroup have been reported previously (Chanit et al., 2009;Hansman et al., 2007;Hansman et al., 2005;Katayama et al., 2004;Oka et al., 2015;Phan et al., 2006). In the present study, intragenogroup recombinant GII.1/GII.4 SaV was detected in four samples, accounting for 8% (4/50) of all SaV infected cases. The recombinant GII.1/GII.4 SaV has been reported previously in Philippines, Vietnam, and USA (Diez-Valcarce et al., 2018;Liu et al., 2015;Nguyen et al., 2008). However, it has not been described elsewhere in Thailand. To our knowledge, this is the first report demonstrating the presence of recombinant SaV in Thailand. Generally, characterization of SaV is based on the nucleotide sequence of capsid gene (Oka et al., 2012) and many other previous studies have identified SaV genotypes based only on this gene. To date, both capsid and polymerase genes are used to classify genotype of noroviruses (Chhabra et al., 2019). Since SaV is a very similar pathogen in many aspects, therefore, future classification and characterization of SaV should rely on both polymerase and capsid sequences to identify the virus diversity. In addition, continued surveillance on SaV is important to monitor the emergence of new virus strains.

CONCLUSIONS
In summary, the results of this study highlight the impact of SaV in diarrheal diseases among children in Chiang Mai, Thailand over the period of nine years and is the first report to describe recombinant SaV infection in Thai children suffering with AGE. The data of nucleotide analysis of both polymerase and capsid genes from this study provide useful information for a better understanding on the caliciviruses other than noroviruses.