European Journal of Clinical Microbiology & Infectious Diseases

, Volume 26, Issue 1, pp 21–27

Emergence of rare sapovirus genotype among infants and children with acute gastroenteritis in Japan

Authors

  • T. G. Phan
    • Department of Developmental Medical Sciences, Institute of International Health, Graduate School of MedicineUniversity of Tokyo
  • Q. D. Trinh
    • Department of Developmental Medical Sciences, Institute of International Health, Graduate School of MedicineUniversity of Tokyo
  • F. Yagyu
    • Department of Developmental Medical Sciences, Institute of International Health, Graduate School of MedicineUniversity of Tokyo
  • S. Okitsu
    • Department of Developmental Medical Sciences, Institute of International Health, Graduate School of MedicineUniversity of Tokyo
    • Department of Developmental Medical Sciences, Institute of International Health, Graduate School of MedicineUniversity of Tokyo
Article

DOI: 10.1007/s10096-006-0235-7

Cite this article as:
Phan, T.G., Trinh, Q.D., Yagyu, F. et al. Eur J Clin Microbiol Infect Dis (2007) 26: 21. doi:10.1007/s10096-006-0235-7
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Abstract

A total of 1,154 fecal specimens from infants and children with acute gastroenteritis in five cities in Japan (Maizuru, Tokyo, Sapporo, Saga, and Osaka), collected from July 2003 to June 2005, were tested for the presence of diarrheal viruses by reverse transcriptase multiplex PCR. Overall, 469 of 1,154 (40.6%) were positive for diarrheal viruses, of which 49 (10.4%) were positive for sapovirus. The peak of sapovirus infection shifted from April–June in 2003–2004 to October–December in 2004–2005. The observations show that maximum sapovirus prevalence can occur during warmer seasons. Sapovirus was subjected to molecular genetic analysis by sequencing. The results indicated that sapovirus genogroup I was a dominant group (100%). Sapovirus strains detected in this study were further classified into four genotypes (GI/1, GI/4, GI/6, and GI/8). Of these, sapovirus GI/1 was the most predominant, followed by sapovirus GI/6; these accounted for 93% (13 of 14) and 7% (1 of 14), respectively, in 2003–2004. However, it was noteworthy that sapovirus GI/6 suddenly emerged to become the leading genotype, accounting for 77% (27 of 35) of isolates in 2004–2005. This is believed to be the first report of the changing distribution of sapovirus genotypes and of the emergence of the rare sapovirus GI/6.

Introduction

Acute gastroenteritis is a common illness in humans worldwide and has a great impact on public health [1]. It is recognized as one of the leading causes of death by infectious disease [1, 2]. The mortality of infants and children due to acute gastroenteritis is greater in developing than in developed countries [3]. While rotavirus is recognized as the most important cause of severe gastroenteritis in infants and young children worldwide, sapovirus (SaV) is also considered a significant global enteropathogen of acute gastroenteritis [47].

SaV, previously referred to as Sapporo-like virus, is a distinct genus within the family Caliciviridae. The prototype strain of SaV is the Sapporo virus (Hu/SaV/Sapporo virus/1977/JP), which was derived from an outbreak in a home for infants in Sapporo, Japan, in 1977 [8]. SaV has a positive-sense single-strand RNA genome surrounded by an icosahedral capsid. The SaV genome is 7.3–7.5 kb long and contains two main open reading frames. The open reading frame 1 encodes a polyprotein that undergoes protease processing to produce several nonstructural proteins, including an RNA-dependent RNA polymerase and a capsid protein. The open reading frame 2 encodes a small basic protein with unknown function [8]. On the basis of the sequence analysis of the capsid gene, SaV is divided into five genogroups, among which only genogroups I, II, IV, and V are known to infect humans [912]. Recently, the diversity of SaV was described by Akihara et al. [13], who found that SaV genogroup I and genogroup II could be classified into eight and five genotypes, respectively. Immunological and seroepidemiologic studies have indicated a worldwide distribution of SaV. The age-related prevalence of antibody against this virus also has shown that infections commonly occur in children under 5 years of age. Furthermore, it was found that serum antibody level to SaV was lowest in the first year of life but increased after 2 years of age [1416].

The objectives of this study were to determine the incidence of diarrheal virus infections in infants and children with acute gastroenteritis in five different cities in Japan during 2003–2005; to characterize the SaV detected according to genogroup and genotype; and to describe the genetic diversity among them. Additionally, the age-related distribution and the seasonal pattern of SaV infection were determined.

Materials and methods

Fecal specimens

A total of 1,154 fecal specimens were collected from sporadic cases of acute gastroenteritis in pediatric clinics from five different cities (Maizuru, Tokyo, Sapporo, Saga, and Osaka) in Japan during the period of July 2003 to June 2005. The ages of the subjects ranged from 2 months to 15 years, the median age being 28 months. This age distribution in the periods 2003–2004 and 2004–2005 was similar. The fecal specimens were suspended in distilled water to prepare 10% suspensions, which were clarified by centrifugation at 10,000×g for 10 min; the supernatants were stored at −30°C.

Extraction of viral genome

The genomes of both RNA and DNA viruses were extracted from 140 μl of 10% fecal suspensions using a single QIAamp spin-column kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

Reverse transcription

In reverse transcription (RT), 4 μl of extracted viral genome was added to a reagent mixture consisting of 5× first strand buffer (Invitrogen, Carlsbad, CA, USA), 10 mM dNTPs (Roche, Mannheim, Germany), 10 mM DTT (Invitrogen), superscript reverse transcriptase III (200 U/μl) (Invitrogen), random primer (hexa-deoxyribonucleotide mixture) (1 μg/μl) (Takara, Shiga, Japan), RNase inhibitor (33 U/μl) (Toyobo, Osaka, Japan), and MilliQ water. The total volume of reaction mixture was 8 μl. The RT step was carried out at 50°C for 1 h, then 99°C for 5 min, after which it was stored at 4°C [17].

Viral detection and polymerase chain reaction

Two multiplex polymerase chain reaction (PCR) procedures using mixtures of primers previously reported were used to identify two groups of diarrheal viruses. The first group includes astrovirus, norovirus (GI, GII), and SaV, and the second group includes group A, B, and C rotaviruses and adenovirus [17]. In the first multiplex PCR, the primer set included equimolar mixes of PreCAP1 and 82 b for astrovirus, G1SKF and G1SKR for norovirus genogroup I, COG2F and G2SKR for norovirus genogroup II, and SLV5317 and SLV5749 for SaV. The respective products of these reactions consisted of amplicons of 719, 330, 387, and 434 bp for astrovirus, norovirus (GI, GII), and SaV, respectively. In the second multiplex PCR, the primer set included equimolar mixes of Beg9 and VP7-1, B5-2 and B3-3, and G8NS1 and G8NA2 for group A, B, and C rotaviruses, respectively, and Ad1 and Ad2 for adenovirus. The respective products of these reactions consisted of amplicons of 395, 814, 352, and 482 bp for group A, B, and C rotaviruses and adenovirus, respectively. The PCR was performed at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 7 min, after which the products were stored at 4°C.

Genotype of group A rotavirus

Genotyping of group A rotavirus was conducted using the G-type specific primers from the method described previously by Das et al. in 1994 [18]. The full-length of VP7 gene was reversely transcribed and then further amplified with Beg9 and End9 primers. The expected size of the PCR product generated from the full-length VP7 gene was 1,062 bp in length. The second amplification was performed using the first PCR product as the template with G-type specific mixed primers (9T1-1, 9T1-2, 9T-3P, 9T-4, and 9T-B) for downstream priming and 9con1 for upstream priming in an amplification of VP7 genes of G1–G4 and G9, respectively. These primers specifically generated five different sizes of amplicons of 158, 224, 466, 403, and 110 bp for G1, G2, G3, G4, and G9, respectively.

Electrophoresis

The PCR products were electrophoresed in a 1.5% agarose gel, after which they were stained with ethidium bromide for 20 min and then examined under ultraviolet light. The results were photographed.

Latex agglutination test

The Diarlex test (Orion Diagnostica, Espo, Finland), a commercial latex agglutination test, was used for the detection of group A rotavirus infection as a confirmatory test in the fecal specimens that were found by RT-PCR to harbor coinfection with SaV and group A rotavirus. A drop of the fecal supernatant was mixed with a drop of test latex on a slide, and the reaction was observed after 2 min. Development of distinct agglutination in the Diarlex reagent was considered a positive result. If agglutination was seen in the negative control latex, the result was considered uninterpretable.

Nucleotide sequencing and phylogenetic analysis

The nucleotide sequences of RT-PCR products (DNA) positive for SaV and norovirus were determined with the Big-Dye terminator cycle sequencing kit and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed using Clustal X software, version 1.6. A phylogenetic tree with 1,000 bootstrap resamples of the nucleotide alignment datasets was generated using the neighbor-joining method with Clustal X. The genetic distance was calculated using Kimura’s two-parameter method (PHYLIP). The sequences of SaV strains detected in the present study—5786/Osaka/JP, 5800/Osaka/JP, 5836/Osaka/JP, 5862/Osaka/JP, 5862/Osaka/JP, and 5821/Osaka/JP—had been submitted to GenBank and had been assigned accession numbers AB242322, AB242323, AB242324, DQ401095, AB242325, and AB242326, respectively. Reference SaV strains and accession numbers used in this study were as follows: PEC (AF182760), Lyon/598/97/F (AJ271056), London/92 (U95645), Mex340/90 (AF435812), Cruiseship/00 (AY289804), Hou7-1181/90 (AF435814), Arg39 (AY289803), Parkville/94 (U73124), Sapporo/82 (U65427), Manchester/93 (X86560), Karachi/730/1992 (AB126249), Karachi/874/1992 (AB181129), Karachi/928/1994 (AB181128), Karachi/1017/1990 (AB181227), Karachi/876/1993 (AB181132), Houston/90 (U95644), Stockholm/97 (AF194182), 12/DCC/Tokyo/Japan/44 (AB236380), Karachi/872/1991 (AB181231), 4408/Maizuru/JP (AB180209), 4724/Osaka/JP (AB180212), and Mex14917/2000 (AF435813).

Results

Molecular epidemiology of viral infections

RT-PCR analysis revealed the presence of viruses in 469 fecal specimens (Table 1). The ages of the infants and children with viral gastroenteritis ranged from 2 months to 11 years. The highest rate of viral infection was in the age group 12–23 months (42%) and the lowest rate was in the age group <6 months (2%). There was no difference in age distribution between the periods 2003–2004 and 2004–2005. In 2003–2004, group A rotavirus was the most prevalent (43.3%, 78 of 180), followed by norovirus (GI and GII) (29.5%, 53 of 180). Interestingly, norovirus (GI and GII) dominated over group A rotavirus and became the leading cause of acute gastroenteritis in 2004–2005, accounting for 45.3% (131 of 289) of cases. Moreover, a rather high rate (4.3%, 20 of 469) of viral coinfections was shown in this study during 2003–2005. It was found that the number of SaV infections increased sharply from 14 cases in 2003–2004 to 35 cases in 2004–2005. The incidence of SaV was highest in the 12–23-month-old group (45%, 22 of 49) and lowest in infants aged <6 months (2%, 1 of 49). Of note, most of the SaV infections (82%, 40 of 49) occurred in infants and children <3 years of age.
Table 1

Distribution of viral infection in infants and children with acute gastroenteritis in five cities in Japan

Year

No. of specimens tested

No. (%a) positive for viruses

Monoinfection (%b)

Mixed infection (%b)

RV

NoV

SaV

Ade

AsV

Ade+AsV

SaV + RA

Ade+NoV GII

RA + NoV GII

Ade + RA

SaV + NoV GII

AsV + RA

A

B

C

I

II

2003–2004

402

180 (44.8)

78 (43.3)

0 (0)

1 (0.6)

1 (0.6)

52 (28.9)

12 (6.7)

22 (12)

5 (2.8)

1 (0.6)

2 (1.1)

3 (1.7)

2 (1.1)

1 (0.6)

0 (0)

0 (0)

2004–2005

752

289 (38.4)

77 (26.6)

0 (0)

0 (0)

2 (0.7)

129 (44.6)

30 (10.4)

32 (11.2)

8(2.8)

0 (0)

0 (0)

1 (0.3)

2 (0.7)

1 (0.3)

5 (1.7)

2 (0.7)

Total

1,154

469 (40.6)

155 (33)

0 (0)

1 (0.2)

3 (0.6)

181 (38.6)

42 (9)

54 (11.5)

13 (2.8)

1 (0.2)

2 (0.4)

4 (0.9)

4 (0.9)

2 (0.4)

5 (1.1)

2 (0.4)

RV rotavirus, NoV norovirus, SaV sapovirus, Ade adenovirus, AsV astrovirus, NoV GI norovirus genogroup I, NoV GII norovirus genogroup II, RA group A rotavirus

aRefers to total number of specimens tested

bRefers to number of viral-positive specimens tested

Seasonal variation of sapovirus infection

A comparison of rate of detection of SaV between the periods 2003–2004 and 2004–2005 is shown in Fig. 1. The monthly mean temperature in the five cities in Japan is also shown. The summer lasts from June to September, and the hottest temperature was recorded in August. The coldest month is January, when the temperature might dip as low as 4°C. The incidence of SaV was highest in April–June (ten cases) and was second highest in January–March (three cases). The lowest rate of detection was recorded in October–December (zero cases) in 2003–2004 (p < 0.001). In contrast, SaV infection was identified continuously from August to February in 2004–2005, and the highest number of cases was recorded in October–December (25 cases), followed by July–September (six cases) (p < 0.001). The peak of SaV infection shifted from April–June in 2003–2004 to October–December in 2004–2005.
https://static-content.springer.com/image/art%3A10.1007%2Fs10096-006-0235-7/MediaObjects/10096_2006_235_Fig1_HTML.gif
Fig. 1

Monthly distribution of SaV infection in infants and children with acute gastroenteritis in five different cities of Japan, July 2003 to June 2005. The mean temperature in Japan during the molecular epidemiology study of SaV infection is shown

Coinfection with sapovirus and other viral enteropathogens

In total, 49 fecal specimens were determined to be positive for SaV by RT-PCR. Seven (14%) of these specimens harbored coinfection with other viral enteropathogens: norovirus in five cases and group A rotavirus in two. The five coinfections with norovirus were detected in Osaka and the two with group A rotavirus in Maizuru. All diarrheal viruses detected as coinfections with SaV (norovirus and group A rotovirus) were confirmed and further characterized by G typing, latex agglutination test, or sequencing analysis. The five noroviruses belonged to genogroup I, genotype 4 in four cases and genogroup I, genotype 3 in one case, while both group A rotaviruses were identified as G3 genotype 3 (Table 2).
Table 2

Characteristics of seven cases of mixed infection with SaV and other enteropathogens among the Japanese pediatric population

Case no.

Patient no.

Sex

Age (months)

City

Month

Year

Sapovirus

Other diarrheal viruses detected

Genogroup

Genotype

RT-PCR

Diarlexa

Sequenceb

Genotypec

1

5,060

F

14

Maizuru

Apr

2004

I

1

Group A rotavirus

Positive

ND

G3

2

5,299

F

17

Maizuru

Apr

2004

I

1

Group A rotavirus

Positive

ND

G3

3

5,720

F

46

Osaka

Dec

2004

I

6

Norovirus

ND

GII/4

ND

4

5,721

F

31

Osaka

Dec

2004

I

6

Norovirus

ND

GII/4

ND

5

5,735

M

49

Osaka

Dec

2004

I

6

Norovirus

ND

GII/4

ND

6

5,797

F

10

Osaka

Oct

2004

I

6

Norovirus

ND

GII/3

ND

7

5,806

M

11

Osaka

Nov

2004

I

6

Norovirus

ND

GII/4

ND

ND not done

aUsed only to detect group A rotavirus

bUsed only to determine genotype of norovirus

cUsed only to genotype group A rotavirus

Nucleotide sequencing and phylogenetic analysis of sapovirus

The PCR products of SaV were sequenced in order to further characterize the genetic relationship among the SaV strains detected in infants and children with acute gastroenteritis in Japan. The sequence of the 5′ end of the SaV capsid gene was targeted for genotyping [10, 13]. Their partial amino acid sequences were compared to each other as well as to those of reference SaV strains available in GenBank by BLAST. A total of 49 SaV sequences were analyzed by phylogenetic analysis and were grouped using the recent SaV capsid region classification scheme of Akihara et al. [13]. All of SaV sequences clustered into a single genogroup, genogroup I (GI). Most of the SaV GI sequences (93%, 13 of 14) in 2003–2004 belonged to genotype 1 (SaV GI/1) (typified by the Manchester virus cluster). Only one SaV GI sequence (7%, 1 of 14) clustered into the 4724/Osaka/JP virus cluster (known as SaV GI/6) (Fig. 2). In 2004–2005, SaV strains were classified into four distinct genotypes (GI/1, GI/4, GI/6, and GI/8). Interestingly, SaV GI/6 emerged as the predominant genotype in 2004–2005, representing 77% (27 of 35) of the strains, followed by SaV GI/1 (11%, 4 of 35) (Table 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs10096-006-0235-7/MediaObjects/10096_2006_235_Fig2_HTML.gif
Fig. 2

Phylogenetic tree of nucleotide sequences of SaV strains detected in infants and children with acute gastroenteritis in five different cities in Japan. All of the SaV sequences were classified into only one distinct genogroup (I), and the SaV genogroup I sequences were clustered into four genotypes (GI/1, GI/4, GI/6, and GI/8). The tree was constructed from partial amino acid sequences of the capsid region of SaV strains. Reference SaV strains were selected from GenBank under the accession numbers indicated in the text. Japanese SaV is highlighted in italic. The PEC strain was used as an outgroup strain for phylogenetic analysis. The scale indicates amino acid substitutions per position. The numbers in the branches indicate the bootstrap values. *Genotype contains Japanese SaV detected in the study

Table 3

Distribution of SaV genotypes based on the sequencing genetic analysis among infants and children with acute gastroenteritis in five different cities in Japan

Date of fecal specimen collection

No. of specimens positive for sapovirus

No. (%) identified as sapovirus genogroup I

Genotype 1

Genotype 4

Genotype 6

Genotype 8

July 2003–June 2004

14

13 (93)

0 (0)

1 (7)

0 (0)

July 2004–June 2005

35

4 (11)

2 (6)

27 (77)

2 (6)

Discussion

SaV infection causes acute gastroenteritis in all age groups, through it occurs predominantly in infants and young children [5, 6, 19, 20]. Overall, 49 of 1,154 fecal specimens tested were positive for SaV, and positive specimens were found in all age groups of the subjects included in the study. However, most (82%) of the SaV infections occurred in infants and children <3 years of age. These results were in line with previously published reports on SaV epidemiology worldwide, in which SaV prevalence was shown to range from 0.3 to 9.3%, far below the prevalence of either rotavirus or norovirus [1922]. Our findings also confirmed SaV as one of the important enteropathogens responsible for viral gastroenteritis among infants and children in Japan. According to surveillance on pediatric cases of viral gastroenteritis in Japan, the main peak of SaV infection was in winter [6, 9]. Interestingly, the highest number of SaV infections in our study was identified in the April–June period in 2003–3004 and in the October–December period in 2004–2005. The observations show that maximum SaV prevalence can occur during warmer seasons.

To date, coinfections with various enteric viruses have been widely reported [12, 20]. Interestingly, we found a rather high rate (14%, 7 of 49) of coinfections with SaV and other viral pathogens. Coinfection of SaV with rotavirus and with norovirus was confirmed by the Diarlex test and G typing. These results underscore that coinfection with SaV and other enteropathogens is not rare.

All of the Japanese SaV sequences belonged to only one SaV genogroup I with four distinct genotypes (GI/1, GI/4, GI/6, and GI/8). The findings clearly indicated that SaV GI was the dominant group to cause acute gastroenteritis among the pediatric population in Japan. In strong agreement with previous reports [7, 10, 13], SaV strains most frequently detected in sporadic gastroenteritis in infants and children in 2003–2004 belonged to the Manchester cluster (GI/1). Of note, SaV GI/6 strains were the most predominant (77%) in 2004–2005 and closely homologous to each other, suggesting that they came from the same source of infection. Moreover, only two SaV GI/6 strains were found in 2002–2003 during a 7-year (1996–2004) survey of SaV infection in diarrheal fecal specimens from Japanese infants and children [20]. In the present study, only one SaV GI/6 was detected in 2003–2004. Taken together, there was an emergence of rare SaV GI/6, and this was the first evidence of the changing distribution of the SaV genotype in association with acute gastroenteritis in Japan. This sudden emergence of SaV GI/6 indicates that the pediatric population in Japan might lack antibody protection to these strains and that these rare strains could be more virulent than those usually associated with pediatric gastroenteritis. Continuous surveillance of SaV infection in Japan is recommended in order to determine whether these rare strains remain dominant in the coming years.

Acknowledgements

This study was supported by grants-in-aid from the Ministry of Education and Sciences and the Ministry of Health, Labor and Welfare, Japan.

We are grateful to Drs. Kunio Kaneshi, Toshimasa Kuroiwa, Yuichi Ueda, Shigekazu Nakaya, Shuichi Nishimura, Kumiko Sugita, Tadashi Nishimura, and Atsuko Yamamoto for collecting the fecal specimens.

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