Department of Virology, Niigata Prefectural Institute of Public Health and Environmental Sciences, Niigata ; 3

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1 Jpn. J. Infect. Dis., 63, , 2010 Original Article Molecular Epidemiological Study of Rotavirus and Norovirus Infections among Children with Acute Gastroenteritis in Nha Trang, Vietnam, December 2005 June 2006 Tsutomu Tamura 1,3 *, Makoto Nishikawa 1,3,DangDucAnh 2, and Hiroshi Suzuki 3 1 Department of Virology, Niigata Prefectural Institute of Public Health and Environmental Sciences, Niigata ; 3 Department of Public Health, Graduate School of Medical and Dental Sciences, Niigata University, Niigata , Japan; and 2 National Institute for Hygiene and Epidemiology, Hanoi, Vietnam (Received April 30, Accepted August 25, 2010) SUMMARY: A molecular epidemiological study of rotavirus (RV) and norovirus (NoV) infections was carried out in Nha Trang city in Vietnam between December 2005 and June RV and NoV were detected in 87 (47.5z) and12(6.6z) of the 183 fecal specimens from children hospitalized with acute gastroenteritis, respectively. The majority of patients with RV and NoV were children younger than 2 years of age. The most frequent RV genotypes detected were G3 (n = 37, 42.5z) andg1(n = 28, 32.2z) for G type, P[8] (n = 61, 70.1z) for P type, and G3P[8] (n = 33, 38.0z) andg1p[8](n = 18, 20.7z) for the G and P genotype combination. GII.12 was the most common genotype (6/12, 50z) for NoV, followed by GII.4 (4/12, 33.3z), and we also identified a rare type (GII.19). The results of this study highlight the increased incidence of G3P[8] and the presence of many OP354-like P[8] RVs, as well as the GII Asia variant of NoVs. Furthremore, the first case of GII.19 of NoV in Vietnam is reported. INTRODUCTION Diarrheal disease remains the second most common cause of death among children younger than 5 years of age (1), with group A rotavirus (RV) and norovirus (NoV) being the main causative agents of acute gastroenteritis in both developed and developing countries. An estimated 527,000 children younger than 5 years of age die of RV-related diarrhea each year, with more than 85z of these deaths occurring in developing countries in Africa and Asia (2). Vietnam is estimated to account for 5,300 6,800 of such children (3). RV infections in this country occur throughout the year, with a slight peak during the winter dry season in both southern and northern Vietnam (4). RVs have two outer shell proteins, VP4 and VP7, against which protective immunity is mainly targeted. RVs are classified into 28 P and 20 G genotypes on the basis of the VP4 and VP7 gene sequences, respectively (5). Five combinations of G and P genotypes (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) make up 90z of the human RV strains worldwide (6), with the G1 strain currently being the most prevalent G genotype strain (6). The predominant G genotype in Vietnam between July 1998 and June 2000 was G2, although G1 then *Corresponding author: Mailing address: Department of Virology, Niigata Prefectural Institute of Public Health and Environmental Sciences, Sowa, Nishi-Ku, Niigata-Shi, Niigata , Japan. Tel: , Fax: , tamura.tsutomu@ pref.niigata.lg.jp became dominant (4,7). As far as P genotypes are concerned, P[8] is responsible for most RV infections in the world (6). Strain OP354, a rare genotype of P[8] RV, was first detected in Malawi in 1998 (10) and was recentlysuggestedtobesubtypep[8]b(8).p[8]brvhasbeen isolated recently in some Asian countries (8,9,11,12). NoV is the second most common cause of severe childhood gastroenteritis, after RV (13), and its prevalence in children with acute gastroenteritis ranges from 6to48z (14). NoV can be classified into five genogroups, GI to GV, and subdivided into many genotypes (15 18). Strains GI, GII, and GIV are associated with human gastroenteritis, whereas GIII and GV are only found in animals. Worldwide outbreak surveillance data show a prominent role for GII strains, especially after the emergence of new GII.4 variants between 1995 and 2007 (14,19). In this study we clarified the etiological role of RV and NoV in sporadic diarrhea and analyzed their genetic diversity in Nha Trang, which is located in southern Vietnam. MATERIALS AND METHODS Samples: A total of 183 fecal specimens were collected from children with gastroenteritis at hospitals in Nha Trang in Vietnam between December 2005 and June Stool samples were stored at -209C until further analysis. RNA extraction and reverse transcription: The fecal specimens were diluted with phosphate-buffered saline ph 7.2 to form a 10z suspensions then centrifuged at 8,000 g for 20 min. Viral RNA genomes were extract- 405

2 Table 1. Primer list for rotavirus and norovirus used in this study Primer Type F/R Sequence (5? 3?) Amplicon Reference Rotavirus primer Detection of RV Beg9 common F GGC TTT AAA AGA GAG AAT TTC CGT CTG G End9 common R GGT CAC ATC ATA CAA TTC TAA TCT AAG 20 VP7 typing abt1 G1 F CAA GTA CTC AAA TCA ATG ATG G act2 G2 F CAA TGA TAT TAA CAC ATT TTC TGT G aet3 G3 F CGT TTG AAG AAG TTG CAA CAG adt4 G4 F CGT TTC TGG TGA GGA GTT G aat8 G8 F GTC ACA CCA TTT GTA AAT TCG aft9 G9 F CTA GAT GTA ACT ACA ACT AC RVG9 common R GGT CAC ATC ATA CAA TTC T 20 Alternative primer for G3 typing G3 G3 F ACG AAC TCA ACA CGA GAG G VP4 typing con3 common F TGG CTT CGC CAT TTT ATA GAC A con2 common R ATT TCG GAC CAT TTA TAA CC 21 1T-1 P[8] R TCT ACT TGG ATA ACG TGC T-1 P[4] R CTA TTG TTA GAG GTT AGA GTC T-1 P[6] R TGT TGA TTA GTT GGA TTC AA T-1 P[9] R TGA GAC ATG CAA TTG GAC T-1 P[10] R ATC ATA GTT AGT AGT CGG Alternative primer for P[8] typing 1T-1D P[8] TCT ACT GGR TTR ACN TGC Detection of P[8]b VP4-F103G P[8]b F TGG ATC AGA AAA AAC TCA AG 550 This paper VP4-R612C P[8]b R CGG TAT TAT GTA AAA CTC AGA G This paper Norovirus primer Detection of NoV G1SKF F CTG CCC GAA TTY GTA AAT GA G1SKR R CCA ACC CAR CCA TTR TAC A 25 COG2F F CAR GAR BCN ATG TTY AGR TGG ATG AG G2SKR R CCR CCN GCA TRH CCR TTR TAC AT 25 Analysis of P2 region L1F F TTC CCY ATY CCT TTG GAA AAG 743 Shinohara (personal communication) L7R R TTG CAC TCA AAY AGA ACC CT 27 ed from these fecal suspensions using Extragen II (Tosoh, Tokyo, Japan) according to the manufacturer's instructions. The extracted RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, Wis., USA) and random hexamer (GE Healthcare, Piscataway, N.J., USA), with preincubation at 259C for 5 min, followed by treatment at 379C for 50 min and 709C for 15 min. RV detection and genotyping: The PCR assay was performed with Beg9 and End9 primers, as described elsewhere (20). Representative amplified PCR products were sequenced to ensure that only desired the PCR product was analyzed. The primers used in this study are shown in Table 1. G and P typing: RV-positive PCR samples were subjected to G and P genotyping by nested PCR assay with type-specific primers, as described previously (19,20). For G typing, 0.5 ml of the 1st round PCR products was added to 24.5 ml of the 2nd round PCR product containing the primer mix with all six serotype-specific forward primers (abt1, act2, aet3, adt4, aat8, and aft9) and a common reverse primer (RVG9) (20). P genotyping was performed with the 1st round PCR product using con2 and con3 primers to amplify the VP4 gene, and the 2nd round PCR using primers 1T-1 (P[8]), 2T-1 (P[4]), 3T-1 (P[6]), 4T-1 (P[9]), 5T-1 (P[10]), and a common primer (con3), as described previously (21). Alternative primers (22,23) were used when the G and P type could not be determined using the aforementioned primers. G and P types were determined on the basis of the size of the amplicon after electrophoresis. When coexistence of types was suspected in a sample by multiplex typing PCR, singleplex PCR was performed using primers for the suspected types. We designed a pair of primers to detect P[8]b as VP4- F103G and VP4-R612C (Table 1), based on strain MMC38 G9P8/05/Bangladesh (EU979379) from Gen- Bank, for P[8]a and P[8]b subtyping. This primer pair was used after first PCR with the con3 and con2 primer pair (product size, 550 bp). Representative amplified PCR products were sequenced to ensure that only the desired PCR products had been analyzed. The PCR assay was conducted in the presence of 1.5 mm MgCl 2, 1 mm/ml of each primer, and Taq polymerase (Biotech 406

3 International, Sydney, Australia) in a final volume of 25 ml. The thermal cycling conditions were 949C for3 min, followed by 30 cycles of 949C, 559C, and 729C for 1 min each, and final incubation at 729C for7min. NoV detection and typing: The presence of NoV in the fecal specimens was determined by multiplex PCR with two primer sets: G1SKF/G1SKR for GI NoV and COG2F/G2SKR for GII NoV (Table 1), as described previously (23,25,26). Since GI and GII NoV were the prevalent genogroups of gastroenteritis, only these two genogroups were included in the analysis. Analysis of the VP1 gene of GII.4 NoV: The VP1 gene of GII.4 strains was analyzed further to determine the variant type. A PCR assay was performed using L1 and L7 (24) primers to detect the P2 region of the VP1 gene of GII.4 NoV (Table 1). The thermal cycling conditions were 949C for 3 min, followed by 40 cycles of 949C, 509C, and 729C for 1 min, 1 min, and 2 min, respectively, and final incubation at 729C for7min. Nucleotide sequencing and phylogenetic analysis: Direct sequencing of the VP7 gene of G3 strains and the VP4 gene of P[8] strains of RV, and sequencing of the 5? region of NoV ORF2 and the P2 region of the VP1 gene of GII.4 NoV, was performed using ABI Prism 310 and 3130 Genetic Analyzer and Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems, Foster City, Calif., USA) according to the manufacturer's instructions. Sequence alignment and phylogenetic analysis were carried out by the neighborjoiningmethodusingmega3.1(28).novgenotyping was based on Kageyama and Okada's scheme using the nucleotide sequence of the N-terminal region of the capsid gene (17,18). The nucleotide sequence accession numbers in GenBank for the samples sequenced in this study are AB AB and AB AB Reference sequences for the phylogenetic analysis of NoV and RV were obtained from GenBank. RESULTS RV and NoV were detected in 87 (47.5z) and12 (6.6z) of 183 fecal specimens from children with gastroenteritis collected in Nha Trang, Vietnam between November 2005 and June 2006, respectively. Dual infection with NoV and RV was detected in only one sample. The age distribution of the patients ranged from 1 month up to 44 months, although 88z of patients were younger than 24 months old. The majority of patients Table 2. Monthly distribution of rotavirus and norovirus infections in patients with acute gastroenteritis Virus Genotype 2005 Dec. Month 2006 Jan. Feb. Mar. Apr. May Jun. Total (z) Rotavirus G (32.2) G ( 6.9) G ) (42.5) G4 1 1 ( 1.1) G1/G (16.1) G1/G2 1 1 ( 1.1) Subtotal Norovirus GII (33.3) GII ( 8.3) GII ) 6 (50.0) GII ( 8.3) Subtotal Total 4/10 17/29 17/39 26/45 8/23 24/32 3/5 99/183 1) : One sample of mixed infection with G3 rotavirus and GII.12 norovirus was included. Table 3. Distribution of rotavirus G and P genotypes among patients with acute gastroenteritis Ptype Gtype P[4] P[6] P[8] Mixed 1) NT Total (z) P[4]/P[6] P[4]/P[8] P[4]/P[6]/P[8] P[6]/P[8] G (32.2) G2 6 6 ( 6.9) G (42.5) G4 1 1 ( 1.1) G1/G2 1 1 ( 1.1) G1/G (16.1) Total (z) 11 (12.6) 1 (1.1) 61 (70.1) 1 (1.1) 1 (1.1) 2 (2.3) 9 (10.3) 1 (1.1) 87 1) : co-infected with two or three G or P genotypes. NT, non-typable samples. 407

4 with RV or NoV infection were also younger than 24 months old. RV was detected throughout the study period, with a peak observed in March (24/87, 27.6z) (Table2).G types among the 87 RV-positive samples were G3 (n = 37, 42.5z), G1 (n = 28, 32.2z), G2 (n = 6, 6.9z), and G4 (n = 1, 1.1z) (Table 3). Mixed G-type RV infections (G1/G3 and G1/G2) were seen in 14 (16.1z) and 1 (1.1z) of the 87 cases, respectively. P[8] represented was the major P type (61/87, 70.1z), with a further 11 cases being P[4] (12.6z) and one P[6] (1.1z). Mixed P-type RV infections were seen in 9 (P[6]/P[8]), 2 (P[4]/P[6]/P[8]), one (P[4]/P[6]), and one (P[4]/P[8]) cases. G3P[8] and G1P[8] were the most common G and P genotype combinations, accounting for 33 (38z) and18(21z) of the RV infections, respectively. The predominant G type was found to vary with time, with G1 predominating from December 2005 to January 2006 and G3 from February to June 2006 (Table 2). As G3 was the most predominant type in this study, we sequenced the VP7 gene from five randomly selected G3 strains, all of which were grouped in the same clade as Asian reference strains, including other Vietnamese strains collected from the database (Fig. 1). Likewise, as P[8] was the most common P genotype, we conducted a genetic analysis of the VP4 gene of 10 randomly selected samples of G1 or G3 genotypes. These were grouped into two clusters: an OP601-like cluster for P[8]a and an OP354-like cluster for P[8]b (Fig. 2). Our design of a specific primer for P[8]b allowed 14 further strains to be differentiated from 49 tested samples out of 73 P[8] strains. These were grouped with G1 of the VP7 gene and classified into G1P[8]b. Only one case of NoV was detected in December Fig. 1. Phylogenetic analysis of VP7 nucleotide sequences of rotavirus G3 strains. The phylogenetic tree was constructed based on 1,007 nucleotide sequences of the G3 VP7 genes. Percentage bootstrap values above 70z were shown at the branch nodes., collected in Nha Trang, Vietnam, ; #, collected during ;, collected in Haiphong, Vietnam, September Fig. 2. Phylogenetic analysis of partial VP4 nucleotide sequences of the rotavirus P[8] strains. The phylogenetic tree was constructed based on 645 nucleotide sequences of the VP4 genes. Percentage bootstrap values above 70z were shown at the branch nodes., collected in Nha Trang, Vietnam; $, reference strains collected in Vietnam. 408

5 Fig. 3. Phylogenetic analysis of norovirus capsid gene. (A) Phylogenetic analysis of partial capsid nucleotide sequences of norovirus strains. The phylogenetic tree was constructed based on 218 nucleotide sequences of capsid gene. Percentage bootstrap values above 70z are shown at the branch nodes. Reference strains and their accession numbers used in Fig. 3(A) are as follows: GII.4: Bristol/93/UK (X76716), VN915/2003/VNM (DQ377174), VN800/2003/VNM (DQ377172), Hokkaido/194/2004/JP (AB240180), Sakai2/2006/JP (AB447448), Beijing/ 274/2005/CHN (EU839585), Chiba/040974/2004/JP (AB294782), Hunter 284E/04O/AU (DQ078794), Terneuzen70/2006/NL (EF126964), Farmington Hills/2002/USA (AY502023), Nijmegen115/2006/NL (EF126966); GII.13: M7/99/US (AY130761); GII.19: NLV/J23/1999/US (AY130762), Shaibah-2/2007/IRQ (EU138878), 299/JPN (EF630529), Hiroshima/ /2006/JP (AB360387), CMH148/01/2001/THA (EU363866); GII.6: SaitamaU3/97/JP (AB039776), Miami/292/94/US (AF414410), HCMC204/2006/VNM (EU137732), HCMC311/2006/VNM (EU137733); GII.10: Mc37/99/Thai (AY237415); GII.15: SaitamaKU80aGII/99/JP (AB058582); GII.12: SaitamaU1/97/JP (AB039775), Hiroshima/25-583/2002/JP (AB354292), NZ587/2006/ NZL (EF187600), HCMC91/2006/VNM (EU137734), NSW021E/2006/AUS (EF187525), CMH145/05/2005/ THA (EU872289), NSW330F/2006/AUS (EF187578); GI.1: Norwalk/68/US2 (M87661)., collected in NhaTrang, Vietnam; ( ), number of strains. (B) Phylogenetic analysis of capsid amino acid sequences include P2 region of the norovirus GII.4 strains. The phylogenetic tree was constructed based on 200 amino acids sequences of the capsid gene include P2 region. Percentage bootstrap values above 70z are shown at the branch nodes. Reference strains and their accession numbers used in Fig. 3(B) are as follows: Beijing/79/2004/CHN (EU839583), Chiba/040974/2004/JP (AB294782), Sakai/04 179/2005/JP (AB220922), Sakai2/2006/JP (AB447448), Beijing/274/2005/CHN (EU839585), Chiba/040110/2004/JP (AJ844477), Chiba/031038/2003/JP (AJ844476), Nijmegen115/2006/NL (EF126966), Kobe034/2006/JP (AB291542), Terneuzen70/2006/NL (EF126964), Hunter 284E/04O/AU (DQ078794), Farmington Hills/2002/USA (AY502023), Bristol virus/b493/ 93 (X76716)., collected in NhaTrang, Vietnam. 2005, with a further 8 in February, 2 in March, and one in April (Table 2). All these cases were identified as GII (12/12, 100z). Phylogenetic analysis based on the nucleotide sequences of the N-terminal region of the capsid gene showed that half (6/12) these GII specimens were GII.12, with a further 4 being GII.4 and one each for was GII.6 and GII.19 (Fig. 3). A phylogenetic analysis of the P2 region of the VP1 amino acid sequences allowed us to classify two GII.4 samples into the GII Asian clade. DISCUSSION RV has been detected essentially year round throughout the country, with a slight peak in the dry winter season, in a six-hospital surveillance in four cities in Viet- 409

6 nam (Hanoi, Haiphong, Nha Trang, and Ho Chi Minh) (4). Although the present study in Nha Trang, which has a tropical climate, was conducted during the dry season, and RVs were also detected throughout the study period, with a peak in March. In accordance with previous reports, our results indicate that RV is one of the leading viruses responsible for childhood gastroenteritis in Vietnam (7,29 35). The predominant G type for RV in Vietnam was G2 from 1998 to 2000 and G1 from 2000 to 2003, whereas genotypes G3 and G3P[8] RV were rare (7,31). However, G3 was isolated as the predominant genotype as of February 2006 in this study, as well as in Haiphong in north Vietnam during the season (36). These observations therefore suggest that G3 infections occur throughout the country. Phylogenetic analysis of the VP7 gene of RV showed that the G3 strains in this study clustered with the strains collected from Ho Chi Minh (Vietnam), Kunming (China), Khabarovsk (Russia), Chiang Mai (Thailand) and Japan between 2001 and 2003 (37), Wuhan (China) in (38), and Malaysia in Thus, these observations also suggest that isolation of the G3 strain has increased in frequency both in Vietnam and in neighboring countries, although the reasons for this increase remain unclear. Since a clinical trial of RotaTeq} (Merck, Whitehouse Station, N.J., USA) vaccine has been underway in Vietnam since 2007, continuous monitoring of the change of RV genotype will be required to understand the epidemiology of RV infection and to evaluate the efficacy of this vaccine in the future. The P[8] strain is the most predominant VP4 gene in the world (7), and this situation was mirrored in our study. Furthermore, phylogenetic analysis showed that P[8] could be grouped into two subclusters: an OP601- like cluster for P[8]a and an OP354-like cluster for P[8]b. Strain OP354 was first detected in Malawi in 1998 (10) as a rare genotype of P[8] RV and was named as subtype P[8]b (8). This genotype was also isolated recently in some Asian countries, including Vietnam (8,9,11,12). All P[8]b strains in our study were classified into G1P[8]b and are therefore closely related to the CU20 strain, which was reported in Thailand during Thus, our observations suggest that the G1P[8]b strain was circulating in Vietnam and neighboring countries during the same period. We designed a specific primer for P[8]b to detect more such strains and found 14 (28.6z) from a total of 49 P[8] strains. This result suggests that our specific primer for P[8]b may be effective for identifying and monitoring P[8]b, although this aspect warrants further study. The prevalence of NoV infection in our study was 7z, with GII.4 and GII.12 being predominant genotypes. These findings are consistent with a previous study performed in Ho Chi Minh, Vietnam during 2005 and 2006 (39) and suggest that these NoV genotypes were prevalent in southern Vietnam in that period. In accordance with previous reports (33,39), NoV GI was not detected in our study, although we identified one rare NoV GII.19 using partial capsid sequence analysis, which was the first case in Vietnam. This genotype had previously only been reported in the USA (15) and Japan (40). Phylogenetic analysis of the P2 region of GII.4 strains allowed us to classify two representative samples intogii.42003asiavariant,suggestingthatanepidemic of this GII.4 variant occurred throughout Asia, including Vietnam. In conclusion, we have analyzed the prevalence of RV and NoV and shown that these viruses are important causal agents of acute viral gastroenteritis in children in Vietnam. 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