Vaccine 27S (2009) F75–F80 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Molecular epidemiology of rotavirus diarrhoea among children in Haiphong, Vietnam: The emergence of G3 rotavirus Ngo Tuan Cuong a , Nguyen Binh Minh a , Dang Duc Anh a , Nguyen Hoai Thu a , Nguyen Tuan Tu b , Tran Van Nam b , Vu Thi Thuy b , Michiko Ogino c , Md Mahbub Alam c , Toyoko Nakagomi c , Osamu Nakagomi c , Tetsu Yamashiro d,∗ a National Institute of Hygiene and Epidemiology, Vietnam Haiphong Children’s Hospital, Vietnam c Division of Molecular Epidemiology, Graduate School of Biomedical Sciences and Global Center of Excellence, Nagasaki University, Nagasaki, Japan d Department of Virology, Institute of Tropical Medicine and Global Center of Excellence, Nagasaki University, 1-12-4 Sakamoto Nagasaki city, Nagasaki 852-8523, Japan b a r t i c l e i n f o a b s t r a c t Keywords: Rotavirus Diarrhoea Variant rotavirus G3 From September 2006- October 2007 hospital-based surveillance was conducted in Haiphong, Vietnam among children less than age years hospitalized for diarrhoea to determine the distribution of G and P types and electropherotypes of rotavirus Of note, the emergence of G3P[8] was identified and the strain was predominant among rotaviruses detected More than 90% of G3P[8] electropherotyped strains shared an identical electropherotype, indicating they were of a single origin and their VP7 sequences were similar to those reported from Japan and China This abrupt emergence of a novel G3 strains underscores the continued need for quality rotavirus surveillance © 2009 Elsevier Ltd All rights reserved Introduction gens, VP7 and VP4, which define independent G and P serotype specificities, respectively Four common G types (G1, G2, G3, and G4) in conjunction with P[8] or P[4] have been commonly identified in human populations worldwide [4–6] Initial protective immunity against rotavirus is thought to be primarily mediated by serotype-specific antibody, but after repeated infections heterotypic protection develops Characterization of strains circulating in the community will allow an understanding of potential overall vaccine effectiveness [6] Vietnam is a developing country with a population of 86 million and is one of the countries where disease burden and economic impact of rotavirus diarrhoea have been studied under the generic protocol provided by the World Health Organization (WHO) It is estimated that in Vietnam each year approximately 125,000 hospitalizations, and 5,300 to 6,800 deaths occur due to rotavirus among children less than years of age representing 8% to 11% of deaths due to any cause in this age group [7,8] This burden of the disease results in an estimated economic burden of US$3.1 million in medical direct cost, US$685,000 in non medical direct cost, and US$1.5 million in indirect costs [9] Such magnitude of disease and economic burden justifies the introduction of rotavirus vaccine into the national childhood immunization schedule of Vietnam In Vietnam and other developing countries, the anticipated introduction of rotavirus vaccines underscores the value of rotavirus strain characterization to properly evaluate the impact of vaccine on the possible shift of circulating strains The Asian Rotavirus Surveillance Network (ARSN) has already provided meaningful information for Globally, acute gastroenteritis is a major public health concern, since each year it claims approximately 1.8 million lives of children less than years of age in both developed and developing countries [1] The global mortality under age five attributable to rotavirus infection was estimated to be approximately 527,000 in 2004, the majority of which occurred in developing countries [2] The level of hygiene and sanitation is not associated with incidence of the disease, because nearly all children experience rotavirus infection during the first years of life regardless of whether they live in developing or developed countries [1,3] Different from bacterial and parasitic diarrhoea, antimicrobial therapies are not effective for rotavirus infections Taken together, it is important to introduce rotavirus vaccines into the countries where they are most needed to minimize the disease burden of rotavirus Rotavirus is a non-enveloped virus with 11 segments of doublestranded RNA genome encased within a triple-layered capsid The outer most capsid is composed of two neutralization anti- Abbreviations: ARSN, Asian Rotavirus Surveillance Network; ELISA, enzymelinked immunosorbent assay; NIHE, National Institute of Hygiene and Epidemiology Vietnam; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptionpolymerase chain reaction; WHO, World Health Organization ∗ Corresponding author Tel.: +81 95 819 7876; fax: +81 95 819 7830 E-mail address: tyamashi@nagasaki-u.ac.jp (T Yamashiro) 0264-410X/$ – see front matter © 2009 Elsevier Ltd All rights reserved doi:10.1016/j.vaccine.2009.08.074 F76 N.T Cuong et al / Vaccine 27S (2009) F75–F80 Materials and methods VP7 gene was purified with QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced by using the ABI PRISM BigDye Termination Cycle Sequencing Reaction kit ver 3.1 (Applied Biosystems, Foster city, CA, USA) on an automated sequencer The constant forward primer Beg9 and reverse primer End9 were used for sequencing Nucleotide sequence alignment was carried out using CLUSTAL X (ver1.82) [18] and phylogenetic tree was constructed based on deduced amino acid sequences by using the neighbor-joining method with bootstrap analysis [19] Reference rotavirus strains and their accession numbers used in the study were as follows: for SA11, V01546; for R142, DQ873676; for Z235, DQ873669; for Y111, DQ873678; for Z033, DQ873673; for 5091, DQ779050; for 107E1B, AB081594; for RMC437, AY603153; for ITO, D86278; for YO, D86284; for CH927, D86275; for AU-1, D86271; for 02/92, D86264 2.1 Surveillance site Results Haiphong, one of Vietnam’s principal port cities with a population of approximately 1.8 million, is located 100 km east of Vietnam’s capital city, Hanoi Haiphong Children’s Hospital is a 330bed teaching hospital which receives approximately 450 patients per day and serves as the sole tertiary medical care institution for children in and around the Haiphong city The hospital participated in rotavirus surveillance conducted by the ARSN for years since 1998 3.1 Age distribution of rotavirus infection public health purposes [7,8,10,11] In Vietnam, preceding ARSN studies revealed that rotavirus infection has several distinctive epidemiologic features that are not usually observed in other developing countries such as a high detection proportion of rotavirus among all diarrhoeal diseases, children becoming infected at an older age, and the circulation of routine strains with few mixed infections [7,8,12] We report findings obtained from a rotavirus surveillance study conducted in the 12 months from September 2006 through August 2007 in Haiphong, Vietnam to compare the findings with those of previously reported in Vietnam and other countries, and describe the abrupt emergence of an unusual G3 strain in Haiphong area and its genetic background 2.2 Specimen collection The study followed the case definition, surveillance system, laboratory procedures, data analysis, and monitoring data quality described in the generic protocols of the WHO [13] Briefly, a single stool specimen was collected from all patients less than years of age who were admitted for treatment of diarrhoea to the surveillance site from September 2006 through August 2007 for 12 months This study was approved by the ethics committees of the Institute of Tropical Medicine, Nagasaki University, and of the National Institute of Hygiene and Epidemiology of Vietnam 2.3 Rotavirus antigen detection and G and P genotyping Rotavirus was detected by enzyme-linked immunosorbent assay (ELISA) (Rotaclone, Meridian Bioscience, Inc., Cincinnati, OH, USA) and genomic RNA was extracted from all rotavirus-positive specimens using the QIAamp Viral RNA Mini kit (QIAGEN, Hilden, Germany) and used to determine VP7 and VP4 genotypes by reverse transcription-polymerase chain reaction (RT-PCR) [14,15] 2.4 Electropherotyping All RNAs extracted from specimens containing G3P[8] rotavirus were further analyzed for their electropherotypes by polyacrylamide gel electrophoresis (PAGE) [16] The extracted viral RNAs were separated on a gel containing 10% acrylamide in the buffer system of Laemmli [17] using an SE600 Ruby gel apparatus (GE Healthcare Biosciences Piscataway NJ, USA) The gel was 1.50 mm in thickness and 12 cm in length The separated RNAs were visualized by ethidium bromide with ultraviolet illumination after electrophoresis for approximately 13 h at a constant current of 15 mA (BioRad PowerPac system) The RNA from the strain Wa (G1P1A[8]) was used for a reference 2.5 Nucleotide sequencing Sequencing analysis was performed on the VP7 gene amplification product with Beg9 and End9 primers The PCR amplified Of 982 persons from whom diarrhoeal specimens were collected, 978 (99.6%) were eligible for the study criteria and their specimens tested for rotavirus The mean age of children from whom eligible samples were collected was 12.3 ± 7.8 (SD) months and 54% were less than 12 months of age Six hundred thirty-six (65%) were boys Of 978, 509 (52%) specimens tested positive for rotavirus The age distribution of rotavirus infections revealed that less than a half (48%) of cases occurred in children less than year of age Most (78%) children hospitalized with rotavirus infections were between and 23 months of age (Table 1) Of note, the detection rates of rotavirus in children 24 months or older was relatively high for a developing country and more resembled that seen in developed countries, although the absolute number of patients in the age group were small (Table 1) 3.2 Monthly distribution of rotavirus infections Rotavirus was detected year-round in Haiphong with a trace of seasonality In Haiphong rotavirus detection rates (52%) were highest in winter months (November to February) with a maximum detection of 81% found in January 2007 and rates declined in summer months (Fig 1) 3.3 Distribution of G and P genotypes Approximately 99% of 509 rotavirus-positive specimens were successfully characterized by G and P genotypes Overall, 88% of the 509 rotavirus-positive specimens had the G and P genotype combinations commonly found in humans: G1P[8], G2P[4], G3P[8], and G4P[8] Of those, G3P[8] was the most frequently found genotype (83%), followed by G1P[8] (5.3%) The high prevalence of G3 rotaviruses was unusual in Vietnam where no G3 rotavirus had Table Age distribution of diarrhoeal and rotavirus patients among children less than age who were hospitalized for diarrhoea in Haiphong Children’s Hospital, Haiphong, Vietnam from September 2006 through August 2007 Age group (month) No of patients Rotavirus positive (%) to to to 11 12 to 23 24 to 35 36 to 47 48 to 59 29 129 371 374 58 12 (24) 50 (39) 187 (50) 208 (56) 44 (76) (75) (80) Total 978 509 (52) N.T Cuong et al / Vaccine 27S (2009) F75–F80 F77 Table Distribution of G and P genotypes of all 509 specimens tested positive for rotavirus (%) G genotype P genotype P[8] P [6] P [4] Mixed NT Total G1 G2 G3 G4 G9 Mixed NT 27 (5.3) 421 (82.7) (0.2) b 24 (4.7) (0.8) 23 (4.5) 0 c (0.2) 0 0 0 0 0 3a (0.6) 0 0 0 (0.8) (0.2) 0 31 (6.1) 451 (88.6) (0.2) (0.2) 25 (4.9) Total 473 (92.9) 28 (5.5) (0.6) (1.0) 509 (100) NT, nontypeable a P[6] and P[8] were detected in these specimens b G1, G2, and G3 were detected in specimen; G1 and G3 were detected in 15 specimens; G2 and G3 were detected in specimens c G1 and G3 were detected in the specimen been detected during the nationwide rotavirus surveillance program conducted in 2000–2003 In contrast, G2 rotavirus which had been the most prevalent strain in Haiphong in 2002–2003, was found only in nine specimens together with other genotypes as mixed infections, and no G2 rotavirus were detected as a sole pathogen (Table 2) Unusual genotypes, G1P[6] and G3P[6], were found in 27 of 509 strains (5.3%) of the rotavirus-positive specimens The rate of mixed infections were low accounting for 5.5% of all rotavirus positive specimens 3.4 PAGE analysis To understand whether these predominant G3P[8] rotaviruses were similar to each other, genomic RNAs from all 421 G3P[8] rotavirus positive specimens were separated on PAGE and 243 (58%) were successfully electropherotyped The analyses identified 11 distinct electropherotypes in the 243 specimens (Fig 2), and 91% of those had an identical electropherotype, g3p8-a (Table 3) Specimens that carried G3P[8] rotavirus with the g3p8-a electropherotype were found year round 3.5 Nucleotide sequencing and phylogenetic analysis To establish the genetic relationship of the G3P[8] VP7 gene of the dominant rotavirus among the G3 VP7 genes available in databases, we chose two amplified VP7 fragments which were identified as the g3p8-a electropherotype, and sequenced to determine that they had an identical sequence These two fragments, represented by NHP0100Haiphong, shared a 99% nucleotide sequence Fig Electropherotypes of G3P[8] rotavirus strains identified in Haiphong, northern Vietnam between September 2006 and August 2007 are shown Lane 1, g3p8-a; lane 2, g3p8-b; lane 3, g3p8-i; lane 4, g3p8-j; lane 5, g3p8-e; lane 6, g3p8-g; lane 7, g3p8-k; lane 8, g3p8-f; lane 9, g3p8-d; lane 10, g3p8-c; lane 11, g3p8-h Electropherotype was successfully determined in 243 out of 421 specimens carrying G3P[8] rotavirus, and 91% of those (222 specimens) had the g3p8-a type shown in the lane identity in the VP7 of G3P[8] strains that recently emerged in Japan and China and they were clustered together with a high bootstrap probability when a phylogenetic tree was constructed (Fig 3) When amino acid sequences were compared Table Distribution of electropherotypes of 243 specimens that carried G3P[8] rotavirus Fig Monthly detection of rotavirus among children less than years hospitalized for diarrhoea in Haiphong, northern Vietnam, from September 2006 to August 2007 is shown Numeral in each column indicates the number of cases involved RV, rotavirus Electropherotype No (%) g3p8-a g3p8-b g3p8-c g3p8-d g3p8-e g3p8-f g3p8-g g3p8-h g3p8-i g3p8-j g3p8-k 222 (91.2) (1.2) (0.4) (0.4) (0.8) (1.6) (1.2) (0.8) (0.8) (0.4) (0.8) Total 243 (100) Data on 243 out of 421 specimens that carried G3P[8] rotavirus were shown RNA patterns could not be determined in the remaining 178 specimens, since RNA contained in those samples were insufficient or degraded F78 N.T Cuong et al / Vaccine 27S (2009) F75–F80 Fig Phylogenetic tree constructed from the deduced amino acid sequences of the VP7 genes of a representative rotavirus strain with g3p8-a electropherotype detected in the study (NHP0100Haiphong) and those of reference strains which carry G3 is shown A simian G3 rotavirus strain SA11 (accession No V01546) was used as an out-group The VP7 gene of strain NHP0100Haiphong segregated into a cluster together with the G3P[8] VP7 genes that emerged in Japan (5091 Japan) and in Wuhan, China (Y111 Wuhan, Z033 Wuhan) The vertical branch lengths are proportional to the similarities of the sequences The scale at the top represents an analysis of the genetic distances among sequences The numbers in the branches indicate the bootstrap values R142 Wuhan (accession no.: DQ873676, country where the strain was detected: China, year when the strain was detected: 2003); Z235 Wuhan (DQ873669, China, 2004); NHP0100Haiphong (GQ129459, Vietnam, 2006); Y111 Wuhan (DQ873678, China, 2004), Z033 Wuhan (DQ873673, China, 2004); 5091 Japan (DQ779050, Japan, 2003–2004), 107E1B (AB081594, India, 1993), RMC437 (AY603153, India, 2004); ITO (D86278, Japan, 1980); YO (D86284, Japan, 1977); CH927 (D86275, China, 1992); AU-1 (D86271, Japan, 1982); 02/92 (D86264, Japan, 1992) Fig Comparison of amino acids sequences in antigenic regions A, B, C, and F of the VP7 protein between a rotavirus G3 strain identified in the study (NHP0100Haiphong) and reference rotavirus G3 strains is shown The strain NHP0100Haiphong showed the complete identity to those of novel G3 strains recently emerged in China (Y111 Wuhan, Z033 Wuhan) and in Japan (5091 Japan), but was different from those of strains that were detected in the 1990s or earlier NHP0100Haiphong (accession no.: GQ129459, country where the strain was detected: Vietnam, year when the strain was detected: 2006); Y111 Wuhan (DQ873678, China, 2004), Z033 Wuhan (DQ873673, China, 2004); 5091 Japan (DQ779050, Japan, 2003–2004), 107E1B (AB081594, India, 1993), RMC437 (AY603153, India, 2004); ITO (D86278, Japan, 1980); YO (D86284, Japan, 1977); CH927 (D86275, China, 1992); AU-1 (D86271, Japan, 1982) in the regions previously identified as antigenic regions A, B, C, and F, NHP0100Haiphong showed the complete identity to those emerged in Japan and China, however, was different from those of G3 strains that were detected in the 1990s or earlier with some variations in amino acid substitutions (Fig 4) The DNA sequence of NHP0100Haiphong was deposited to GenBank under accession number GQ129459 Discussion Several epidemiological features of rotavirus infection among children less than years of age with acute severe diarrhoea in Haiphong, northern Vietnam is described in this study The detection rate of rotavirus in the study was relatively high (52%) with less than a half of cases (48%) occurring in the first year of life Global common rotavirus strains were detected in 88% of all the 509 rotavirus-positive specimens with low rate (5.5%) of mixed infections The profile of rotavirus infection obtained in the study is more similar to that observed in developed countries as described in the previous studies [7,8,12] G and P genotypes were determined successfully in 99% of all rotavirus-positive specimens in the study Procedures including RNA extraction, a series of RTPCR with multiple and a single primer sets were repeated, which may have resulted in attaining the high rate of successful rotavirus genotyping Globally, G1P[8], G2P[4], G3P[8], and G4P[8] are the four most common G, P combinations accounting for 89% of all the rotavirus diarrhoea among children [4] However, the ratio of rotavirus infections caused by the common genotypes varies from continent to continent For example, while cases with the four common G, P genotypes have been as high as 90% of all rotavirus infections in developed countries in North America and Europe, they have been responsible for only 70% of all rotavirus infections in developing countries in Asia [4] Of the four common rotaviruses, G1P[8] has played a significant role in childhood diarrhoea, causing more than 70% of rotavirus infections in Europe [20–23] The predominance N.T Cuong et al / Vaccine 27S (2009) F75–F80 of G1P[8] rotavirus has also been reported in developing countries in Asia [7,12,24], where a variety of unusual rotavirus strains have been frequently identified, such as G5 strains from Vietnam [25] and G11 and G12 strains from Nepal [14,26] In contrast, G3P[8] rotavirus has not been a predominant strain until recently in developed countries and in several developing countries Rotavirus surveillance in the United Kingdom in 1995–1998 demonstrated that G3P[8] accounted for only 3.3% of all rotavirus infections [21] Ten years of rotavirus surveillances in Hungary (1992–2000 and 2000–2003) detected G3P[8] in only 1.3% of all the rotavirus infections [27,28] A 3-year French study from 1995 identified G3P[8] in 1.6% of rotavirus infections [20] In Vietnam, the nationwide surveillance detected G3 strains only in 3.6% of rotavirus cases in 1998–1999 [8] and no G3 strains were detected in 2000–2003 [7] A number of studies conducted in other Asian countries also reported that G3 was the least frequent rotavirus among common strains showing its proportion ranging from 0% to 5.8% to the all rotaviruses genotyped [14,24,29,30] One study in Japan, however, reported an abrupt increase of G3P[8] to over 90% together with a sudden disappearance of G1, G4, and G9 genotypes in the 2003–2004 season [31] A recent report from Wuhan, China also reported that G3P[8] was the most common G-type/P-type combination (approximately 85% both in children and adults) in 2000–2006 seasons, while in the year of emergence of the strain was not precisely stated In this regard, the abrupt emergence of G3P[8] strains in Haiphong Vietnam appears to follow the pattern witnessed in Japan and China Of particular significance in this study is that G3P[8] rotavirus was predominantly detected as a causative agent of diarrhoea, accounting for 83% of all rotavirus infections among hospitalized children less than years of age in Haiphong, Vietnam (Table 2) The predominance of G3P[8] rotavirus has been observed throughout the study period keeping a high proportion in a range between 60% at September 2006 and 96% at January 2007 (data not shown) PAGE is regarded as a useful method for strain identification because even a single nucleotide substitution is reflected in a relative migration rate of a particular segment on the gel [32,33] The method has been used in many epidemiological studies to examine the genetic background of rotavirus strains [14,16,34] In PAGE analysis, 11 genomic rotavirus RNA segments were successfully visualized in 243 out of 421 specimens carrying G3P[8] rotavirus, and 91% of those shared an identical RNA migration pattern, termed g3p8-a Our results suggest that over 90% of G3P[8] strains circulating in Haiphong, Vietnam during the study period were likely to be of a single strain origin The widespread distribution of G3 strains observed during the study may be driven by the lack of G3 specific protective immunity among children in Haiphong city, induced probably by a prolonged absence of G3 strains in the community since 2000 [7] A hypothesis that the rotaviruses bearing electropherotype g3p8-a might have acquired a trait that enables them to transmit among humans more efficiently than strains of other electropherotypes, needs to be examined This abrupt emergence of a novel G3 strains in Vietnam and other parts of Asia underscores the continued need for high-quality rotavirus surveillance even after the burden of rotavirus disease has been established and rotavirus vaccine is introduced in a given country Conflict of interest Nil declared Funding sources This study was supported by the “Program of Founding Research Centers for Emerging and Re-emerging Infectious Diseases” funded F79 by the Ministry of Education, Culture, Sports, Science and Technology, Japan References [1] Cunliffe NA, Nakagomi O A critical time for rotavirus vaccines: a review Expert Rev Vaccines 2005;4(4):521–32 [2] World Health 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precisely stated In this regard, the abrupt emergence of G3P[8] strains in Haiphong Vietnam. .. features of rotavirus infection among children less than years of age with acute severe diarrhoea in Haiphong, northern Vietnam is described in this study The detection rate of rotavirus in the study... termed g3p8-a Our results suggest that over 90% of G3P[8] strains circulating in Haiphong, Vietnam during the study period were likely to be of a single strain origin The widespread distribution of