Journal of Medical Virology 80:298–305 (2008)

Identification of Human Astrovirus InfectionsAmong Children With Acute Gastroenteritisin the Southern Part of VietnamDuring 2005–2006

Tuan Anh Nguyen,1,2 LePhuc Hoang,2 Le Duc Pham,2 Kim Trong Hoang,2 Misashi Mizuguchi,1

Shoko Okitsu,1,3 and Hiroshi Ushijima1,3*1Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine,The University of Tokyo, Tokyo, Japan2Children’s Hospital 1, Ho Chi Minh City, Vietnam3Aino Health Research Center, Aino University, Tokyo, Japan

A molecular epidemiological study on commondiarrheal viruses was conducted in a children’shospital in Ho Chi Minh City between December2005 and November 2006. Fecal samples werecollected from 502 pediatric patients with acutegastroenteritis, and were screened for eight typesof viral agents. Human astroviruses (HAstVs),which were detected with a prevalence of 13.9%,became the second most frequent viral enter-opathogen. Although detected in both dry andrainy seasons, the majority (92.8%) of HAstVsin this study were found in the rainy season.Patients aged between 6 and 23 months werefound to be more infected from astroviruswhen compared to other age groups. The ratiobetween boys and girls was 2.3:1. The over-all mean severity score of astrovirus positivepatients was 11.8�2.45. The mean severity scoreof the inpatients was statistically higher thanthat of the outpatients; however, there was nodifference in severity scores between the agegroups observed. The clinical manifestationsof astrovirus infections among the Vietnamesechildren were diarrhea, vomiting, fever, cough-ing, and coryza. All of the detected astrovirusesbelonged to genotype 1. The phylogenetic treeclearly indicated that HAstVs-1 worldwide couldbe classified into four different lineages, in which,Vietnamese astroviruses and other recentlyisolated strains from other countries clusteredinto the distinct lineage, 1d. These results provideimportant information on astrovirus infectionsamong Vietnamese children. J. Med. Virol.80:298–305, 2008. � 2007 Wiley-Liss, Inc.

KEY WORDS: astrovirus; acute gastroenteritis;Vietnam

INTRODUCTION

Human astroviruses (HAstVs) are members of thefamily Astroviridae. They were first identified byelectron microscopy in stool specimens from childrenwithdiarrhea [AppletonandHiggins, 1975], and recentlydetermined as the second or third most common cause ofacute gastroenteritis in infants and young children, witha prevalence varying from2% to 16.5% [Glass et al., 1996;Gaggero et al., 1998; Mustafa et al., 2000]. The mainsymptom of astrovirus infection is usually watery andmilddiarrhea [Matsuietal., 1993].Outbreaksofdiarrheaassociated with astrovirus have been reported in com-munities, schools, and child care and aged-care center[Glass et al., 1996; Mitchell et al., 1999; Marshall et al.,2007].

HAstV has a positive-sense, single-stranded RNAgenomeofabout6,800nucleotide (nt) in length, excludingthe poly (A) tail at the 30 end. Three overlapping openreading frames (ORFs) were determined, in which,ORF1a and ORF1b encode nonstructural proteins, andORF2 encodes the capsid precursor protein [Willcocksand Carter, 1993; Lewis et al., 1994; Sanchez-Fauquieret al., 1994]. HAstVs are classified into eight serotypes byantigenic reactivityof the capsidproteins [KurtzandLee,1984; Lee andKurtz, 1994; Taylor et al., 2001], and there

Grant sponsor: The Ministry of Education, Culture, Sports,Science, and Technology, Japan; Grant sponsor: Ministry ofHealth, Labor, and Welfare, Japan.

*Correspondence to: Hiroshi Ushijima, MD, PhD, Departmentof Developmental Medical Sciences, Institute of InternationalHealth, Graduate School of Medicine, The University of Tokyo,7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.E-mail: ushijima-hiroshi@jcom.home.ne.jp

Accepted 30 August 2007

DOI 10.1002/jmv.21032

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2007 WILEY-LISS, INC.

is a perfect correlation between serotype and genotype[Noel et al., 1995]. Epidemiological studies aroundthe world have demonstrated that serotype 1 is res-ponsible for most astrovirus infections, and serotypes7 and 8 are rare compared to other serotypes [Lee andKurtz, 1994; Palombo andBishop, 1996;Koopmanset al.,1998; Mustafa et al., 2000; Espul et al., 2004]. Recently,molecular analysis of partial nucleotide sequence at the50 end of the capsid gene revealed thatHAstVs-1 could beclassified into four lineages, from 1a to 1d [Guix et al.,2002; Gabbay et al., 2007].

Vietnam is a developing country of 82 million people,in which diarrhea is considered a burden on its youngpopulation, and HAstV has been determined as one ofthe viral agents causing acute gastroenteritis in chil-dren [Nguyen et al., 2007]. HAstV infections have beenreported in only as sporadic cases in Vietnam [Landaetaet al., 2003; Nguyen et al., 2007]. In this study, wereported the detection of HAstV among diarrhoeicpediatric patients in a Ho Chi Minh City hospital, anddescribed the clinical manifestations of astrovirus-associated acute gastroenteritis as well as analyzingthe molecular characteristics of the astrovirus strainsdetected.

MATERIALS AND METHODS

Surveillance Site and Patients

Children’sHospital 1 is oneof twopediatrichospitals inHo Chi Minh City, with a capacity of 850 beds, receivingpediatric patients from most parts of the city as well asother provinces in the Mekong Delta, South Vietnam.Patients with acute gastroenteritis are screened atthe out-clinic ward, and admitted to the Department ofGastroenterology, if necessary. The symptoms of de-hydration are assessed by pediatricians, based on theWHO guideline [World Health Organization, 1995], andthe severity of diarrhoeic patients is evaluated basedon the 20-point numerical score [Ruuska and Vesikari,1990].

Fecal Sample Collection and Virus Detection

A total of 502 samples were collected from pediatricpatients, who visited the hospital from December2005 to November 2006 with a clinical diagnosis ofacute gastroenteritis. The fecal specimens from theoutpatients were collected at the out-clinic ward orfrom the inpatients within 24 hr after admission andstored at �208C until use. They were prepared as a10% suspension in distilled water and the viral RNAgenomes were extracted from the fecal suspension witha QIAamp Viral RNA Mini Kit (QIAGEN1, Hilden,Germany) according to the manufacturer’s instruction.The presence of eight types of common viral agents inthe fecal specimens was determined by RT-multiplexPCR [Yan et al., 2003, 2004]. Briefly, two sets of primerswere used: set A for the detection of groups A, B, andC rotavirus, and adenovirus, and set B was for thedetection of norovirus GI, GII, sapovirus, and astro-

virus. The primer pair used for the detection of HAstVwas PreCAP1 (sense, nt �82 to �62, GGACTGCAAAG-CAGCTTCGTG) and 82b (anti-sense, nt 637–617, GTG-AGCCACCAGCCATCCCT) [Matsui et al., 1998; Saka-moto et al., 2000], amplifying a 719 bp amplicon (startpositions of primers were based on the 50 end of thecapsid gene of the prototype H1 strain, accessionnumber L 23513). Neither bacteria nor parasite wasscreened in this study.

Genotyping of Astroviruses

Astrovirus genotyping was carried out by using RT-PCR analysis of extracted viral RNA, with serotypespecificprimers forHAstV-1 to -8, asdescribedpreviously[Sakamoto et al., 2000]. All PCR products were electro-phoresed in 2.0% agarose gel, followed by staining withethidium bromide (0.5 mg/ml), and then visualized underultraviolet light. For strains that could not be typed bythese processes, the genotype was identified further bysequence analysis.

Nucleotide Sequencingand Phylogenetic Analysis

The 719-bp amplicon of selected astrovirus positivesamples was subjected to sequencing by using the BigDye Terminator Cycle SequencingKit version 3.1 and anABI Prism 310 Genetic Analyzer (Applied Biosystems,Inc., Foster City, CA) according to the manufacturer’sinstruction. The primers, PreCAP1 and 82b, were usedfor sequencing, and generating a partial nucleotidesequence that included a small part of the 30 end ofORF1b, and approximately a 550 bp part of the 50 end ofORF2. Since a number of reference HAstV strains had anucleotide sequence starting from nt 247 of the 50 end ofORF2, a comparison with a shorter region (300 bp)spanning nt 247–546 and amino acid 83–182 wasperformed. Multiple sequence alignments were calcu-lated using the CLUSTALX program, and the phyloge-netic tree was constructed by the neighbor-joiningmethod with the MEGA3.1 software package [Kumaret al., 2004], based on different astrovirus sequencesavailable from GenBank.

Nucleotide Sequence Accession Numbers

The nucleotide sequences of the capsid gene of theselected Vietnamese astroviruses described in this studywere deposited into GenBank under accession numbersEU030287–EU030319. The reference strains and acces-sion numbers used for sequence analysis were as follows:A2/88 (Z25771), Arg158 (AY324858), Bcn1.1 (AF348753),Bcn1.3 (AF348755), BrG1-5 (DQ139825), Dresden (AY720892), H1 (L23513), H2 (L13745), H3 (AF117209), H4(Z33883), H5 (U15136), H6 (Z46658), H8 (Z66541),JAPAS115 (AB000287), Melb1E (AF175253), Melb1F(AF175254), PA-COD379-BR (DQ917390), PAK437 (AB000285), PAstV (AB037272), RJ8479/BR (DQ381498), S7(AB000300), Ven835 (AF211956), and WH2009 (DQ788612).

J. Med. Virol. DOI 10.1002/jmv

Human Astrovirus Infections in Vietnam 299

Statistical Analysis

SPSS was used for statistical analysis. The t-test wasused to compare the prevalence rates among differentage groups, as well as mean severity scores between thedifferent populations studied. P< 0.05 was consideredto be significant.

RESULTS

Detection of Target Viruses

Five out of eight target viral agents were detected in347 of 502 (69.1%) fecal specimens. As expected, groupArotavirus was the most predominant cause, with anoverall prevalence of 53.2%. Interestingly, astroviruswas the second most common viral enteropathogen inthis study (70/502, 13.9%), followed by norovirus GII,adenovirus, and sapovirus,with an overall prevalence of6.4%, 2.4%, and 1.2%, respectively.Regarding monoinfection, group A rotavirus, astro-

virus, genogroup II norovirus, adenovirus, and sapovi-rus were detected in 46.8%, 9.8%, 3.4%, 1.2%, and 0.4%of samples, respectively. Mixed infection between morethan one target virus was found in 38 samples (7.6%), inwhich, coinfection between group A rotavirus and theother viral agents was dominant (Table I).

Seasonal Pattern of Human Astrovirus Infections

The southern part of Vietnam, including Ho Chi MinhCity, has a tropical climate, in which the dry seasonusually begins in November and ends in April of thefollowing year. The remaining months are the rainyseason. The average temperature and mean monthlyrelative humidity does not changemuch during the year.In this study, HAstVswere found in two distinct periods;one being from March to May, which falls during theend of the dry season and beginning of the rainy season,and the other during the rainy season from Augustto November. However, 65 out of 70 (92.8%) HAstVsdetected in this study were identified in the rainy season(Table II). It is noteworthy that the detection rate ofHAstV inOctoberwas 70%,whichwasmuchhigher thanthat of rotavirus—the most common viral agent in thisstudy.

Characteristics of HAstV Positive Patients

Patients who enrolled in this study were classified intofive age groups:<6, 6–11, 12–23, 24–35, and>35monthsold. Among 502 patients, 477 medical records wereavailable for analysis of the age distribution, in which,43,168,198,46,and22patientsbelonged toeachof thefiveage groups, respectively. Diarrhea due toHAstVwas seenin all five age groups, and astrovirus infections weremorelikely found in patients 6–11 and 12–23 months old(P< 0.001) (Table III). The ratio between boys and girlswas 2.3:1. Thirty out of 70 HAstV-positive cases (42.9%)lived inHoChiMinhCity,andotherpatients (57.1%)camefrom 17 provinces in the southern part of Vietnam.

J. Med. Virol. DOI 10.1002/jmv

TABLE

I.Distribution

ofTarget

ViralAgen

tsDetectedFrom

502ChildrenWithAcu

teGastroen

teritisin

theChildren’sHospital1,HoChiMinhCity,During2005–2006

Mon

oinfection

Multiple

infection

RAV

HAstV

NoV

IIAdV

SaV

RAV—HAstV

RAV—NoV

IIRAV—SaV

RAV—AdV

NoV

II—HAstV

AdV—HAstV

NoV

II—AdV

NoV

II—SaV

RAV—NoV

II—HAstV

RAV—AdV—HAstV

No.

(%)of

specim

ens

235(46.8)

49(9.8)

17(3.4)

6(1.2)

2(0.4)

15(3.0)

10(2.0)

3(0.6)

2(0.4)

2(0.4)

2(0.4)

1(0.2)

1(0.2)

1(0.2)

1(0.2)

RAV,groupArotavirus;HAstV,humanastrovirus;NoV

II,norov

irusgen

ogroupII;AdV,aden

ovirus;

SaV,sa

pov

irus.

300 Nguyen et al.

J. Med. Virol. DOI 10.1002/jmv

TABLE

II.Mon

thly

Distribution

ofViralEnterophatogen

sDetectedFrom

ChildrenWithAcu

teGastroen

teritisin

theChildren’sHospital1,HoChiMinhCity,During

2005–2006

Sea

sonality

Dry

season

Rainyseason

Dry

Total(%

)Dec

2005

Jan2006

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

No.

ofsp

ecim

ens

20

30

43

57

30

53

32

17

30

32

60

98

502(100)

RAV

(%)

7(35.0)

15(50.0)

23(53.5)

44(77.2)

24(80.0)

29(54.7)

19(59.4)

9(52.9)

12(40.0)

7(21.9)

16(26.7)

62(63.3)

267(53.2)

HAstVa(%

)0(0)

0(0)

0(0)

1(1

.8)

2(6

.6)

12

(22.6

)0(0)

0(0)

7(2

3.3

)4

(12.5

)42

(70.0

b)

2(2

.0)

70(13.9)

NoV

II(%

)2(10.0)

1(3.3)

2(4.7)

4(7.0)

0(0)

3(5.7)

2(6.3)

0(0)

6(20.0)

4(12.5)

5(8.3)

3(3.1)

32(6.4)

AdV

(%)

0(0)

0(0)

2(4.7)

2(3.5)

0(0)

3(5.7)

1(3.1)

0(0)

0(0)

1(3.1)

3(5.0)

0(0)

12(2.4)

SaV

(%)

0(0)

1(3.3)

2(4.7)

0(0)

1(3.3)

1(1.9)

0(0)

0(0)

1(3.3)

0(0)

0(0)

0(0)

6(1.2)

aThedata

forHAstVare

inboldface.

bDetection

rate

ofHAstVin

Octob

erwashigher

thanthatof

RAV.

TABLE

III.

Attributesof

70HAstV

PositiveCasesandMea

nSev

erityScore

ofPatien

tsin

Each

Group

Distribution

ofpatien

tsby

Age(m

onth)

Gen

der

Place

ofliving

Patien

tstatus

<6

6–11

12–23

24–35

>35

Male

Fem

ale

HCMCa

Others

Inpatien

tOutpatien

t

No.

(%)of

casesb

4(5.7)

27(38.6

c )33(47.1

c)

3(4.3)

3(4.3)

49(70)

21(30)

30(42.9)

40(57.1)

62(88.6)

8(11.4)

Vesikari’sscored

10.8�3.0

11.8�2.7

11.9

�2.4

11.5�2.1

11.5�0.5

11.6

�2.5

12.1�2.4

11.6�2.5

11.9

�2.7

12.2�2.3

e9.4

�2.2

e

aHCMC,HoChiMinhCity.

bData

basedon

70HAstVpositivecases.

c P<0.001.

dData

basedon

44ou

tof

49cases,whichsh

owed

mon

oinfectionwithHAstV.

eP<0.01.

Human Astrovirus Infections in Vietnam 301

Clinical Signs and Symptomsof Astrovirus Infections

Among 49 patients showing monoinfection withHAstV, 44medical records were enough data to analyzethe clinical signs and symptoms of astrovirus infections.The main clinical manifestations observed in childrenwith HAstV infection were diarrhea (100%), vomiting(84.1%), highest temperature �38.58C (50.0%), cough-ing (40.1%), and coryza (25.0%). Themajority of patients(79.5%) presented watery stool, whereas 13.6% hadmucus and 6.9%mucus and blood in the stool. Only 4 outof 44 (9.9%) patients showed moderate dehydration,while the other patients had no dehydration during theentire course of the disease. Evaluation of severity inpatients by using the 20-point numerical score showedthat the scores ranged between 6 and 17 points, with theoverall mean score of 11.8�2.45.The severity scores were analyzed further by age

groups, gender, place of living, and status of patients(Table III). Obviously, the mean score of patients<6 months of age was lower than that of patients inthe other age groups; however, the difference was notstatistically significant (P> 0.05). Similarly, the meanscores of severity between male and female patients,as well as between patients who lived in Ho ChiMinh City and other provinces were not statisticallydifferent. However, the inpatients in this study sufferedfrom a severer disease than the outpatients, with themean severity scores in each group being 12.1� 2.3 and9.4� 2.2, respectively, and the difference was statisti-cally significant (P< 0.01).

Genotyping of Astrovirus

Fifty-six out of 70 specimens, which presentedpositive for HAstV, were successfully typed by nestedPCR. All of them showed the band of PCR product at theposition of the genotype 1 (212 bp). Then, 14 untypeablesamples were subjected to sequencing at the 50 end ofthe capsid gene, and a BLAST search clearly showedthat they had best identities (99%) with a genotype1 human astrovirus, Dresden strain. Altogether, all ofthe 70 HAstVs detected in this study were genotype 1.

Nucleotide Sequence Analysis of Astrovirus

Thirty-three specimens, including 14 untypeable and19selected typeable specimens, successfully determinedthe nucleotide sequences. The phylogenetic tree, basedon the 300 bp region of the 50 end of ORF2, revealed thatall of the Vietnamese astroviruses were grouped intothe HAstV-1 cluster [Guix et al., 2002]. Furthermore,the phylogenetic tree clearly showed that HAstVs-1could be classified into four lineages (1a–1d), in which,Vietnamese astrovirus strains clustered into the line-age, 1d, together with other astrovirus strains recentlyisolated fromSpain, Germany, Brazil, andChina. OtherHAstVs isolated during the early 1990s, including theprototype H1, clustered into the other three lineages,1a–1c (Fig. 1).

The nucleotide comparison showed that Vietnameseastrovirus strains were 99.0–100% identical to eachother. The similarities between Vietnamese strains andother astrovirus strains in the lineage, 1d, were from97.3% to 99.6%, in which Vietnamese strains and theDresden strain were most similar. The divergencesbetween Vietnamese strains and other HAstVs fromlineage 1a–1c varied from 8.0% to 12.1%. Nucleotidesequence changes resulting in amino acid substitutionsin relation to the prototypeH1were observed in only onestrain, HCMC201/2006, with two residues TS insteadofNPat position aa109-110.Wider observation of aminoacid comparison at the 50 end of ORF2 revealed thatthe HCMC220/2006 strain shared a substitution Tinstead of A at position aa 46 with the PAK437/91strain. Furthermore, six strains (HCMC169/2006,HCMC201/2006, HCMC215/2006, HCMC216/2006, HCMC225/2006, andHCMC365/2006) shared a unique residueW, instead of R, at position aa 20. Interestingly, allVietnamese astrovirus strains showed a distinct sub-stitution of S instead of N at position aa 44, whichwas not found in any other astrovirus strains amonggenotype 1.

DISCUSSION

In this study, we described a 1-year surveillance ofcommon viral agents causing acute gastroenteritis inchildren at a pediatric hospital in Ho Chi Minh City.More than half (57.1%) of the patients who enrolled inthis surveillance came from various provinces in theMekong Delta, therefore, the results of this studymightreflect thepattern of astrovirus infection in the southernpart of Vietnam. Many of the target viruses weredetected in concordance with the previous epidemiolog-ical study conducted at the same hospital during 2002–2003 [Nguyen et al., 2007], thus confirming the diversityof viral enterophathogens and the burden of viraldiarrhea in the area. As expected, group A rotaviruswas still the most frequent cause, however, HAstVbecame the second most common agent with an overallprevalence of 13.9%, which was much higher than thatof the previous study, 0.6% [Nguyen et al., 2007]. Asimilar detection method was used in both studies,confirming the emergence of HAstV during 2005–2006.On the otherhand, althoughdetected inboth thedryandrainy season, the majority (92.8%) of HAstVs in thisstudy were found in the rainy season. This result was inagreement with the previous study in Vietnam [Nguyenet al., 2007] and other reports fromBrazil [Gabbay et al.,2007; Santos et al., 2007]. Furthermore, HAstVs werefound at an exclusively high detection rate (70%) inOctober frompatientswho lived not only inHoChiMinhCity, but also various provinces in the Mekong Delta(data not shown), suggesting that the viruses werewidespread in the area at that time. Since fecal speci-mens were collected at the out-clinic ward or within24 hr after admission, outbreak of nosocomial infectionwas excluded.

J. Med. Virol. DOI 10.1002/jmv

302 Nguyen et al.

In this study, HAstVs were found in specimenscollected from patients in all five age groups. However,infants aged 6–11 months and children from 12 to23 months old were found to be infected more thanpatients in the other age groups. This result wasconcordant with other astrovirus studies from differentcountries [Medina et al., 2000; Mustafa et al., 2000;Gabbay et al., 2007]. The protection from HAstVinfection might be explained by the passive immunityin infants <6 months, or the active immunity frompreviously acquired infections in children more than2 years of age. Interestingly, although themean severityscore of patients <6 months was lower than that ofpatients in the other age groups, the difference was not

statistically significant. This finding suggests thatpassive immunity only helps to protect from infectionevent, but does not influence the severity of disease.

The clinical manifestations of HAstV infection inpatients in this study were similar to those from otherreports; with diarrhea, vomiting, and fever beingthe main clinical signs and symptoms. However, bodytemperature�38.58Cwas found in 50% of patients, andbloody diarrhea was noted in three patients. Thesefeatures were different from data reported by Walterand Mitchell [2003], in which, only 20–25% of patientshad fever, and no bloody diarrhea was reported. On theother hand, although only 9.9% of patients in this studysuffered from dehydration, the mean severity score was

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. Phylogenetic tree of the 300 bp region of ORF2 of 33 HAstVs-1 from Ho Chi Minh City (HCMC),and other reference astrovirus strains available fromGenBank. The prototypeH1 is circled. Four lineagesofHAstV-1 are indicated,with the lineage, 1d, includingVietnamese strains andother strains isolated fromSpain, Germany, Brazil, and China (Bcn1.1, Bcn1.3, Dresden, BrG1-5, RJ8479/04, and WH2009/04-05).The porcine astrovirus, PAstV, is used as an outgroup.

Human Astrovirus Infections in Vietnam 303

relatively high (11.8�2.45), when compared to themean severity score reported previously (only 5) [WalterandMitchell, 2003]. This high score might result from along duration of diarrheal disease, and/or high numbersof diarrhea or vomiting episodes per day (data notshown). However, it is well known that astrovirus canoccur in mixed infections with a variety of otherinfectious agents such as bacteria. Since bacteria andparasite were not screened in this study, the questionremains whether HAstV was the sole causative agentthat created severity of diarrhea disease among thepatients studied.In agreementwith the previous study conducted inHo

Chi Minh City, all of the HAstVs isolated in this studybelonged to genotype 1, in which, all of the HAstV weredetermined as genotype 1 by RT-multiplex PCR. Thisindicated circulation of genotype 1 HAstVs in the entirearea.Thepredominance ofHAstV-1wasalso found in allepidemiological studies over the world [Palombo andBishop, 1996; Medina et al., 2000; Mustafa et al., 2000;Guix et al., 2002; Gabbay et al., 2007], except for one inMexico, where HAstV-2 was the most common serotype[Guerrero et al., 1998]. On the other hand, Gabbay et al.[2007] reported that the concordance observed betweenRT-PCR and sequencing in genotyping of astroviruswas only 69.2% due to several reasons, in which, thediscordant results were mostly related to HAstVs-4 andHAstVs-7. Our data of 19 typeable HAstV-1 strains,subjected to sequence analysis, indicated that the twotypingmethodsmentionedabovewerehighly agreeable,and it also emphasized the high specificity of the type-specific primer, AST-S1, which was used for genotypingHAstV-1. Data from another study using another set ofprimers also showed an absolute concordance betweenresults ofRT-PCRand sequencingamong typeable cases[Mitchell et al., 1999]. Recently, nucleotide sequencingwas chosen as the typingmethod for astrovirus inmanystudies, thus confirming the usefulness of this method[Mitchell et al., 1999; Medina et al., 2000].This is the first time that data on nucleotide analysis

of Vietnamese astrovirus were reported. In this study,HAstVs-1 could be divided into four lineages, in which,Vietnamese astroviruses and other strains which havebeen isolated since the end of the 20th century, clusteredtogether into the lineage, 1d. This suggests an evolutionof HAstV-1 through nucleotide mutations overtime.Furthermore, the presence of HAstVs-1d from variouscountries on different continents suggests how widelythey spread around the world. Besides, many recentstudies have used a 348 bp region of ORF2, generatingfrom primers Mon269 and Mon270 [Noel et al., 1995],for analyzing the nucleotide characteristics of theastrovirus detected [Medina et al., 2000; Mustafaet al., 2000; Guix et al., 2002; Gabbay et al., 2007]. Witha longer sequence obtained (719 bp), the primer pair,preCAP1 and 82b, were useful in screening HAstV andperforming nucleotide sequence analysis at the capsidregion. Within the 300 bp region, spanning nt 247–546,all of the nucleotide mutations within Vietnameseastroviruses were silent, except for one (HCMC201/

2006). However, a wider observation toward the 50 endof ORF2 showed that more substitutions uniquelyoccurred among Vietnamese astroviruses. This findingconfirmed the difference between HAstVs isolated fromHoChiMinhCity and others in other lineages, aswell asthe prototype H1.

The results of this study highlight the impact ofastrovirus in diarrheal diseases among children in thesouthern part of Vietnam, and are the first to describethe clinical manifestations of astrovirus infections inVietnamese children. The data of nucleotide analysisfrom this study could provide useful information forknowledge on astrovirus characteristics, as well as thedevelopment of an astrovirus vaccine.

ACKNOWLEDGMENTS

We deeply thank the staff of the Department ofGastroenterology, Children’s Hospital 1, Ho Chi MinhCity for collecting samples.

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