84th Ph.D Thesis Supervisor: Prof. Wonyong Kim

Molecular Characterization of Human Group A Rotavirus Infections. Expression of Human Rotavirus Genes and Rotavirus Like Particle Production

Microbiology Major Department of Medical Science, Graduate School Chung-Ang University

LE VAN PHAN

December, 2008

Molecular Characterization of Human Group A Rotavirus Infections. Expression of Human Rotavirus Genes and Rotavirus Like Particle Production

A thesis submitted in accordance with the requirements of Chung-Ang University for the degree of Doctor of Philosophy

Microbiology Major Department of Medical Science, Graduate School Chung-Ang University

LE VAN PHAN

December, 2008 2

THE UNDERSIGNED THESIS COMMITTEE HEREBY APPROVE THE THESIS OF LE VAN PHAN AS QUALIFIED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Chairman Vice-chairman Member Member Member

Department of Medical Science, Graduate School Chung-Ang University

December, 2008 3

ABSTRACT Rotaviruses are the most common etiological agent of severe diarrhea in infants and young children and are responsible, worldwide, for an estimated 454,000–705,000 death annually. In this study, five hundred four fecal specimens, collected between 2004 and 2006 in Seoul, South Korea from young children with acute diarrhea, were screened for rotavirus by ELISA with VP6-specific antibody. Of these samples, 394 (78.2%) were confirmed as group A rotavirus and they underwent G- and P typing using a combination of ELISA, RT-PCR, and sequence analysis methods. The dominant circulating G serotype was G1 (35.6%) followed by G3 (26.4%), G4 (14.7%), and G2 (11.9%). There was a low prevalence of G9 (1.0%) and of unusual G type rotavirus, in particular, G12 (0.5%) and G8 (0.3%). Of the P genotype rotavirus in circulation, P[8] (53.0%) was most common followed by P[6] (15.5%), P[4] (15.2%), and P[9] (2.3%). Determination of G- and P type combinations revealed that G1P[8] strains were most prevalent (25.4%), amid G3P[8] (16.8%), G2P[4] (6.3%), and G4P[6] (6.1%) strains. Unusual or rare combinations such as G2P[6], G2P[8], G3P[4], G2P[9], G1P[9], G3P[9], G12P[6], G1P[4], G3P[6], and G8P[8] were also found. Owing to the recent emergence of G8 and G12 rotavirus, the findings from this study are important since they provide new information concerning the local and global spread of rotavirus genotypes. 4

Previous epidemiological studies reported that G1 to G4 serotypes were common, particularly G1 as the predominant strain during the past years. Recently, increasing numbers of studies have documented that G9 and G12 strains may represent the new epidemiologically important G serotypes. Several reports have revealed considerable genetic diversity within the VP7 gene of predominant G1 strains and G9 strains as well. In this work Korean G1P[8], G9P[8], and unusual G12P[6] human rotavirus strains have also been isolated and characterized for VP7, VP4, VP6, and NSP4 gene. For G1P[8] human rotavirus strains, all strains showed a “long” RNA pattern and VP6 subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that they all clustered into lineage I, the same as reported G1 strains in Japan, China, Vietnam, and Thailand except that one old Korean strain (Kor-64) belonged to lineage IV. Also, phylogenetic analysis of VP4 genes revealed that they were of P[8] genotype with two distinct lineages (P[8]-3 and P[8]-2). With respect to the NSP4 gene, all G1 strains fell into the genotype B. For G9P[8] human rotavirus strain (CAU 202), CAU 202 strain showed a “long” RNA pattern and VP6 subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that CAU 202 and other Korean G9 strains deposited in GenBank databases clustered into genetic lineage III. Also, phylogenetic analysis of VP4 genes revealed that CAU 202 was of P[8] genotype with lineages P[8]-3. With respect to the NSP4 gene, CAU 202 fell into the 5

genotype B. Also for G12 human rotavirus strains, CAU 195 and CAU 214 were isolated and characterized. Both CAU 195 and CAU 214 strain showed a “long” RNA pattern and subgroup II specificity. VP7 gene of CAU 195 and CAU 214 strains exhibited 89.8% to 99.7% at the nucleotide similarities and 92.3% to 100% at the amino acid identities respectively, to those of rotaviruses with G12 specificity in GenBank. VP4 gene sequences of CAU 195 and CAU 214 showed the highest identity with P[6] specificity to porcine strain Gottfried with homologies of 83.2 to 83.3% at the nucleotide and 89.5 to 89.7% at the amino acid levels. In addition, the NSP4 gene of these strains were most closely related to the human and porcine branch within the Wa lineage, and revealed that they belong to genotype B. Finally, four structural proteins of VP2, VP4, VP6, and VP7 of human rotavirus have been cloned and expressed successfully using baculovirus expression system, respectively. The double-layered rotavirus like particles (2/6 VLPs) and triple-layered rotavirus like particles (2/4/6/7 VLPs) were then generated and characterized, respectively.

Keywords: Human rotavirus; Phylogenetic analysis; Virus like particles

6

ABBREVIATIONS A

Adenine

a.a.

Amino acid

bp

Base pair

C

Cytosine

DNA

Deoxyribonucleic acid

CPE

Cytopathic effect

dNTP

Deoxyribonucleotide triphosphate

dsRNA

Double-stranded RNA

EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunoSorbent assay

EM

Electron microscopy

FBS

Fetal bovine serum

G

Guanine

kDa

Kilodalton

LB

Lysogeny broth (or Luria Bertani medium)

Leu

Leucine

M

Molar

MAb

Monoclonal antibody

Met

Methionine

nm

Nanometer 7

nt

Nucleotide

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline

PCR

Polymerase chain reaction

RNA

Ribonucleic acid

HRV

Human rotavirus

RT-PCR

Reverse transcription-Polymerase chain reaction

Sf9

Spodoptera frugiperda cell

T

Thymine

Taq

Thermus aquaticus DNA polymerase

Tris

Tris (hydroxymethyl) aminomethane

U

Unit

UV

Ultra-violet

VLP

Virus like particle

8

CONTENTS CHAPTER I. General Introduction…………………………………...14 CHAPTER II. Molecular Epidemiology of Human Group A Rotavirus Infections. Detection of Unusual Rotavirus Genotype G8P[8] and G12P[6]………18 1. INTRODUCTION……………………………………………..…….18 2. MATERIAL AND METHODS……………………………………21 Stool specimens……………………………………….…..…………21 VP7 (G) serotyping by ELISA…………………………….………..21 RNA extraction for genotyping…………………………….……….21 VP7 (G) Genotyping by RT-PCR………………….……….…….22 VP4 (P) genotyping by RT-PCR……………………………………23 Nucleotide sequencing and phylogenetic analysis…………………23 3. RESULTS…………………………………………………….……..25 Determination of G serotypes……………………….…….………..25 Determination and phylogenetic analysis of G12 serotypes…….…27 Determination of P genotypes………………………………………30 Distribution of G- and P types……………………………………30 4. D I S C U S S I O N … … … … … … … … … … … … . … … … … … . 3 2 CHAPTER III. Molecular Characterization of Prevalent G1P[8] Human Rotavirus Strains Isolated from Children Hospitalized with Acute Gastroenteritis………….39 9

1. INTRODUCTION…………………………………….……………39 2. MATERIAL AND METHODS………………………….………….42 Stool specimens……………………………………….……………42 Isolation of rotavirus…………………………….………………….42 Polyacrylamide gel electrophoresis (PAGE)……………………..43 Reverse transcription-Polymerase chain reaction…………………..44 Nucleotide sequencing and phylogenetic analysis…………………45 Nucleotide sequence accession numbers…………………………...46 3. RESULTS……………………………………………………………49 Isolation and RNA electropherotype of six G1P[8] strains………...49 Phylogenetic analysis of VP7, VP4, VP6, and NSP4 gene of six G1P[8] strains…………………………………...………………….49 4. D I S C U S S I O N … … … … … … … … … … … … … … … … … . . 5 8 CHAPTER IV. Molecular Characterization of A Human Rotavirus Isolate of G9P[8] strain……………………………………….64 1. INTRODUCTION………………………………………….………..64 2. MATERIALS AND METHODS…………………………………….67 Stool specimens……………………………………………………...67 Isolation of rotavirus………………………………………………..67 Immunofluorescence assay (IFA) for detection of CAU 202………68 Polyacrylamide gel electrophoresis (PAGE)……………………….68 10

Reverse transcription- Polymerase chain reaction………………69 Nucleotide sequencing and phylogenetic analysis…….…………70 Nucleotide sequence accession numbers………………………..71 3. RESULTS…………………………………………………………….73 Isolation of G9 rotaviruses and indirect immunofluorescence assay (IFA)………………………………………...…………………73 RNA Electropherotype of CAU 202 (G9) rotaviruses……..………78 Phylogenetic analysis of VP7, VP4, VP6, and NSP4 gene of CAU 202………………………………………..………………….78 4. DISCUSSION………………………………………………………..85 CHAPTER V. Molecular Characterization of Unusual Human Rotavirus Strains G12 with P[6] Detected in South Korea………………………………………..90 1. INTRODUCTION………………..………………………………….90 2. MATERIALS AND METHODS…………………………………….93 Stool specimens……………………..……………………………….93 Isolation of rotaviruses in cell culture…………………….…………93 Indirect immunofluorescence assay (IFA)…………….………….....94 Polyacrylamide gel electrophoresis (PAGE)…………….………….94 Reverse transcription-Polymerase chain reaction………….……….95 Nucleotide sequencing and phylogenetic analysis…………….……96 11

3. RESULTS……………………………………………………………98 Detection of G12 human rotaviruses………………..……………….98 Isolation of G12 rotaviruses and indirect immunofluorescence assay (IFA)…………………………………………..……………….98 RNA electropherotype of G12 rotaviruses………………………….103 Sequence determination of G12 rotaviruses……………….…….…103 4. DISCUSSION…………………………………………………..109 CHAPTER VI. E x p r e s s i o n o f H u m a n R o t a v i r u s G e n e s and Rotavirus Like Particle Production…………………..116 1. INTRODUCTION…………………………………………...……..116 2. MATERIALS AND METHODS……………………………...……120 Viruses and RNA extraction…………….…………………………120 Reverse transcription-Polymerase chain reaction…………………..120 Generation of recombinant baculoviruses expressing VP2, VP4, VP6, and VP7 proteins………………………………………..……121 Plaque forming assay……………………………………………....122 Immunofluorescence assay (IFA)………………………………… 123 Western immunoblotting…………………………………………...123 Rotavirus like particle (VLP) production and purification…...…….124 3. RESULTS…………………………………………..………………126 Expression of VP2, VP4, VP6, and VP7 gene……………………126 12

Confirmation of recombinant baculoviruses expressing VP2, VP4, VP6, and VP7 protein of HRV.........................................................130 Rotavirus- virus like particle production…………………………130 4. DISCUSSION………………………………………..…………….136 CHAPTER VII. General Discussion……………………………139 REFERENCES…………………………………........................ 145 ACKNOWLEGMENTS………………………………………………175

13

I GENERAL INTRODUCTION Rotaviruses, which form one genus of the family Reoviridae, are now recognized as the most important cause of severe viral gastroenteritis in humans and animals [Estes and Cohen, 1989]. Rotaviruses are classified into seven groups (A to G) on the basis of their distinct antigenic and genetic properties. Human infection has been reported with group A, B, and C rotavirus [Yoshinaga et al., 2006]. Of these, group A rotaviruses are the most important, being a major cause of severe gastroenteritis in infants and young children and are responsible, worldwide, for an estimated 454,000 - 705,000 deaths annually [Barril et al., 2006; Glass et al., 1996; Khamrin et al., 2006; Parashar et al., 2006]. Rotaviruses are composed of three concentric protein layers surrounding 11 segments of doublestranded RNA [Estes, 2001]. The innermost layer is composed mainly of VP2, which comprises about 90% of the core protein mass and binds to viral RNA [Labbe et al., 1991] and may participate in the replication and encapsidation of the RNA genome. The middle layer is composed entirely of VP6, which comprises >80% of the protein mass of the particle and bears group- and subgroup-specific epitopes [Greenberg et al., 1983], and its removal from subviral particles has been associated with loss of viral 14

transcriptase activity [Sandino et al., 1986], although VP6 by itself shows no polymerase activity. The outer layer is composed of glycoprotein VP7 and dimeric spikes of VP4. The glycoprotein VP7 and the spike protein VP4 independently elicit neutralizing antibodies and induce protective immunity [Estes, 2001; Hoshino et al., 1985]. Group A rotaviruses are differentiated by serotype based on the diversity of the VP7 (a glycoprotein, which defines the G-type) and VP4 (a protease-cleaved protein, which defines the P-type) antigens present on the outer capsid protein. To date, 16 G serotypes [Gulati et al., 2007] and at least 27 P genotypes have been reported [Bozdayi et al., 2008; Khamrin et al., 2007a; Martella et al., 2007]. As with other genome-segmentedviruses, rotaviruses can reassort their genes independently [Gouvea and Brantly, 1995], many combinations of G and P genotypes are possible. There are now 42 different combinations of G and P genotypes detected, but five of them (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) are the most prevalent in humans globally and comprise more than 90% of the human cases detected worldwide. Among them, the G1P[8] strain is by far the most commonly found worldwide [Espinola et al., 2008; Gentsch et al., 2005; Parra et al., 2007a; Santos and Hoshino, 2005] This study is an extension of an earlier molecular epidemiological investigation [Song et al., 2003] into the distribution of G- and P type 15

strains of rotavirus in Seoul, South Korea. Conducted between 2004 and 2006, a combination of enzyme- linked immunosorbent assay (ELISA), multiplex reverse transcription polymerase chain reaction (RT-PCR), and sequence analysis methods were employed. An update on the most prevalent rotavirus strains currently circulating South Korea is reported. The genetic characterization with molecular analysis of VP7, VP4, VP6, and NSP4 genes of rotavirus strains is very important and a key step to acquiring an in-depth understanding of the ecology and evolution of rotaviruses [Li et al., 2008]. Although rotavirus surveillances are conducted annually in Korea, few sequence data of common circulating rotavirus strains such as G1, G2, G3, G4, G9, and unusual G12 strains are available in GenBank databases. So, based on the results of molecular epidemiological investigation, the most prevalent G1P[8] (25.4%) together with rare G9P[8] (1%), and unusual G12P[6] (0.5%) human rotavirus strains were selected for isolation and genetic characterization. The full length VP7, VP4, VP6, and NSP4 genes were sequenced and compared with those of human rotaviruses available on public databases in order to know whether any change in the VP7 genes occurred. Finally, four structural proteins of VP2, VP4, VP6, and VP7 of human rotavirus have been cloned and expressed using baculovirus expression system. The recombinant proteins were then characterized and used as specific 16

materials for rotavirus like particle production- a specific class of subunit vaccine that mimic the structure of authentic virus particles.

17

II Molecular Epidemiology of Human Group A Rotavirus Infections. Detection of Unusual Rotavirus Genotypes G8P[8] and G12P[6] 1.

INTRODUCTION Rotaviruses are the most common etiological agent of severe

diarrhea in infants and young children [Kapikian et al., 2001]. To date, 16 G serotypes [Gulati et al., 2007] and at least 27 P genotypes have been reported [Khamrin et al., 2007b; Martella et al., 2007; Steyer et al., 2007a]. Of these variants, epidemiological studies have shown that four G (G1 G4) and three P types (P[4], P[6], and P[8]) are the most frequent VP7 and VP4 types associated with global human rotavirus infection [Santos and Hoshino, 2005]. Despite the high prevalence of G1, G2, G3, and G4, a fifth, new genotype, G9, has emerged as a causative agent of diarrhea in children from the United States, Canada, Australia, United Kingdom, Europe, Latin America, Africa, and many Asian countries including South Korea [Banerjee et al., 2006; Chen et al., 2007; Clark et al., 2004; Doan et al., 2003; Hung et al., 2006; Iturriza-Gomara et al., 2000; Khamrin et al., 2006; Kim et al., 2005; Kostouros et al., 2003; Lin et al., 2006; Linhares 18

et al., 2006; Steele and Ivanoff, 2003; Van Damme et al., 2007; Wang et al., 2007; Yoshinaga et al., 2006]. Another rotavirus genotype, G8, was first detected in Indonesian children [Matsuno et al., 1985] and an increase in its occurrence has been reported recently in studies worldwide [Adah et al., 2001; Cunliffe et al., 2000; Gerna et al., 1990; Holmes et al., 1999; Matthijnssens et al., 2006; Pietruchinski et al., 2006; Steyer et al., 2007b; Uchida et al., 2006; Volotao et al., 2006]. The unusual human strain, G12 (L26 and L27) was first identified in 1987 from strains causing gastroenteritis in children from the Philippines [Taniguchi et al., 1990; Urasawa et al., 1990]. Since then, G12 has appeared in the United States and Thailand, and shortly thereafter, in India and Japan [Das et al., 2003; Griffin et al., 2002; Pongsuwanna et al., 2002; Shinozaki et al., 2004] such that today, G12 rotavirus has been detected in many countries across the world [Rahman et al., 2007a]. Even though G9 was first detected in the rural provinces of South Korea [Kim et al., 2005], G8 strains have not yet appeared and only a few reports are available on the occurrence of G12 [Kang et al., 2005; Santos and Hoshino, 2005]. Socioeconomic status and environmental conditions have improved but rotavirus is still the most common viral agent of acute diarrhea in young children aged between 6 and 24 months in South Korea [Kim et al., 1990; Seo and Sim, 2000]. South Korean surveillance studies 19

reveal significantly different changes in rotavirus types across recent years. G4, G2, and G9 strains were most frequent during the period from 1998 to 2004 [Kang et al., 2005; Kim et al., 2005; Min et al., 2004; Moon et al., 2007; Song et al., 2003] while previous epidemiological reports spanning 1987 - 1999, indicate that G1 - G4 were common, G1 being the predominant strain [Kim et al., 1999; Kim, 1993; Seo and Sim, 2000]. These distinct changes in the prevalence of G- and P type rotavirus make it essential that a thorough understanding is gained of the relative importance of rotavirus strains circulating locally. Surveillance studies are necessary since they allow a comprehensive evaluation of evolving rotavirus and the resultant data can be used, in part, for assessing the capacity of vaccines to provide heterotypic protection. Furthermore, they provide the basis for any argument that may be advanced in support of new vaccination programs. This study is an extension of an earlier molecular epidemiological investigation [Song et al., 2003] into the distribution of G- and P type strains of rotavirus in Seoul, South Korea. Conducted between 2004 and 2006, a combination of enzyme-linked immunosorbent assay (ELISA), multiplex reverse transcription polymerase chain reaction (RT-PCR), and sequence analysis methods were employed. An update on the most prevalent rotavirus strains currently circulating South Korea is reported. 20

2.

MATERIALS AND METHODS

2.1.

Stool Specimens A total of 504 fecal specimens were obtained from young

children less than 5 years old who presented with acute diarrhea in five general hospitals located in Seoul during the period between January 2004 and February 2006. All specimens were diluted 10-fold with phosphate buffered saline (PBS; pH 7.4) and clarified by centrifugation 10,000g for 10 min. The supernatants were tested for group A rotavirus antigen by ELISA with VP6-group-specific antibody (Dako Diagnostics, Cambridgeshire, UK). 2.2.

VP7 (G) Serotyping by ELISA Fecal specimens were used for serotyping by ELISA with

serotype

specific

monoclonal

antibodies

(mAb)

following

the

manufacturer’s protocol (rotaMA; Serotec Company, Sapporo, Japan). G1- to G4-specific neutralizing mAbs to HRV included G1-specific KU, G2-specific S2, G3-specific YO, and G4-specific ST3. When the A492 value of a specimen was greater than 0.2, and 2 times greater than those of other serotypes, the specimen was determined as positive. 2.3.

RNA Extraction for Genotyping Rotavirus dsRNA was extracted using Trizol reagent (Life 21

Technologies, Grand Island, NY). In brief, 0.3 ml of supernatant of a fecal suspension in PBS was mixed with 0.7 ml of Trizol reagent and 0.2 ml of chloroform/isoamylalcohol (24:1). After centrifugation at 12,000g for 10 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol. The RNA precipitate was collected by centrifugation at 12,000g for 10 min, washed with 70% ethanol and finally dissolved in 20 μl of RNase-free water. 2.4.

VP7 (G) Genotyping by RT-PCR RT-PCR of the VP7 gene was performed using primers specific

for genotypes G1-G6, G8-G10, and G11 [Das et al., 1994; Gouvea et al., 1990; Gouvea et al., 1994]. Also, three different sets of G-typing primers (H1, C and A pools) [Santos et al., 2003] were used to analyze specimens that were untyped by ELISA and to confirm those that were typed by ELISA. The reaction was carried out with one cycle of reverse transcription at 450C for 30 min, followed by 35 cycles of amplification (30 sec at 940C, 30 sec at 480C, 1 min at 720C), and a final extension of 7 min at 720C in a GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA). Electrophoresis of each PCR product in 1.2% SeaKem LE agarose gel (FMC Bioproducts, Rockland, ME) was performed and following ethidium bromide staining, the results were viewed under the GelDoc 2000 image-analysis system (BioRad, Hercules, CA). 22

2.5.

VP4 (P) Genotyping by RT-PCR PCR typing for the VP4 gene was performed using step-

amplification as in the VP7 genotyping. The first step amplified gene 4 with con3 and con2 primers, and the second amplification was performed with P type-specific primers (1T-1, 2T-1, 3T-1, and 4T-1) and con3. The result was confirmed with an alternative set of type-specific primer pairs (1C-1 and 1C-2; 2C-1 and 2C-2; 3C-1 and 3C-2; and 4C-1 and 4C-2) [Gentsch et al., 1992]. 2.6.

Nucleotide Sequencing and Phylogenetic Analysis The VP7 genes untyped by RT-PCR genotyping were examined

using sequence analysis. Each amplified product was inserted into a pCR 2.1 cloning vector, transformed to E. coli TOP 10F’ (Invitrogen, Carlsbad, CA), and sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems) and ABI PRISM 310 automated DNA sequencer. The resultant VP7 gene sequences were aligned using the CLUSTAL X program [Thompson et al., 1997] against corresponding sequences of representative rotavirus G types from the NCBI GenBank. An unrooted phylogenetic tree based on nucleotide sequences was constructed using neighbor-joining algorithms [Saitou and Nei, 1987] from the PHYLIP suite of programs [Felsenstein, 1993]. Evolutionary distance matrices were generated by the neighbor-joining method described by Jukes and 23

Cantor [Jukes and Cantor, 1969] and tree topology was evaluated using a bootstrap analysis [Felsenstein, 1985] of the neighbor-joining dataset with the SEQBOOT and CONSENSE programs from the PHYLIP package. The nucleotide sequences obtained in this study were deposited in the NCBI GenBank (EF059916 and EF059917).

24

3.

RESULTS

3.1.

Determination of G Serotypes The distribution of human rotavirus G types by ELISA and RT-

PCR is presented in Table 1.1. Of a total of 504 fecal specimens obtained from young children with acute diarrhea, 394 (78.2%) samples were positive for group A rotavirus and were subjected to serotyping using G1 G4 specific mAbs and the RT-PCR method. As shown in Table 1.1, all four major G types are represented G1 (35.6%, n = 140) was the dominant circulating serotype, followed by G3 (26.4%, n = 104), G4 (14.7%, n = 58), and G2 (11.9%, n = 47). Minor or unusual serotypes, G9 (1.0%, n = 4) and G8 (0.3%, n = 1) were detected as well as mixed serotypes such as G1/3 (1.0%, n = 4), G1/4 (0.5%. n = 2), G2/9 (0.5%, n = 2), and G3/4 (0.5%, n = 2). Twenty-eight samples (7.1%) that underwent full length VP7 gene amplification with G serotype-specific primers were negative but were positive with VP4 specific primers.

25

Table 1.1. Distribution of Human Group A Rotavirus G and P Types From Young Children between 2004 and 2006 in Seoul, South Korea G-serotype G1

G2

G3

G4

G8

G9

G12

G1/3

G1/4

G2/9

G3/4

NTa

Total (%)

P[4]

7

25

6

14

-

-

-

1

-

2

-

5

60 (15.2)

P[6]

16

10

1

24

-

-

2

-

-

-

1

7

61 (15.5)

P[8]

100

7

66

17

1

2

-

1

2

-

-

13

209 (53.0)

P[9]

2

3

2

-

-

1

-

-

-

-

-

1

9 (2.3)

P[4]/[8]

6

-

13

1

-

-

-

1

-

-

-

2

23 (5.8)

P[6]/[8]

-

-

-

1

-

1

-

-

-

-

1

-

3 (0.8)

NTa

9

2

16

1

-

-

-

1

-

-

-

-

29 (7.4)

Total

140

47

104

58

1

4

2

4

2

2

2

28

394

(%)

(35.6)

(11.9)

(26.4)

(14.7)

(0.3)

(1.0)

(0.5)

(1.0)

(0.5)

(0.5)

(0.5)

(7.1)

(100)

P-genotype

a

NT: Nontypeable, samples were not amplified in first RT-PCR using primers detecting the VP4 or VP7 gene.

26

3.2.

Determination and Phylogenetic Analysis of G12 Serotypes The complete VP7 gene sequences of 1,062 nucleotides from two

isolates, CAU 195 and CAU 214, could not be typed by ELISA and RTPCR methods. These sequences were analyzed and compared against the corresponding sequences of representative serotypes from the genus Rotavirus in the NCBI GenBank database. A phylogenetic tree based on nucleotide sequences was constructed using neighbor-joining algorithms and the tree topology, supported by high bootstrap values, shows the positions of CAU 195 and CAU 214 (Fig. 1.1). Figure 1.1 clearly indicates that CAU 195 and CAU 214 isolates form a tight group with the prototype G12 human rotavirus, L26 (M58290) as their closest phylogenetic relative with nucleotide sequence similarities of 90.7% and 90.3%, respectively (Table 1.2). In addition, this cluster can be separated distinctly from clusters of other G serotype rotaviruses. VP7 gene nucleotide sequence similarities of CAU 195 and CAU 214 with other G serotype clusters were much lower, ranging between 47.7% (G5 porcine rotavirus OSU, X06722) and 77.9% (G9 human rotavirus L169, DQ873674). It is evident, therefore, that isolates CAU 195 and CAU 214 belong to the G12 serotype cluster.

27

G6 IND-Bovine (U15000) G2 DS-1-Human (AB118023) G11 YM-Porcine (M23194)

G10 Mc35-Human (D14033)

G7 PO-13-Avian (D82979)

G8 B37-Human (J04334)

G4 cr117-Human (AF450294)

G1 Wa-Human (M21843) G9 L169-Human (DQ873674)

82 53 100

100

G12 CAU 195-Human (EF059916) G12 CAU 214-Human (EF059917) G12 L26-Human (M58290)

G5 OSU-Porcine (X06722)

G3 SA11-Simian (K02028) G13 L338-Equine (D13549) 0.1

Fig. 1.1. Unrooted phylogenetic tree based onVP7genenucleotide sequences shows relationships between two Korean isolates CAU 195, CAU 214 and representative serotypes of rotavirus. Numbers at nodes indicate the level of bootstrap support (%) based on the neighbor-joining analysis of 1,000 re-sampled datasets; only values above 50% are given.Bar represents 0.1 substitutions per nucleotide position.Porcine rotavirus OSU (X06722) was used as the out-group. 28

Table 1.2. Percent Similarity of the Nucleotide Sequence Encoding Gene VP7 in Korean Isolates CAU 195 and CAU 214 with the Corresponding Nucleotide Sequence of Other Rotavirus Serotypes

CAU 214 Wa DS-1 SA11 cr117 OSU IND PO-13 B37 L169 Mc35 YM L26 L338

CAU 195

CAU 214

99.6 75.8 74.1 77.8 73.9 47.7 73.0 64.9 73.4 77.9 74.6 74.9 90.7 75.7

75.5 73.8 77.6 73.5 49.0 72.7 64.6 73.2 77.6 74.4 74.5 90.3 75.7

Wa

DS-1

SA11

cr117

OSU

IND

PO-13

B37

L169

Mc35

YM

L26

74.9 76.3 77.1 49.8 74.0 65.6 72.5 77.1 74.5 74.4 75.1 74.1

73.9 71.3 48.8 73.4 62.7 73.9 75.1 73.7 76.0 73.8 73.4

75.3 47.1 76.4 65.9 74.7 79.0 76.8 77.7 77.1 77.6

49.9 74.6 64.6 71.4 77.0 74.4 75.9 74.2 73.4

49.5 44.7 50.3 50.0 47.7 46.9 49.6 48.7

64.1 74.5 74.9 74.3 74.5 73.5 73.9

64.2 66.2 64.3 63.7 66.3 63.8

75.7 75.8 76.1 73.5 73.4

77.3 78.7 76.8 76.2

74.8 72.9 74.0

74.2 74.4

75.2

29

3.3.

Determination of P Genotypes The distribution of VP4 genotypes is presented in Table

1.1 and all four major human rotavirus P genotypes are represented. Table 1.1 shows that the most common circulating genotype was P[8] (53.0%, n = 209) followed by P[6] (15.5%, n = 61), P[4] (15.2%, n = 60), and P[9] (2.3%, n = 9). The mixed genotypes of P[4]/[8] and P[6]/[8] occurred with a prevalence of 5.8% (n = 23) and 0.8% (n = 3), respectively. Twenty nine samples

(7.4%)

that

underwent

full

length

VP4

gene

amplification with P genotype-specific primers were negative but were positive with VP7-specific primers. 3.4.

Distribution of G- and P Types The distribution of G- and P type combinations is shown

in Table 1.1. The results indicate that G1P[8] strains are most prevalent (25.4%, n = 100) followed by strains of G3P[8] (16.8%, n = 66), G2P[4] (6.3%, n = 25), G4P[6] (6.1%, n = 24), and G4P[8] (4.3%, n = 17). Unusual or rare combinations such as G1P[6] (4.1%, n = 16), G4P[4] (3.6%, n = 14), G2P[6] (2.5%, n = 10), G1P[4] (1.8%, n = 7), G2P[8] (1.8%, n = 7), G3P[4] (1.5%, n = 6), G2P[9] (0.8%, n = 3), G1P[9] (0.5%, n = 2), G3P[9] (0.5%, n = 2), G12P[6] (0.5%, n = 2), G3P[6] (0.3%, n = 1), and G8P[8] (0.3%, n = 1) were also detected.

30

Table 1.3. Prevalence of G9 Serotypes in South Korea Authors

Kim et al. Song et al. Min et al. Kim et al. Kang et al. Kang et al. Moon et al.

Year Published

2002 2003 2004 2005 2005 2006 2007

Period of

Percentage

Sample Collection

No. of G9 / No. of Total Samples

2001–2002 1999–2000 2000–2001 2002–2003 2002–2003 2005–2006 1999–2002

0/89 0/205 1/322 79/203 49/461 1/81 0/115

0 0 0.3 38.9 11 1.2 0

31

Province

Seoul/Urban Seoul/Urban 6 large cities/Urban Jeongeub District/Rural Urban and Rural Cheju/Urban Seoul suburb/Urban

4.

DISCUSSION Four major G serotypes (G1 - 4) have been documented

worldwide [Beards et al., 1989; Santos and Hoshino, 2005] and until 1996, G1 appeared to be the most prevalent strain followed by strains G4, G2, and G3 [Gentsch et al., 1996]. A similar trend in the prevalence of G serotype strains is evident from studies conducted over the past 10 years in South Korea. Until 1997, G1 was also the most prevalent strain (45 - 81%) regardless of geographical area or season [Kim et al., 1999; Kim, 1993; Kim et al., 1990; Seo and Sim, 2000]. Since then, the predominant G type strain became G4 (28.0 - 40.9%) [Kang et al., 2005; Kim et al., 2002; Song et al., 2003], then G2 (40.9- 50.6%) [Min et al., 2004; Moon et al., 2007], and more recently, G9 (39%) [Kim et al., 2005]. Study data from this 2-year investigation indicates that the G1 strain (35.6%) is again, the most prevalent serotype replacing the G4 (40.5 - 40.9%) strain that was dominant in the previous Seoul survey [Kim et al., 2002; Song et al., 2003]. The prevalence of G1 is slightly higher than that currently recorded in other nation-wide studies, 30.1% [Min et al., 2004] and 18% [Kang et al., 2005], and in other rural surveys, 25% [Kim et al., 2005] and 27.8% [Moon et al., 2007]. Minor G serotypes documented as common in developing countries include G5, G8,

32

G9, G10, and G12 [Duan et al., 2007; Gulati et al., 2007; Pietruchinski et al., 2006; Steyer et al., 2007b; Wang et al., 2007] and in several countries around the world, the G9 serotype is most prevalent [Clark et al., 2004; Kostouros et al., 2003; Linhares et al., 2006; Steele and Ivanoff, 2003; Van Damme et al., 2007]. In this study, the G9 prevalence rate of 1.0% is much lower than that reported for some Asian countries: 54.8 - 91.6% in Thailand [Jiraphongsa et al., 2005; Khamrin et al., 2006], 78.3% in Malaysia [Hung et al., 2006], 24.1% in Taiwan [Chen et al., 2007], and 19.1% in South India [Banerjee et al., 2006]. This 1.0% prevalence rate is, however, similar to the rates found in Vietnam, 0.5% [Doan et al., 2003], in China, 0.9 - 4.0% [Fang et al., 2005; Fang et al., 2002], in Japan, 1.0- 5.9% [Yoshinaga et al., 2006; Zhou et al., 2003], in eastern India, 2.1% [Samajdar et al., 2006], and in Hong Kong China, 5.1% [Lo et al., 2005]. This difference in prevalence may be accounted for by factors such as study population demographics, periods of the study, seasonal initiation times and analytical methods. Concerning the latter, it has been observed that some G9 strains can be mistyped as G3 [Santos et al., 2003] owing to the type-specific primers used [Gouvea et al., 1990]. Therefore, all study samples identified as G3 and G4 strains in this investigation, were confirmed as these serotypes using an alternative set of G-typing primers [Das et al., 1994]. 33

Table 1.3 presents the prevalence of G9 strains in South Korea. Although G9 was not detected until 2002 [Min et al., 2004; Moon et al., 2007; Song et al., 2003], its prevalence, especially in rural provinces, has increased from 11% to 39% [Kang et al., 2005; Kim et al., 2005]. In urban areas, however, G9 occurs

with far lower prevalence, 1.2% [Kim et al., 2002;

Min et al., 2004; Moon et al., 2007; Song et al., 2003], a finding consistent with the observation in this study, that G9 strains were found in only four samples from Seoul (1.0%). One possible explanation for the higher prevalence in small rural areas, 39% in the Jeongeub District [Kim et al., 2005], may be the occurrence of outbreaks of infection. Alternatively, geographical area and season may play a role. To date, several reports have been made on the emergence of rare G type strains, G8 and G12. Since rotavirus G8 was first detected in Indonesian children [Matsuno et al., 1985], it has been found sporadically all over the world. Its appearance in the human population is thought to be a possible consequence of bovine-human rotavirus genome reassortment

[Adah

et

al.,

2003;

Browning

et

al.,

1992;

Matthijnssens et al., 2006; Steyer et al., 2007b]. Combinations of G8 with P[4] and P[6] are most frequently reported [Adah et al., 2001; Cunliffe et al., 2000; Fischer et al., 2003; Steele et al., 2002] and in this South Korean study, one strain, G8[P8] was identified for the first time. 34

The unusual human strain, G12 (L26 and L27) was first identified in 1987 from strains causing gastroenteritis in children from the Philippines [Taniguchi et al., 1990; Urasawa et al., 1990]. Since then, G12 has appeared in the United States and Thailand, and shortly thereafter, in India and Japan [Das et al., 2003; Griffin et al., 2002; Pongsuwanna et al., 2002; Shinozaki et al., 2004] such that today, G12 rotavirus has been detected in many countries across the world [Rahman et al., 2007a] In this study, the phylogenetic analysis of nucleotide sequences from two G12 isolates, CAU 195 and CAU 214 showed that they clustered tightly with the G12 serotype with similarities in excess of 90%. This article is the first to report on the comprehensive validation of G12 strains in South Korea, an analysis that included relevant type strains from the NCBI GenBank. Although an earlier report exists of a sample suspected of being G12 [Kang et al., 2005], the data from this report are inconclusive because the G12 serotype cannot be detected using the

multiplex

RT-PCR

employed

[Santos

et

al.,

2003].

Furthermore, there is no evidence of sequence analysis, or of an immunological

assay

that

uses

G12-specific

antibody.

A

description of four other G12 strains has been presented [Santos and Hoshino, 2005] but evidence related to P-specificity status is lacking and sequence information on the VP7 gene is not available on public databases. The findings discussed here, 35

provide important information concerning the spread of G8 and G12 type rotavirus and they may either represent the early emergence of extant Korean strains or be isolates newly introduced to South Korea from abroad. The prevalence of mixed infections (G type, 2.5%; P type, 6.6%) was similar to reports from the developed countries [Gentsch et al., 1996; Griffin et al., 2000; Iturriza-Gomara et al., 2000] but was lower than the rates seen in developing countries such as India, Bangladesh, and Brazil [Jain et al., 2001; Timenetsky Mdo et al., 1994; Unicomb et al., 1999]. A global rotavirus surveillance study indicated that the G/P combinations most frequently reported in humans were G1P[8], G3P[8], G4P[8], G2P[4], G9P[8], and G9P[6] [Santos and Hoshino, 2005]. Recent Korean studies uncovered a temporal change in the most common G type combinations of rotavirus that is dependent on season and geography. The dominant combination shifts from G1P[8] (25.4%) to G4P[8] (7.8%; 1999 - 2000) [Song et al., 2003] to G2P[4] (52.4; 1999 - 2002) [Moon et al., 2007], (45.7%; 2000 - 2001) [Min et al., 2004], (28.1%; 2001- 2002) [Kim et al., 2002] to G4P[6] (27%; 2002 -2003) [Kang et al., 2005], and then, G9P[8] (39%; 2002 -2004) [Kim et al., 2005]. In this Seoul study, 210 (53.3%) samples comprised these common G/P combinations and the G1P[8] combination (25.4%) was dominant. Although G4P[6] is considered to be rare globally, 36

P[6] strains combined with G1, G2, G3, or G4 have been reported as a cause of outbreaks of nosocomial rotavirus infection in hospitals and newborn nurseries [Kilgore et al., 1996; Lee et al., 2001; Linhares et al., 2002; Steele et al., 1995]. The implication is that the prevalence of formerly common G/P combinations is falling in South Korea while that of uncommon G/P combinations is on the increase. Of note, is the first appearance in South Korea of rare combinations that include G8P[8] (0.3%, n = 1), G12P[6] (0.5%, n = 2), and G2P[9] (0.8%, n = 3). In this study, co-infections with two different G types and a single P type (5.6%, n = 22), or with two different P types and a single G type (1.8%, n = 7) were detected but their occurrence was limited. In addition, the finding that 28 samples (7.1%) that were negative upon full length VP7 gene amplification but positive

with

VP4

gene-specific

primers

was

unexpected.

Similarly, the 29 samples (7.4%) that were negative upon fulllength VP4 gene amplification but positive with VP7 genespecific primers were unusual. Further investigation of these findings is needed to confirm whether the rotavirus is undergoing genetic diversification. The data from this study demonstrate that in Seoul, South Korea, there is a high level of diversity among G- and P type rotavirus, and that the prevalence of wellestablished or newly, emerging serotypes such as G8P[8] and 37

G12P[6] changes relatively quickly. More surveillance studies are

vital

for

epidemiology

amassing of

unusual

information serotypes,

on and

the the

molecular further

characterization of G8 and G12 rotavirus is essential. Only by collating large volumes of stringent data, can an efficient, evidence based program for vaccine development be progressed

38

III Molecular Characterization of Prevalent G1P[8] Human Rotavirus Strains Isolated from Children Hospitalized with Acute Gastroenteritis 1.

INTRODUCTION Rotaviruses are the most common etiological agent of

severe diarrhoea in infants and young children [Kapikian et al., 2001]. Recent estimates indicate that worldwide 611,000 deaths occur

annually

due

to

severe

rotavirus

diarrhea

and

approximately 85% of these deaths occur in the developing countries [Bozdayi et al., 2008; Parashar et al., 2006]. The virus is a member of family Reoviridae and its genome is composed of 11 segments of double stranded RNA that encode for six structural and six nonstructural proteins [Estes, 2001; Kapikian et al., 2001]. Based on the diversity of the VP7 and VP4 antigens present on the outer capsid, 16 G serotypes [Gulati et al., 2007] and at least 27 P genotypes have been reported to date [Bozdayi et al., 2008; Khamrin et al., 2007a; Martella et al., 2007]. As with other genome-segmented-viruses, rotaviruses can reassort their genes independently [Gouvea and Brantly, 1995], many combinations of G and P genotypes are possible. To date, 42 different combinations of G and P genotypes have been detected, but five of them (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) 39

are the most prevalent in humans globally and comprise more than 90% of the human cases detected worldwide. Among them, the G1P[8] strain is by far the most commonly found worldwide [Espinola et al., 2008; Gentsch et al., 2005; Parra et al., 2007a; Santos and Hoshino, 2005]. In Korea, rotavirus is still the most common viral agent of acute diarrhoea in young children aged between 6 and 24 months [Kim et al., 1990; Seo and Sim, 2000]. There were significantly different results in G typing in recent years. Until 1997, G1 was also the most prevalent strain regardless of geographical area or season [Kim et al., 1999; Kim, 1993; Kim et al., 1990; Seo and Sim, 2000]. Since then, the predominant G type strain shifted G4 [Kang et al., 2005; Kim et al., 2002; Song et al., 2003], G2 [Min et al., 2004; Moon et al., 2007], and G9 strain [Kim et al., 2005]. More recently, the study data from a 2year investigation indicated that the G1 strains were again [Le et al., 2008]. Although rotavirus surveillances are conducted annually in Korea, few sequence data of common circulating rotavirus strains such as G1, G2, G3, G4, and G9 are available in GenBank databases. The genetic characterization with molecular analysis of VP7, VP4, VP6, and NSP4 genes of rotavirus strains is

important

and

a

key

step

to

acquiring

an

in-depth

understanding of the ecology and evolution of rotaviruses [Li et al., 2008]. In this study, six G1P[8] human rotavirus strains, 40

which had been previously genotyped as G1P[8] by RT-PCR, were selected randomly from the prevalent G1P[8] strains in the previous study [Le et al., 2008] for isolation and genetic characterization. The full length VP7, VP4, VP6, and NSP4 genes were sequenced and compared with those of human rotaviruses available on public databases in order to know whether any change in the VP7 genes occurred.

41

2.

MATERIALS AND METHODS

2.1.

Stool Specimens The fecal specimens were obtained from young children

(<5 years of age) hospitalized with acute diarrhea in five different general hospitals located in Seoul during the period between January 2004 and February 2006. All specimens were 10-fold diluted with PBS (pH 7.4) and clarified by centrifugation 10,000rpm for 10 min. The supernatants were tested and genotyped by RT-PCR method

[Das et al., 1994; Gentsch et al.,

1992; Gouvea et al., 1990; Santos et al., 2003] and nucleotide sequence analysis. 2.2.

Isolation of Rotavirus Six G1 rotavirus strains, which were selected randomly

from the prevalent G1P[8]-positive stool specimens in the previous study [Le et al., 2008], were isolated and propagated in MA104 cells. Briefly, the specimen was diluted 10-fold with phosphate buffered saline (PBS; pH 7.4) and clarified by centrifugation 10,000 g for 10 min. The supernatant filtered using a 0.45 μm sterile syringe filter (Corning, NY, USA) was pretreated with 10 μg ml -1 Trypsin (Difco TM Trypsin 250, Becton, Dickinson & Company Sparks, MD, USA) for 30 min at 37 o C and inoculated onto MA104 cells in glass tubes with Minimum Essential Alpha Medium (Gibco, Invitrogen, Carlsbad, 42

CA, USA) in the presence of Trypsin (5 μg ml -1 ) followed by incubation in rolling culture using a rotator. The culture cells were harvested 5–7 days after infection and subsequently passed onto MA104 cells until CPE was appeared. The culture fluid of the

final

passage

was

examined

for

rotavirus

by

RNA

polyacrylamide gel electrophoresis and RT-PCR using specific primer [Gentsch et al., 1992; Gouvea et al., 1990]. 2.3

Polyacrylamide gel electrophoresis (PAGE) The presence of rotavirus strains in MA104 cells was

determined by detection of dsRNA segments of rotavirus in polyacrylamide gel electrophoresis (PAGE). Viral RNAs were extracted from viral infected cell culture using Trizol reagent (Invitrogen, Carlsbad, CA, USA). In brief, 0.3 ml of suspension of viral infected cell culture was mixed with 0.7 ml of Trizol reagent and 0.2 ml of chloroform/isoamylalcohol (24:1). After centrifugation at 10,000 rpm for 10 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol.

The

RNA

precipitate

was

collected

by

centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol and finally dissolved in 20 μl of RNase-free water. The electrophoretic migration pattern of dsRNA of each rotavirus was determined by 10% acrylamide slab gel with a 3.5% acrylamide stacking gel for 16 h at 20 mA [Jones et al., 2003]. 43

The dsRNA segments were visualized by staining gel with silver nitrate [Herring et al., 1982]. 2.4.

Reverse Transcription-Polymerase Chain Reaction (RT-

PCR) Rotavirus dsRNA was extracted from infected MA104 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as described in previous study [Le et al., 2008]. The VP7 fulllength gene (1,062 bp) from dsRNA genome was amplified using the Beg9 and End9 primers as described previously [Gouvea et al., 1990]. The specific primers for full genes of VP4 (2,359 bp), VP6 (1,356 bp), and NSP4 (750 bp) amplification were designed by aligning sequences of representative rotavirus strains obtained from GenBank databases by using Primer 3 software (Rozen & Skaletsky,

2000)

with

default

settings.

Primer

sets

were

presented in table 2.1. RT-PCR for amplification of VP4, VP6, and NSP4 gene was carried out, respectively, with initial reverse transcription step at 42 o C for 30 min, followed by 30 cycles of amplification of 94 o C for 1 min, 52 o C for 1 min, 72 o C for 1 min (for NSP4), 2 min (for VP6), and 3min (for VP4), and a final extension

of

electrophoresed

10 in

min

at

1.2%

72 o C. SeaKem

The LE

PCR

products

agarose

gel

were (FMC

Bioproducts, Rockland, ME) and viewed under the GelDoc 2000 image-analysis system (BioRad, Hercules, CA, USA) following

44

ethidium bromide staining. 2.5.

Nucleotide Sequencing and Phylogenetic Analysis The VP7, VP4, VP6, and NSP4 genes of G1P[8] isoaltes

were examined using comprehensive sequence analysis. Each amplified PCR product was inserted into a pCR 2.1. cloning vector, transformed to E. coli TOP 10F’ (Invitrogen), and sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and ABI PRISM 310 automated DNA sequencer. M13 reverse and T7 promoter primers were used for sequence analysis. In addition, three VP4 internal primers (G1VP4F1, G1VP4F2, and G1VP4F3), and one VP6 internal primer (VP6-F1) were designed and used for sequenceing. The resultant gene sequences were aligned using the CLUSTAL X 1.81 (Thompson et al., 1997) and MegAlign (DNASTAR,

Madison,

WI)

against

corresponding

gene

nucleotide sequences of representative rotaviruses from GenBank database. The rooted trees were constructed using the neighbourjoining algorithms (Saitou & Nei, 1987) from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distance matrices were generated by the neighbour-joining method described by Jukes and Cantor (1969) and tree topology evaluated using a bootstrap analysis (Felsenstein, 1985) of the neighbour-joining dataset with the SEQBOOT and CONSENSE programs from the

45

PHYLIP package. 2.6.

Nucleotide Sequence Accession Numbers The nucleotide sequence data of six G1P[8] isolates

reported in this study was submitted to GenBank with accession numbers EU679389- EU679394 (VP7); EU679395- EU679400 (VP4); EU679383- EU679388 (VP6), and EU679377- EU679382 (NSP4). Referenced sequences deposited in GenBank databases are

following:

For

VP7:

Kor-64

(U26378),

KUMS02-11

(DQ478436),

KUMS01-21

(DQ478430),

KUMS01-27

(DQ478433),

KUMS01-13

(DQ478427),

KUMS00-93

(DQ478425),

KUMS00-91

(DQ478424),

KUMS00-76

(DQ478421), KUMS00-36 (DQ478418), KUMS02-1 (DQ478415), KUMS01-2 (DQ478413), KUMS00-54 (DQ478409), Mvd9814 (AF480291), Py9856

Mvd9813

(DQ015681),

(AF480290), Py9855

Mvd9815

(AF480292),

(DQ015680),

Mvd9614

(AF480296), Mvd9616 (AF480266), Py99365 (DQ015682), Cos69 (U26369), Cos-70 (U26370), Wa (M21843), Isr-56 (U26376), Brz-6 (U26368), PA10/90 (DQ377587), PA5/90 (DQ377573), PA78/89 (DQ377572), Va-12 (U26394), AU19 (AB018697), C95 (L24165),

C60

(L24164),

88H249

(AB081795),

89H452

(AB081796), 87Y1397 (AB081793), Thai-2104 (DQ512982), VN-355 Cau202

(DQ512968), (EF059923),

Dhaka16-03 KMR787

46

(DQ492674); (EF077355),

For

VP4:

KMR023

(EF077350), KMR058

KMR670 (EF077342),

(EF077338), KMR541

KMR012 (EF077333),

(EF077330), KMR751

KMR773 (EF077326),

(EF077320),

KMR267

(AB222784),

ITO

(EF077346),

KMR061

KMR733

(EF077339),

(EF077336),

KMR580

KMR538

KMR101 (EF077335),

(EF077331),

(EF077328),

KMR766

KMR748

KMR004 (EF077327),

(EF077325),

(EF077319),

(AB008280),

(EF077343),

Wa

Odelia

KMR419

(M96825),

KU

(AB008296),

L8

(AF061358), L5 (AF052450), H8 (U41006), F45 (U30716), Py9856 (EU045216), OP351 (AJ302147), Py99371 (EU045220), Py00469 (EU045226), TF101 (AF183870), OP530 (AJ302152), OP354 (AJ302148), MW670 (AJ302146), CAU195 (EF059920); For VP6: A253 AF317122), H1 (AF242394), KJ44 (DQ494413), JL94

(AY538664),

NCDV

(AF317127),

OSU

(AF317123),

RMC321 (AF531913), S2 (Y00437), SA11 (AY187029), UK (X53667), WC3 (AF411322), YM (X69487), FI-14 (D00323), L338 (D82974), 116E (U85998), TK159 (AY661888), E210 (U36240), Gottfried (D00326), Wa (K02086), H-2 (D00324), FI23 (D82971); For NSP4: DS-1 (AF174305), L26 (AJ311732), KUN

(D88829),

UK

(K03384),

NCDV

(X06806),

SA-11

(K01138), B223 (AF144805), ST3 (U59110), 116E (U78558), OSU

(D88831),

(AF165219),

AU1

WA

(K02032),

(D89873),

GRV

H-1

(AF144800),

(AB055968),

A34

CMH222

(DQ288660), CU-1 (AF144806), EW (U96335), EHP (U96336), 47

EC (U96337), Ty-1 (AB065285), Ty-3 (AB065286), Ch-1 (AB065287)

48

3.

RESULTS

3.1.

Isolation and RNA electropherotype of six G1P[8]

strains The dsRNA electrophoretic migration pattern of six human rotavirus G1P[8] strains was analyzed by PAGE and compared to that of other referenced rotavirus strains namely, Wa (“long” dsRNA pattern) and DS-1 (“short” dsRNA pattern). All six human rotavirus G1P[8] strains displayed a ‘‘long’’ RNA pattern like Wa HRV strain, but quite distinct from that of DS-1 (Fig. 2.1). The isolation of G1 strains was demonstrated by electron microscopy using the method of negative staining (Fig 2.2). The result shown that referenced human rotavirus Wa train and G1 isolate strains were similar in size (100 nm) and morphology. 3.2.

Phylogenetic analysis of VP7, VP4, VP6, and NSP4 gene

of six G1P[8] strains The phylogenetic tree for the nucleotide sequences of the VP7 gene of six G1P[8] HRV strains isolated in this study has been made in order to compare with those of other representative G1 rotavirus strains collected from GenBank databases . As shown in Fig. 2.3, the human rotavirus G1 strains segregated into six lineages (I to VI) and all six G1P[8] strains clustered into VP7 genetic lineage I. In addition to six G1 strains isolated in 49

this study (CAU136, CAU163, CAU160, CAU200, CAU164, and CAU219), twelve VP7 gene sequences of other Korean G1 strains deposited in the GenBank databases were included in this study to assess genetic diversity. The analysis revealed that they also clustered into VP7 genetic lineage I except one strain (Kor64) belonged to VP7 lineage IV. This Korean G1 strain (Kor-64) was obtained from an infant with diarrhea in 1988 while others obtained in 1999-2002. Within the VP7 lineage I, three distinct sublineages (A-C) were defined. The result showed that most of them clustered into VP7 sublineage IA, and IB except one Korean G1 strain (KUMS02-1) clustered into sublineage IB, the same as reported G1 strains obtained from Thailand (Thai-2104) and from Vietnam (VN-355). The phylogenetic analysis of P[8] genotype of VP4 gene clustered all six G1P[8] strains into genetic lineage P[8]-3 from a total of four different lineages described to date for this genotype (Fig. 2.4). Beside six G1P[8] isolates analyzed in this study, nineteen gene sequences of VP4 with P[8] genotype of other Korean strains deposited in the GenBank databases were also analysed and shown that they belonged to two distinct lineages (P[8]-2 and P[8]-3). Also, phylogenetic analysis of VP6 and NSP4 gene of all six G1P[8] isolates has been made and shown that they all belonged to VP6 subgroup II specificity and NSP4 genotype B, respectively (Fig. 2.5 and 2.6). 50

Table 2.1. Oligonucleotide primers used for amplification and sequencing of the VP7, VP4, VP6, and NSP4 gene of G1P[8] human rotavirus strain.

Gene

Primer

Sequence 5’to 3’

Sense

Position

Reference

VP7

Beg9

GGCTTTAAAAGAGAGAATTTCCGTCTGG

+

1–28

[Gouvea et al., 1990]

End9

GGTCACATCATACAATTCTAATCTAAG

-

1062–1036

[Gouvea et al., 1990]

Con3

TGGCTTCGCCATTTTATAGACA

+

11-32

[Gentsch et al., 1992]

G1VP4F1

CGGTCTACCACCAATTCAAAATAC

+

672-695

This study

G1VP4F2

GTTACATTATCTACGCAATTCAC

+

1231-1253

This study

G1VP4F3

CAACACAAACATCTACGATCAGT

+

1832-1854

This study

G1VP4R1

GGTCACATCCTCAATAGCYTTCTCAC

-

2359-2334

This study

VP6-F

GGCTTTWAAACGAAGTCT

+

1-18

This study

VP6-F1

ATAGATCTCAACCAATGCATG

+

523-543

This study

VP6-R

GGTCACATCCTCTCACTA

-

1339-1356

This study

NSP4-F

GGCTTTTAAAAGTTCTGTTC

+

1-20

This study

NSP4-R

GGTCACACTAAGACCATTCC

-

750-731

This study

VP4

VP6

NSP4

51

Fig 2.1. Electrophoretic migration patterns of genomic RNA of celladapted G1 rotavirus strains. Lane 1: DS1, Lane 2: Wa, Lane 3: CAU136, Lane 4: CAU160, Lane 5: CAU163, Lane 6: CAU164, Lane 7: CAU200; Lane 8: CAU219

52

Fig 2.2. Electron micrograph of referenced human rotavirus Wa strain (A) and

representative

G1P[8]

human

Magnification bar equals 100 nm

53

rotavirus

isolate

strain

(B).

88

• CAU136 • CAU163

99

• CAU160 68

• CAU200 Kor/KUMS01-2 Kor/KUMS01-27

99 68

A

Kor/KUMS01-13

Py9855 61

Mvd9814 Mvd9815 Py9856 Mvd9813 Kor/KUMS02-1

60

VN-355 63

B

Thai-2104

100

99

Lineage I

Dhaka16-03 • CAU164

67

Kor/KUMS02-11

71

Kor/KUMS00-36 • CAU219 99

C

Kor/KUMS00-91 Kor/KUMS00-76

74

Kor/KUMS01-21 KorKUMS00-93 66

Kor/KUMS00-54

Mvd9614

97 90

Mvd9616 Va-12

98

94

Lineage II

Py99365 Cos-69

74 99

Cos-70 Wa

96 100

Brz-6

Lineage III

Isr-56 67 100

PA10/90

Lineage V

PA78/89 PA5/90 87Y1397

64

Lineage IV

88H249

100

Kor-64

96 67

89H452 PorcineC95 100

PorcineC60 AU19

Lineage VII Lineage VI

0.02

Fig 2.3. Phylogenetic tree for the nucleotide sequences of the VP7 gene of G1P[8] HRV strains in comparison with those of other representative G1 rotavirus strains. Bootstrap values (expressed as percentages of 1000 replications) greater than 60% are shown at branch points. The tree was generated based on the neighbor-joining method using the MEGA3.1 program. The scale bar represents 2% of nucleotide changes between close relatives. 54

Kor/KMR023 Kor/KMR267 Kor/KMR748 83

Kor/KMR419 Kor/KMR670

97

Kor/KMR004 Kor/KMR061 • CAU219 62 Kor/KMR101

TF101 OP351

76

Kor/Cau202

63

Py00469 • CAU164 97 • CAU163

61

• CAU136 97

• CAU160

81

• CAU200

P[8]- 3

Py99371 Kor/KMR058

100

Kor/KMR733

72 99

Kor/KMR580 Kor/KMR751

Kor/KMR787 96

Kor/KMR773

100

Kor/KMR766 Kor/KMR012 KU Kor/KMR541

99 91

83

Kor/KMR538 F45

P[8]- 2

Py9856

95

L5

78

H8

80

ITO L8

100

P[8]- 1

Wa Odelia

92

MW670 99 64

OP530

P[8]- 4

OP354 G12P[6]/CAU195

0.05

Fig 2.4. Phylogenetic tree for the nucleotide sequences of the VP4 gene of G1P[8] HRV strains in comparison with those of other representative P[8] rotavirus strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points. 55

Hu/CAU160 99 Hu/CAU163 Hu/CAU200 100

Hu/CAU136 Hu/CAU164 87

II

Hu/TK159 Hu/Wa Hu/CAU219

78 100

99

Av/E210 Hu/116E

60

Po/Gottfried Eq/FI-14 99

I+II

Eq/L338 Bo/KJ44

100 93

Po/JL94 Hu/RMC321 I

Bo/YM

100

Po/A253

100

Eq/H1 86

Po/OSU

100

Eq/H-2

89

Non-I+II

Eq/FI-23 Hu/S2

99

Si/SA11 Bo/UK

I

Bo/NCDV

99 65

Po/WC3

0.02

Fig 2.5. Phylogenetic tree for the nucleotide sequences of the VP6 gene of G1P[8] HRV strains in comparison with those of other representative rotavirus strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points.

56

Hu/CAU136 100 Hu/CAU163 Hu/CAU160 100

84

Hu/CAU200 Hu/CAU164

100 Hu/CAU219 B

Hu/116E 9171

Hu/ST3

92 Hu/WA Po/A34 Po/OSU 96 96 Eq/H-1 Ca/CU-1 Hu/CMH222

74

C

Hu/AU1 76

Ca/GRV Bo/NCDV

68

Bo/B223 Si/SA-11 60

Hu/L26

A

Bo/UK

69

Hu/DS-1 95

Hu/KUN Mu/EC Mu/EW

100 95

D

Mu/EHP

Av/Ch-1 Av/Ty-1

100 87

E Av/Ty-3

0.2

Fig 2.6. Phylogenetic tree for the nucleotide sequences of the NSP4 gene of G1P[8] HRV strains in comparison with those of other representative rotavirus strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points.

57

4.

DISCUSSION The study carried out during 19 consecutive years in Italy

showed that the emergence or introduction of new variants (new lineages or sub-lineages) of G1 strains could explain the continuous circulation of G1 rotavirus in a given geographic area [Arista et al., 2006]. A higher variability was demonstrated in the VP7 sequences of the G1 rotaviruses when compared to G2 and G4 strains. Thus, intraserotypic heterogenicity is a possible mechanism by which G1 strains have become the most prevalent strain of rotavirus worldwide [Arista et al., 2006; Parra et al., 2007a]. By sequencing the VP7 gene and deducing the amino acid sequence from rotaviruses with G1 genotype, both [Xin et al., 1993] and [Diwakarla and Palombo, 1999] found temporal variation in the VP7 sequence and in the antigenicity of the strains. VP7 gene analysis of G1 strains has been reported and revealed considerable genetic diversity and at least four major global lineages of serotype G1 VP7 occurred within rotaviruses collected from diverse geographical locations [Araujo et al., 2007; Arista et al., 2006; Berois et al., 2003; Jin et al., 1996; Maunula and von Bonsdorff, 1998; Parra et al., 2005; Parra et al., 2007a; Trinh et al., 2007; Xin et al., 1993]. In this study, the phylogenetic analysis revealed differences in the VP7 genetic lineages among Korean G1 strains. While all Korean G1 strains circulating in recent years [Le et al., 2008; Moon et al., 2007] 58

clustered into VP7 genetic lineage I, the old Korean G1 strain (Kor-64) belonged to VP7 genetic lineage IV. This strain is the unique G1 strain found in GenBank databases and obtained from an infant with diarrhea in 1988. The different VP7 genetic lineages were also observed between Korean G1 strains and referenced Wa strain (G1P[8]) that Wa strain was originally obtained in the 1970s in the USA and defined as VP7 lineage III [Jin et al., 1996]. This result is similar to other studies that also fail to detect Wa-like strains circulating in the field in recent years. All observations showed a substantial diversity between the G1 rotaviruses collected throughout the world in recent years and the G1 rotaviruses detected in the 1970s in the USA and Asia (Wa-like) that apparently are no longer circulating in the field, suggesting that this lineage is extinct [Arista et al., 2006; Berois et al., 2003; Diwakarla and Palombo, 1999; Jin et al., 1996; Maunula and von Bonsdorff, 1998; O'Halloran et al., 2002; Rodriguez-Castillo et al., 2006; Xin et al., 1993]. Three distinct sublineages (A-C) of lineage I have been described in the VP7 gene of G1 strains [Arista et al., 2006]. In this study, Korean G1 strains also clustered into three sublineages IA, IB, and IC. Seventy four G1 rotavirus strains isolated in Japan, China, Vietnam, and Thailand have been characterized and reported that they all clustered into VP7 genetic lineage III [Trinh et al., 2007]. Because all studied G1 strains from the four countries clustered 59

into VP7 lineage III, only two representative strains (VN-355 and Thai-2104) out of 74 G1 rotavirus strains were selected and analyzed in this study. The result revealed that all Korean G1 strains and two representative strains clustered into VP7 lineage I (Fig. 2.3). Interestingly, there is somewhat difference found between nomenclature of VP7 lineage of referenced Wa strain (G1P[8]) in the phylogenetic tree made by Trinh, in which Wa strain belonged to VP7 lineage IV [Trinh et al., 2007], and phylogenetic trees made in this study and other studies that Wa strain was widely defined as the representative of VP7 lineage III [Arista et al., 2006; Berois et al., 2003; Jin et al., 1996; Parra et al., 2005; Parra et al., 2007a]. This finding suggests that VP7 lineage III of all 74 G1 strains isolated in Japan, China, Vietnam, and Thailand reported by Trinh [Trinh et al., 2007] and VP7 lineage I of Korean G1 strains in this report are actually the same. If our hypothesis is true, all studied G1 strains isolated in Japan, China, Vietnam, Thailand, and Korea should be clustered into VP7 lineage I. Previous data of sequencing and phylogenetic analysis of the VP4 gene has shown distinct diversity within P[8] genotypes and four distinct lineages have been described [Arista et al., 2005; Arista et al., 2006; Cunliffe et al., 2001; Espinola et al., 2008; Gouvea et al., 1999]. Genotype P[8] of VP4 was found to be undergone genetic and antigenic drift [Jin et al., 1996]. 60

Phylogenetic analysis of all Korean P[8] genotypes found in GenBank databases to date revealed that most of them clustered into P[8]-3 except two strains (KMR541 and KMR538) clustered into P[8]-2 (Fig. 2.4). This P[8]-3 observation is in accordance with numerous studies reported to date that P[8] genotypes mainly clustered into P[8]-3 [Ansaldi et al., 2007; Araujo et al., 2007; Arista et al., 2006; Espinola et al., 2008]. Co-circulation of different P[8] lineages was observed in some reports [Ansaldi et al., 2007; Arista et al., 2006; Cunliffe et al., 2001; Espinola et al., 2008]. This study showed the co-circulation of lineages P[8]2 and P[8]-3, similar to observation in Paraguay [Espinola et al., 2008]. Korean P[8] genotypes are different when compared to Wa strain, which is representative of lineage P[8]-1. Because there is no previous available data of Korean P[8] genotypes found in GenBank databases, it is difficult to assess the temporal genetic diversity within P[8] genotypes. All Korean P[8] genotypes used in this study were obtained recently [Le et al., 2008; Min et al., 2004]. VP6 protein is encoded by gene six and is the most abundant protein of the virus, making up more than half the viral mass. In addition to its role in classification, the VP6 antigen is also a primary target for group A rotavirus diagnosis using a variety of techniques including ELISA, immunofluorescence, and electron microscopy [Estes, 2001]. VP6 genes of both SGI and 61

SGII strains have been shown substantial divergence. VP6 genes of most current US rotaviruses have evolved substantially compared to isolates from decades earlier [Iturriza Gomara et al., 2002; Kerin et al., 2007]. Along with VP6 protein, the nonstructural protein NSP4, encoded by gene segment 10, is a glycoprotein anchoring in the membrane of the endoplasmic reticulum

[Estes,

2001].

This

protein

has

been

studied

extensively due to its role in viral morphogenesis and its potential enterotoxigenic property [Ball et al., 1996; Kirkwood and Palombo, 1997; Tavares Tde et al., 2008; Tian et al., 1995]. The human rotaviruses NSP4 genotype A has been associated usually with VP6 subgroup I specificity, whereas NSP4 genotype B with VP6 subgroup II specificity [Cunliffe et al., 1997; Kirkwood and Palombo, 1997]. Usually G1P[8] strains are associated with a VP6 subgroup II [Iturriza Gomara et al., 2002]. In this study, all Korean G1P[8] isolates belong to VP6 subgroup II specificity and NSP4 genotype B (Fig. 2.5 and 2.6), the same as referenced Wa strain (G1P[8]). Since several

vaccine

formulations are being developed to obtain specific protection against rotavirus disease, it is very important to investigate the circulating strains to anticipate possible antigenic changes that might affect the effectiveness of the vaccine. Studies of intragenotype diversity of rotavirus infections are the key model to understand how rotaviruses evolve and to measure how 62

genetic and antigenic differences in genotypes. Taken together, the phylogenetic analysis of VP7, VP7, VP6, and NSP4 gene of Korean G1 strains suggests that the new variant G1P[8] strains may have been emerged and circulated in south Korea.

63

IV Molecular Characterization of A Human Rotavirus Isolate of G9P[8] strain 1.

INTRODUCTION Rotaviruses are classified into seven groups (A to G) on

the basis of their distinct antigenic and genetic properties. Human infection has been reported with group A, B, and C rotavirus [Yoshinaga et al., 2006]. Of these, group A rotaviruses are the most important, being a major cause of severe gastroenteritis in infants and young children worldwide [Barril et al., 2006; Glass et al., 1996; Khamrin et al., 2006]. The infections with rotavirus are responsible for about 454,000705,000 deaths worldwide annually [Parashar et al., 2006]. There is so far no effective universal vaccine for prevention of rotavirus disease [Mulholland, 2004; Parashar et al., 2003; Yoshinaga et al., 2006]. Previous studies have shown that patterns of G type distribution appear to have regional and local particularities, and ‘unusual’ G serotypes are now detected throughout the world, especially in developing countries [Barril et al., 2006]. Recently, the G9 reported as a causative agent of diarrhea in children is recognized as one of the most widespread emerging genotypes in

64

the world including USA, Canada, Australia, United Kingdom, Germany, Brazil, Africa, China, India, Vietnam, Japan, Thailand, Argentina, Malaysia, Northern Ireland, Taiwan, south Korea, and so on [Barril et al., 2006; Carmona, 2006; Carvalho-Costa et al., 2006; Feeney et al., 2006; Hung et al., 2006; Kang et al., 2005; Khamrin et al., 2006; Kim et al., 2005; Kirkwood et al., 2003; Kirkwood et al., 1999; Laird et al., 2003; Lin et al., 2006; Marques et al., 2007; Stupka et al., 2007; Yoshinaga et al., 2006; Zhou et al., 2003]. In Korea, rotavirus is still the most common viral agent of acute diarrhoea in young children aged between 6 and 24 months [Kim et al., 1990; Seo and Sim, 2000]. There were significantly different results in G typing in recent years. The G4, G2, and G9 were determined as the most frequent G type during the period from 1998 to 2004, respectively, [Kang et al., 2005; Kim et al., 2005; Min et al., 2004; Moon et al., 2007; Song et al., 2003] while the previous epidemiological studies reported that G1 to G4 were common, particularly G1 as the predominant strain during the period from 1987 to 1999 [Min et al., 2004; Seo and Sim, 2000; Song et al., 2003]. Although human rotavirus G9 strains have been reported in some where in South Korea recently, there is so far no information of isolation of G9 strain. So, having isolates of G9 strain, a previously uncommon strain worldwide, is very important for further study on genomic 65

characterization and other characteristics as well. In this study, a G9 human rotavirus strain, CAU202 was isolated from the previous genotyped diarrheic stools of an infant in Korea. The full length VP7, VP4, VP6, and NSP4 genes of CAU 202 were then sequenced and compared with those of human rotaviruses available on public databases.

66

2.

MATERIALS AND METHODS

2.1.

Stool Specimens The fecal specimens were obtained from young children

(<5 years of age) hospitalized with acute diarrhea in five different general hospitals located in Seoul during the period between January 2004 and February 2006. All specimens were 10-fold diluted with PBS (pH 7.4) and clarified by centrifugation 10,000 rpm for 10 min. The supernatants were tested and genotyped by RT-PCR method

[Das et al., 1994; Gouvea et al.,

1990; Santos et al., 2003] and nucleotide sequence analysis. 2.2.

Isolation of Rotavirus Rotavirus strain was isolated and propagated from a G9

stool specimen in MA104 cells. Briefly, the specimen was diluted 10-fold with phosphate buffered saline (PBS; pH 7.4) and clarified by centrifugation 10,000 g for 10 min. The supernatant filtered using a 0.45 μm sterile syringe filter (Corning, NY, USA) was pretreated with 10 μg ml -1 Trypsin (Difco TM Trypsin 250, Becton, Dickinson & Company Sparks, MD, USA) for 30 min at 37 o C and inoculated onto MA104 cells in glass tubes with Minimum Essential Alpha Medium (Gibco, Invitrogen, Carlsbad, CA, USA) in the presence of Trypsin (5 μg ml -1 ) followed by incubation in rolling culture using a rotator. The culture cells were harvested 5–7 days after infection and subsequently passed 67

onto MA104 cells until CPE was observed. The culture fluid of the

final

passage

was

examined

for

rotavirus

by

RNA

polyacrylamide gel electrophoresis, immunofluorescence assay and RT-PCR using specific primer [Gouvea et al., 1990]. 2.3.

Immunofluorescence Assay (IFA) for Detection of G9

strain (CAU202) Indirect immunofluorescence assay was used to detect rotavirus antigen in MA104 cells. CPE appeared rotaviruses infected MA104 cells in a 96-well plate (NUNC TM ) were fixed with 80% acetone at room temperature for 10 min. The fixed cells were washed three times in PBS (pH 7.2) and added 50 µl of monoclonal antibody specific for VP6 of Wa strain human rotavirus (1:50 in PBS). The plate was incubated at 37 o C for 1 hr followed by three washes in PBS and 50 µl of fluorescein isothiocyanate

(FITC)-conjugated

goat

anti-mouse

immuno-

globulin (Invitrogen, Carlsbad, CA, USA) diluted 1:100 in PBS was added to each well. The plate was incubated at 37°C for 1hr, washed three times with PBS and mounted in buffered glycerol (80%). The cells were examined with fluorescence microscope (Leica, DM-2500). 2.4.

Polyacrylamide Gel Electrophoresis (PAGE) The presence of rotavirus strains in cell was determined

by detection of dsRNA segments of rotavirus in polyacrylamide 68

gel electrophoresis (PAGE). Viral RNAs were extracted from viral infected cell culture using Trizol reagent (Invitrogen, Carlsbad, CA, USA). In brief, 0.3 ml of suspension of viral infected cell culture was mixed with 0.7 ml of Trizol reagent and 0.2 ml of chloroform/isoamylalcohol (24:1). After centrifugation at 10,000 rpm for 10 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol. The RNA precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol and finally dissolved in 20 μl of RNase-free water. The electrophoretic migration pattern of dsRNA of each rotavirus was determined by 10% acrylamide slab gel with a 3.5% acrylamide stacking gel for 16 h at 20 mA [Jones et al., 2003]. The dsRNA segments were visualized by staining gel with silver nitrate and compared to those of other strains of Wa and DS-1 [Herring et al., 1982]. 2.5.

Reverse Transcription- Polymerase Chain Reaction (RT-

PCR) Rotavirus dsRNA was extracted from infected MA104 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The VP7 full-length gene (1,062 bp) from dsRNA genome was amplified using the Beg9 and End9 primers as described previously [Gouvea et al., 1990]. The RT-PCR primers for full genes of VP4 (2,359 bp), VP6 (1,356 bp), and NSP4 (750 bp)

69

amplification

were

designed

by

aligning

sequences

of

representative rotavirus strains from GenBank nonredundant database by using Primer 3 software (Rozen & Skaletsky, 2000) with default settings. The primer sequences are as follows; VP411F [Gentsch et al., 1992] (5’- TGGCTTCGCCATTTTATAGA CA -3’), and VP4-2359R (5’- GGTCACATCCTCAATAGCY TTCTCAC -3’), VP6-1F (5’- GGCTTTWAAACGAAGTCT - 3’) and VP6-1356R (5’- GGTCACATCCTCTCACTA- 3’), NSP4-1F (5’-GGCTTTTAAAAGTTCTGTTC-3’),

and

NSP4-750R

(5’-

GGTCACACTAAGACCATTCC-3’). RT-PCR reaction for amplification of VP4, VP6, and NSP4 gene was carried out respectively with initial reverse transcription step at 42 o C for 30 min, followed by 30 cycles of amplification of 94 o C for 1 min, 52 o C for 1 min, 72 o C for 1 min (for NSP4), 2 min (for VP6), and 3 min (for VP4), and a final extension

of

electrophoresed

10 in

min

at

1.2%

72 o C. SeaKem

The LE

PCR

products

agarose

gel

were (FMC

Bioproducts, Rockland, ME) and viewed under the GelDoc 2000 image-analysis system (BioRad, Hercules, CA, USA) following ethidium bromide staining. 2.6.

Nucleotide Sequencing and Phylogenetic Analysis The VP7, VP4, VP6, and NSP4 genes of rotavirus strain

CAU 202 were examined using comprehensive sequence analysis. 70

Each amplified product was inserted into a pCR 2.1. cloning vector, transformed to E. coli TOP 10F’ (Invitrogen), and sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and ABI PRISM 310 automated DNA sequencer. M13 reverse and T7 promoter primers were used for sequence analysis. In addition, VP4 internal

primers

(VP4-703F:

5’-

GTAGTACCATTATCATT

ATCATC -3’ and VP4-1231F: 5’- GTTACATTATCTACGCAA TTCAC -3’) were designed and used for sequenceing. The resultant gene sequences were aligned using the CLUSTAL X 1.81 (Thompson et al., 1997) and MegAlign (DNASTAR, Madison, WI) against corresponding gene nucleotide or deduced amino

acid

sequences

of

representative

rotaviruses

from

GenBank database. The rooted trees were constructed using the neighbour-joining algorithms (Saitou & Nei, 1987) from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distance matrices were generated by the neighbour-joining method described by Jukes and Cantor (1969) and tree topology evaluated using a bootstrap analysis (Felsenstein, 1985) of the neighbour-joining dataset with the SEQBOOT and CONSENSE programs from the PHYLIP package. 2.7.

Nucleotide Sequence Accession Numbers Nucleotide sequence data described in this report have

71

been submitted to the GenBank database and have been assigned the accession numbers: EF059922 (VP7), EF059923 (VP4), EU556223 (VP6), and EF059924 (NSP4).

72

3.

RESULTS

3.1.

Isolation of G9 Rotaviruses and Indirect Immuno-

fluorescence Assay (IFA) The sample of CAU 202, which was previously screened positive for rotavirus by ELISA and RT-PCR, was inoculated on MA104 cells in roller tube culture in the presence of Trypsin for rotavirus isolation. The infected cells were harvested 6 days after infection and five passages were done. The results revealed that the cytopathic effect (CPE) of infected cells was appeared by the third passages (Fig 3.1). The positive isolate (CAU 202) was confirmed for G9 rotavirus by RT-PCR using specific primers [Gentsch et al., 1992; Gouvea et al., 1990] that P and G type of strain CAU202 were of P[8] and G9 genotype with 356bp and 306bp in size , respectively (data not shown). The isolate strain (CAU 202) was also demonstrated by electron microscopy using the method of negative staining (Fig 3.2). The result shown that referenced human rotavirus Wa train and isolate strain (CAU 202) were similar in size (100 nm) and morphology. Adaptation of rotavirus strain CAU 202 in MA104 cells was then determined by indirect immunofluorescence assay (IFA). Cell-associated viruses fluoresced brightly showing a distinctive granular appearance and no host cellular reactivity 73

was noted (Fig 3.3). The infection level per cell varied from a few virus particles to large clusters filling the cytoplasm. The negative control sample showed no specific binding against monoclonal antibody specific for VP6 of human rotavirus.

74

Fig 3.1. Cytopathic effect (CPE) of uninfected cells (A) and infected cells (B) with CAU202 (G9P[8]) isolate. The CPE was observed after three days pos-infection of the third passages

75

Fig 3.2. Electron micrograph of referenced human rotavirus Wa strain (A) and human rotavirus isolate strain CAU 202 (B). Magnification bar equals 100 nm.

76

Fig 3.3. Detection of Human rotavirus strain CAU202 by indirect immunofluorescence assay (IFA) using specific monoclonal antibody against VP6 protein of HRV Wa strain. Uninfected cells (A) and infected cells (B) with HRV isolate CAU202 strain (G9P[8])

77

3.2.

RNA Electropherotype of CAU202 (G9) Rotaviruses The RNA electrophoretic migration pattern of the cell

culture grown strain CAU 202 was analyzed by PAGE and compared to that of other rotavirus strains namely, Wa and DS-1. G9 isolates displayed a ‘‘long’’ RNA pattern like Wa but quite distinct from that of DS-1 (Fig. 3.4). The mobility of genome segments 5 and 6 of this strain is slower and 10 and 11 is widely spaced

than

those

in

strain

DS-1

which

is

a

peculiar

characteristic commonly found in the long RNA pattern, and subgroup II specificity. 3.3.

Phylogenetic Analysis of VP7, VP4, VP6, and NSP4

Gene of CAU202 The resultants of RT-PCR showed that VP7, VP4, VP6, and NSP4 gene sequences were of 1062bp, 2359bp, 1,356 bp, and 750 bp in size, respectively, which were in agreement with respected nucleotide sequences of VP7, VP4, VP6, and NSP4 gene

of

human

group

A

rotavirus.

Complete

nucleotide

sequences for VP7, VP4, VP6, and NSP4 genes of strain CAU 202 were then subjected to nucleotide sequencing analysis As shown in Fig. 3.5, the human rotavirus G9 strains segregated into four lineages (I to IV) and strain CAU 202 clustered into VP7 genetic lineage III. In addition to strain CAU 202 isolated in this study, nine VP7 gene sequences of other 78

Korean G9 strains deposited in the GenBank databases were included in this study to assess genetic diversity. The analysis revealed that they also clustered into VP7 genetic lineage III. With respect to the VP4 gene, CAU 202 was of P[8] genotype and clustered into genetic lineage P[8]-3 from a total of four different lineages described to date for this genotype (Fig 3.6). In this phylogenetic analysis of P[8] genotype, twenty four gene sequences of VP4 with P[8] genotype of other Korean strains deposited in the GenBank databases were also analysed and shown that most of them (22/24) belonged to genetic lineage P[8]-3 except two strains belonged to genetic lineage P[8]-2. Also, phylogenetic analysis of VP6 and NSP4 gene of strain CAU 202 has been made and shown that CAU 202 belonged to VP6 subgroup II specificity and NSP4 genotype B, respectively (Fig. 3.7 and 3.8)

79

Fig 3.4. Electrophoretic migration patterns of genomic RNA of celladapted rotavirus strains CAU 202.

80

Fig 3.5. Phylogenetic tree for deduced amino acid sequences of the VP7 gene of CAU202 HRV strain isolated in this study in comparison with those of other global G9 strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points.

81

Fig 3.6. Phylogenetic tree for the nucleotide sequences of the VP4 gene of CAU202 (G9P[8]) strains in comparison with those of other representative P[8] rotavirus strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points. 82

Fig 3.7. Phylogenetic tree for the nucleotide sequences of the VP6 gene of CAU202 strains in comparison with those of other representative rotavirus strains. The bar indicates the variation scale. Bootstrap values greater than 60% are shown at branch points.

83

Fig 3.8. Phylogenetic tree for the nucleotide sequences of the NSP4 gene of CAU202 in comparison with those of other representative rotavirus strains. The bar indicates the variation scale

84

4.

DISCUSSION Rotaviruses have been now considered as the most

important cause of severe viral gastroenteritis in humans and animals with high morbidity and mortality in developed and developing countries. The best hope for its prevention is the development of an effective vaccine but there is so far no effective universal vaccine for prevention of rotavirus disease [Mulholland, 2004; Parashar et al., 2003; Yoshinaga et al., 2006]. G9 human rotavirus strains have been recently emerged and reported as a causative agent of diarrhea in children. Which are also recognized as one of the most widespread emerging genotypes in the world including USA, Canada, Australia, United Kingdom, Germany, Brazil, Africa, China, India, Vietnam, Japan, Ireland, Thailand, Argentina, Malaysia, Northern Ireland, Taiwan [Barril et al., 2006; Carmona, 2006; Carvalho-Costa et al., 2006; Feeney et al., 2006; Hung et al., 2006; Khamrin et al., 2006; Kirkwood et al., 2003; Kirkwood et al., 1999; Laird et al., 2003; Lin et al., 2006; Reidy et al., 2005; Yoshinaga et al., 2006; Zhou et al., 2003], and so on. Previous data of sequencing and phylogenetic analysis of the VP7 gene has shown distinct genetic diversity among G9 rotavirus strains and six lineages with eleven sublineages have been described to date [Bozdayi et al., 2008; Phan et al., 2007; Rahman et al., 2005; Ramachandran et al., 2000; Stupka et al., 2007]. In this study, the phylogenetic 85

analysis revealed that CAU 202 and all nine other Korean G9 strains deposited in the GenBank databases clustered into VP7 genetic lineage III. In Korea, there were significantly different results in G typing in recent years in some parts of Korea. The G4, G2, and G9 were determined as the most frequent G type during the period from 1998 to 2004, respectively, [Kang et al., 2005; Kim et al., 2005; Min et al., 2004; Song et al., 2003] while the previous epidemiological studies reported that G1 to G4 were common, particularly G1 as the predominant strain during the period from 1987 to 1999 [Min et al., 2004; Seo and Sim, 2000; Song et al., 2003]. Interestingly, the study on the distribution of human group A rotavirus VP7 and VP4 types circulating in Seoul, Korea between 2004 and 2006 [Le et al., 2008] revealed that G1 serotypes were the most prevalent in comparison to others G serotypes. In such a complicated situation of rotavirus emergence circulating in Korea, it is essential to gain the updated and comprehensive information about the relative importance of rotavirus strains circulating locally before applying vaccination programs. The identification of CAU 202 strain in this study together with other G9 strains that isolated in different geographic parts in the world one more highlights the global presence of serotype G9. Although G9 human rotavirus strains have been recently found in some parts of Korea [Kang et al., 86

2005; Kim et al., 2005], there is no information of isolation and characterization as well of G9 human rotavirus strains to date. So, the isolation and characterization of G9 strain (CAU 202) done in this study are very necessary and significant to better understanding the genetic information and relationship as well among G9 strains isolated in different countries. Unlike other globally common G1, G2, G3 or G4 strains, which are detected almost exclusively in conjunction with P[8] or P[4], the G9 strains have been detected in association with variety of P types including P[4], P[6], P[8], P[9], P[11], and P[19] [Carmona et al., 2006; Hoshino et al., 2004; Khamrin et al., 2006; Kirkwood et al., 1999; Mphahlele et al., 1999; Okada et al., 2000; Ramachandran et al., 2000; Zhou et al., 2003; Zhou et al., 2001]. The VP4 gene sequence analysis of strain CAU 202 in this study showed the highest identity to other P[8] VP4 rotaviruses. This meant that CAU 202 strain was of G9P[8] genogroup. The present study was in agreement with the results obtained recently that G9P[8] combinations were found in Ireland, Paraguay Thailand, Taiwan, Brazil, Malaysia, India, Northern Ireland, Japan, Australia [Banerjee et al., 2006; Carmona et al., 2006; Carvalho-Costa et al., 2006; Feeney et al., 2006; Hung et al., 2006; Khamrin et al., 2006; Kirkwood et al., 2003; Lin et al., 2006; Parra et al., 2005; Reidy et al., 2005; Zhou et al., 2001]. Phylogenetic analysis of the VP4 gene has 87

shown genetic diversity within P[8] genotypes and four genetic lineages have been described [Arista et al., 2005; Arista et al., 2006; Cunliffe et al., 2001; Espinola et al., 2008; Gouvea et al., 1999]. This study revealed that CAU 202 (G9P[8]) and all Korean rotaviruses with P[8] genotype found in GenBank databases to date clustered into P[8]-3 except two strains (KMR541

and

KMR538)

clustered

into

P[8]-2.

Rotavirus

nonstructural protein NSP4, which is encoded by gene segment 10, was found to be a viral enterotoxin related to pathogenicity and also has multiple functions in rotavirus morphogenesis [Estes, 2001; Kaga et al., 1994]. The NSP4 gene has been differentiated into five genotypes (A–E) [Ciarlet et al., 2000; Iturriza-Gomara et al., 2003], among which human rotaviruses belong to genotypes A, B, or C. In this study, NSP4 gene of the strain CAU 202 was found to be NSP4 genetic genotype B. Polyacrylamide

gel

electrophoresis

(PAGE)

has

developed and become a common procedure for characterization of rotavirus strains [Jones et al., 2003; Kobayashi et al., 1989; Lin et al., 2006; Pietruchinski et al., 2006; Pongsuwanna et al., 2002]. The rotavirus genome consists of 11 double-stranded RNA segments and there is a great variety in the migration patterns of the RNA of different strains in PAGE. The RNA patterns of group A rotaviruses can be classified into two major distinctive groups of "long" and "short" RNA patterns in which 88

the migration of segment 11 is rapid and slow, respectively [Espejo et al., 1979; Kalica et al., 1981]. The G9 strains have been found to have either "long" or "short" RNA patterns [Kirkwood et al., 2003; Lin et al., 2006; Zhou et al., 2001]. In present study, strain CAU 202 (G9P[8]) was analyzed by PAGE and compared with that of other rotavirus strains namely, Wa and DS-1. CAU 202 strain displayed a ‘‘long’’ RNA pattern like Wa but quite distinct from that of DS-1 (Fig 3.3). This ‘‘long’’ RNA pattern of CAU 202 strain was similar with that of G9P[8] strains isolated in Australia [Kirkwood et al., 2003], in Japan [Zhou et al., 2001], in Taiwan [Lin et al., 2006]. Immunofluorescence assay (IFA) was the most reliable method

for

the

detection

of

virus

replication,

although

characteristic cytopathic effects were produced sporadically by most isolates [Birch et al., 1983]. IFA has been applied widely for detection of rotavirus [Ellens et al., 1978; Moosai et al., 1979; Schirrmeier and Heinrich, 1981]. In this study the IFA has been used and shown that cell-associated viruses fluoresced brightly showing a distinctive granular appearance and no host cellular reactivity was noted. The findings of G9 strains recently in south Korea and other countries imply that G9 human rotavirus should be incorporated into candidate rotavirus vaccine.

89

V Molecular Characterization of Unusual Human Rotavirus Strains G12 with P[6] Detected in South Korea 1.

INTRODUCTION Rotaviruses are the most common etiological agent of

severe diarrhoea in infants and young children [Kapikian et al., 2001] and are responsible, worldwide, for an estimated 454,000705,000 deaths annually [Parashar et al., 2006]. In South Korea, rotavirus is also the most common viral agent of acute diarrhea which is estimated 46% of hospitalized children aged 6–24 months with acute gastroenteritis [Seo and Sim, 2000]. The outer capsid of rotavirus is composed of two proteins, glycoprotein VP7 and protease-sensitive VP4 that define G- and P- types of rotavirus as well as confer protective immunity [Estes, 2001]. Based on these proteins, a dual classification system of group A rotaviruses has been introduced. To date, 16 G serotypes [Gulati et al., 2007] and at least 27 P genotypes have been reported [Khamrin et al., 2007a; Martella et al., 2007; Parra et al., 2007b; Steyer et al., 2007a]. Of these variants, epidemiological studies have shown that four G (G1–G4) and three P (P[4], P[6], and P[8]) are the most frequent VP7 and VP4 types associated with global human rotavirus infection [Santos and Hoshino, 2005]. Recently, one of the G serotypes that is considered 90

unusual rotavirus G12, which was reported as a causative agent of diarrhea in children has been documented in many countries [Rahman et al., 2007a]. The first G12 strains, L26 and L27 were identified in children less than 2 years old in 1987 in the Philippines [Taniguchi et al., 1990; Urasawa et al., 1990]. Since then, G12 has appeared in the United States [Griffin et al., 2002], Thailand [Pongsuwanna et al., 2002], India [Das et al., 2003], Japan [Shinozaki et al., 2004], Italy [Grassi et al., 2006], Australia [Kirkwood et al., 2006], Argentina [Castello et al., 2006], Brazil [Pietruchinski et al., 2006], Nepal [Uchida et al., 2006], Slovenia [Steyer et al., 2007b], Hungary [Banyai et al., 2007], Belgium [Rahman et al., 2007a], and Bangladesh [Rahman et al., 2007b], including strains or RT-PCR products with as yet unpublished G12 VP7 sequences in the GenBank database. Today, G12 rotavirus is considered as a sixth most important global genotype mirroring the rise of G9 in the late 1990s [IturrizaGomara et al., 2000]. To date, extensive rotavirus surveillance studies reported that G1 to G4 were common in South Korea. Even though G9 was first detected in the rural provinces [Kim et al., 2005], but only a report is available on the occurrence of G12 [Kang et al., 2005] and characteristics and phylogenetic relatedness among these strains have not yet been investigated. Rotavirus vaccines could fail to protect children from rotavirus gastroenteritis because of insufficient cross-protection against 91

the disease caused by new emerging rotavirus genotypes not covered by the vaccine. Thus, the development of a safe and effective rotavirus vaccine has been an important global public health goal. In this study , two unusual G12 human rotavirus strains, which were detected from our rotavirus surveillance study between 2004 and 2006 in Seoul, South Korea [Le et al., 2008], have been isolated and characterized for the first cases in south Korea.

92

2.

MATERIALS AND METHODS

2.1.

Stool Specimens A total of 394 rotavirus faecal specimens were collected

from young children with acute diarrhea at five hospitals in Seoul, South Korea between 2004 and 2006. Two G12 rotavirus stool specimens were detected by type-specific primer-dependent reverse transcription–PCR (RT–PCR) [Das et al., 1994; Gouvea et al., 1990; Santos et al., 2003] and nucleotide sequence analysis. 2.2.

Isolation of Rotaviruses in Cell Culture Rotavirus strains were isolated and propagated from two

G12 stool specimens in MA104 cells. Briefly, the specimens were diluted 10-fold with phosphate buffered saline (PBS; pH 7.4) and clarified by centrifugation 10,000 g for 10 min. The supernatants filtered using a 0.45 μm sterile syringe filter (Corning, NY, USA) were pretreated with 10 μg ml -1 Trypsin (Difco TM Trypsin 250- Becton, Dickinson & Company Sparks, MD, USA) for 30 min at 37 o C and inoculated onto MA104 cells in glass tubes with Minimum Essential Alpha Medium (Gibco, Invitrogen, Carlsbad, CA, USA) in the presence of Trypsin (5 μg ml -1 ) followed by incubation in rolling culture using a rotator. The culture cells were harvested 5–7 days after infection and subsequently passed onto MA104 cells until CPE was appeared. 93

The culture fluid of the final passage was examined for rotavirus by RNA polyacrylamide gel electrophoresis and immunofluorescence assay. 2.3.

Indirect Immunofluorescence Assay (IFA) Indirect immunofluorescence assay was used to detect

rotavirus antigen in MA104 cells. CPE appeared rotaviruses infected MA104 cells in a 96-well plate (NUNC) were fixed with 80% acetone at room temperature for 10 min. The fixed cells were washed three times in PBS (pH 7.2) and added 50 µl of monoclonal antibody specific for VP6 of human rotavirus (1:50 in PBS). The plate was incubated at 37 o C for 1 hr followed by three washes in PBS and 50 µl of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Invitrogen, Carlsbad, CA, USA) diluted 1:100 in PBS was added to each well. The plate was incubated at 37°C for 1hr, washed three times with PBS and mounted in buffered glycerol (80%). The cells were examined with fluorescence microscope (Leica, DM2500). 2.4.

Polyacrylamide gel electrophoresis (PAGE) The presence of rotavirus strains in cell was determined

by detection of dsRNA segments of rotavirus in polyacrylamide gel electrophoresis (PAGE). Viral RNAs were extracted from viral infected cell culture using Trizol reagent (Invitrogen, 94

Carlsbad, CA, USA). In brief, 0.3 ml of suspension of viral infected cell culture was mixed with 0.7 ml of Trizol reagent and 0.2 ml of chloroform/isoamylalcohol (24:1). After centrifugation at 10,000 rpm for 10 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol. The RNA precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol and finally dissolved in 20 μl of RNase-free water. The electrophoretic migration pattern of dsRNA of each rotavirus was determined by 10% acrylamide slab gel with a 3.5% acrylamide stacking gel for 16 h at 20 mA [Jones et al., 2003]. The dsRNA segments were visualized by staining gel with silver nitrate and compared to those of other strains of Wa and DS-1 [Herring et al., 1982]. 2.5.

Reverse Transcription-Polymerase Chain Reaction (RT-

PCR) Rotavirus dsRNA was extracted from infected MA104 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The VP7 full-length gene (1,062 bp) from dsRNA genome was amplified using the Beg9 and End9 primers as described previously [Gouvea et al., 1990]. The specific primers for full genes of VP4 (2,359 bp), VP6 (1,356 bp), and NSP4 (750 bp) amplification representative

were

designed

rotavirus

strains

95

by

aligning obtained

sequences from

of

GenBank

databases by using Primer 3 software (Rozen & Skaletsky, 2000) with default settings. The primer sequences designed are as follows;

VP4F

(5’-TGGCTTCRCTCATTTATAGACAGCT),

VP4R (5’GGTCACATCCTCTATRGAGCTCTCA-3’), VP6F (5’GGCTTTWAAACGAAGTCT - 3’), VP6R (5’- GGTCACATCC TCTCACTA- 3’); NSP4F (5’-GGCTTTTAAAAGTTCTGTTC-3’), NSP4R

(5’-GGTCACACTAAGACCATTCC-3’).

RT-PCR

for

amplification of VP4, VP6, and NSP4 gene was carried out, respectively, with initial reverse transcription step at 42 o C for 30 min, followed by 30 cycles of amplification of 94 o C for 1 min, 52 o C for 1 min, 72 o C for 1 min ( for NSP4), 2 min (for VP6), and 3 min (for VP4), and a final extension of 10 min at 72 o C. The PCR products were electrophoresed in 1.2% SeaKem LE agarose gel (FMC Bioproducts, Rockland, ME) and viewed under the GelDoc 2000 image-analysis system (BioRad, Hercules, CA, USA) following ethidium bromide staining. 2.6.

Nucleotide Sequencing and Phylogenetic Analysis The VP7, VP6, VP4, and NSP4 genes of rotavirus CAU

195 and CAU 214 strains were examined using comprehensive sequence analysis. Each amplified product was inserted into a pCR 2.1. cloning vector, transformed to E. coli TOP 10F’ (Invitrogen), and sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and

96

ABI PRISM 310 automated DNA sequencer. M13 reverse and T7 promoter primers were used for sequence analysis. In addition, VP4 internal primers (VP4-738F: 5’-TTATCAACGTGCTCA GGTTAATGA-3’, VP4-1243F: 5’-ACTCAATTTACTGACTTCG TATCAC-3’) designed using the corresponding sequences of the strain DS-1 (P[4]), ST3 (P[6]), KU (P[8]), and AU-1 (P[9]) were used as sequencing. The resultant gene sequences were aligned using the CLUSTAL X 1.81 (Thompson et al., 1997) and MegAlign (DNASTAR, Madison, WI) against corresponding gene

nucleotide

or

deduced

amino

acid

sequences

of

representative rotaviruses from GenBank database. The rooted trees were constructed using the neighbour-joining algorithms (Saitou & Nei, 1987) from the PHYLIP suite of programs (Felsenstein,

1993).

Evolutionary

distance

matrices

were

generated by the neighbour-joining method described by Jukes and Cantor (1969) and tree topology evaluated using a bootstrap analysis (Felsenstein, 1985) of the neighbour-joining dataset with the SEQBOOT and CONSENSE programs from the PHYLIP package.

97

3.

RESULTS

3.1.

Detection of G12 Human Rotaviruses Among 394 rotavirus specimens collected from rotavirus

surveillance, rotavirus strains CAU 195 and CAU 214 with G12 specificity were detected by sequence analysis of the VP7 gene obtained by RT-PCR. The patient with strain CAU 195 and CAU 214 were both female infants with 6 and 11 months old, respectively. They were hospitalized for 7–8 days with symptoms of cough, nasal discharge, anorexia, high fever, watery diarrhea (8–10

times/day),

vomiting

(7–8

times/day),

and

notified

relatively high level of liver enzymes (SGOT and SGPT) and ketone. 3.2.

Isolation of G12 Rotaviruses and Indirect Immuno-

fluorescence Assay (IFA) Adaptation of rotavirus strains CAU 195 and CAU 214 in MA104 cells were determined by indirect immunofluorescence assay. Cell-associated viruses fluoresced brightly showing a distinctive granular appearance and no host cellular reactivity was noted (Fig 4.1). The infection level per cell varied from a few virus particles to large clusters filling the cytoplasm. The negative control sample showed no specific binding against monoclonal antibody specific for VP6 of human rotavirus.

98

Human rotavirus G12 isolate strains (CAU 195 and CAU 214) were also demonstrated by electron microscopy using the method of negative staining (Fig 4.2). The result showed that referenced human rotavirus Wa strain and both G12 isolate strains were similar in size (100 nm) and morphology.

99

Fig. 4.1. Detection of Human rotavirus strain CAU195 and CAU214 by indirect immunofluorescence assay (IFA) using specific monoclonal antibody against VP6 protein of Wa strain HRV. Uninfected cells (A) and infected cells (B) with HRV strain CAU195. Uninfected cells (C) and infected cells (D) with HRV strain CAU214.

100

Fig 4.2. Electron micrograph of referenced human rotavirus Wa strain (A) and human rotavirus G12 isolate strains of CAU 195 (B) and CAU 214 (C). Magnification bar equals 100 nm.

101

Fig 4.3. Electrophoretic migration patterns of genomic RNA of celladapted rotavirus strains CAU 195 and CAU 214.

102

3.3.

RNA Electropherotype of G12 Rotaviruses The RNA electrophoretic migration pattern of the cell

culture grown strain CAU 195 and CAU 214 were analyzed by PAGE and compared to that of other rotavirus strains namely, Wa and DS-1. Two G12 isolates displayed a ‘‘long’’ RNA pattern like Wa but quite distinct from that of DS-1 (Fig. 4.3). The mobility of genome segments 5 and 6 of these strains are slower and 10 and 11 is widely spaced than those in strain DS-1 which is a peculiar characteristic commonly found in the long RNA pattern, 3.4.

Sequence Determination of G12 Rotaviruses Complete nucleotide sequences for VP7, VP4, and NSP4

genes of CAU195 and CAU214 strains were subjected to nucleotide and amino acid sequence analysis. The resultants of VP7 gene sequences of CAU 195 and CAU 214 strains were both found to be 1,062 bp in length and code 326 amino acids. The VP7 gene sequences of the two strains were almost identical to each other (99.6% nucleotides, 99.4% amino acids). When compared with G12 isolates or VP7 gene RT-PCR products in GenBank database, these two strains showed highest identity with G12 strains; BP1503/05 and BP875/05 in Hungary, SI403/06 in Slovenian, and Se585 in the USA. The highest identity was found in recently reported BP1503/05 and BP875/05 strains with 99.3-99.7% at the nucleotide and 99.4-100% at the amino 103

acid levels (Table 3.1). However, the lowest homologies revealed from India strain RU172 isolated from porcine with 90.1% and 89.8% at the nucleotide and 94.2% and 93.6% at the amino acids, respectively. In addition, these strains presented low homologies to G12 prototype strain L26 (90.7% and 90.3% in nucleotide, 92.9% and 92.3% in amino acids, respectively) reported in 2000 from Philippines. The VP4 gene sequences of strains CAU195 and CAU214 were both 2,359 bp in length and code 775 amino acids. The VP4 gene sequences of the two strains were very similar to each other (99.8% nucleotides, 99.6% amino acids). These strains were compared with reference strains of various P types (Table 3.2), and were found to be the most similar to P[6] rotavirus Gottfried strain, with 83.2% and 83.3% identity at the nucleotide level and 89.5% and 89.7% identity at the amino acid level, respectively. For NSP4 gene, the resultant sequencing data shows that these strains were highly homologous each other (99.6% in nucleotide, 99.4% in amino acid). These two strains showed highest identity with human G1 strain Wa with 93.7% and 93.6% at the nucleotide and 96.6% and 96.0% at the amino acid levels (Table 3.3), respectively. The lowest homologies revealed from murine strain EHP with 67.6% and 67.2% at the nucleotide and 60.6-60.0% at the amino acids, respectively. Among G12 strains, these strains were distinct to L26 and Se585 human strains (81.8104

84.3% in nucleotide, 84.0-85.7% in amino acids), but relatively high with porcine strain RU172 (89.6% and 89.2% in nucleotide, 92.0% and 92.6% in amino acids, respectively).

105

Table 3.1. The VP7 nucleotide and amino acid sequence identities of the Korean CAU 195 and CAU 214 strains from other G12 rotaviruses obtained from GenBank Strain

CAU 195

CAU 214

nt

aa

nt

aa

99.6

99.4

-

-

P genotype

CAU 195 CAU 214

GenBank No.

Reported year

Country

Notice

P[6]

EF059916

2006

S. Korea

Isolate

P[6]

EF059917

2006

S. Korea

Isolate

L26

90.7

92.9

90.3

92.3

P[4]

M58290

1990

Philippines

Isolate

Se585

99.1

99.4

98.7

98.8

P[6]

AJ311741

2001

USA

Isolate

T152

97.4

97.9

97.0

97.2

P[9]

AB071404

2002

Thailand

Isolate

ISO1

98.4

99.4

98.0

98.8

P[4]

AY206861

2003

India human

Isolate

ISO2

98.8

99.1

98.4

98.5

P[6]

AY098669

2003

India human

Isolate

ISO5

98.2

99.4

97.8

98.8

P[6]

AF508734

2003

India human

Isolate

CP727

97.6

97.5

97.2

96.9

P[9]

AB125852

2004

Japan

Isolate

CP1030

97.5

97.5

97.1

96.9

P[9]

AB125853

2004

Japan

Isolate

K12

97.8

97.2

97.4

96.6

*

AB186120

2004

Japan

Isolate Isolate

HC91

97.5

97.8

97.0

97.1

P[9]

AY855065

2006

Brazil

RU172

90.1

94.2

89.8

93.6

P[7]

DQ204743

2006

India porcine

Isolate

Arg720

97.5

97.5

97.1

96.9

P[9]

DQ111868

2006

Argentina

Isolate

BP1503/05

99.7

100

99.3

99.4

P[8]

AM397928

2007

Hungary

*

BP875/05

99.7

100

99.3

99.4

P[8]

AM397926

2007

Hungary

*

SI-264/06

98.8

98.1

98.4

97.4

P[8]

DQ995173

2007

Slovenia

PCR product

SI-403/06

99.4

99.2

98.9

98.5

P[8]

DQ995174

2007

Slovenia

PCR product

05K066

98.7

99.4

98.3

98.8

*

AB275301

2007

Nepal

PCR product

05N054

99.4

99.4

99.0

98.8

*

AB275291

2007

Nepal

PCR product

RV176/00

99.0

99.1

98.6

98.5

P[6]

DQ490556

2007

Bangladesh

Isolate

RV161/00

99.0

99.1

98.6

98.5

P[6]

DQ490550

2007

Bangladesh

Isolate

N26-02

98.6

98.5

98.2

97.9

P[6]

DQ146687

2007

Bangladesh

Isolate

MV404/02

99.2

98.8

98.7

98.1

P[6]

DQ501280

2007

United Kingdom

Isolate

B4633/03

99.1

99.1

98.7

98.5

P[8]

DQ146643

2007

Belgium

Isolate

MD844

99.3

99.7

98.9

99.1

P[8]

AB269689

2006

Saudi Arabia

*

Dhaka12-03

97.9

98.5

97.6

97.9

P[6]

DQ146665

2007

Bangladesh

Isolate

Dhaka25-02

98.6

98.8

98.2

98.2

P[8]

DQ146654

2007

Bangladesh

Isolate

Matlab13-03

97.7

97.9

97.4

97.2

P[6]

DQ146676

2007

Bangladesh

Isolate

*: not clear

106

Table 3.2. The VP4 nucleotide and amino acid sequence identities of the Korean CAU195 and CAU214 strains from other rotavirus strains with different P genotypes P genotype

Strain

CAU 195

CAU 214 nt

aa

GenBank No

Origin

nt

aa

CAU 214

99.8

99.6

P[1]

NCDV

67.5

68.5

P[2]

SA11

71.7

74.1

71.7

74.1

X14204

Simian

P[3]

CU1

71.1

75.0

71.1

75.0

L20876

Canine

P[4]

DS-1

74.0

77.0

74.1

76.9

AJ540227

Human

P[5]

UK

67.1

69.9

67.1

69.9

M22306

Bovine

P[6]

Gottfried

83.2

89.5

83.3

89.7

M33516

Porcine

P[7]

OSU

69.8

71.7

69.7

71.7

X13190

Porcine

P[8]

KU

75.8

78.5

75.9

78.6

AB222784

Human

P[9]

K8

65.2

65.2

65.2

65.0

D90260

Human

P[10]

69M

70.8

74.3

70.8

74.3

M60600

Human

P[11]

B223

60.7

55.6

60.6

55.6

M92986

Bovine

P[12]

H-2

70.5

74.5

70.5

74.5

L04638

Equine

P[13]

A46

69.6

70.2

69.5

70.2

AY050274

Porcine

P[14]

PA169

65.8

66.7

65.7

66.3

D14724

Human

P[15]

Lp14

71.0

74.7

71.0

74.6

L11599

Lamb

P[16]

EDIM

65.9

70.3

65.8

70.3

AF039219

Murine

P[17]

PO-13

62.1

58.3

62.1

58.3

AB009632

Avian

P[18]

L338

71.0

73.0

71.0

73.0

D13399

Equine

P[19]

Mc345

77.0

83.9

77.1

84.0

D38054

Human

EF059920

CAU 195 67.4

68.5

Human

EF059921

Human

M63267

Bovine

P[20]

EHP

68.2

71.7

68.2

71.7

U08424

Murine

P[21]

Hg18

69.8

72.6

69.8

72.6

AF237665

Bovine

P[22]

160/01

64.0

58.6

64.1

58.6

AF526374

Rabbit

P[23]

A34

66.7

61.9

66.8

61.9

AY174094

Porcine

P[24]

TUCH

70.7

74.8

70.8

74.8

AY596189

Simian

P[25]

Dhaka6

66.1

66.1

66.2

66.1

AY773004

Human

P[26]

134/04-15

69.1

71.1

69.1

71.0

DQ061053

Porcine

*

P21-5

68.9

70.3

69.0

70.4

DQ629926

Porcine

*

344-04-1

69.4

71.2

69.4

71.4

DQ242615

Porcine

*

CMP034

68.7

72.0

68.8

72.2

DQ534016

Porcine

*: Reported in 2007.

107

Table 3.3. The NSP4 nucleotide and amino acid sequence identities of Korean CAU 195 and CAU 214 strains from other rotavirus strains with different NSP4 genotypes NSP4 Genotype

CAU 195

Strain nt

CAU 214 aa

nt

GenBank No

Origin

aa

CAU 195

EF059918

Human

EF059919

Human

CAU 214

99.6

99.4

A

DS-1

82.2

84.6

82.1

84.0

AF174305

Human

A

SA11

79.6

84.0

79.6

83.4

AF087678

Simian

A

UK

83.3

84.6

83.0

84.0

M21885

Bovine

A

B223

75.9

82.9

75.8

82.3

AF144805

Bovine

A

L26

82.0

84.6

81.8

84.0

AJ311732

Human

A

H-2

82.4

84.0

82.2

83.4

AF144801

Equine

A

BAP-2

81.4

85.7

81.3

85.1

AF144795

Lapine

A

Se585

84.3

85.7

84.0

85.1

AJ311731

Human

B

Wa

93.7

96.6

93.6

96.0

AF093199

Human

B

ST3

91.6

92.0

91.5

91.4

U59110

Human

B

OSU

89.4

92.6

89.3

92.0

D88831

Porcine

B

AU32

91.8

93.7

91.7

93.1

D88830

Human

B

YM

90.4

93.1

90.3

92.6

X69485

Porcine

B

RU172

89.6

92.6

89.2

92.0

DQ204740

Porcine

C

CU-1

77.8

84.6

77.7

84.0

AF144806

Canine

C

FRV64

81.3

82.3

81.1

81.7

D88833

Feline

C

AU1

82.6

83.4

82.6

82.9

D89873

Human

D

EW

68.3

61.1

67.9

60.6

AB003805

Murine

D

EHP

67.6

60.6

67.2

60.0

U96336

Murine

*

ADRV

42.0

13.1

42.6

13.1

AY548957

Human

* Group B rotavirus

108

4.

DISCUSSION Rotavirus is the major cause of morbidity and mortality in

developed and developing countries and now considered the most important cause of severe viral gastroenteritis in humans and animals. The best hope for its prevention is the development of an effective vaccine. Several candidate rotavirus vaccines have been formulated to match the most prevalent serotypes, but there is a need to define the currently circulating serotypes and their temporal and geographic variations. Such studies will be important to identify changes in the prevalence of circulating strains and the emergence of novel strains that may impact the efficacy of vaccines [Castello et al., 2006]. In this study, two unusual G12 human rotaviruses (CAU 195 and CAU 214) have been isolated and characterized. VP7 gene sequencing analysis of strain CAU 195 and CAU 214 was found to be high identity to each other (99.6%) and to other G12 rotavirus strains, identity ranging from 89.8% to 99.7%. VP7 gene sequences of strain CAU 195 and CAU 214 showed highest identity with that of G12 strains BP1503/05 and BP875/05 in Hungary [Banyai et al., 2007], SI-403/06 in Slovenia [Steyer et al., 2007b] and Se585 isolated in United States [Griffin et al., 2002] rather than G12 prototype strain (L26) in Philippines [Taniguchi et al., 1990; Urasawa et al., 1990] and other G12 isolates in other countries (Table 3.1). Also, the gene sequence 109

analysis was done in order to compare VP4 gene sequence of strain CAU 195 and CAU 214 with that of different P genotype specificities of rotavirus strains (Table 3.2). The result showed that strain CAU 195 and CAU 214 belonged to P[6] rotavirus. Rotavirus nonstructural protein NSP4, which is encoded by gene segment 10, was found to be a viral enterotoxin related to pathogenicity and also has multiple functions in rotavirus morphogenesis [Estes, 2001; Kaga et al., 1994]. The NSP4 gene has been differentiated into five genotypes (A–E) [Ciarlet et al., 2000; Iturriza-Gomara et al., 2003], among which human rotaviruses belong to genotypes A, B, or C. In this study, the NSP4 gene of CAU 195 and CAU 214 has been sequenced and compared with other NSP4 genes of different rotavirus strains (Table 3.3). Interestingly, the NSP4 gene sequences of CAU 195 and CAU 214 were completely distinct from NSP4 gene sequences of other G12 strains available on GenBank such as Se585 and L26 strain. They belong to genotype B while NSP4 gene of G12 strains of Se585 and L26 belong to genotype A. So, the differences of NSP4 gene sequences of CAU 195 and CAU 214 in this study to other G12 rotavirus strains could be considered as the newest information. The deduced amino acid and gene sequencing analysis of VP7, VP4, and NSP4 of strain CAU 195 and CAU 214 were all found to be high identity to each other, 99.6% nucleotides and 110

99.4% amino acids in VP7, 99.8% nucleotides and 99.6% amino acids in VP4, 99.6% nucleotides and 99.4% amino acids in NSP4 (Table 3.1, 3.2, and 3.3). Both NSP4 genes of CAU 195 and CAU 214 belonged to genotype B. This high identity may be understood that both CAU 195 and CAU 214 may probably have originated from an identical clone of G12 or the same source infection from abroad. The patient with strain CAU 195 and CAU 214 were both female infants with 6 and 11 months old, respectively. They were hospitalized for 7–8 days with symptoms of cough, nasal discharge, anorexia, high fever, watery diarrhea (8–10 times/day), vomiting (7–8 times/day) and notified relatively high level of liver enzymes (SGOT and SGPT) and ketone. This observation was consistent with all other G12 reports that G12 strain caused diarrhea in infants and children at the different age [Banyai et al., 2007; Pietruchinski et al., 2006; Pongsuwanna et al., 2002; Samajdar et al., 2006; Shinozaki et al., 2004; Steyer et al., 2007b]. This meant that that G12 strain was really an important pathogen causing gastroenteritis. This is an interesting finding given that G12 human rotavirus has emerged and could be regarded as the first report of comprehensively analyzed G12 strains in South Korea that included relevant type strains from the NCBI GenBank. Although, a reported sample was suspected of being G12 [Kang et al., 111

2005], the data were inconclusive because the G12 strains could not be typed using current multiplex RT-PCR methods [Das et al., 1994; Gouvea et al., 1990; Isegawa et al., 1993; Santos et al., 2003; Taniguchi et al., 1992]; only G1-, G2-, G3-, G4-, G5-, G6-, G8-, G9-, G10-, and G11 serotypes can be detected using these techniques. Furthermore, there appears to be no evidence of sequence

analysis

incorporating

relevant

G

type

strains,

including G12, for comparison or evidence of immunological assay using G12-specific antibody. The prevalence rate of G12 strains (0.5%) detected in this study [Le et al., 2008] was very low when compared with that of recently other G12 strains recorded in other countries, such as 17.1% in India [Samajdar et al., 2006], 1.6% in Brazil [Pietruchinski et al., 2006], 14.3%, 3.1%, 3.8%, 12.5% in the year of 1999, 2000, 2001, and 2002, respectively, in Argentina [Castello et al., 2006], 1.7% in Slovenia [Steyer et al., 2007b], 23.0% and 20.0% in Nepal [Pun et al., 2007; Uchida et al., 2006], 6.9% in Hungary [Banyai et al., 2007], and 11.1 % in Bangladesh [Rahman et al., 2007b]. This low prevalence rate and having no more information of case infections of G12 strains in infants and young children in all parts of South Korea to date may reflect the quality of available healthcare and hospitals, especially in Seoul. The health care system of children’s hospitals in Seoul may be good and provide better control and eradication the infection of 112

G12 strains right after their introduction to two female infants living in Seoul, respectively, from abroad. Detection of G12 strains at a low percentage (0.5%) indicated that G12 strain was still rare genotype in South Korea. Of course, the finding of G12 human rotavirus in

this

study

may

present

the

possible

emergence of a new strain in South Korea. This may be a big problem for rotavirus vaccine developing strategies in near future. The first finding of G12 strains (L26 and L27) in the Philippines [Taniguchi et al., 1990; Urasawa et al., 1990], and then in the United States [Griffin et al., 2002], Thailand [Pongsuwanna et al., 2002], India [Das et al., 2003], Japan [Shinozaki et al., 2004], Italy [Grassi et al., 2006], Australia [Kirkwood et al., 2006], Argentina [Castello et al., 2006], Brazil [Pietruchinski et al., 2006], Nepal [Uchida et al., 2006], Slovenia [Steyer et al., 2007b], Hungary [Banyai et al., 2007], Belgium [Rahman et al., 2007a], Bangladesh [Rahman et al., 2007b] and now in South Korea [Le et al., 2008] were clearly evidences that G12 strain, an unusual human rotavirus strain, has been spreading worldwide and raises the question about the possibility of G12 emergence as a new global genotype. Polyacrylamide

gel

electrophoresis

(PAGE)

has

developed and become a common procedure for characterization of rotavirus strains [Jones et al., 2003; Kobayashi et al., 1989; 113

Pietruchinski et al., 2006; Pongsuwanna et al., 2002]. The rotavirus genome consists of 11 double-stranded RNA segments and there is a great variety in the migration patterns of the RNA of different strains in PAGE. The RNA patterns of group A rotaviruses can be classified into two major distinctive groups of "long" and "short" RNA patterns in which the migration of segment 11 is rapid and slow, respectively [Espejo et al., 1979; Kalica et al., 1981]. PAGE has been applied to characterize the G12 findings in other countries, such as the prototype strain L26 (G12P[4])

in

Philippines

has

a

‘‘long’’

electropherotype

[Taniguchi et al., 1990; Urasawa et al., 1990], strain Se585 (G12[6]) in the United state has a ‘‘short’’ electropherotype [Griffin et al., 2002], strain HC91 (G12P[9]) in Brazil has a ‘‘long’’ electropherotype [Pietruchinski et al., 2006], strain T152 (G12P[9])

in

Thailand

has

a

‘‘long’’

electropherotype

[Pongsuwanna et al., 2002], strains of ISO1 (G12P[4]), ISO2 (G12P[6]) have a ‘‘long’’ electropherotype, while strain ISO5 (G12P[6]) has a ‘‘short’’ electropherotype in India [Das et al., 2003], strain CP727 (G12P[9]) and CP1030 (G12P[9]) in Japan have a ‘‘long’’ electropherotype [Shinozaki et al., 2004]. In present study, strain CAU 195 and CAU 214 were also analyzed by PAGE and compared to that of other rotavirus strains namely, Wa and DS-1. Two G12 isolates displayed a ‘‘long’’ RNA pattern like Wa but quite distinct from that of DS-1 (Fig. 4.2). 114

Interestingly, two G12P[6] strains in this study have a ‘‘long’’ electropherotype while strain Se585 (G12[6]) in the United state has

a

‘‘short’’

electropherotype.

The

difference

of

electropherotype between G12P[6] of strain CAU 195 and CAU 214 in this study [Le et al., 2008] and G12P[6] of strain Se585 in United state [Griffin et al., 2002] may be explained that strain CAU 195 and CAU 214 were probably reassortant strains based on G12 strain, which was introduced to South Korea from abroad, with a donor VP4 segment from a P[6] strain. Nucleotide Sequence Accession Numbers Nucleotide sequence data described in this report have been submitted to the GenBank database and have been assigned the accession numbers EF059916 (CAU 195, VP7), EF059917 (CAU 214, VP7), EF059920 (CAU 195, VP4), EF059921 (CAU 214, VP4), EF059918 (CAU 195, NSP4), EF059919 (CAU 214, NSP4). EU556221 (CAU 195, VP6), and EU556222 (CAU 214, VP6).

115

VI Expression of Human Rotavirus Genes and Rotavirus- Like Particle Production. 1.

INTRODUCTION Rotavirus disease causes the death of approximately half

a million children annually, affecting mainly children in developing

countries,

but

accounts

for

one

third

of

hospitalizations for diarrhoea worldwide [Istrate et al., 2008; Parashar et al., 2006; Parashar et al., 2003]. The development and use of a rotavirus vaccine for childhood immunization programs would reduce substantially the mortality rates from rotavirus gastroenteritis in developing countries and to virtually eliminate hospitalizations due to rotavirus gastroenteritis, a heavy burden in developed countries. Vaccines have also been identified

as

the

prime

means

of

prevention

because

improvements in water supply, hygiene or sanitization are unlikely to decrease the spread of this disease [Cunliffe and Nakagomi, 2005; Istrate et al., 2008; Peixoto et al., 2007]. To date, all candidate rotavirus vaccines tested for infants have been live attenuated rotaviruses delivered orally and have shown variable or partial protection against rotavirus reinfection and diarrhea [Kapikian et al., 2005]. Some vaccines produced side effects in infants such as pyrogenesis, irritability, decreased 116

appetite, abdominal cramping and intussusception [Joensuu et al., 1998]. It was recently estimated that the risk of intussusception was higher after the first dose of Rhesus rotavirus tetravalent vaccine, but was still evident after the second dose [Kapikian et al., 2005]. The important questions remaining for these oral live vaccines are still related to safety and side effects as well as production costs. So, development of second generation of nonreplicating rotavirus vaccine should be considered as an alternative to live vaccines [Iosef et al., 2002]. Although two new live, oral RV vaccines have recently been licensed for use in children showing good efficacy and safety profiles after two or three doses, respectively [Kapikian et al., 2005; Ruiz-Palacios et al., 2006], the results from clinical trials in developing countries where the vaccine is most needed are still awaiting. Moreover, oral delivery of vaccines may be associated with certain limitations, such as poor long-term immunologic memory [John and Jayabal, 1972], inhibition of vaccine take by maternal antibodies

in

the

youngest

children,

the

possibilities

of

development of reassortments between vaccine strains and wildtype strains [Brandtzaeg, 2007; Istrate et al., 2008]. Subunit vaccines based on recombinant proteins can suffer from poor immunogenicity owing to incorrect folding of the target protein or poor presentation to the immune system. Virus-like particles (VLPs) represent a specific class of subunit 117

vaccine that mimic the structure of authentic virus particles. They are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit

vaccines.

VLPs

have

therefore

shown

dramatic

effectiveness as candidate vaccines [Noad and Roy, 2003]. VLPbased vaccines have previously been evaluated in humans for different diseases with great impact on public health such as candidate vaccines for human immunodeficiency virus [Doan et al., 2003] or licensed vaccines for hepatitis B virus [Adkins and Wagstaff, 1998] and human papillomavirus [Group, 2007; Paavonen et al., 2007]. In the case of rotavirus, rotavirus-like particle, a candidate vaccine against rotavirus infection, is composed by different proteins among the main rotavirus structural proteins (VP2, VP6, VP7, and VP4) that self-assemble in insect cells coinfected with recombinant baculoviruses expressing the rotavirus structural proteins. Co-expression of VP2 and VP6 results in the production of double-layered particles (2/6-VLPs), whereas coexpression of VP2, VP6, and VP7, with or without VP4, results in the production of triple-layered particles of 2/6/7 VLPs or 2/4/6/7 VLPs, respectively [Conner et al., 1996; Crawford et al., 1994; Istrate et al., 2008; Jiang et al., 1999; Labbe et al., 1991; Madore et al., 1999; Peixoto et al., 2007; Vieira et al., 2005]. These rotavirus-like particles were shown to be effective in 118

inducing immune responses in mice [Bertolotti-Ciarlet et al., 2003; Coste et al., 2000; Crawford et al., 1999; Istrate et al., 2008; Madore et al., 1999; O'Neal et al., 1998; O'Neal et al., 1997], rabbits [Ciarlet et al., 1998; Crawford et al., 1999], cows [Fernandez et al., 1998; Kim et al., 2002], and pigs [Iosef et al., 2002; Yuan et al., 2004]. In this study, four structural proteins of VP2, VP4, VP6, and VP7 of human rotavirus have been cloned and expressed using baculovirus expression system, respectively. The double-layered rotavirus like particles (2/6 VLPs) and triplelayered rotavirus like particles (2/4/6/7 VLPs) were then generated and characterized, respectively.

119

2.

MATERIALS AND METHODS

2.1.

Viruses and RNA Extraction Human rotavirus Wa strain (G1P[8]) was cultivated in

fetal rhesus monkey kidney (MA104) cells in the presence of trypsin as previously described [Estes and Graham, 1980]. Rotavirus dsRNA was extracted from infected MA104 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). In brief, 0.3 ml of suspension of viral infected cell culture was mixed with 0.7 ml of Trizol reagent and 0.2 ml of chloroform/isoamylalcohol (24:1). After centrifugation at 10,000 rpm for 10 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol. The RNA precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol and finally dissolved in 50µl of RNase-free water. 2.2.

Reverse Transcription-Polymerase Chain Reaction (RT-

PCR) RT-PCR for amplification of the full ORF (open reading frame) of VP2 (2,670 bp), VP4 (2,325 bp), VP6 (1,191 bp), and VP7 (978 bp) gene of HRV strain Wa was carried out, respectively, using specific primers as described in table 4.1. The reactions were carried out with initial reverse transcription step at 42 o C for 30 min, followed by 30 cycles of amplification of 94 o C for 1 min, 52 o C for 2 min, 72 o C for 3 min (for VP2 and 120

VP4), 2 min (for VP6), and 1 min (for VP7), and a final extension

of

electrophoresed

10 in

min

at

1.2%

72 o C. SeaKem

The LE

PCR

products

agarose

gel

were (FMC

Bioproducts, Rockland, ME) and viewed under the GelDoc 2000 image-analysis system (BioRad, Hercules, CA, USA) following ethidium bromide staining. 2.3.

Generation of Recombinant Baculoviruses Expressing

VP2, VP4, VP6, and VP7 Proteins. The fresh PCR products of VP2, VP4, VP6, and VP7 gene were inserted directly into pBlueBac4.5/V5-His-TOPO R vector, respectively, according to Manual of pBlueBac4.5/V5-HisTOPO R TA Expression Kit (Invitrogen). The ligation mixtures were then transformed into competent E. coli DH5ɑ (Invitrogen), grown on LB agar plate containing antibiotic. The recombinant vectors were collected from blue colonies and checked the insert by restriction enzyme and sequencing analysis. Recombinant

baculoviruses

were

generated

by

co-

transfection of Spodoptera frugiperda (Sf9) insect cells with Bac-N-Blue TM DNA (Invitrogen) and a baculovirus transfer vector containing the gene of interest. Recombination will occur between homologous sequences in viral DNA and transfer vector to yield recombinant viral DNA that is circular and will replicate and infect cells. The procedure of recombinant baculovirus 121

production

was

performed

according

to

the

Bac-N-Blue

Transfection and Expression Guide (Invitrogen). In brief, a 1.5 ml microcentrifuge tube containing 10 µl (0.5 µg) of the Bac-NBlue TM DNA was mixed with 4 µl of recombinant transfer plasmid (1 µg/µl) containing VP2, VP4, VP6, and VP7 gene, respectively,

1ml

of

Grace’s

insect

medium

(without

supplements or FBS), 20 µl of Cellfectin R reagent. The mixture of each was mixed gently and transfected with prepared monolayer of Spodoptera frugiperda (Sf9) cells in a 60 mm dish and incubated at 27°C. Seventy two hours after transfection, the CPE (cytopathic effect) of cells was checked and the supernatant was harvested and stored at 4°C for isolation of desire recombinant baculovirus. 2.4.

Plaque Forming Assay Recombinant baculoviruses expressing of VP2, VP4, VP6,

and VP7 protein, respectively, were plaque-purified by the following procedures. In brief, the prepared monolayer of Sf9 cells in a 60mm plate (5x10 6 cells/plate) was infected with 10 -1 , 10 -2 , 10 -3 , and 10 -4 dilution of transfection viral stock and incubated at 27°C for 1 h. The inoculum was removed and the overlay media containing 1% agarose and 0.16 mg/ml of X-gal were added. The plates were incubated at 27°C until the plaques were formed.

122

2.5.

Immunofluorescence Assay (IFA) Each recombinant baculovirus expressing VP2, VP4, VP6,

and

VP7

proteins,

respectively,

was

inoculated

into

the

monolayer of Sf9 cells prepared in a 96-well plate. When the CPE appeared, the cells were fixed with 80% acetone for 10 min, washed the plate three times with PBS (pH 7.2) and then air dried. The fixed cells were reacted with 50 µl of diluted anti-His monoclonal antibody (IG Therapy Co., Ltd) and incubated at 37°C for 1h. After washing three times with PBS, the cells were then stained with 50 µl of diluted FITC-conjugated goat antimouse IgG (Kirkegaard Perry Laboratories) and incubated at 37°C for 1h.

Finally, the cells were washed three times with

PBS, mounted in buffered glycerol (80%) and examined by fluorescence microscopy (Olympus). 2.6.

Western Immunoblotting The infected insect cells with recombinant baculovirus

expressing VP2, VP4, VP6, and VP7 proteins, respectively, were electrophoresed on 10% vertical SDS-PAGE under denaturing conditions and were then transferred to nitrocellulose membrane. The membrane containing proteins was washed one time with TBST (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20, pH 8.0) and then blocked in blocking buffer (5% skim milk in TBST) for 30 min at 25°C. After washing with TBST, the membrane was 123

reacted with human rotavirus Wa strain-positive rabbit sera or anti-His monoclonal antibody for 1h at 37°C with shaking and then washed three times with TBST. After washing, the diluted alkaline phosphatase-conjugated secondary antibody (Kirkegaard Perry Laboratories) was added and incubated for 1h at 37°C. After washing the membrane three times with TBST, BCIP/NBT solution (Kirkegaard Perry Laboratories) was added as a substrate and waited for about 15 min for bands to appear. 2.7.

Rotavirus

Like

Particle

(VLP)

Production

and

Purification. VLPs

were

expressed

and

purified

as

previously

described [Labbe et al., 1991; Tosser et al., 1992]. The doublelayered rotavirus like particles (2/6 VLPs) and triple-layered rotavirus like particles (2/4/6/7 VLPs) were generated by coinfection of Spodoptera frugiperda (Sf9) insect cells with different combinations of recombinant baculoviruses (5 PFU per cell) which code for the rotavirus proteins VP2, VP4, VP6, and VP7, respectively. The infected cells were collected at 5 or 7 days post-infection. For purification of VLPs, CsCl was added to the aqueous phase containing VLPs to obtain a refractive index of 1.3620 and the mixture was centrifuged for 18 h at 35,000 rpm in a Beckman SW55 rotor. The bands containing the VLPs were then subjected to a second CsCl gradient centrifugation. The gradient fractions containing the VLPs were kept at 4°C. 124

Table. 4.1. Oligonucleotide primers used for full amplification of ORF (open reading frame) of VP2, VP4, VP6, and VP7 gene of Human rotavirus Wa strain. Gene

Primer

Sequence 5’to 3’

Sense

VP2

VP2-F

ATGGCGTACAGGAAGCGCGGAGCTA

+

17-41

VP2-R

CAGTTCGTTCATAATGCGCATATTGT

-

2661-2686

VP4-F

ATGGCTTCGCTCATTTATAGACAGC

+

10-34

VP4-R

CAATTTACATTGTAGTATTAACTGT

-

2310-2334

VP6-F

ATGGAGGTTCTGTACTCAC

+

24-42

VP6-R

CTTAATCAACATGCTTCT

-

1197-1214

VP7-F

ATGTATGGTATTGAATATACC

+

49-69

VP7-R

AATTCTGTAGTAAAAAGCAGC

-

1006-1026

VP4 VP6 VP7

125

Position

Size (bp) 2,670 2,325 1,191 978

3.

RESULTS

3.1.

Expression of VP2, VP4, VP6 and VP7 Gene Amplification of the VP2, VP4, VP6, and VP7 gene of

Human rotavirus Wa strain was achieved by using RT-PCR with specific primer sets. The amplified gene for full length of open reading frame (ORF) of VP2, VP4, VP6, and VP7 had 2,670bp, 2,325bp, 1,191bp and 978 bp in size, respectively, which were in agreement with expected nucleotides of VP2, VP4, VP6, and VP7 gene of Human rotavirus group A (Fig. 5.1). The PCR products of VP2, VP4, VP6, and VP7 gene were then inserted directly into pBlueBac4.5/V5-His-TOPO R vector, respectively. Recombinant baculoviruses have been generated by cotransfection between Bac-N-Blue TM DNA and pBlueBac4.5/V5His-TOPO R transfer vector containing VP2, VP4, VP6, and VP7 gene, respectively, in Sf9 cells. The CPE was detected after 72h (Fig. 5.2). After 72h of transfection, the supernatant of infected Sf9 cells was subjected to plaque assay and recombinant plaques were easily distinguished from non-recombinant, because the transfer vector with lacZ gene made recombinant baculovirus blue plaque (Fig. 5.3). Recombinant baculoviruses, which expectedly contained VP2, VP4, VP6, and VP7 gene and expressed VP2, VP4, VP6, and VP7 protein, were cultured respectively in Sf9 cells for further assays.

126

Fig. 5.1. Amplification of the VP2 (2,670bp), VP4 (2,325bp), VP6 (1,191bp), and VP7 (978bp) gene of Human rotavirus Wa strain by RT-PCR. M: Gene RulerTM 1kb DNA Ladder.

127

A

B

Fig. 5.2. Representative pictures of uninfected Sf9 cells (A) and infected Sf9 cells (B) with recombinant baculovirus. The cytopathic effect (CPE) was detected after 72h of infection.

128

A

B

C

Fig. 5.3. Plaque forming assay was used for isolation of recombinant baculoviruses expressing of VP2, VP4, VP6, and VP7 protein, respectively. Monolayer of Sf9 cells in a 60mm plate (5x106 cells/plate) was infected with 10-2 (A), 10-3 (B), and 10-4 (C) dilution of transfection viral stock.

129

3.2.

Confirmation of Recombinant Baculoviruses Expressing VP2, VP4, VP6, and VP7 Protein of HRV. VP2, VP4, VP6, and VP7 recombinant proteins expressed

in baculovirus expression system were confirmed by indirect immunofluorescence assay (IFA) using anti His- monoclonal antibody and Western blotting method using anti V5- monoclonal antibody (MAb) as well as specific polyclonal antibody of rabbit anti human rotavirus Wa strain. IFA analysis showed that all four recombinant baculoviruses expressing VP2, VP4, VP6, and VP7 protein, respectively, reacted strongly with anti His-MAb (Fig. 5.4). Also, Western blotting analysis showed that protein bands of 97,9 (for VP2), 85,25 (for VP4), 43,67 (for VP6), and 35,86 (for VP7) kDa were presented in the infected Sf9 cell lysates with recombinant baculoviruses respectively (Fig. 5.5). These observations were in agreement with the expected molecular mass of VP2, VP4, VP6, and VP7 protein of human group A rotavirus. 3.3.

Rotavirus- Virus Like Particle Production The production of double-layered rotavirus like particles

(2/6 VLPs) and triple-layered rotavirus like particles (2/4/6/7 VLPs) generated by co-infection of Spodoptera frugiperda (Sf9) insect

cells

with

different

combinations 130

of

recombinant

baculoviruses, respectively, were analyzed by Western blot (Fig. 5.6). The result showed that recombinant protein bands of VP2 and VP6 in the composition of 2/6 VLPs as well as VP2, VP4, VP6, and VP7 of the composition of 2/4/6/7 VLPs were reacted strongly with a polyclonal antibody rabbit anti HRV Wa strain. The

conformation

of

double-layered

rotavirus

like

particles (2/6 VLPs) and triple-layered rotavirus like particles (2/4/6/7 VLPs) were also confirmed by electron microscopy with the use of negative staining method (Fig 5.7). The observations revealed that rotavirus like particles were produced and they were similar in size (100 nm) and morphology with that of native human rotavirus Wa strain

131

1

2

3

4

5

Fig. 5.4. Detection of expressed VP2, VP4, VP6, and VP7 protein of recombinant baculoviruses in Sf9 cells by indirectimmunofluorescence assay (IFA) using anti His- MAb. Column 1: Mock-infected Sf9 cells as a negative control. Column 2, 3, 4 and 5: Sf9 cells infected with VP2, VP4, VP6, and VP7-recombinant baculoviruses, respectively.

132

A

B

Fig. 5.5. Detection of expressed VP2 (97,9 kDa), VP4 (85,2 kDa), VP6 (43,6 kDa), and VP7 (35,8 kDa) protein of recombinant baculoviruses in Sf9 cell lysates by Western blotting. The recombinant proteins VP2, VP4, VP6, and VP7 were separated on a 10% polyacrylamide gel and then transferred to nitrocellulose membrane and immunostained with anti V5- monoclonal antibody (A) and specific polyclonal antibody rabbit of anti human rotavirus Wa strain (B). M: Precision Plus ProteinTM Standard.

133

Fig. 5.6. Virus like particle composition was confirmed by Western blot using a polyclonal antibody rabbit anti human rotavirus Wa strain. M: Precision Plus ProteinTM Standard. A: Double-layered rotavirus like particles (2/6 VLPs). B: Triple-layered rotavirus like particles (2/4/6/7 VLPs).

134

Fig 5.7. Electron micrograph of rotavirus like particle production. A: native human rotavirus Wa strain, B: Double- layered rotavirus like particles (2/6 VLPs). C: Triple-layered rotavirus like particles (2/4 /6/7 VLPs). Magnification bar equals 100 nm.

135

4.

DISCUSSION Rotaviruses

protein

layers

are

composed

surrounding

11

of

three

segments

concentric of

double-

stranded RNA [Estes, 2001]. The innermost layer is composed mainly of VP2, which comprises about 90% of the core protein mass and binds to viral RNA [Labbe et al., 1991] and may participate in the replication and encapsidation of the RNA genome. The middle layer is composed entirely of VP6, which comprises >80% of the protein

mass

of

the

particle

and

bears

group-

and

subgroup-specific epitopes [Greenberg et al., 1983], and its removal from subviral particles has been associated with loss of viral transcriptase activity [Sandino et al., 1986], although VP6 by itself shows no polymerase activity. The outer layer is composed of glycoprotein VP7 and dimeric spikes of VP4. The glycoprotein VP7 and

the

spike

protein

VP4

independently

elicit

neutralizing antibodies and induce protective immunity [Estes, 2001; Hoshino et al., 1985]. Gene

cloning

and

expression

have

been

successfully used to assess the structural and functional roles of individual viral proteins. The in vivo assembly of virus-like particles after the simultaneous expression of their component proteins offers the possibility of 136

studying

viral

replication, particles

protein

and

functions

assembly.

carrying

relevant

in

virus

structure,

Furthermore, viral

antigens

virus-like may

prove

useful for the development of a vaccine [Gonzalez et al., 2004; Gonzalez and Affranchino, 1995]. In this study, recombinant proteins of four structural proteins of human rotavirus (VP2, VP6, VP7, and VP4) have been expressed successfully using baculovirus expression system. The recombinant proteins all revealed positively immunoreaction

when

reacted

with

antisera

against

human

rotavirus strain Wa. The molecular weight of VP2-, VP4-, VP6-, and VP7- recombinant protein observed in this study was in agreement with the expected molecular mass of their native structural proteins of human group A rotavirus.

These

recombinant

materials

will

be

very

important and reliable source for studying their protein structures and functions in order to better understanding of human rotavirus. It has been shown that recombinant rotavirus VP2 assembles into core-like particles, and that co-expression of this protein with VP6 results in the formation of double-layered virus-like particles [Labbe et al., 1991; Tosser et al., 1992]. Recently, triplelayered rotaviruslike particles were obtained in vivo after simultaneous 137

expression of the capsid proteins VP2, VP4, VP6 and VP7 [Crawford et al., 1994]. Here we have produced double-layered rotavirus-like particles (2/6 VLPs) and triplelayered rotavirus-like particles (2/4/6/7 VLPs). The protein compositions of 2/6 VLPs and 2/4/6/7 VLPs have been confirmed positively by western blot and electron microscopy

(EM)

with

the

use

of

negative

staining

method. Virus like particles represent a novel class of subunit

vaccine

that

are

able

to

stimulate

efficient

cellular and humoral immune responses for both viral and non-viral diseases

[Noad and Roy, 2003]. They are

safer than many live virus preparations because they are usually free of viral genetic material. In addition, they are more effective than many subunit vaccines because they are more conformationally authentic.

138

VII GENERAL DISCUSSION The development of new, efficient, and better rotavirus

vaccines

depends

on

a

library

of

good

surveillance data that monitors change in the populations of

circulating

rotavirus.

Even

though

current

epidemiological data have been collected from nationwide or individual studies from provinces in South Korea [Kang et al., 2005; Kim et al., 2005; Min et al., 2004; Moon et al., 2007], the continuous study in Seoul, is particularly significant because this capital city has a very

high

density

of

inhabitants.

Approximately

10

million people reside here nearly a quarter of the entire population of South Korea. In this study, fecal samples from children with acute infectious diarrhea were collected and tested in the same hospitals in Seoul as those participating in a previous surveillance study conducted between 1998 and 2000 [Song et al., 2003]. Four major G serotypes (G1 4) have been documented worldwide [Beards et al., 1989; Santos and Hoshino, 2005] and until 1996, G1 appeared to be the most prevalent strain followed by strains G4, G2, and G3 [Gentsch et al., 1996]. A similar 139

trend in the prevalence of G serotype strains is evident from studies conducted over the past 10 years in South Korea. Until 1997, G1 was also the most prevalent strain (45 - 81%) regardless of geographical area or season [Kim et al., 1999; Kim, 1993; Kim et al., 1990; Seo and Sim, 2000]. Since then, the predominant G type strain became G4 (28.0 - 40.9%) [Kang et al., 2005; Kim et al., 2002; Song et al., 2003], then G2 (40.9- 50.6%) [Min et al., 2004; Moon et al., 2007] and more recently, G9 (39%) [Kim et al., 2005]. Study data from this 2-year investigation indicates that the G1 strain (35.6%) is again, the most prevalent serotype replacing the G4 (40.5 - 40.9%) strain that was dominant in the previous Seoul survey [Kim et al., 2002; Song et al., 2003]. It is known that the antigenic variation within a serotype is a mechanism by which variants of rotavirus emerge

to

escape

host

immunity.

Studies

of

intragenotype diversity are the key model to define genetic

and

antigenic

variation

of

rotavirus

by

classification of genotypes into lineages and sublineages, providing a profile of strains that could potentially change vaccine efficacy [Araujo et al., 2007; Bok et al., 2002; Martella et al., 2005; Wen et al., 1995]. VP7 gene analysis of G1 strains has been reported and revealed 140

considerable genetic diversity and at least four major global lineages of serotype G1 VP7 occurred within rotaviruses

collected

from

diverse

geographical

locations [Araujo et al., 2007; Arista et al., 2006; Berois et al., 2003; Jin et al., 1996; Maunula and von Bonsdorff, 1998; Parra et al., 2005; Parra et al., 2007a; Trinh et al., 2007; Xin et al., 1993]. In this work, the phylogenetic analysis revealed differences in the VP7 genetic lineages among prevalent Korean G1 strains. While all Korean G1 strains circulating in recent years [Le et al., 2008; Moon et al., 2007] clustered into VP7 genetic lineage I, the old Korean G1 strain (Kor-64) belonged to VP7 genetic lineage IV. Although G9 was not detected until 2002 [Min et al., 2004; Moon et al., 2007; Song et al., 2003], its prevalence, especially in rural provinces, has increased from 11% to 39% [Kang et al., 2005; Kim et al., 2005]. In urban areas, however, G9 occurs with far lower prevalence, 1.2% [Kim et al., 2002; Min et al., 2004; Moon

et

al.,

2007;

Song

et

al.,

2003],

a

finding

consistent with the observation in this study, that G9 strains were found in only four samples from Seoul (1.0%).

One

possible

explanation

for

the

higher

prevalence in small rural areas, 39% in the Jeongeub 141

District [Kim et al., 2005], may be the occurrence of outbreaks of infection. Previous data of sequencing and phylogenetic analysis of the VP7 gene has shown distinct genetic diversity among G9 rotavirus strains and six lineages with eleven sublineages have been described to date

[Bozdayi et al., 2008; Phan et al., 2007; Rahman

et al., 2005; Ramachandran et al., 2000; Stupka et al., 2007]. Our phylogenetic analysis revealed that CAU 202 and all other Korean G9 strains deposited in the GenBank databases clustered into VP7 genetic lineage III. The G9 human rotavirus strains have been recently found in some parts of Korea [Kang et al., 2005; Kim et al., 2005], however,

there

is

no

information

of

isolation

and

characterization as well of G9 human rotavirus strains to date. So, the isolation and characterization of G9 strain (CAU 202) done in this study are very necessary and significant

to

better

understanding

the

genetic

information and relationship as well among G9 strains isolated in different countries. G12 rotaviruses were first detected in 1987 among children with diarrhea from the Philippines [Taniguchi et al., 1990], but no further cases were reported until 1998. Nonetheless, G12 rotaviruses have spread globally and have been detected in the United States [Griffin et 142

al., 2002], Thailand [Pongsuwanna et al., 2002], India [Das et al., 2003], Japan [Shinozaki et al., 2004], Italy [Grassi et al., 2006], Argentina [Castello et al., 2006], Brazil [Pietruchinski et al., 2006], Nepal [Uchida et al., 2006], Slovenia [Steyer et al., 2007b], Hungary [Banyai et al., 2007], and Bangladesh [Rahman et al., 2007b]. A striking increase in the prevalence of G12 from 4.2% (2003) to 30% (2005) was reported in India [Samajdar et al., 2006], mirroring the rise of G9 in the late 1990s [Iturriza-Gomara et al., 2000]. As a result, G12 is now the sixth most important genotype in the world [Rahman et al., 2007a]. In this study, the phylogenetic analysis of nucleotide sequences from two G12 isolates, CAU 195 and CAU 214 showed that they clustered tightly with the G12 serotype with similarities in excess of 90%. This article

is

the

first

to

report

on

the

comprehensive

validation of G12 strains in South Korea. The data from this molecular investigation demonstrates that in Seoul, South Korea, there is a high level of diversity among Gand P type rotavirus, and that the prevalence of wellestablished or newly, emerging serotypes such as G8P[8] and

G12P[6]

changes

relatively

quickly.

More

surveillance studies are vital for amassing information on the molecular epidemiology of unusual serotypes. 143

Only by collating large volumes of stringent data, can an efficient,

evidence

based

program

for

vaccine

development be progressed. Gene cloning and expression have been successfully used to assess the structural and functional roles of individual viral proteins. The in vivo assembly of virus-like particles after the simultaneous expression

of

their

component

proteins

offers

the

possibility of studying viral protein functions in virus structure, replication, and assembly. Furthermore, viruslike particles carrying relevant viral antigens may prove useful for the development of a vaccine. In this study, the recombinant proteins of four structural proteins of VP2, VP4, VP6 and VP7 of human rotavirus have been produced respectively and used as specific materials for generation of rotavirus like particle. This may be a new approach for developing a new generation of subunit vaccine.

144

REFERENCES Adah MI, Nagashima S, Wakuda M, Taniguchi K. 2003. Close relationship between G8-serotype bovine and human rotaviruses

isolated

in

Nigeria.

J

Clin

Microbiol

41(8):3945-3950. Adah MI, Wade A, Taniguchi K. 2001. Molecular epidemiology of rotaviruses in Nigeria: detection of unusual strains with G2P[6]

and

G8P[1]

specificities.

J

Clin

Microbiol

39(11):3969-3975. Adkins JC, Wagstaff AJ. 1998. Recombinant hepatitis B vaccine: a review of its immunogenicity and protective efficacy against hepatitis B. BioDrugs 10(2):137-158. Ansaldi F, Pastorino B, Valle L, Durando P, Sticchi L, Tucci P, Biasci P, Lai P, Gasparini R, Icardi G. 2007. Molecular characterization of a new variant of rotavirus P[8]G9 predominant in a sentinel-based survey in central Italy. J Clin Microbiol 45(3):1011-1015. Araujo IT, Assis RM, Fialho AM, Mascarenhas JD, Heinemann MB, Leite JP. 2007. Brazilian P[8],G1, P[8],G5, P[8],G9, and P[4],G2 rotavirus strains: nucleotide sequence and phylogenetic analysis. J Med Virol 79(7):995-1001. Arista S, Giammanco GM, De Grazia S, Colomba C, Martella V. 2005. Genetic variability among serotype G4 Italian human rotaviruses. J Clin Microbiol 43(3):1420-1425. Arista S, Giammanco GM, De Grazia S, Ramirez S, Lo Biundo C, Colomba C, Cascio A, Martella V. 2006. Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population. J Virol 80(21):10724145

10733. Ball JM, Tian P, Zeng CQ, Morris AP, Estes MK. 1996. Agedependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272(5258):101-104. Banerjee I, Ramani S, Primrose B, Moses P, Iturriza-Gomara M, Gray JJ, Jaffar S, Monica B, Muliyil JP, Brown DW, Estes MK,

Kang

G.

2006.

Comparative

study

of

the

epidemiology of rotavirus in children from a communitybased birth cohort and a hospital in South India. J Clin Microbiol 44(7):2468-2474. Banyai K, Bogdan A, Kisfali P, Molnar P, Mihaly I, Melegh B, Martella V, Gentsch JR, Szucs G. 2007. Emergence of serotype G12 rotaviruses, Hungary. Emerg Infect Dis 13(6):916-919. Barril PA, Martinez LC, Giordano MO, Castello AA, Rota RP, Isa MB, Masachessi G, Ferreyra LJ, Glikmann G, Nates SV. 2006. Detection of group a human rotavirus G9 genotype circulating in Cordoba, Argentina, as early as 1980. J Med Virol 78(8):1113-1118. Beards GM, Desselberger U, Flewett TH. 1989. Temporal and geographical distributions of human rotavirus serotypes, 1983 to 1988. J Clin Microbiol 27(12):2827-2833. Berois M, Libersou S, Russi J, Arbiza J, Cohen J. 2003. Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay

from

1996-1999.

J

Med

Virol

71(3):456-462. Bertolotti-Ciarlet A, Ciarlet M, Crawford SE, Conner ME, Estes MK. 2003. Immunogenicity and protective efficacy of 146

rotavirus 2/6-virus-like particles produced by a dual baculovirus

expression

vector

and

administered

intramuscularly, intranasally, or orally to mice. Vaccine 21(25-26):3885-3900. Birch CJ, Rodger SM, Marshall JA, Gust ID. 1983. Replication of human rotavirus in cell culture. J Med Virol 11(3):241250. Bok K, Matson DO, Gomez JA. 2002. Genetic variation of capsid protein VP7 in genotype g4 human rotavirus strains: simultaneous emergence and spread of different lineages in Argentina. J Clin Microbiol 40(6):2016-2022. Bozdayi G, Dogan B, Dalgic B, Bostanci I, Sari S, Battaloglu NO, Rota S, Dallar Y, Nishizono A, Nakagomi O, Ahmed K. 2008. Diversity of human rotavirus G9 among children in Turkey. J Med Virol 80(4):733-740. Brandtzaeg P. 2007. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 25(30):5467-5484. Browning GF, Snodgrass DR, Nakagomi O, Kaga E, Sarasini A, Gerna G. 1992. Human and bovine serotype G8 rotaviruses may be derived by reassortment. Arch Virol 125(1-4):121128. Carmona RC. 2006. Human rotavirus serotype g9, sao paulo, Brazil, 1996-2003. Emerg Infect Dis 12(6):963-968. Carmona RC, Timenetsky Mdo C, Morillo SG, Richtzenhain LJ. 2006. Human rotavirus serotype G9, Sao Paulo, Brazil, 1996-2003. Emerg Infect Dis 12(6):963-968. Carvalho-Costa FA, Assis RM, Fialho AM, Boia MN, Alves DP, Martins CM, Leite JP. 2006. Detection and molecular 147

characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004. Mem Inst Oswaldo Cruz 101(3):291-294. Castello AA, Arguelles MH, Rota RP, Olthoff A, Jiang B, Glass RI,

Gentsch

JR,

Glikmann

G.

2006.

Molecular

Epidemiology of Group A Rotavirus Diarrhea among Children in Buenos Aires, Argentina, from 1999 to 2003 and Emergence of the Infrequent Genotype G12. J Clin Microbiol 44(6):2046-2050. Chen SY, Chang YC, Lee YS, Chao HC, Tsao KC, Lin TY, Ko TY, Tsai CN, Chiu CH. 2007. Molecular epidemiology and clinical

manifestations

of

viral

gastroenteritis

in

hospitalized pediatric patients in Northern Taiwan. J Clin Microbiol 45(6):2054-2057. Ciarlet M, Crawford SE, Barone C, Bertolotti-Ciarlet A, Ramig RF, Estes MK, Conner ME. 1998. Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity. J Virol 72(11):9233-9246. Ciarlet M, Liprandi F, Conner ME, Estes MK. 2000. Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. Arch Virol 145(2):371-383. Clark HF, Lawley DA, Schaffer A, Patacsil JM, Marcello AE, Glass RI, Jain V, Gentsch J. 2004. Assessment of the epidemic potential of a new strain of rotavirus associated with the novel G9 serotype which caused an outbreak in the United States for the first time in the 1995-1996 season. J Clin Microbiol 42(4):1434-1438. 148

Conner ME, Zarley CD, Hu B, Parsons S, Drabinski D, Greiner S, Smith R, Jiang B, Corsaro B, Barniak V, Madore HP, Crawford S, Estes MK. 1996. Virus-like particles as a rotavirus subunit vaccine. J Infect Dis 174 Suppl 1:S88-92. Coste A, Sirard JC, Johansen K, Cohen J, Kraehenbuhl JP. 2000. Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea. J Virol 74(19):8966-8971. Crawford SE, Estes MK, Ciarlet M, Barone C, O'Neal CM, Cohen J, Conner ME. 1999. Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles. J Virol 73(6):4813-4822. Crawford SE, Labbe M, Cohen J, Burroughs MH, Zhou YJ, Estes MK.

1994.

Characterization

of

virus-like

particles

produced by the expression of rotavirus capsid proteins in insect cells. J Virol 68(9):5945-5952. Cunliffe NA, Gentsch JR, Kirkwood CD, Gondwe JS, Dove W, Nakagomi O, Nakagomi T, Hoshino Y, Bresee JS, Glass RI, Molyneux ME, Hart CA. 2000. Molecular and serologic characterization of novel serotype G8 human rotavirus strains detected in Blantyre, Malawi. Virology 274(2):309-320. Cunliffe NA, Gondwe JS, Graham SM, Thindwa BD, Dove W, Broadhead RL, Molyneux ME, Hart CA. 2001. Rotavirus strain diversity in Blantyre, Malawi, from 1997 to 1999. J Clin Microbiol 39(3):836-843. Cunliffe NA, Nakagomi O. 2005. A critical time for rotavirus vaccines: a review. Expert Rev Vaccines 4(4):521-532. 149

Cunliffe NA, Woods PA, Leite JP, Das BK, Ramachandran M, Bhan MK, Hart CA, Glass RI, Gentsch JR. 1997. Sequence analysis

of

NSP4

gene

of

human

rotavirus

allows

classification into two main genetic groups. J Med Virol 53(1):41-50. Das BK, Gentsch JR, Cicirello HG, Woods PA, Gupta A, Ramachandran M, Kumar R, Bhan MK, Glass RI. 1994. Characterization of rotavirus strains from newborns in New Delhi, India. J Clin Microbiol 32(7):1820-1822. Das S, Varghese V, Chaudhury S, Barman P, Mahapatra S, Kojima K, Bhattacharya SK, Krishnan T, Ratho RK, Chhotray GP, Phukan AC, Kobayashi N, Naik TN. 2003. Emergence of novel human group A rotavirus G12 strains in India. J Clin Microbiol 41(6):2760-2762. Diwakarla CS, Palombo EA. 1999. Genetic and antigenic variation of capsid protein VP7 of serotype G1 human rotavirus isolates. J Gen Virol 80 ( Pt 2):341-344. Doan LT, Okitsu S, Nishio O, Pham DT, Nguyen DH, Ushijima H. 2003. Epidemiological features of rotavirus infection among hospitalized children with gastroenteristis in Ho Chi Minh City, Vietnam. J Med Virol 69(4):588-594. Duan ZJ, Li DD, Zhang Q, Liu N, Huang CP, Jiang X, Jiang B, Glass R, Steele D, Tang JY, Wang ZS, Fang ZY. 2007. Novel human rotavirus of genotype G5P[6] identified in a stool specimen from a Chinese girl with diarrhea. J Clin Microbiol 45(5):1614-1617. Ellens DJ, de Leeuw PW, Straver PJ, van Balken JA. 1978. Comparison of five diagnostic methods for the detection 150

of rotavirus antigens in calf faeces. Med Microbiol Immunol 166(1-4):157-163. Espejo RT, Calderon E, Gonzalez N, Salomon A, Martuscelli A, Romero P. 1979. Presence of two distinct types of rotavirus in infants and young children hospitalized with acute gastroenteritis in Mexico City, 1977. J Infect Dis 139(4):474-477. Espinola EE, Amarilla A, Arbiza J, Parra GI. 2008. Sequence and phylogenetic

analysis

of

the

VP4

gene

of

human

rotaviruses isolated in Paraguay. Arch Virol 153(6):10671073. Estes MK. 2001. Rotaviruses and their replication. In: Knipe DM, Howley RM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors Fields Virology, 4th edn, vol 2Philadelphia: Lippincott, Williams and Wilkins:p. 1747– 1785. Estes MK, Cohen J. 1989. Rotavirus gene structure and function. Microbiol Rev 53(4):410-449. Estes MK, Graham DY. 1980. Identification of rotaviruses of different origins by the plaque-reduction test. Am J Vet Res 41(1):151-152. Fang ZY, Wang B, Kilgore PE, Bresee JS, Zhang LJ, Sun LW, Du ZQ, Tang JY, Hou AC, Shen H, Song XB, Nyambat B, Hummelman E, Xu ZY, Glass RI. 2005. Sentinel hospital surveillance for rotavirus diarrhea in the People's Republic of China, August 2001-July 2003. J Infect Dis 192 Suppl 1:S94-99. Fang ZY, Yang H, Qi J, Zhang J, Sun LW, Tang JY, Ma L, Du 151

ZQ, He AH, Xie JP, Lu YY, Ji ZZ, Zhu BQ, Wu HY, Lin SE, Xie HP, Griffin DD, Ivanoff B, Glass RI, Gentsch JR. 2002. Diversity of rotavirus strains among children with acute diarrhea in China: 1998-2000 surveillance study. J Clin Microbiol 40(5):1875-1878. Feeney SA, Mitchell SJ, Mitchell F, Wyatt DE, Fairley D, McCaughey C, Coyle PV, O'Neill HJ. 2006. Association of the G4 rotavirus genotype with gastroenteritis in adults. J Med Virol 78(8):1119-1123. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783 - 791. Felsenstein

J.

1993.

PHYLIP

(Phylogenetic

Inference

Package), version 3.5c. Seattle, USA: Department of Genetics, University of Washington. Fernandez FM, Conner ME, Hodgins DC, Parwani AV, Nielsen PR, Crawford SE, Estes MK, Saif LJ. 1998. Passive immunity to bovine rotavirus in newborn calves fed colostrum

supplements

from

cows

immunized

with

recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines. Vaccine 16(5):507-516. Fischer TK, Page NA, Griffin DD, Eugen-Olsen J, Pedersen AG, Valentiner-Branth P, Molbak K, Sommerfelt H, Nielsen NM.

2003.

Characterization

of

incompletely

typed

rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections. Virology 311(1):125-133. Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, Das BK, Bhan MK. 1992. Identification of group A 152

rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol 30(6):1365-1373. Gentsch JR, Laird AR, Bielfelt B, Griffin DD, Banyai K, Ramachandran M, Jain V, Cunliffe NA, Nakagomi O, Kirkwood CD, Fischer TK, Parashar UD, Bresee JS, Jiang B, Glass RI. 2005. Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis 192 Suppl 1:S146-159. Gentsch JR, Woods PA, Ramachandran M, Das BK, Leite JP, Alfieri A, Kumar R, Bhan MK, Glass RI. 1996. Review of G and P typing results from a global collection of rotavirus strains: implications for vaccine development. J Infect Dis 174 Suppl 1:S30-36. Gerna G, Sarasini A, Zentilin L, Di Matteo A, Miranda P, Parea M, Battaglia M, Milanesi G. 1990. Isolation in Europe of 69 M-like (serotype 8) human rotavirus strains with either subgroup

I

or

II

specificity

and

a

long

RNA

electropherotype. Arch Virol 112(1-2):27-40. Glass RI, Kilgore PE, Holman RC, Jin S, Smith JC, Woods PA, Clarke MJ, Ho MS, Gentsch JR. 1996. The epidemiology of rotavirus diarrhea in the United States: surveillance and estimates of disease burden. J Infect Dis 174 Suppl 1:S511. Gonzalez AM, Nguyen TV, Azevedo MS, Jeong K, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. 2004. Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine 153

strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM. Clin Exp Immunol 135(3):361-372. Gonzalez SA, Affranchino JL. 1995. Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6. J Gen Virol 76 ( Pt 9):2357-2360. Gouvea V, Brantly M. 1995. Is rotavirus a population of reassortants? Trends Microbiol 3(4):159-162. Gouvea V, Glass RI, Woods P, Taniguchi K, Clark HF, Forrester B,

Fang

ZY.

1990.

Polymerase

chain

reaction

amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol 28(2):276-282. Gouvea V, Lima RC, Linhares RE, Clark HF, Nosawa CM, Santos N. 1999. Identification of two lineages (WA-like and F45-like) within the major rotavirus genotype P[8]. Virus Res 59(2):141-147. Gouvea V, Santos N, Timenetsky Mdo C. 1994. Identification of bovine and porcine rotavirus G types by PCR. J Clin Microbiol 32(5):1338-1340. Grassi T, De Donno A, Guido M, Gabutti G. 2006. G-genotyping of rotaviruses in stool samples in Salento, Italy. J Prev Med Hyg 47(4):138-141. Greenberg H, McAuliffe V, Valdesuso J, Wyatt R, Flores J, Kalica A, Hoshino Y, Singh N. 1983. Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infect Immun 39(1):91-99. Griffin DD, Kirkwood CD, Parashar UD, Woods PA, Bresee JS, 154

Glass RI, Gentsch JR. 2000. Surveillance of rotavirus strains in the United States: identification of unusual strains. The National Rotavirus Strain Surveillance System collaborating laboratories. J Clin Microbiol 38(7):27842787. Griffin DD, Nakagomi T, Hoshino Y, Nakagomi O, Kirkwood CD,

Parashar

UD,

Glass

RI,

Gentsch

JR.

2002.

Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related to bovine rotaviruses. Virology 294(2):256-269. Group

TFIS.

2007.

Quadrivalent

vaccine

against

human

papillomavirus to prevent high-grade cervical lesions. N Engl J Med 356(19):1915-1927. Gulati BR, Deepa R, Singh BK, Rao CD. 2007. Diversity in Indian equine rotaviruses: identification of genotype G10,P6[1] and G1 strains and a new VP7 genotype (G16) strain in specimens from diarrheic foals in India. J Clin Microbiol 45(7):2354. Herring AJ, Inglis NF, Ojeh CK, Snodgrass DR, Menzies JD. 1982. Rapid diagnosis of rotavirus infection by direct detection

of

viral

nucleic

acid

in

silver-stained

polyacrylamide gels. J Clin Microbiol 16(3):473-477. Holmes JL, Kirkwood CD, Gerna G, Clemens JD, Rao MR, Naficy AB, Abu-Elyazeed R, Savarino SJ, Glass RI, Gentsch

JR.

1999.

Characterization

of

unusual

G8

rotavirus strains isolated from Egyptian children. Arch Virol 144(7):1381-1396. 155

Hoshino Y, Jones RW, Ross J, Honma S, Santos N, Gentsch JR, Kapikian

AZ.

2004.

Rotavirus

serotype

G9

strains

belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than

those

belonging

to

lineage

2

or

3.

J

Virol

78(14):7795-7802. Hoshino Y, Sereno MM, Midthun K, Flores J, Kapikian AZ, Chanock RM. 1985. Independent segregation of two antigenic

specificities

(VP3

and

VP7)

involved

in

neutralization of rotavirus infectivity. Proceedings of the National Academy of Sciences, USA 82:8701 - 8704. Hung LC, Wong SL, Chan LG, Rosli R, Ng AN, Bresee JS. 2006. Epidemiology and strain characterization of rotavirus diarrhea in Malaysia. Int J Infect Dis. Iosef C, Van Nguyen T, Jeong K, Bengtsson K, Morein B, Kim Y, Chang KO, Azevedo MS, Yuan L, Nielsen P, Saif LJ. 2002. Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-likeparticles associated with immunostimulating complexes. Vaccine 20(13-14):1741-1753. Isegawa Y, Nakagomi O, Nakagomi T, Ishida S, Uesugi S, Ueda S. 1993. Determination of bovine rotavirus G and P serotypes by polymerase chain reaction. Mol Cell Probes 7(4):277-284. Istrate C, Hinkula J, Charpilienne A, Poncet D, Cohen J, Svensson L, Johansen K. 2008. Parenteral administration of RF 8-2/6/7 rotavirus-like particles in a one-dose 156

regimen induce protective immunity in mice. Vaccine 26(35):4594-4601. Iturriza-Gomara M, Anderton E, Kang G, Gallimore C, Phillips W, Desselberger U, Gray J. 2003. Evidence for genetic linkage between the gene segments encoding NSP4 and VP6 proteins in common and reassortant human rotavirus strains. J Clin Microbiol 41(8):3566-3573. Iturriza-Gomara M, Cubitt D, Steele D, Green J, Brown D, Kang G, Desselberger U, Gray J. 2000. Characterisation of rotavirus G9 strains isolated in the UK between 1995 and 1998. J Med Virol 61(4):510-517. Iturriza Gomara M, Wong C, Blome S, Desselberger U, Gray J. 2002. Molecular characterization of VP6 genes of human rotavirus

isolates:

correlation

of

genogroups

with

subgroups and evidence of independent segregation. J Virol 76(13):6596-6601. Jain V, Das BK, Bhan MK, Glass RI, Gentsch JR. 2001. Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India. J Clin Microbiol 39(10):3524-3529. Jiang B, Estes MK, Barone C, Barniak V, O'Neal CM, Ottaiano A, Madore HP, Conner ME. 1999. Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles. Vaccine 17(7-8):1005-1013. Jin Q, Ward RL, Knowlton DR, Gabbay YB, Linhares AC, Rappaport R, Woods PA, Glass RI, Gentsch JR. 1996. Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses. 157

Arch Virol 141(11):2057-2076. Jiraphongsa C, Bresee JS, Pongsuwanna Y, Kluabwang P, Poonawagul U, Arporntip P, Kanoksil M, Premsri N, Intusoma U. 2005. Epidemiology and burden of rotavirus diarrhea in Thailand: results of sentinel surveillance. J Infect Dis 192 Suppl 1:S87-93. Joensuu J, Koskenniemi E, Vesikari T. 1998. Symptoms associated

with

rhesus-human

reassortant

rotavirus

vaccine in infants. Pediatr Infect Dis J 17(4):334-340. John TJ, Jayabal P. 1972. Oral polio vaccination of children in the tropics. I. The poor seroconversion rates and the absence of viral interference. Am J Epidemiol 96(4):263269. Jones RW, Ross J, Hoshino Y. 2003. Identification of parental origin of cognate dsRNA genome segment(s) of rotavirus reassortants by constant denaturant gel electrophoresis. J Clin Virol 26(3):347-354. Jukes TH, Cantor CR. 1969. Evolution of protein molecules: In: Munro HN, editor. Mammalian protein metabolism. Vol. 3. New York: Academic Press:pp 21-132. Kaga E, Tobita M, Saito T, Iizuka M, Urayama O, Nakagomi T, Uesugi S, Nakagomi O. 1994. Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children. Clin Diagn Virol 2(6):359-366. Kalica AR, Greenberg HB, Espejo RT, Flores J, Wyatt RG, Kapikian AZ, Chanock RM. 1981. Distinctive ribonucleic 158

acid patterns of human rotavirus subgroups 1 and 2. Infect Immun 33(3):958-961. Kang JO, Kilgore P, Kim JS, Nyambat B, Kim J, Suh HS, Yoon Y, Jang S, Chang C, Choi S, Kim MN, Gentsch J, Bresee J, Glass R. 2005. Molecular epidemiological profile of rotavirus in South Korea, July 2002 through June 2003: emergence of G4P[6] and G9P[8] strains. J Infect Dis 192 Suppl 1:S57-63. Kapikian AZ, Hoshino Y, Chanock RM. 2001. Rotaviruses. In: Knipe DM, Howley RM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors Fields Virology, 4th edn,

vol

2

Philadelphia:

Lippincott,

Williams

and

Wilkins:p. 1787–1825. Kapikian AZ, Simonsen L, Vesikari T, Hoshino Y, Morens DM, Chanock RM, La Montagne JR, Murphy BR. 2005. A hexavalent

human

rotavirus-bovine

reassortant

vaccine

designed

for

use

rotavirus in

(UK)

developing

countries and delivered in a schedule with the potential to eliminate the risk of intussusception. J Infect Dis 192 Suppl 1:S22-29. Kerin

TK,

Kane

EM,

Glass

RI,

Gentsch

JR.

2007.

Characterization of VP6 genes from rotavirus strains collected in the United States from 1996-2002. Virus Genes 35(3):489-495. Khamrin P, Maneekarn N, Peerakome S, Chan-it W, Yagyu F, Okitsu S, Ushijima H. 2007a. Novel porcine rotavirus of genotype P[27] shares new phylogenetic lineage with G2 porcine rotavirus strain. Virology 361(2):243-252. 159

Khamrin P, Peerakome S, Tonusin S, Malasao R, Okitsu S, Mizuguchi

M,

Ushijima

H,

Maneekarn

N.

2007b.

Changing pattern of rotavirus G genotype distribution in Chiang Mai, Thailand from 2002 to 2004: decline of G9 and reemergence of G1 and G2. J Med Virol 79(11):17751782. Khamrin P, Peerakome S, Wongsawasdi L, Tonusin S, Sornchai P, Maneerat V, Khamwan C, Yagyu F, Okitsu S, Ushijima H, Maneekarn N. 2006. Emergence of human G9 rotavirus with an exceptionally high frequency in children admitted to hospital with diarrhea in Chiang Mai, Thailand. J Med Virol 78(2):273-280. Kilgore PE, Unicomb LE, Gentsch JR, Albert MJ, McElroy CA, Glass

RI.

1996.

Neonatal

rotavirus

infection

in

Bangladesh: strain characterization and risk factors for nosocomial infection. Pediatr Infect Dis J 15(8):672-677. Kim DS, Park BS, Jung DH, Ahn JM, Kim CJ, Kang SY. 1999. Prevalence and identification of rotaviruses in stool specimens of G and P Typing of Korean Rotavirus 327 patients with acute diarrhoea from several regions of Korea. J Korean Pediatr Soc 42:501–509. Kim EJ, Seo BT, Park SG, Lee JJ. 2002. VP4 and VP7 genotyping of group A rotavirus isolated from diarrhoea patients in Seoul by multiplex PCR. J Bacteriol Virol 32:291–297. Kim JS, Kang JO, Cho SC, Jang YT, Min SA, Park TH, Nyambat B, Jo DS, Gentsch J, Bresee JS, Mast TC, Kilgore PE. 2005. Epidemiological profile of rotavirus infection in the 160

Republic of Korea: results from prospective surveillance in the Jeongeub District, 1 July 2002 through 30 June 2004. J Infect Dis 192 Suppl 1:S49-56. Kim KH. 1993. VP7 typing of group A rotaviruses (Rv) using reverse transcription-polymerase chain reaction (RT-PCR). J Korean Soc Virol 23:39–45. Kim KH, Yang JM, Joo SI, Cho YG, Glass RI, Cho YJ. 1990. Importance of rotavirus and adenovirus types 40 and 41 in acute gastroenteritis in Korean children. J Clin Microbiol 28(10):2279-2284. Kirkwood C, Bogdanovic-Sakran N, Palombo E, Masendycz P, Bugg H, Barnes G, Bishop R. 2003. Genetic and antigenic characterization of rotavirus serotype G9 strains isolated in Australia between 1997 and 2001. J Clin Microbiol 41(8):3649-3654. Kirkwood CD, Cannan D, Bogdanovic-Sakran N, Bishop RF, Barnes

GL.

2006.

National

Rotavirus

Surveillance

Program annual report, 2005-06. Commun Dis Intell 30(4):434-438. Kirkwood CD, Gentsch JR, Hoshino Y, Clark HF, Glass RI. 1999. Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the United States. Virology 256(1):45-53. Kirkwood CD, Palombo EA. 1997. Genetic characterization of the

rotavirus

nonstructural

protein,

NSP4.

Virology

236(2):258-265. Kobayashi N, Lintag IC, Urasawa T, Taniguchi K, Saniel MC, Urasawa S. 1989. Unusual human rotavirus strains having 161

subgroup I specificity and "long" RNA electropherotype. Arch Virol 109(1-2):11-23. Kostouros E, Siu K, Ford-Jones EL, Petric M, Tellier R. 2003. Molecular

characterization

of

rotavirus

strains

from

children in Toronto, Canada. J Clin Virol 28(1):77-84. Labbe M, Charpilienne A, Crawford SE, Estes MK, Cohen J. 1991. Expression of rotavirus VP2 produces empty corelike particles. J Virol 65(6):2946-2952. Laird AR, Gentsch JR, Nakagomi T, Nakagomi O, Glass RI. 2003. Characterization of serotype G9 rotavirus strains isolated in the United States and India from 1993 to 2001. J Clin Microbiol 41(7):3100-3111. Le VP, Kim JY, Cho SL, Nam SW, Lim I, Lee HJ, Kim K, Chung SI, Song W, Lee KM, Rhee MS, Lee JS, Kim W. 2008. Detection of unusual rotavirus genotypes G8P[8] and G12P[6] in South Korea. J Med Virol 80(1):175-182. Lee CN, Lin CC, Kao CL, Zao CL, Shih MC, Chen HN. 2001. Genetic characterization of the rotaviruses associated with a nursery outbreak. J Med Virol 63(4):311-320. Li DD, Duan ZJ, Zhang Q, Liu N, Xie ZP, Jiang B, Steele D, Jiang

X,

Wang

ZS,

Fang

ZY.

2008.

Molecular

characterization of unusual human G5P[6] rotaviruses identified in China. J Clin Virol 42(2):141-148. Lin YP, Chang SY, Kao CL, Huang LM, Chung MY, Yang JY, Chen HY, Taniguchi K, Tsai KS, Lee CN. 2006. Molecular epidemiology of G9 rotaviruses in Taiwan between 2000 and 2002. J Clin Microbiol 44(10):3686-3694. Linhares AC, Mascarenhas JD, Gusmao RH, Gabbay YB, Fialho 162

AM, Leite JP. 2002. Neonatal rotavirus infection in Belem, northern Brazil: nosocomial transmission of a P[6] G2 strain. J Med Virol 67(3):418-426. Linhares AC, Verstraeten T, Wolleswinkel-van den Bosch J, Clemens R, Breuer T. 2006. Rotavirus serotype G9 is associated with more-severe disease in Latin America. Clin Infect Dis 43(3):312-314. Lo JY, Szeto KC, Tsang DN, Leung KH, Lim WW. 2005. Changing epidemiology of rotavirus G-types circulating in Hong Kong, China. J Med Virol 75(1):170-173. Madore HP, Estes MK, Zarley CD, Hu B, Parsons S, Digravio D, Greiner S, Smith R, Jiang B, Corsaro B, Barniak V, Crawford

S,

Conner

ME.

1999.

Biochemical

and

immunologic comparison of virus-like particles for a rotavirus subunit vaccine. Vaccine 17(19):2461-2471. Marques AM, Diedrich S, Huth C, Schreier E. 2007. Group A rotavirus genotypes in Germany during 2005/2006. Arch Virol. Martella V, Ciarlet M, Banyai K, Lorusso E, Arista S, Lavazza A, Pezzotti G, Decaro N, Cavalli A, Lucente MS, Corrente M, Elia G, Camero M, Tempesta M, Buonavoglia C. 2007. Identification of group A porcine rotavirus strains bearing a novel VP4 (P) Genotype in Italian swine herds. J Clin Microbiol 45(2):577-580. Martella V, Ciarlet M, Baselga R, Arista S, Elia G, Lorusso E, Banyai K, Terio V, Madio A, Ruggeri FM, Falcone E, Camero M, Decaro N, Buonavoglia C. 2005. Sequence analysis of the VP7 and VP4 genes identifies a novel VP7 163

gene allele of porcine rotaviruses, sharing a common evolutionary origin with human G2 rotaviruses. Virology 337(1):111-123. Matsuno S, Hasegawa A, Mukoyama A, Inouye S. 1985. A candidate for a new serotype of human rotavirus. J Virol 54(2):623-624. Matthijnssens J, Rahman M, Yang X, Delbeke T, Arijs I, Kabue JP, Muyembe JJ, Van Ranst M. 2006. G8 rotavirus strains isolated in the Democratic Republic of Congo belong to the DS-1-like genogroup. J Clin Microbiol 44(5):18011809. Maunula L, von Bonsdorff CH. 1998. Short sequences define genetic

lineages:

rotaviruses

based

phylogenetic on

partial

analysis sequences

of

group

of

A

genome

segments 4 and 9. J Gen Virol 79 ( Pt 2):321-332. Min BS, Noh YJ, Shin JH, Baek SY, Kim JO, Min KI, Ryu SR, Kim BG, Kim do K, Lee SH, Min HK, Ahn BY, Park SN. 2004. Surveillance study (2000 to 2001) of G- and P-type human rotaviruses circulating in South Korea. J Clin Microbiol 42(9):4297-4299. Moon SS, Green YS, Song JW, Ahn CN, Kim H, Park KS, Song KJ, Lee JH, Baek LJ. 2007. Genetic distribution of group A human rotavirus types isolated in Gyunggi province of Korea, 1999-2002. J Clin Virol 38(1):57-63. Moosai RB, Gardner PS, Almeida JD, Greenaway MA. 1979. A simple immunofluorescent technique for the detection of human rotavirus. J Med Virol 3(3):189-194. Mphahlele MJ, Peenze I, Steele AD. 1999. Rotavirus strains 164

bearing the VP4P[14] genotype recovered from South African children with diarrhoea. Arch Virol 144(5):10271034. Mulholland EK. 2004. Global control of rotavirus disease. Adv Exp Med Biol 549:161-168. Noad R, Roy P. 2003. Virus-like particles as immunogens. Trends Microbiol 11(9):438-444. O'Halloran F, Lynch M, Cryan B, Fanning S. 2002. Application of restriction fragment length polymorphism analysis of VP7-encoding genes: fine comparison of Irish and global rotavirus isolates. J Clin Microbiol 40(2):524-531. O'Neal CM, Clements JD, Estes MK, Conner ME. 1998. Rotavirus 2/6 viruslike particles administered intranasally with cholera toxin, Escherichia coli heat-labile toxin (LT), and LT-R192G induce protection from rotavirus challenge. J Virol 72(4):3390-3393. O'Neal CM, Crawford SE, Estes MK, Conner ME. 1997. Rotavirus virus-like

particles administered mucosally

induce protective immunity. J Virol 71(11):8707-8717. Okada J, Urasawa T, Kobayashi N, Taniguchi K, Hasegawa A, Mise K, Urasawa S. 2000. New P serotype of group A human rotavirus closely related to that of a porcine rotavirus. J Med Virol 60(1):63-69. Paavonen J, Jenkins D, Bosch FX, Naud P, Salmeron J, Wheeler CM, Chow SN, Apter DL, Kitchener HC, Castellsague X, de Carvalho NS, Skinner SR, Harper DM, Hedrick JA, Jaisamrarn U, Limson GA, Dionne M, Quint W, Spiessens B, Peeters P, Struyf F, Wieting SL, Lehtinen MO, Dubin G. 165

2007. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 369(9580):2161-2170. Parashar UD, Gibson CJ, Bresse JS, Glass RI. 2006. Rotavirus and

severe

childhood

diarrhea.

Emerg

Infect

Dis

12(2):304-306. Parashar UD, Hummelman EG, Bresee JS, Miller MA, Glass RI. 2003. Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 9(5):565-572. Parra GI, Bok K, Martinez V, Russomando G, Gomez J. 2005. Molecular characterization and genetic variation of the VP7 gene of human rotaviruses isolated in Paraguay. J Med Virol 77(4):579-586. Parra GI, Espinola EE, Amarilla AA, Stupka J, Martinez M, Zunini M, Galeano ME, Gomes K, Russomando G, Arbiza J. 2007a. Diversity of group A rotavirus strains circulating in Paraguay from 2002 to 2005: detection of an atypical G1 in South America. J Clin Virol 40(2):135-141. Parra GI, Galeano ME, Arbiza J. 2007b. Genetic relationship between porcine rotavirus strains bearing a new P-type. Vet Microbiol 125(1-2):193-195. Peixoto C, Sousa MF, Silva AC, Carrondo MJ, Alves PM. 2007. Downstream processing of triple layered rotavirus like particles. J Biotechnol 127(3):452-461. Phan TG, Okitsu S, Maneekarn N, Ushijima H. 2007. Genetic heterogeneity, evolution and recombination in emerging 166

G9 rotaviruses. Infect Genet Evol 7(5):656-663. Pietruchinski E, Benati F, Lauretti F, Kisielius J, Ueda M, Volotao EM, Soares CC, Hoshino Y, Linhares RE, Nozawa C, Santos N. 2006. Rotavirus diarrhea in children and adults in a southern city of Brazil in 2003: distribution of G/P types and finding of a rare G12 strain. J Med Virol 78(9):1241-1249. Pongsuwanna Y, Guntapong R, Chiwakul M, Tacharoenmuang R, Onvimala N, Wakuda M, Kobayashi N, Taniguchi K. 2002. Detection of a human rotavirus with G12 and P[9] specificity in Thailand. J Clin Microbiol 40(4):1390-1394. Pun SB, Nakagomi T, Sherchand JB, Pandey BD, Cuevas LE, Cunliffe NA, Hart CA, Nakagomi O. 2007. Detection of G12 human rotaviruses in Nepal. Emerg Infect Dis 13(3):482-484. Rahman M, Matthijnssens J, Goegebuer T, De Leener K, Vanderwegen L, van der Donck I, Van Hoovels L, De Vos S, Azim T, Van Ranst M. 2005. Predominance of rotavirus G9

genotype

in

children

hospitalized

for

rotavirus

gastroenteritis in Belgium during 1999-2003. J Clin Virol 33(1):1-6. Rahman M, Matthijnssens J, Yang X, Delbeke T, Arijs I, Taniguchi K, Iturriza-Gomara M, Iftekharuddin N, Azim T, Van Ranst M. 2007a. Evolutionary history and global spread of the emerging g12 human rotaviruses. J Virol 81(5):2382-2390. Rahman M, Sultana R, Ahmed G, Nahar S, Hassan ZM, Saiada F, Podder

G,

Faruque

AS, 167

Siddique

AK,

Sack

DA,

Matthijnssens J, Van Ranst M, Azim T. 2007b. Prevalence of G2P[4] and G12P[6] rotavirus, Bangladesh. Emerg Infect Dis 13(1):18-24. Ramachandran M, Kirkwood CD, Unicomb L, Cunliffe NA, Ward RL, Bhan MK, Clark HF, Glass RI, Gentsch JR. 2000. Molecular

characterization

of

serotype

G9

rotavirus

strains from a global collection. Virology 278(2):436-444. Reidy N, O'Halloran F, Fanning S, Cryan B, O'Shea H. 2005. Emergence

of

G3

and

G9

rotavirus

and

increased

incidence of mixed infections in the southern region of Ireland 2001-2004. J Med Virol 77(4):571-578. Rodriguez-Castillo A, Ramirez-Gonzalez JE, Padilla-Noriega L, Barron BL. 2006. Analysis of human rotavirus G1P[8] strains by RFLP reveals higher genetic drift in the VP7 than the VP4 gene during a 4-year period in Mexico. J Virol Methods 138(1-2):177-183. Ruiz-Palacios GM, Perez-Schael I, Velazquez FR, Abate H, Breuer T, Clemens SC, Cheuvart B, Espinoza F, Gillard P, Innis BL, Cervantes Y, Linhares AC, Lopez P, MaciasParra M, Ortega-Barria E, Richardson V, Rivera-Medina DM, Rivera L, Salinas B, Pavia-Ruz N, Salmeron J, Ruttimann R, Tinoco JC, Rubio P, Nunez E, Guerrero ML, Yarzabal JP, Damaso S, Tornieporth N, Saez-Llorens X, Vergara RF, Vesikari T, Bouckenooghe A, Clemens R, De Vos B, O'Ryan M. 2006. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 354(1):11-22. Saitou N, Nei M. 1987. The neighbor-joining method: a new 168

method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406-425. Samajdar S, Varghese V, Barman P, Ghosh S, Mitra U, Dutta P, Bhattacharya SK, Narasimham MV, Panda P, Krishnan T, Kobayashi N, Naik TN. 2006. Changing pattern of human group A rotaviruses: emergence of G12 as an important pathogen among children in eastern India. J Clin Virol 36(3):183-188. Sandino AM, Jashes M, Faundez G, Spencer E. 1986. Role of the inner

protein

capsid

on

in

vitro

human

rotavirus

transcription. J Virol 60(2):797-802. Santos N, Hoshino Y. 2005. Global distribution of rotavirus serotypes/genotypes

and

its

implication

for

the

development and implementation of an effective rotavirus vaccine. Rev Med Virol 15(1):29-56. Santos N, Volotao EM, Soares CC, Albuquerque MC, da Silva FM,

Chizhikov

V,

Hoshino

Y.

2003.

VP7

gene

polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR. Virus Res 93(1):127-138. Schirrmeier H, Heinrich HW. 1981. [Use of immunofluorescence technic for the diagnosis of rotavirus infection in the calf]. Arch Exp Veterinarmed 35(2):187-198. Seo JK, Sim JG. 2000. Overview of rotavirus infections in Korea. Pediatr Int 42(4):406-410. Shinozaki K, Okada M, Nagashima S, Kaiho I, Taniguchi K. 2004. Characterization of human rotavirus strains with G12 and P[9] detected in Japan. J Med Virol 73(4):612169

616. Song MO, Kim KJ, Chung SI, Lim I, Kang SY, An CN, Kim W. 2003. Distribution of human group a rotavirus VP7 and VP4 types circulating in Seoul, Korea between 1998 and 2000. J Med Virol 70(2):324-328. Steele AD, Ivanoff B. 2003. Rotavirus strains circulating in Africa during 1996-1999: emergence of G9 strains and P[6] strains. Vaccine 21(5-6):361-367. Steele AD, Nimzing L, Peenze I, De Beer MC, Geyer A, Angyo I, Gomwalk NE. 2002. Circulation of the novel G9 and G8 rotavirus strains in Nigeria in 1998/1999. J Med Virol 67(4):608-612. Steele AD, van Niekerk MC, Mphahlele MJ. 1995. Geographic distribution of human rotavirus VP4 genotypes and VP7 serotypes in five South African regions. J Clin Microbiol 33(6):1516-1519. Steyer A, Poljsak-Prijatelj M, Barlic-Maganja D, Jamnikar U, Mijovski JZ, Marin J. 2007a. Molecular characterization of a new porcine rotavirus P genotype found in an asymptomatic pig in Slovenia. Virology 359(2):275-282. Steyer A, Poljsak-Prijatelj M, Bufon TL, Marcun-Varda N, Marin J. 2007b. Rotavirus genotypes in Slovenia: unexpected detection of G8P[8] and G12P[8] genotypes. J Med Virol 79(5):626-632. Stupka JA, Parra GI, Gomez J, Arbiza J. 2007. Detection of human rotavirus G9P[8] strains circulating in Argentina: phylogenetic analysis of VP7 and NSP4 genes. J Med Virol 79(6):838-842. 170

Taniguchi K, Urasawa T, Kobayashi N, Gorziglia M, Urasawa S. 1990. Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: implication for new G serotype specificity. J Virol 64(11):5640-5644. Taniguchi K, Wakasugi F, Pongsuwanna Y, Urasawa T, Ukae S, Chiba S, Urasawa S. 1992. Identification of human and bovine rotavirus serotypes by polymerase chain reaction. Epidemiol Infect 109(2):303-312. Tavares Tde M, Brito WM, Fiaccadori FS, Freitas ER, Parente JA, Costa PS, Giugliano LG, Andreasi MS, Soares CM, Cardoso DD. 2008. Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil. Mem Inst Oswaldo Cruz 103(3):288-294. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):48764882. Tian P, Estes MK, Hu Y, Ball JM, Zeng CQ, Schilling WP. 1995. The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J Virol 69(9):57635772. Timenetsky Mdo C, Santos N, Gouvea V. 1994. Survey of rotavirus

G

and

P

types

associated

with

human

gastroenteritis in Sao Paulo, Brazil, from 1986 to 1992. J Clin Microbiol 32(10):2622-2624. 171

Tosser G, Labbe M, Bremont M, Cohen J. 1992. Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses. J Virol 66(10):5825-5831. Trinh QD, Nguyen TA, Phan TG, Khamrin P, Yan H, Hoang PL, Maneekarn N, Li Y, Yagyu F, Okitsu S, Ushijima H. 2007. Sequence analysis of the VP7 gene of human rotavirus G1 isolated in Japan, China, Thailand, and Vietnam in the context of changing distribution of rotavirus G-types. J Med Virol 79(7):1009-1016. Uchida R, Pandey BD, Sherchand JB, Ahmed K, Yokoo M, Nakagomi

T,

Cuevas

LE,

Cunliffe

NA,

Hart

CA,

Nakagomi O. 2006. Molecular epidemiology of rotavirus diarrhea among children and adults in Nepal: detection of G12 strains with P[6] or P[8] and a G11P[25] strain. J Clin Microbiol 44(10):3499-3505. Unicomb LE, Podder G, Gentsch JR, Woods PA, Hasan KZ, Faruque AS, Albert MJ, Glass RI. 1999. Evidence of highfrequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. J Clin Microbiol 37(6):1885-1891. Urasawa S, Urasawa T, Wakasugi F, Kobayashi N, Taniguchi K, Lintag IC, Saniel MC, Goto H. 1990. Presumptive seventh serotype of human rotavirus. Arch Virol 113(3-4):279-282. Van Damme P, Giaquinto C, Maxwell M, Todd P, Van der Wielen M. 2007. Distribution of rotavirus genotypes in Europe, 2004-2005: the REVEAL Study. J Infect Dis 195 Suppl 1:S17-25. 172

Vieira HL, Estevao C, Roldao A, Peixoto CC, Sousa MF, Cruz PE, Carrondo MJ, Alves PM. 2005. Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production. J Biotechnol 120(1):72-82. Volotao EM, Soares CC, Maranhao AG, Rocha LN, Hoshino Y, Santos N. 2006. Rotavirus surveillance in the city of Rio de Janeiro-Brazil during 2000-2004: detection of unusual strains with G8P[4] or G10P[9] specificities. J Med Virol 78(2):263-272. Wang YH, Kobayashi N, Zhou DJ, Yang ZQ, Zhou X, Peng JS, Zhu ZR, Zhao DF, Liu MQ, Gong J. 2007. Molecular epidemiologic analysis of group A rotaviruses in adults and children with diarrhea in Wuhan city, China, 20002006. Arch Virol 152(4):669-685. Wen L, Ushijima H, Kakizawa J, Fang ZY, Nishio O, Morikawa S, Motohiro T. 1995. Genetic variation in VP7 gene of human rotavirus serotype 2 (G2 type) isolated in Japan, China, and Pakistan. Microbiol Immunol 39(11):911-915. Xin KQ, Morikawa S, Fang ZY, Mukoyama A, Okuda K, Ushijima H. 1993. Genetic variation in VP7 gene of human rotavirus serotype 1 (G1 type) isolated in Japan and China. Virology 197(2):813-816. Yoshinaga M, Phan TG, Nguyen TA, Yan H, Yagyu F, Okitsu S, Muller WE, Ushijima H. 2006. Changing distribution of group A rotavirus G-types and genetic analysis of G9 circulating in Japan. Arch Virol 151(1):183-192. Yuan L, Ishida S, Honma S, Patton JT, Hodgins DC, Kapikian 173

AZ, Hoshino Y. 2004. Homotypic and heterotypic serum isotype-specific

antibody

responses

to

rotavirus

nonstructural protein 4 and viral protein (VP) 4, VP6, and VP7 in infants who received selected live oral rotavirus vaccines. J Infect Dis 189(10):1833-1845. Zhou Y, Li L, Okitsu S, Maneekarn N, Ushijima H. 2003. Distribution of human rotaviruses, especially G9 strains, in

Japan

from

1996

to

2000.

Microbiol

Immunol

47(8):591-599. Zhou Y, Supawadee J, Khamwan C, Tonusin S, Peerakome S, Kim B, Kaneshi K, Ueda Y, Nakaya S, Akatani K, Maneekarn N, Ushijima H. 2001. Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997. J Med Virol 65(3):619-628.

174

ACKNOWLEGMENTS My dear parents not only raised me with love but also taught me how to be an independent, successful, and good person. I am here to express my deeply gratitude to their endless love, support, encouragement and patience throughout my life. I would like to give deeply thanks to my beautiful wife, parents in law, elder brothers and sister

who

have

always

given

me

fully

mental

and

material life. I would especially like to express my heartfelt gratitude and profound respect to Professor Wonyong Kim of the Department of Microbiology, College of Medicine, Chung-Ang University, south of Korea, for his invaluable academic supervision, financial supports, and kindly helps. It is obvious that I would not have been able to follow out my ambition without his supports and academic guidance. My special thanks are given to all Professors of the College of Medicine, Chung-Ang University who have given me useful lectures, suggestions, and advices. Especially, many thanks are directed to Prof. Chung Sang-In, Prof. Kim Ki-Jeong, Prof. Yoon Yoo-Sik of the Department

of

Microbiology, 175

College

of

Medicine,

Chung-Ang University and Prof Kang Shien- Young of the College of Veterinary Medicine, Chungbuk National University

for

their

agreement

to

act

as

the

thesis

examiner. Life would have been just too tedious without the daily contact with my friends Mr Park Hee- Kuk, Mr Kang Sung-Hoon, Mr Jung Yeon-Chang, Miss Cho SungLim, Miss Park Mi-Hark, Miss Jung Min-Young, Miss Togloom Ariunaa, Mr Nguyen Hoai Bac and Miss Nguyen Thi Hai Van. I especially appreciate their friendship, support and helps. I would like to give them my sincere thanks. The last but not the least, I would like to express my infinite gratitude to Professor Truong Van DungDirector of National Institute of Veterinary ResearchVietnam, who has always given me the best advices and assistances during my scientific work in Vietnam and Korea. Above people are very remarkable and significant for me. Their contributions constitute a debt that I may never hope to repay.

LE VAN PHAN 176