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
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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