J Bacteriol Virol.  2007 Dec;37(4):231-240. 10.4167/jbv.2007.37.4.231.

Isolation and Characterization of G9 Human Rotaviruses

• Lee KB
• Kim JW
• Lee SW
• Cheon DS
• Jee YM
• Kang SY

• KMID: 1513583
• DOI: http://doi.org/10.4167/jbv.2007.37.4.231

Abstract

Group A rotaviruses are the most common causes of gastroenteritis among infants and young children. The outer capsid layer of the virus is composed of two structural proteins, VP4 and VP7, and they play important roles in protection by eliciting neutralization antibodies. Group A rotaviruses are subdivided into distinct G and P serotypes according to the antigenic differences of the VP7 and VP4, respectively. Rotavirus G9 serotype was thought to be the fifth most common serotype circulating among the population worldwide. In this study, G9 human rotaviruses (HRV) were isolated from fecal samples using MA104 cells and characterized. Characteristic cytopathic effects of rotavirus were observed and rotaviral antigens were confirmed by indirect immunofluorescence antibody test in MA104 cells inoculated with isolated HRV strains. The nucleotide sequences of the VP7 gene of Korean G9 HRV isolated in this study were determined and compared with those of other recent and prototype G9 rotavirus strains from other parts of the world. Also, the nucleotide sequences of VP4 and NSP4 gene of Korean G9 HRV were determined and compared with those of other rotavirus strains from other countries. The results showed that the Korean HRV isolates belong to a G9, P[8] and NSP4 B genotype. The Korean G9 HRV isolates and their nucleotide sequence data would be usefully applied for the vaccine development of HRV in the near future.

Keyword

G9 Rotavirus; VP4; VP7; NSP4

MeSH Terms

Antibodies
Base Sequence
Capsid
Child
Fluorescent Antibody Technique, Indirect
Gastroenteritis
Genotype
Humans*
Infant
Rotavirus*
Antibodies

Figure

• 

  Figure 1. Electron microscopic detection of rotavirus KNIH-7 in MA104 cells.

• 

  Figure 2. Electrophoretic migration patterns of genomic RNA of cell-adapted Korean G9 human rotavirus isolates. Lane 1, Wa; lane 2, DS-1; lane 3, KNIH-7; lane 4, KNIH-9; lane 5, KNIH-13.

• 

  Figure 3. Phylogenetic analysis of VP4 gene of Korean G9 human rotavirus isolates in comparison with those of other representative P type rotavirus strains; DS-1 (P[4]/P1B, human, AB118025), KO-2 (P[4]/P1B, human, AF401755), RV-5 (P[4]/P1B, human, M32559), 1076 (P[6]/P2A, human, L20877), Wa (P[8]/P1A, human, L34161), VA70 (P[8]/P1A, human, AJ540229), Hochi (P[8]/P1A, human, AB039943), KU (P[8]/P1A, human, M21014), YO (P[8]/P1A, human, AB008279), MO (P[8]/P1A, human, AB008278), P (P[8]/P1A, human, AJ050228), F45 (P[8]/P1A, human, U30716), OP351 (P[8]/P1A, human, AJ302147), OP354 (P[8]/P1A, human, AJ302148), AU-1 (P[9]/P3, human, D10970), and 69M (P[10]/P4, human, M60600). The bar indicates 0.1 substitutions per nucleotide.

• 

  Figure 4. Phylogenetic analysis of NSP4 gene of Korean G9 human rotavirus isolates in comparison with those of other representative NSP4 rotavirus strains; DS-1 (A/G2, human, AF174305), SA11 (A/G3, simian, AF087678), H-2 (A/G3, equine, AF14481), BAP-2 (A/G3, lapine, AF144795), B223 (A/G10, bovine, AF144805), L26 (A/G12, human, AJ311732), E210 (A/G2, human, U59107), CBNU-2 (A/G2, human), MD844 (B/G12, human, AF269688), Wa (B/G1, human, AF093119), ST-3 (B/G4, human, U59110), OSU (B/G5, porcine, D88831), AU32 (B/G9, human, D88830), CBNU-1 (B/G1, human), CBNU-3 (B/G3, human), AU-1 (C/G3, human, D89873), CU-1 (C/G3, canine, AF144806), FRV64 (C/G3, feline, D88833), EW (D/G3, murine, AB003805), and EHP (D/G3, murine, U06336). The bar indicates 0.1 substitutions per nucleotide.

• 

  Figure 5. Phylogenetic analysis of VP7 gene of Korean G9 human rotavirus isolates in comparison with those of other representative G9 rotavirus strains; WI61 (I, human, USA, AB180969), F45 (I, human, Japan, AB180970), AU32 (I, human, Japan, AB045372), 116E (II, human, India, L14072), SP2747VP7 (III, human, Japan, AB045372), 95H115 (III, human, Japan, AB045373), K-1 (III, human, Japan, AB045374), SP1904VP7 (III, human, Japan, AB091754), A2 (III, porcine, USA, AB180971), US1205 (III, human, USA, AF060487), R143 (III, human, Brazil, AF274969), R146 (III, human, Brazil, AF274970), R160 (III, human, Brazil, AF274971), R44 (III, human, Brazil, AF438227), R136 (III, human, Brazil, AF438228), 5001DB/97 (III, human, South Africa, AF529864), INL-1 (III, human, India, AJ250227), BD524 (III, human, Bangladesh, AJ250543), MW47 (III, human, Malawi, AJ250544), CC117 (III, human USA, AJ491153), At694 (III, human USA, AJ491159), BP7 (III, human, India, AJ491161), DL73 (III, human, India, AJ491165), DL38 (III, human, India, AJ491165), EM39 (III, human, USA, AJ491169), In826 (III, human, USA, AJ491173), N23 (III, human, India, AJ491177), NE413 (III, human, USA, AJ491178), NE458 (III, human, USA, AJ491180), Om526 (III, human, USA, AJ491182), Ph158 (III, human, USA, AJ491183), SE121 (III, human, USA, AJ491192), T203 (III, human, China, AY003871), MG9–06 (III, human, Australia, AY307805), Melb-09.12 (III, human, Australia, AY307088), TK2082VP7 (III, human, Japan, AB091755), and Mc345 (III, human, Thailand, D38055). The bar indicates 0.1 substitutions per nucleotide.

Reference

References

1). 박선경, 허연선, 빙혜선, 천두성, 초가기, 전무형. 대전 지역 설사환자 분변 유래 group A rotavirus의 P 및 G 유전자 분포. J Bacteriol Virol. 37:169–175. 2007.

2). 조성림, 안장훈, 김기정, 정상인, 임인석, 김원용. 서울 지역에서 분리한 사람 로타바이러스 비구조단백질 NSP4 의 유전적 변이. J Bacteriol Virol. 36:79–87. 2006.

3). 지영미, 안정배, 천두성, 최우영, 김운호, 이정수, 이강 범, 이회규, 윤재득. 국내 유행 바이러스성 장염의 역학. 소아감염. 11:17–19. 2004.

4). Bon F, Fromantin C, Aho S, Pothier P, Kohli EG. P geno-typing of rotavirus strains circulating in France over a three-year period: detection of G9 and P strain at low frequencies. J Clin Microbiol. 38:1681–1683. 2000.

5). Ciarlet M, Liprandi F, Conner ME, Estes MK. Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. Arch Virol. 145:371–383. 2000.

6). Cohen J, Charpilienne A, Chilmonczyk S, Estes MK. Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus. Virology. 171:131–140. 1989.

7). Estes MK, Cohen J. Rotavirus gene structure and function. Microbiol Rev. 53:410–449. 1989.

8). Estes MK, Palmer EL, Obijeski JF. Rotaviruses: A review. Curr Top Microbiol Immunol. 105:123–184. 1983.

9). Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, Das BK, Bhan MK. Identification of group A rotavirus gene 4 type by polymerase chain reaction. J Clin Microbiol. 30:1365–1373. 1992.

10). Gouvea V, Glass RI, Woods P, Taniguchi K, Clark HF, Forrester B, Fang ZY. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 28:276–282. 1990.

11). Graham DY, Estes MK. Proposed working serologic classification system for rotaviruses. Ann Inst Pasteur. 136:5–12. 1985.

12). Herring AJ, Inglis NF, Ojeh CK, Snodgrass DR, Menzies JD. Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels. J Clin Microbiol. 16:473–477. 1982.

13). Hoshino Y, Sereno MM, Midthun K, Flores J, Kapikian AZ, Chanock RT. Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity. Pro Natl Acad Sci. 82:8701–8704. 1985.

14). Hoshino Y, Wyatt RG, Greenberg HB, Flores J, Kapikian AZ. Serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization. J Infect Dis. 149:694–702. 1984.

15). Iturriza-Gomara M, Gagandeep K, Gray J. Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol. 31:259–265. 2004.

16). Jain V, Das BK, Bhan MK, Glass RI, Gentsch JR. Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India. J Clin Microbiol. 39:3524–3529. 2001.

17). Kang JO, Kilgore P, Kim JS, Nyambat B, Kim JG, Suh HS, Yoon YM, Jang SJ, Chang CH, Choi SW, Kim MN, Gentsch J, Bresee J, Glass R. Molecular epidemiological profile of rotavirus in South Korea, July 2002 through June 2003: Emergence of G4P[6] and G9P[8] strains. J Infect Dis. 192(Suppl 12):S57–S63. 2005.

18). Kang SY, Benfield DA, Gorziglia M, Saif LJ. Characterization of the neutralizing epitopes of the VP7 of the Gottfried strain of porcine rotavirus. J Clin Microbiol. 31:2291–2297. 1993.

19). Kang SY, Nagaraja KV, Newman JA. Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA104 cell line. Avian Dis. 30:494–499. 1986.

20). Kapikian AZ, Hoshino Y, Chanock RM. Rotaviruses. Knipe DM, Howley PM, editors. Fields Virology. 4th ed.p. 1787–1833. Lippincott Williams & Wilkins;Philadelphia: 2001.

21). Kattoura MD, Clapp LL, Patton JT. The rotavirus non-structural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Virology. 191:698–708. 1992.

22). Kirkwood CD, Palombo EA. Genetic characterization of the rotavirus nonstructural protein, NSP4. Virology. 236:258–265. 1997.

23). Laird AR, Gentsch JR, Nakagomi T, Nakagomi O, Glass RI. Characterization of serotype G9 rotavirus strains isolated in the United States and India from 1993 to 2001. J Clin Microbiol. 41:3100–3111. 2003.

24). Lo JY, Szeto KC, Tsang DN, Leung KH, Lim WW. Changing epidemiology of rotavirus G-type circulating in Hong Kong. J Med Virol. 75:170–173. 2004.

25). Mattion NM, Cohen J, Aponte C, Estes MK. Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34. Virology. 190:68–83. 1992.

26). Meyer JC, Bergmann CC, Bellamy AR. Interaction of rota-virus cores with the nonstructural glycoprotein NS28. Virology. 171:98–107. 1989.

27). Poncet D, Aponte C, Cohen J. Rotavirus protein NSP3 (NS34) is bound to the 3′ end consensus sequence of viral mRNAs in infected cells. J Virol. 67:3159–3165. 1993.

28). Santos N, Hoshino Y. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol. 15:29–56. 2005.

29). Santos N, Volotao EM, Soares CC. VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR. Virus Res. 93:127–138. 2003.

30). Sato K, Inaba Y, Shinozaki T, Fujii R, Matumoto M. Isolation of human rotavirus in cell cultures. Arch Virol. 69:155–160. 1981.

31). Tafazoli F, Zeng CQY, Estes MK. The NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized epithelial cells. J Virol. 75:1540–1546. 2001.

32). Yolken R, Arango-Jaramillo S, Eiden J, Vonderfecht S. Lack of genomic reassortment following infection of infant rats with group A and group B rotaviruses. J Infect Dis. 158:1120–1123. 1988.

33). Yoshinaga M, Phan TG, Nquven TA, Yan H, Okitsu S, Muller WE, Ushijima H. Changing distribution of group A rotavirus G-type and genetic analysis of G9 circulating in Japan. Arch Virol. 151:183–192. 2006.