We detected rotavirus G5P[6] with a long RNA pattern in a Vietnamese child with diarrhea. Viral outer capsid protein VP7 and VP4 genes suggest that it likely originated from porcine rotavirus either by genetic reassortment or as whole virions. To our knowledge, this is the first report of human rotavirus G5 in Asia

Ahmed Kamruddin

Anh Dang Duc

Nakagomi Osamu

Nagasaki University's Academic Output SITE: NAOSITE

DISPATCHES1232 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 8, August 2007Rotavirus G5P[6] in Child with Diarrhea, VietnamKamruddin Ahmed,* Dang Duc Anh,† and Osamu Nakagomi*We detected rotavirus G5P[6] with a long RNA pat-tern in a Vietnamese child with diarrhea. Viral outer capsid protein VP7 and VP4 genes suggest that it likely originated from porcine rotavirus either by genetic reassortment or as whole virions. To our knowledge, this is the fi rst report of human rotavirus G5 in Asia.Rotaviruses cause 352,000–592,000 deaths per year in children <5 years of age (1). More than 80% of these deaths occur in developing countries (1). In multicenter tri-als, monovalent and pentavalent rotavirus vaccines were safe and highly effi cacious for children in developing coun-tries (2). Among developing countries in Asia, Vietnam is progressing rapidly toward introducing rotavirus vaccine (3). Rotavirus accounts for 55% of diarrheal diseases in Vietnam, prevalent serotypes are G1, G2, G3, G4, and G9, and 3% of rotaviruses were untypeable (3). This low per-centage of untypeable strains was unanticipated because it is assumed that developing countries are the source of un-usual rotavirus strains (4). Many people in Vietnam live in close contact with domestic animals, which may promote interspecies transmission of rotaviruses and reassortment of human and animal rotaviruses. Therefore, we hypothesized that in rural Vietnam, some rotavirus infections are caused by animal rotaviruses or animal–human reassortants.The StudyWe conducted a study by using double-stranded RNA obtained from 38 untypeable samples collected from chil-dren <5 years of age with rotavirus infections who were ad-mitted to Khanh Hoa General Hospital and Ninh Hoa Hos-pital, Khanh Hoa Province, Vietnam. RNA was extracted from rotavirus-positive stool samples by using RNaid kits (MP Biomedicals/Qbiogene, Solon, OH, USA). Rotavirus-positive stool samples were detected by using Rotaclone ELISA kits (Meridian Bioscience. Inc., Cincinnati, OH, USA). Stool samples were collected from October 2003 through March 2004 as part of a rotavirus surveillance proj-ect conducted by 1 of the authors (D.D.A.) in collaboration with the Centers for Disease Control and Prevention, At-lanta, Georgia, USA.The G and P types were determined by using reverse transcription–PCR (5) Samples that could not be typed were subjected to nucleotide sequencing. Amplifi cation of the nonstructural protein NSP4 gene was conducted as de-scribed (6).Rotavirus genomic RNAs were visualized by poly-acrylamide gel electrophoresis (7) and staining with a Silver Stain II kit (Wako Pure Chemical Industries Ltd., Osaka, Japan) with a few modifi cations. The gel was incu-bated in staining solution for 45 min instead of 15 min, and in each washing step, the temperature of deionized water was maintained at 25°C.VP7, the VP8* portion of outer capsid VP4, and NSP4 genes were sequenced by using the method previ-ously described (5). Sequence similarity was searched with BLAST (www.ncbi.nlm.nih.gov/blast), multiple sequence alignment was conducted with ClustalW (www.ebi.ac.uk/clustalw), and phylogenetic trees were constructed by the neighbor-joining method using MEGA version 3.1 (8). Bootstrap analysis of 1,000 replicates was used to inves-tigate branching of constructed trees. The N-glycosylation site of VP7 was predicted by using a NetNGlyc 1.0 Server (www.cbs.dtu.dk/services/netnglyc).A total of 38 samples were obtained (31 from Khanh Hoa General Hospital and 7 from Ninh Hoa Hospital). G and P types were determined for 35 samples. G type could not be determined for 3 samples (KH142, KH228, and KH210). However, these samples were typed as G2, G4, and G5, respectively, by a homology search of VP7 gene sequence. Thirty-three samples were typed as G1P[8], 2 as G2P[4], 1 as G4P[6], 1 as G5P[6], and 1 as G9P[8]. Three different electropherotypes with a long pattern were iden-tifi ed in 20 electrophoretic-positive samples. Of these, 18 samples showed the same electropherotype and all were typed as G1P[8]. Each of the G4P[6] and G5P[6] rotavi-ruses showed different electropherotypes. The sample that contained G5 rotavirus was isolated from a 7-month-old girl admitted to Khanh Hoa General Hospital in 2004 with diarrhea, fever, and malnutrition.Sequence analyses of the VP7 gene showed that KH210 had more than 80% nucleotide and 90% amino acid identities with representative G5 rotaviruses from humans and pigs (Table 1). When we compared VP7 antigenic re-gions A (aa 87–101), B (aa 143–152), C (aa 208–223), and F (aa 235–242) (9) with representative strains of human G5 rotaviruses, unique substitutions were observed at aa posi-tions 217 (V) of region C and 235 (Y) in region F. Other unique substitutions observed in KH210 were C, I, S, and N at positions 6, 29, 112, and 119, respectively.Phylogenetic analysis of the amino acid sequence of the VP7 gene (Figure 1A) of G5 strains showed 2 geneti-cally distinct groups; KH210 clustered with a porcine G5 strain from Australia. This group also contained a human *Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and †National Institute of Hygiene and Epidemi-ology, Hanoi, VietnamRotavirus G5[P6] in Child with Diarrhea, Vietnam Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 8, August 2007 1233 G5 strain from Cameroon. The human G5 strain from Brazil may belong to the other group. Similar to other G5 strains, VP7 of KH210 showed 1 potential N-glycosylation site at position 69.The nucleotide and amino acid sequences of VP8* of strains KH210 and KH228 showed high identity (89%–93% and 90%–95%) with lineage I and relatively low identity (81%–86% and 85%–90%) with other lineages of P[6] (Table 2). Phylogenetic analysis of the amino acid se-quence of VP8* of KH210, KH228, and other P[6] strainsshowed that Vietnamese strains clustered with Italian por-cine strains of lineage Ic (Figure 1B).Phylogenetic analysis of the amino acid sequence of NSP4 showed that KH210 was closely related to porcine Table 1. Percentage identity of amino acid and nucleotide sequences of VP7 genes of KH210 and selected rotavirus G5 strains* Strain Species (country) Deduced amino acid identity, % Nucleotide identity, % MRC 3105† Human (Cameroon) 92 89IAL-28 Human (Brazil) 91 85CC117 Porcine (Argentina) 93 86C134 Porcine (Argentina) 93 86TFR-41 Porcine (Australia) 93 NA‡JL94 Porcine (People’s Republic of China) 91 85OSU Porcine (USA) 91 84A34 Porcine (Venezuela) 92 84A46 Porcine (Venezuela) 92 82134/04–15 Porcine (Italy) 91 83H1 Equine (USA) 91 85*GenBank accession nos.: MRC3105/2000, AY327107; IAL-28, L79916; CC117, L35056; C134, L35058; TFR-41, P32547; JL94, AY538665; OSU,X04613; A34, L35059; A46, L35054; 134/04–15, DQ062572; H1, AF242393. †Partial sequence. ‡NA, not available. Figure 1. A) Phylogenetic tree constructed from deduced amino acid sequences of the VP7 gene of animal and human G5 rotaviruses. Strain 4695G5, an equine G3 strain, was used as an outgroup. Bootstrap values are expressed as percentages. Bootstrap value <50 is not shown. Strain KH210 clustered with the human G5 rotavirus from Cameroon (MRC3105) and other G5 rotaviruses of porcine origin from Australia and Argentina. The Brazilian human isolate of G5 rotavirus clustered with G5 rotaviruses of porcine and equine origin. Species of origin followed by country of isolation is shown in parentheses after the strain name. B) Phylogenetic tree constructed from the deduced amino acid sequences of the VP8* gene of rotaviruses representing all P[6] lineages. Strain OSU was used as an outgroup. Bootstrap values are expressed as percentages. A bootstrap value <50 is not shown. Strains KH210 and KH228 clustered with lineage Ic. Species of origin followed by country of isolation is shown in parentheses after the strain name. Scale bar shows genetic distance expressed as amino acid substitutions per site. DISPATCHES1234 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 8, August 2007rotaviruses and belonged to genotype B (Figure 2), with identity of 94% to 96% to porcine stains and 91% to 95% to human strains of genotype B. Similar to the structure of most rotaviruses, 2 potential N-glycosylation sites were located at aa positions 8 and 18 (8).ConclusionsG5 rotaviruses are isolated mainly from pigs. However, in 1994 these viruses were reported in samples from Bra-zilian children with diarrhea (10). Subsequently, G5 was identifi ed as a cause of human infection in several states in Brazil (11), which suggests a broader distribution of this unusual serotype. Human G5 rotaviruses were then identi-fi ed in children with acute diarrhea in Argentina (12) and Paraguay (13). Recently, human G5 rotavirus was reported in Cameroon (4), the fi rst human G5 rotavirus isolated in Africa.The human G5 rotavirus from Cameroon has a short RNA pattern. In contrast, the human G5 rotaviruses de-tected in Vietnam and Brazil have long RNA patterns. However, their VP7 genes are not in the same cluster in the phylogenetic tree. Brazilian G5 rotaviruses isolated from humans are usually found in combination with the P[8] genotype (10). This combination is a result of naturally oc-curring reassortment between human (P[8], Wa-like) and animal (G5, OSU-like) strains (14). We have identifi ed the combination of KH210 with genotype P[6]. This genotype is found predominantly in strains of porcine origin. VP8* of Vietnamese strains clus-tered with porcine strains isolated in Italy and belonged to the Ic lineage of P[6]. The NSP4 gene of KH210 belonged to genotype B and had high amino acid identities with both human and porcine rotaviruses of the same genotype. These fi ndings are consistent with those of RNA-RNA hy-bridization (7) and sequence analysis (15). Human strain Wa and porcine strain OSU were shown to form 3 hybrid bands, including the one that corresponded to the NSP4 gene (7). Rotaviruses from different species were shown by phylogenetic analysis to have similar NSP4 sequences that were clustered into the same NSP4 genotype. However, identifying the origin of the NSP4 gene in KH210 was not possible. On the basis of unique amino acid substitutions in VP7 and phylogenetic analysis of VP7 and VP8*, we believe that Vietnamese G5 strain most likely originated from porcine rotavirus by genetic reassortment or as whole virions. Although 2 strains with different G genotypes (G5 Table 2. Percentage identity of the partial amino acid and nucleotide sequences of VP8* genes of KH210 and selected rotavirus P[6]strains* Strain (lineage) Species (country) Deduced amino acid identity, % Nucleotide identity, % M37 (Ia) Human (Venezuela) 90 89221/04–7 (Ib) Porcine (Italy) 94 92134/04–10 (Ic) Porcine (Italy) 95 93ES51/04 (Id) Porcine (Spain) 92 91Gottfried (II) Porcine (USA) 85 82AU19 (III) Human (Japan) 87 81BP1198/98 (IV) Human (Hungary) 90 86BP1227/02 (V) Human (Hungary) 88 85*The amino acid and nucleotide sequence identity of VP8* genes of KH210 and KH228 were 99%. The amino acid sequence identity of VP8* genes between KH228 and Gottfried was 84%. Otherwise, the identity of amino acid and nucleotide sequences of VP8* genes of KH228 and selected rotavirus P[6] strains were same as for KH210. GenBank accession nos.: M37, L20877; 221/04–7, AY955303; 134/04–10, AY955299; ES51/04, AY955306;Gottfried, M33516; AU19, AB017917; BP1198/98, AJ621504; BP1227/02, AJ621505. Figure 2. Phylogenetic tree constructed from the deduced amino acid sequences of the NSP4 gene of rotaviruses representing all genotypes. Strain Bristol, a group C rotavirus, was used as an outgroup. Bootstrap values are expressed as percentages. Bootstrap value <50 is not shown. Strain KH210 clustered with strains in genotype B. Species of origin is shown in parentheses after the strain name. Scale bar shows genetic distance expressed as amino acid substitutions per site.Rotavirus G5[P6] in Child with Diarrhea, Vietnam Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 8, August 2007 1235 for KH210 and G4 for KH228) have identical VP8* genes (Figure 1B), which suggests a contemporary genetic reas-sortment event, it is not known whether sequence diver-gence between KH210 and porcine rotaviruses indicates interspecies transmission. More information will be obtained from sequencing of contemporary porcine rotavirus strains in Vietnam. Identi-fi cation of a human G5P[6] strain in Vietnam will provide a rationale for expanded surveillance to understand its preva-lence in Vietnam and other Asian countries.AcknowledgmentsThe DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank accession nos. are AB257126 and AB303218 for the VP7 and NSP4 genes of strain KH210 and AB271687 and AB271688 for the VP8* gene of KH210 and KH228.This study was supported by the grant The Collaborative Study on Emerging and Re-emerging Infectious Diseases in Viet-nam: Enhancement of Research Capacity, from the Ministry of Education, Culture, Sports, Science and Technology, Japan.Dr Ahmed is an associate professor at the Institute of Scien-tifi c Research, Oita University, Oita, Japan. His primary research interests are molecular epidemiology and pathogenesis of infec-tious diseases.References  1.  Parashar UD, Hummelman EG, Bresee JS, Miller MA, Glass RI. Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis. 2003;9:565–72.  2.  Glass RI, Parashar UD, Brese JS, Turcios R, Fischer TK, Widdow-son MA, et al. Rotavirus vaccines: current prospects and future chal-lenges. Lancet. 2006;368:323–32.  3.  Van Man N, Luan le T, Trach DD, Thanh NT, Van Tu P, Long NT, et al. Epidemiological profi le and burden of rotavirus diarrhea in Viet-nam: 5 years of sentinel hospital surveillance, 1998–2003. J Infect Dis. 2005;Suppl:S127–32.  4.  Esona MD, Armah GE, Geyer A, Steele AD. Detection of an unusual human rotavirus strain with G5P[8] specifi city in a Cameroonian child with diarrhea. J Clin Microbiol. 2004;42:441–4.  5.  Uchida R, Pandey BD, Sherchand JB, Ahmed K, Yokoo M, Nak-agomi T, et al. Molecular epidemiology of rotavirus diarrhea among children and adults in Nepal: detection of G12 strains with P[6] or P[8] and a G11P[25] strain. J Clin Microbiol. 2006;44:3499–505.  6.  Ciarlet M, Liprandi F, Conner ME, Estes MK. Species specifi city and interspecies relatedness of NSP4 genetic groups by compara-tive NSP4 sequence analyses of animal rotaviruses. Arch Virol. 2000;145:371–83.  7.  Nakagomi O, Nakagomi T. Genetic diversity and similarity among mammalian rotaviruses in relation to interspecies transmission of rotavirus. Arch Virol. 1991;120:43–55.  8.  Kumar S, Tamura K, Nei M. MEGA3: Integrated software for mo-lecular evolutionary genetics analyses and sequence alignment. Brief Bioinform. 2004;5:150–63.  9.  Martella V, Ciarlet M, Baselga R, Arista S, Elia G, Lorusso E, et al. Sequence analysis of the VP7 and VP4 genes identifi es a novel VP7 gene allele of porcine rotaviruses, sharing a common evolutionary origin with human G2 rotaviruses. Virology. 2005;337:111–23.10.  Gouvea V, de Castro L, Timenetsky M, Greenberg H, Santos N. Ro-tavirus serotype G5 associated with diarrhea in Brazilian children. J Clin Microbiol. 1994;32:1408–9.11.  Leite JPG, Alfi eri AA, Woods PA, Glass RI, Gentsch JR. Rotavirus G and P types circulating in Brazil: characterization by RT-PCR, probe hybridization, and sequence analysis. Arch Virol. 1996;141:2365–74.12.  Bok K, Castagnaro N, Borsa A, Nates S, Espul C, Fay O, et al. Sur-veillance for rotavirus in Argentina. J Med Virol. 2001;65:190–8.13.  Coluchi N, Munford V, Manzur J, Vazquez C, Escobar M, Weber E, et al. Detection, subgroup specifi city, and genotype diversity of rotavirus strains in children with acute diarrhea in Paraguay. J Clin Microbiol. 2002;40:1709–14.14.  Alfi eri AA, Leite JPG, Nakagomi O, Kaga E, Woods PA, Glass RI, et al. Characterization of human rotavirus genotype P[8]G5 from Brazil by probe-hybridization and sequence. Arch Virol. 1996;141:2353–64.15.  Horie Y, Masamune O, Nakagomi O. Three major alleles of rota-virus NSP4 proteins identifi ed by sequence analysis. J Gen Virol. 1997;78:2341–6.Address for correspondence: Kamruddin Ahmed, Division of Infectious Diseases, Department of Social and Environmental Medicine, Institute of Scientifi c Research, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu-shi, Oita 879-5593, Japan; email: ahmed@med.oita-u.ac.jpUse of trade names is for identifi cation only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

Rotavirus G5P[6] in child with diarrhea, Vietnam

https://nagasaki-u.repo.nii.ac.jp/?action=repository_action_common_download&item_id=19383&item_no=1&attribute_id=18&file_no=1

Rotavirus G5P[6] in child with diarrhea, Vietnam

Abstract

Similar works

Full text

Available Versions

Nagasaki University's Academic Output SITE: NAOSITE