Limits...
Chikungunya virus was isolated in Thailand, 2010.

Sasayama M, Benjathummarak S, Kawashita N, Rukmanee P, Sangmukdanun S, Masrinoul P, Pitaksajjakul P, Puiprom O, Wuthisen P, Kurosu T, Chaichana P, Maneekan P, Ikuta K, Ramasoota P, Okabayashi T, Singhasivanon P, Luplertlop N - Virus Genes (2014)

Bottom Line: However, no update of CHIKV circulating in Thailand has been published since 2009.In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010.We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

View Article: PubMed Central - PubMed

Affiliation: Mahidol-Osaka Center for Infectious Diseases, Ratchathewi, Bangkok, 10400, Thailand.

ABSTRACT
Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008-2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

Show MeSH

Related in: MedlinePlus

a Phylogenetic tree showing the relationships between the structural genes of different chikungunya virus (CHIKV) strains. The information was obtained from 56 clones derived from 7 infected patients in Ratchaburi Province of Thailand in 2010. The numbers below or above the branch nodes represent the neighbor-joining bootstrap values. Analysis was based on the nucleotide sequences of the structural genes using the MEGA 5.05 program and the maximum likelihood method based on 1,000 bootstrap replications. The 2010 isolates are denoted by black circles and bold type. Scale bars indicate 0.02 nucleotide substitutions per site. GenBank Accession numbers for all isolates were recorded. GenBank Accession nos: CP1 (AB857730–AB857737); CP7 (AB857738–AB857745); CP9 (AB857746–AB857753); CP10 (AB857754–AB857761); CP11 (AB857762–AB857769); CP13 (AB857770–AB857777); and CP16 (AB857778–AB857785), b Three-dimensional structures of chikungunya E1, E2, and E3 monomers by ribbon model were obtained from the Protein Data Bank (ID code 3N41) [16]. The domains are color coded as follows: Domain I of E1 (dark red), domain II of E1 (dark yellow), domain III of E1 (dark blue), the FL region of E1 (orange), domain A of E2 (blue–green), domain B of E2 (dark green), domain C of E2 (pink), the arch region of E2 (purple), and E3 (gray). Mutated residues are depicted as a space-filling model: H456 (red), E572 (magenta), P1113 (light green), N1158 (light blue), and sugar-binding residues (gray). Yellow in space-filling model shows the immunodominant site. This figure was prepared by Molecular Operating Environment (Chemical Computing Group Inc) (Color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4232745&req=5

Fig1: a Phylogenetic tree showing the relationships between the structural genes of different chikungunya virus (CHIKV) strains. The information was obtained from 56 clones derived from 7 infected patients in Ratchaburi Province of Thailand in 2010. The numbers below or above the branch nodes represent the neighbor-joining bootstrap values. Analysis was based on the nucleotide sequences of the structural genes using the MEGA 5.05 program and the maximum likelihood method based on 1,000 bootstrap replications. The 2010 isolates are denoted by black circles and bold type. Scale bars indicate 0.02 nucleotide substitutions per site. GenBank Accession numbers for all isolates were recorded. GenBank Accession nos: CP1 (AB857730–AB857737); CP7 (AB857738–AB857745); CP9 (AB857746–AB857753); CP10 (AB857754–AB857761); CP11 (AB857762–AB857769); CP13 (AB857770–AB857777); and CP16 (AB857778–AB857785), b Three-dimensional structures of chikungunya E1, E2, and E3 monomers by ribbon model were obtained from the Protein Data Bank (ID code 3N41) [16]. The domains are color coded as follows: Domain I of E1 (dark red), domain II of E1 (dark yellow), domain III of E1 (dark blue), the FL region of E1 (orange), domain A of E2 (blue–green), domain B of E2 (dark green), domain C of E2 (pink), the arch region of E2 (purple), and E3 (gray). Mutated residues are depicted as a space-filling model: H456 (red), E572 (magenta), P1113 (light green), N1158 (light blue), and sugar-binding residues (gray). Yellow in space-filling model shows the immunodominant site. This figure was prepared by Molecular Operating Environment (Chemical Computing Group Inc) (Color figure online)

Mentions: Next, to examine sequence variations within the viral genome, the seven clinical isolates were subjected to plaque purification in Vero cells. Eight plaques were selected for each of the isolates: CP1:1–8, CP7:1–8, CP9:1–8, CP10:1–8, CP11:1–8, CP13:1–8, and CP16:1–8. The region containing the structural genes (capsid–E3–E2–6k–E1) was amplified using primers published by Sreekumar et al. [13]. The amplicons were sequenced and aligned with the reference strain sequences available in GenBank (Fig. 1a). The E1-A226V and E2-I211T mutations, which provide a suitable background for CHIKV adaptation in Ae. albopictus [10, 11, 14, 15], were relatively well conserved among all clones obtained from the seven clinical isolates. Furthermore, several sequence variations were identified. We identified several unique amino acid substitutions that were not present in the strains responsible for the Thai outbreak in 2008–2009: E1-N349I in all CP16 clones; E1-P304L in clone CP7:6; E2-H131Y in clone CP13:2; and E2-E247A in clone CP16:4 (Table 1). These substitutions were conserved when compared to any strains reported in neighboring countries. Moreover, these are exposed on the surface of the molecule, suggesting that it may affect interaction of them with the other E monomers or antibodies (Fig. 1b) [16]. Phylogenetic analysis showed that all of the clinical isolates in 2010 formed a homogeneous cluster within a broad group, which also included the isolates from the Thai outbreak of 2008–2009 (ECSA genotype). Notably, all clones formed a branch that shared >99 % homology with the Indian Ocean Lineage (IOL), a relatively independent cluster within the ECSA genotype that caused an outbreak of unprecedented magnitude in 2005–2006 (Fig. 1a). This conclusion was also supported by the phylogenetic analysis based on E1 gene sequence (data not shown). The CHIKV isolates from China (2010), India (2010–2011), and Cambodia (2011) also clustered within the IOL.Fig. 1


Chikungunya virus was isolated in Thailand, 2010.

Sasayama M, Benjathummarak S, Kawashita N, Rukmanee P, Sangmukdanun S, Masrinoul P, Pitaksajjakul P, Puiprom O, Wuthisen P, Kurosu T, Chaichana P, Maneekan P, Ikuta K, Ramasoota P, Okabayashi T, Singhasivanon P, Luplertlop N - Virus Genes (2014)

a Phylogenetic tree showing the relationships between the structural genes of different chikungunya virus (CHIKV) strains. The information was obtained from 56 clones derived from 7 infected patients in Ratchaburi Province of Thailand in 2010. The numbers below or above the branch nodes represent the neighbor-joining bootstrap values. Analysis was based on the nucleotide sequences of the structural genes using the MEGA 5.05 program and the maximum likelihood method based on 1,000 bootstrap replications. The 2010 isolates are denoted by black circles and bold type. Scale bars indicate 0.02 nucleotide substitutions per site. GenBank Accession numbers for all isolates were recorded. GenBank Accession nos: CP1 (AB857730–AB857737); CP7 (AB857738–AB857745); CP9 (AB857746–AB857753); CP10 (AB857754–AB857761); CP11 (AB857762–AB857769); CP13 (AB857770–AB857777); and CP16 (AB857778–AB857785), b Three-dimensional structures of chikungunya E1, E2, and E3 monomers by ribbon model were obtained from the Protein Data Bank (ID code 3N41) [16]. The domains are color coded as follows: Domain I of E1 (dark red), domain II of E1 (dark yellow), domain III of E1 (dark blue), the FL region of E1 (orange), domain A of E2 (blue–green), domain B of E2 (dark green), domain C of E2 (pink), the arch region of E2 (purple), and E3 (gray). Mutated residues are depicted as a space-filling model: H456 (red), E572 (magenta), P1113 (light green), N1158 (light blue), and sugar-binding residues (gray). Yellow in space-filling model shows the immunodominant site. This figure was prepared by Molecular Operating Environment (Chemical Computing Group Inc) (Color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232745&req=5

Fig1: a Phylogenetic tree showing the relationships between the structural genes of different chikungunya virus (CHIKV) strains. The information was obtained from 56 clones derived from 7 infected patients in Ratchaburi Province of Thailand in 2010. The numbers below or above the branch nodes represent the neighbor-joining bootstrap values. Analysis was based on the nucleotide sequences of the structural genes using the MEGA 5.05 program and the maximum likelihood method based on 1,000 bootstrap replications. The 2010 isolates are denoted by black circles and bold type. Scale bars indicate 0.02 nucleotide substitutions per site. GenBank Accession numbers for all isolates were recorded. GenBank Accession nos: CP1 (AB857730–AB857737); CP7 (AB857738–AB857745); CP9 (AB857746–AB857753); CP10 (AB857754–AB857761); CP11 (AB857762–AB857769); CP13 (AB857770–AB857777); and CP16 (AB857778–AB857785), b Three-dimensional structures of chikungunya E1, E2, and E3 monomers by ribbon model were obtained from the Protein Data Bank (ID code 3N41) [16]. The domains are color coded as follows: Domain I of E1 (dark red), domain II of E1 (dark yellow), domain III of E1 (dark blue), the FL region of E1 (orange), domain A of E2 (blue–green), domain B of E2 (dark green), domain C of E2 (pink), the arch region of E2 (purple), and E3 (gray). Mutated residues are depicted as a space-filling model: H456 (red), E572 (magenta), P1113 (light green), N1158 (light blue), and sugar-binding residues (gray). Yellow in space-filling model shows the immunodominant site. This figure was prepared by Molecular Operating Environment (Chemical Computing Group Inc) (Color figure online)
Mentions: Next, to examine sequence variations within the viral genome, the seven clinical isolates were subjected to plaque purification in Vero cells. Eight plaques were selected for each of the isolates: CP1:1–8, CP7:1–8, CP9:1–8, CP10:1–8, CP11:1–8, CP13:1–8, and CP16:1–8. The region containing the structural genes (capsid–E3–E2–6k–E1) was amplified using primers published by Sreekumar et al. [13]. The amplicons were sequenced and aligned with the reference strain sequences available in GenBank (Fig. 1a). The E1-A226V and E2-I211T mutations, which provide a suitable background for CHIKV adaptation in Ae. albopictus [10, 11, 14, 15], were relatively well conserved among all clones obtained from the seven clinical isolates. Furthermore, several sequence variations were identified. We identified several unique amino acid substitutions that were not present in the strains responsible for the Thai outbreak in 2008–2009: E1-N349I in all CP16 clones; E1-P304L in clone CP7:6; E2-H131Y in clone CP13:2; and E2-E247A in clone CP16:4 (Table 1). These substitutions were conserved when compared to any strains reported in neighboring countries. Moreover, these are exposed on the surface of the molecule, suggesting that it may affect interaction of them with the other E monomers or antibodies (Fig. 1b) [16]. Phylogenetic analysis showed that all of the clinical isolates in 2010 formed a homogeneous cluster within a broad group, which also included the isolates from the Thai outbreak of 2008–2009 (ECSA genotype). Notably, all clones formed a branch that shared >99 % homology with the Indian Ocean Lineage (IOL), a relatively independent cluster within the ECSA genotype that caused an outbreak of unprecedented magnitude in 2005–2006 (Fig. 1a). This conclusion was also supported by the phylogenetic analysis based on E1 gene sequence (data not shown). The CHIKV isolates from China (2010), India (2010–2011), and Cambodia (2011) also clustered within the IOL.Fig. 1

Bottom Line: However, no update of CHIKV circulating in Thailand has been published since 2009.In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010.We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

View Article: PubMed Central - PubMed

Affiliation: Mahidol-Osaka Center for Infectious Diseases, Ratchathewi, Bangkok, 10400, Thailand.

ABSTRACT
Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008-2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

Show MeSH
Related in: MedlinePlus