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Jean Dubuisson - One of the best experts on this subject based on the ideXlab platform.
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development and characterization of a human monoclonal antibody targeting the n terminal region of hepatitis c virus envelope Glycoprotein E1
Virology, 2018Co-Authors: Ahmed Atef Ahmed Abouzeid Mesalam, Jean Dubuisson, Steven K. H. Foung, Isabelle Desombere, Ali Farhoudi, Freya Van Houtte, Lieven Verhoye, Jonathan K Ball, Arvind H PatelAbstract:Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.
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development and characterization of a human monoclonal antibody targeting the n terminal region of hepatitis c virus envelope Glycoprotein E1
Virology, 2018Co-Authors: Ahmed Atef Ahmed Abouzeid Mesalam, Jean Dubuisson, Steven K. H. Foung, Isabelle Desombere, Ali Farhoudi, Lieven Verhoye, Jonathan K Ball, Freya Van Houtte, Arvind H PatelAbstract:Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.
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Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.
Journal of virology, 2017Co-Authors: Juliano G. Haddad, Yves Rouillé, Xavier Hanoulle, Véronique Descamps, Monzer Hamzé, Fouad Dabboussi, Gilles Duverlie, Muriel Lavie, Thomas F. Baumert, Jean DubuissonAbstract:ABSTRACT Hepatitis C virus (HCV) envelope Glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this Glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV Glycoprotein E1 and the genomic RNA during HCV morphogenesis. IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.
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Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly
Journal of Virology, 2017Co-Authors: Juliano Haddad, Thomas Baumert, Yves Rouillé, Xavier Hanoulle, Véronique Descamps, Monzer Hamzé, Fouad Dabboussi, Gilles Duverlie, Muriel Lavie, Jean DubuissonAbstract:Hepatitis C virus (HCV) envelope Glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this Glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV Glycoprotein E1 and the genomic RNA during HCV morphogenesis.
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disulfide bonds in hepatitis c virus Glycoprotein E1 control the assembly and entry functions of e2 Glycoprotein
Journal of Virology, 2013Co-Authors: Véronique Descamps, Gilles Duverlie, Ahmed Wahid, Francois Helle, Francois Penin, Jean DubuissonAbstract:Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane Glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion Glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV Glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.
S Natkunam - One of the best experts on this subject based on the ideXlab platform.
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combined detection and genotyping of chikungunya virus by a specific reverse transcription polymerase chain reaction
Journal of Medical Virology, 2002Co-Authors: Futoshi Hasebe, Maria Del Carmen Parquet, Basu Dev Pandey, Edward Gitau Matumbi Mathenge, Kouichi Morita, V Balasubramaniam, Z Saat, A Yusop, M Sinniah, S NatkunamAbstract:A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and Glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype.
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combined detection and genotyping of chikungunya virus by a specific reverse transcription polymerase chain reaction
Journal of Medical Virology, 2002Co-Authors: Futoshi Hasebe, Basu Dev Pandey, Edward Gitau Matumbi Mathenge, Kouichi Morita, V Balasubramaniam, Z Saat, A Yusop, M Sinniah, Maria Del Carmen Parquet, S NatkunamAbstract:A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and Glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype. J. Med Virol. 67:370–374, 2002. © 2002 Wiley-Liss, Inc.
Gilles Duverlie - One of the best experts on this subject based on the ideXlab platform.
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Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.
Journal of virology, 2017Co-Authors: Juliano G. Haddad, Yves Rouillé, Xavier Hanoulle, Véronique Descamps, Monzer Hamzé, Fouad Dabboussi, Gilles Duverlie, Muriel Lavie, Thomas F. Baumert, Jean DubuissonAbstract:ABSTRACT Hepatitis C virus (HCV) envelope Glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this Glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV Glycoprotein E1 and the genomic RNA during HCV morphogenesis. IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.
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Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly
Journal of Virology, 2017Co-Authors: Juliano Haddad, Thomas Baumert, Yves Rouillé, Xavier Hanoulle, Véronique Descamps, Monzer Hamzé, Fouad Dabboussi, Gilles Duverlie, Muriel Lavie, Jean DubuissonAbstract:Hepatitis C virus (HCV) envelope Glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this Glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV Glycoprotein E1 and the genomic RNA during HCV morphogenesis.
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Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion.
Journal of Virology, 2015Co-Authors: Pierre Falson, Birke Bartosch, Khaled Alsaleh, Birke Andrea Tews, Antoine Loquet, Yann Ciczora, Laura Riva, Cédric Montigny, Claire Montpellier, Gilles DuverlieAbstract:In hepatitis C virus (HCV)-infected cells, the envelope Glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein.
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Hepatitis C virus envelope Glycoprotein E1 forms trimers at the surface of the virion
Journal of virology, 2015Co-Authors: Pierre Falson, Birke Bartosch, Khaled Alsaleh, Birke Andrea Tews, Antoine Loquet, Yann Ciczora, Laura Riva, Cédric Montigny, Claire Montpellier, Gilles DuverlieAbstract:In hepatitis C virus (HCV)-infected cells, the envelope Glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV Glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope Glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.
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disulfide bonds in hepatitis c virus Glycoprotein E1 control the assembly and entry functions of e2 Glycoprotein
Journal of Virology, 2013Co-Authors: Véronique Descamps, Gilles Duverlie, Ahmed Wahid, Francois Helle, Francois Penin, Jean DubuissonAbstract:Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane Glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion Glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV Glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.
Vladimir Brusic - One of the best experts on this subject based on the ideXlab platform.
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A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope Glycoprotein and their cross-reactivity
BMC Medical Genomics, 2015Co-Authors: Jing Sun, Vladimir BrusicAbstract:BackgroundHepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope Glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses.ResultsStructural characterization of E2 core protein and a broadly neutralizing antibody AR3C together with E1E2 sequence information enabled the analysis of B-cell epitope variability. The E2 binding site by AR3C and its surrounding area were identified from the crystal structure of E2c-AR3C complex. We clustered HCV strains using the concept of "discontinuous motif/peptide" and classified B-cell epitopes based on their similarity.ConclusionsThe assessment of antibody neutralizing coverage provides insights into potential cross-reactivity of the AR3C neutralizing antibody across a large number of HCV variants.
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a systematic analysis of a broadly neutralizing antibody ar3c epitopes on hepatitis c virus e2 envelope Glycoprotein and their cross reactivity
BMC Medical Genomics, 2015Co-Authors: Vladimir BrusicAbstract:Background Hepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope Glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses.
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a systematic analysis of a broadly neutralizing antibody ar3c epitopes on hepatitis c virus e2 envelope Glycoprotein and their cross reactivity
BMC Medical Genomics, 2015Co-Authors: Jing Sun, Vladimir BrusicAbstract:Background Hepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope Glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses.
Futoshi Hasebe - One of the best experts on this subject based on the ideXlab platform.
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combined detection and genotyping of chikungunya virus by a specific reverse transcription polymerase chain reaction
Journal of Medical Virology, 2002Co-Authors: Futoshi Hasebe, Maria Del Carmen Parquet, Basu Dev Pandey, Edward Gitau Matumbi Mathenge, Kouichi Morita, V Balasubramaniam, Z Saat, A Yusop, M Sinniah, S NatkunamAbstract:A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and Glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype.
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combined detection and genotyping of chikungunya virus by a specific reverse transcription polymerase chain reaction
Journal of Medical Virology, 2002Co-Authors: Futoshi Hasebe, Basu Dev Pandey, Edward Gitau Matumbi Mathenge, Kouichi Morita, V Balasubramaniam, Z Saat, A Yusop, M Sinniah, Maria Del Carmen Parquet, S NatkunamAbstract:A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and Glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype. J. Med Virol. 67:370–374, 2002. © 2002 Wiley-Liss, Inc.