Novel Isolated And Purified Strains Of Chikungunya Virus And Polynucleotides And Polypetide Sequences, Diagnostic And Immunogenical Uses Thereof
a technology of chikungunya virus and polynucleotide sequence, applied in the field of wild strains of chikungunya virus, can solve the problems of arthralgia or arthritic symptoms that may persist for months or years, and the pathognomonic sign of the disease is very painful
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example 1
Identification and Characterization of CHIK Viruses Causing the Indian Ocean Outbreak
[0107]The inventors (as sometimes referred therein as “we”) report the nearly complete genome sequence of six selected clinical isolates, along with partial sequences of glycoprotein E1 from a total of 60 patients from Réunion, Seychelles, Mauritius, Madagascar and Mayotte Islands. The present results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, three of which being located in alphavirus conserved positions of nsP2 (which contains helicase, protease and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid of a patient showed unique changes in nsP1 (T30...
example 2
Identification and Characterization of a Soluble Form of E2 (sE2) of the CHIK Virus
[0134]The TOPO / CHIK-21.pE2 (CNCM I-3587) plasmid containing the cDNA coding for the pE2 glycoprotein (E3+E2) from the CHIK 21 virus strain (Schuffenecker et al., Plos Med., 3:1058, 2006) was used as a template for the amplification by PCR of the ectodomain sequence of the E2 envelope glycoprotein (FIG. 29). The ectodomain of gp-E2 (E2-1 to E2-364; 85% of E2) is strictly conserved among the CHIK-21, -27, 49 and 115 cell lines isolated in the Indian Ocean during the epidemic outbreak of 2005-06 (see FIG. 29). The soluble form of the sE2 corresponds to the gp-E2 ectodomain which is deleted at the carboxylic terminal of its transmembrane anchor region. It is of interest that the soluble form carries the main epitopes eliciting virus-neutralizing antibodies. The PCR primers are described in FIG. 30 (SEQ ID NO:79 and 80): they allow the cloning of the sE2 sequence between the unique sites Bg / lI and NotI of ...
example 3
Construction of the TRIP Vector Expressing the Soluble Form of E2 (sE2) of the CHIK Virus According to the Present Invention
[0137]The gene coding the CHIK sE2 protein has been optimised by the Genecust firm so as to provide a synthetic DNA with an enriched G+C content in comparison to the cDNA obtained from the viral genomic RNA. The G+C rich codons (amino acids E2-1 to E2-364, soluble gp-E2 ectodomain, sE2) were fused to the signal peptide sequence of the human calreticuline (ssCRT) MLLSVPLLLLGLLGLAA (SEQ ID NO: 77) for translocation of the viral protein into the secretion pathway. The enzyme restriction sites BamHi in 5′ and XhoI in 3′ have been added at their respective ends of the sequences coding for the fusion ssCRT+sE2 protein.
[0138]The synthetic gene was cloned into the TRIP vector between the BamHI and XhoI sites under the transcription of the ieCMV promoter. The non-replicative and integrative TRIP / CHIK.sE2 plasmid thus produced was validated for the expression of the sE2 ...
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