Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2',5'-oligoadenylate synthetase 1b.

The interferon-inducible 2',5'-oligoadenylate synthetase 1b (Oas1b) protein inhibits West Nile virus (WNV) infection by preventing viral RNA (vRNA) accumulation in infected cells. Serial passage of WNV in Oas1b-expressing mouse cells selected a virus variant with improved growth capacity. Two major amino acid substitutions were identified in this Oas1b-resistant WNV variant: NS3-S365G in the ATPase/helicase domain of NS3 and 2K-V9M in the C-terminal segment of NS4A. To assess their effect on antiviral activity of Oas1b, the NS3 and 2K mutations were engineered into an infectious WNV cDNA clone. The NS3 mutation alters requirement of ATP for ATPase activity and attenuates Oas1b-mediated suppression of vRNA accumulation. However, growth of NS3-mutant virus remains impaired in Oas1b-expressing cells. Only the 2K-V9M mutation efficiently rescued viral growth by promoting vRNA replication. Thus, WNV resistance to Oas1b antiviral action could be attributed to the 2K-V9M substitution with a potential role of NS3-S365G through rescue of vRNA accumulation.

Identification of a polyketide synthase coding sequence specific for anatoxin-a-producing Oscillatoria cyanobacteria.

We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins.

Aedes albopictus mosquito: the main vector of the 2007 Chikungunya outbreak in Gabon.

The primary vector at the origin of the 2007 outbreak in Libreville, Gabon is identified as Aedes albopictus, trapped around the nearby French military camp. The Chikungunya virus was isolated from mosquitoes and found to be identical to the A226V circulating human strain. This is the first field study showing the role of the recently arrived species Aedes albopictus in Chikungunya virus transmission in Central Africa, and it demonstrates this species' role in modifying the epidemiological presentation of Chikungunya in Gabon.

Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.

BACKGROUND: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis. METHODS AND FINDINGS: We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our 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, four of which were 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 showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector. CONCLUSIONS: The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.

Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-'Synechococcus' clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon.

PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of 'Synechococcus' (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511(T), although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511(T), PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-'Synechococcus' strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.

rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira.

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria.

PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium NOSTOC: PCC 7120 gave three products. Two represented true ITS regions of different sizes, while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNA(Ile) and tRNA(Ala) genes, separated by a large stem-loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes, and the 5' half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L. The two spacer regions of NOSTOC: PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNA(Ile) and tRNA(Ala) genes, only the tRNA(Ile) gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B-box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.