Flavivirus genome recoding by codon optimisation confers genetically stable in vivo attenuation in both mice and mosquitoes.

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

Correction to: Description of Wenzhouxiangella salilacus sp. nov., a moderate halophilic bacterium isolated from a salt lake in Xinjiang Province, China.

In the original publication of the article, the deposit accession numbers of strain 15181T in the acknowledgment section were incorrectly provided as "KCTC 62172T and MCCC 1K03442T".

Description of Wenzhouxiangella salilacus sp. nov., a moderate halophilic bacterium isolated from a salt lake in Xinjiang Province, China.

A Gram-stain negative, non-motile, strictly aerobic and rod-shaped bacterium, designated as 15181T, was isolated from a salt lake in Xinjiang Province, China. Strain 15181T was able to grow at 10-40 °C (optimum 37 °C), pH 6.0-8.5 (optimum 7.0) and with 1-14% NaCl (optimum 4%, w/v). According to phylogenetic analysis based on 16S rRNA gene sequences, strain 15181T was assigned to the genus Wenzhouxiangella with high 16S rRNA gene sequence similarity of 97.4% to Wenzhouxiangella sediminis XDB06T, followed by Wenzhouxiangella marina KCTC 42284T (95.9%). Strain 15181T exhibited ANI values of 80.0% and 72.0% to W. sediminis XDB06T and W. marina KCTC 42284T, respectively. The in silico DDH analysis revealed that strain 15181T shared 19.1% and 18.7% DNA relatedness with W. sediminis XDB06T and W. marina KCTC 42284T, respectively. Chemotaxonomic analysis showed that the sole respiratory quinone was ubiquinone-8, the major fatty acids included iso-C15:0, iso-C16:0 and summed feature 9 (C16:0 10-methyl and/or iso-C17:1ω9c). The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified glycolipids, two unidentified phospholipids, two unidentified aminophospholipids and an unidentified lipid. On the basis of phenotypic, genotypic and chemotaxonomic characteristics presented in this study, strain 15181T is concluded to represent a novel species in the genus Wenzhouxiangella, for which the name Wenzhouxiangella salilacus sp. nov. is proposed. The type strain is 15181T (=KCTC 62172T=MCCC 1K03442T).

Confluentibacter flavum sp. nov., Isolated from the Saline Lake.

A Gram-stain-negative, rod-shaped, non-motile, bacterial isolate designated 3BT, was isolated from a saline lake, and subjected to a polyphasic taxonomic investigation. The phylogenetic analysis based on 16S rRNA gene sequence clearly showed an allocation to the genus Confluentibacter with similarity ranging from 95.1 to 98%. OrthoANI values between strain 3BT and related strains of Confluentibacter (< 90%) were lower than the threshold value of 95% ANI relatedness recommended for species demarcation. Strain 3BT grew at 4-35 °C and pH 6.0-8.0 (optimum, 28 °C and pH 6.5) and with 0-3% (w/v) NaCl (optimum, 0.5%). The predominant respiratory quinone was menaquinone-6 (MK-6) and the major fatty acids were iso-C15:0, iso-C15:1 G, iso-C15:0 3-OH, and iso-C17:0 3-OH. The polar lipid profile of strain 3BT comprised phosphatidylethanolamine, one unidentified aminolipid, one aminophospholipid, and three unidentified lipids (L1-3). The DNA G+C content was 33.1 mol%. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain 3BT is described as a novel species in genus Confluentibacter, for which the name Confluentibacter flavum sp. nov. (type strain 3BT = CGMCC115960T = KCTC52969T) is proposed.

Kordiimonas pumila sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, translucent-white, aerobic, motile and rod-shaped strain, designated N18T, was isolated from a coastal sediment sample collected in Zhoushan, Zhejiang Province, China. 16S rRNA gene similarity analysis revealed that strain N18T demonstrated highest similarity to the genus Kordiimonas(95.3-97.2 %). Phylogenetic analysis of 16S rRNA gene sequence showed that strain N18T represented a distinct lineage in the clade consisting of the genus Kordiimonas. Strain N18T was found to grow at 10-37 °C (optimum 28 °C), pH 6.0-8.0 (optimum 7.0) and with 1.0-4.0 % (w/v) NaCl (optimum 2.5 %). The G+C content of the genomic DNA was 55.3 mol%. The major cellular fatty acids were identified as summed feature 3 (comprising iso-C15 : 0 2-OH/C16 : 1ω7c), iso-C17 : 1ω9c and iso-C15 : 0. The polar lipid profile of N18T consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unidentified glycolipid, an unidentified aminoglycolipid, an unidentified aminophospholipid and five unidentified lipids. The respiratory quinone was Q-10. Based on chemotaxonomic, morphological and physiological properties, strain N18T could be distinguished from its closest phylogenetic neighbours. Thus, we propose Kordiimonas pumila sp. nov., the type strain is N18T (=MCCC 1K03436T=KCTC 62164T).

Rhodohalobacter barkolensis sp. nov., isolated from a saline lake and emended description of the genus Rhodohalobacter.

A Gram-stain-negative, non-motile, aerobic, rod-shaped bacterium, designated 15182T, was isolated from a saline lake in China. The novel strain 15182T was able to grow at 10-40 °C (optimum, 37 °C), pH 7.0-8.0 (optimum, 7.5) and with 0.5-4 % NaCl (optimum, 2-3 %, w/v). The phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 15182T was most closely related to the genus Rhodohalobacter by sharing the highest sequence similarity of 97.0 % with Rhodohalobacter halophilus JZ3C29T. Chemotaxonomic analysis showed that the sole respiratory quinone was menaquinone 7, the major fatty acids included C16 : 0 N alcohol and C16 : 1ω11c. The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four uncharacterized glycolipids, one uncharacterized phospholipid and two uncharacterized lipids. The genomic DNA G+C content of the strain 15182T was 42.4 mol%. The average nucleotide identity value between 15182T and R. halophilus JZ3C29T was 75.4 %, and the in silico DNA-DNA hybridization value of the two strains was 19.1 %. On the basis of its phenotypic, chemotaxonomic, genotypic and genomic characteristics presented in this study, strain 15182T is suggested to represent a novel species in the genus Rhodohalobacter, for which the name Rhodohalobacter barkolensis sp. nov. is proposed. The type strain is 15182T (=KCTC 62172T=MCCC 1K03442T). An emended description of the genus Rhodohalobacter is also presented.