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09/16/2022

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We obtained the complete genome sequence of the virus isolate (Ebola virus/H. sapiens-tc/SLE/2014/Makona-Italy-INMI1) originating from a health worker evacuated from Sierra Leone to Italy in late November 2014. The virus was isolated on Vero E6 cells. Viral RNA was extracted (Roche) from the first passage; the complete genome was amplified in 45 overlapping fragments with EBOV-specific primers using the One-Step RT-PCR kit (Qiagen). The fragments were sequenced from both ends using Sanger techniques and assembled with the Sequencher software.

All laboratory activities involving the virus isolates was conducted following BSL-2 biosafety practices and procedures in BSL-2 laboratory. Viral RNA was extracted and screened for the CHIKV by using Reverse-transcription PCR (RT-PCR) previously described6. The E1 (N = 19) genes of positive sample were subsequently sequenced and analyzed to determine the virus genotype. Isolates identified as Asian genotype (N = 18) were subjected tofull genome sequencing using the Ion Torrent sequencing platform (Life Technologies, USA).

Contigs produced de novo were blasted against the original chloroplast genome reference in order to exclude contigs of nuclear origin. Contigs with coverage below 10x were eliminated, likely leading to the exclusion of contigs of mitochondrial origin as well. The remaining de novo and reference-guided contigs were assembled into larger contigs in Sequencher5.3.2 (Gene Codes Inc., Ann Arbor, MI) based on at least 20 bps overlap and 98% similarity.

Genomic DNA was extracted from whole blood samples or saliva using standard methods. Mutation analysis was performed by direct sequencing of all eight exons of NPHS2, and the mutation bearing exons 8 and 9 of WT1 using exon-flanking primers. All mutations were confirmed with sequencing of the complementary strands. All sequences were analyzed using the Sequencher software package (Gene Codes Corp, Ann Arbor, MI). Read more...

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