hrvatski

Resekvenciranje genoma virusa Puumala iz WHO arbovirusne kolekcije

The inability of RNA polymerase to perform proofreading during replication causes high mutation rates in Hantaviruses and other RNA viruses. Our goal was to clone and determine the sequence of the Puumala virus strain Sotkamo from WHO arbovirus collection (Dr. R. Tesh, UTMB) and to compare it to the published sequence. RNA from VeroE6 cells infected with Puumala virus strain Sotkamo was isolated using chloroform-phenol extraction and the cDNA was prepared using random hexamers. Viral sequences were amplified using specific primers and cloned into suitable vectors. At least three clones of each fragment were sequenced using dye termination sequencing on Applied Biosystems Model 3100 Genetic Analyzer. The sequence obtained for the three segments showed more than 99% homology compared to the published genome for the Puumala Sotkamo strain. Sequencing showed seven mutations in the S segment, four in the non-coding region and three silent mutations in the open reading frame for the nucleocapsid protein. M segment contained eleven mutations in the coding region for Puumala glycoprotein, all but two caused amino acid changes. Only five mutations were observed in L segment, causing four amino acid changes in the 6471 nucleotides long open reading frame for the viral polymerase. Interestingly, these mutations aren't spread evenly through the genome. The L segment shows proportionally the smallest number of mutations, whereas M and S segments have a higher but proportionally similar amount of changes. However, their potential effects differ within the genome segment. While 80% of mutations cause an amino acid change in the M segment, all mutations are silent in the S segment. The segment's non-coding region probably accumulates more nucleotide changes than the coding region.

PUUV; genomska sekvenca; resekvenciranje; SZO arbovirusna kolekcija

nije evidentirano

engleski

Resequencing of the Puumala virus strain Sotkamo from the WHO Arbovirus collection

The inability of RNA polymerase to perform proofreading during replication causes high mutation rates in Hantaviruses and other RNA viruses. Our goal was to clone and determine the sequence of the Puumala virus strain Sotkamo from WHO arbovirus collection (Dr. R. Tesh, UTMB) and to compare it to the published sequence. RNA from VeroE6 cells infected with Puumala virus strain Sotkamo was isolated using chloroform-phenol extraction and the cDNA was prepared using random hexamers. Viral sequences were amplified using specific primers and cloned into suitable vectors. At least three clones of each fragment were sequenced using dye termination sequencing on Applied Biosystems Model 3100 Genetic Analyzer. The sequence obtained for the three segments showed more than 99% homology compared to the published genome for the Puumala Sotkamo strain. Sequencing showed seven mutations in the S segment, four in the non-coding region and three silent mutations in the open reading frame for the nucleocapsid protein. M segment contained eleven mutations in the coding region for Puumala glycoprotein, all but two caused amino acid changes. Only five mutations were observed in L segment, causing four amino acid changes in the 6471 nucleotides long open reading frame for the viral polymerase. Interestingly, these mutations aren't spread evenly through the genome. The L segment shows proportionally the smallest number of mutations, whereas M and S segments have a higher but proportionally similar amount of changes. However, their potential effects differ within the genome segment. While 80% of mutations cause an amino acid change in the M segment, all mutations are silent in the S segment. The segment's non-coding region probably accumulates more nucleotide changes than the coding region.

PUUV; Genome sequence; Resequencing; WHO Arbovirus collection

nije evidentirano