Naslov
Resequencing of the Puumala virus strain sotkamo from the WHO arbovirus collection

Autori
Kurolt, Ivan-Christian ; Paessler, Slobodan ; Markotić, Alemka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
The 8th International Conference on HFRS, HPS & Hantaviruses : Program & Abstracts Book / Papa, Anna ; Antoniadis, Antonis - Atena : International Society for Hantaviruses and Hantaviral Diseases, 2010, 184-184

ISBN
5-256-54411-2

Skup
International Conference on HFRS, HPS & Hantaviruses (8 ; 2010)

Mjesto i datum
Atena, Grčka, 20.05.2010. - 22.05.2010

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Puumala virus; resequencing; reference strain; mutation

Sažetak
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. These plasmids were sequenced using dye termination sequencing on Applied Biosystems Model 3100 Genetic Analyzer. All detected “mutations” were confirmed by direct sequencing of PCR products. 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. Although only one clone was sequenced, all mutations were confirmed by direct sequencing. 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. Acknowledgements: We thank J.W. LeDuc, PhD (University of Texas Medical Branch, Galveston, USA) for his support during this study. This study was prepared as a part of the following projects: 143-1430115-0103 and J-29-2007 (P.I. Dr. A. Markotić) supported by the Croatian Ministry of Sciences, Education and Sports. These materials are based on a thesis development funded by the National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA