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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Virology Journal 1/2018

Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells

Zeitschrift:
Virology Journal > Ausgabe 1/2018
Autoren:
Julia Wigren Byström, Jonas Näslund, Fredrik Trulsson, Magnus Evander, Olivia Wesula Lwande, Clas Ahlm, Göran Bucht
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12985-018-0932-8) contains supplementary material, which is available to authorized users.

Abstract

Background

Rodent borne viruses of the Orthohantavirus genus cause hemorrhagic fever with renal syndrome among people in Eurasia, and hantavirus cardiopulmonary syndrome in the Americas. At present, there are no specific treatments or efficient vaccines against these diseases. Improved understanding of viral transcription and replication may instigate targeted treatment of Orthohantavirus infections. For this purpose, we investigated the kinetics and levels of viral RNA transcription during an ongoing infection in-vitro.

Methods

Vero E6 cells were infected with Puumala Orthohantavirus (strain Kazan) before cells and supernatants were collected at different time points post infection for the detection of viral RNAs. A plasmid containing primer binding sites of the three Orthohantavirus segments small (S), medium (M) and large (L) was constructed and standard curves were generated to calculate the copy numbers of the individual transcripts in the collected samples.

Results

Our results indicated a rapid increase in the copy number of viral RNAs after 9 h post infection. At peak days, 2–6 days after infection, the S- and M-segment transcripts became thousand and hundred-fold more abundant than the copy number of the L-segment RNA, respectively. The presence of viral RNA in the cell culture media was detected at later time-points.

Conclusions

We have developed a method to follow RNA transcription in-vitro after synchronous infection of Vero cells. The obtained results may contribute to the understanding of the viral replication, and may have implications in the development of antiviral drugs targeting transcription or replication of negative stranded RNA viruses.
Zusatzmaterial
Additional file 1: Figure S1. The nucleotide sequence of the synthetic DNA. The selected sequence (1377 bp) are color coded to show the origin of the sequences in the RNA segments of Puumala Orthohantavirus. The nucleotides 80–326, shown in red letters, originate from the S segment; the black letters indicate the nucleotides 37–438 and 3178–3357 of the M-segment while the blue letters indicate the nucleotides 2640–2736 and 2935–3385 of the L-segment. (PPTM 34 kb)
12985_2018_932_MOESM1_ESM.pptm
Literatur
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