Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells
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RESEARCH
Open Access
Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells Julia Wigren Byström1, Jonas Näslund2, Fredrik Trulsson1,2, Magnus Evander3, Olivia Wesula Lwande3, Clas Ahlm1 and Göran Bucht2*
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. Keywords: Orthohantavirus, RNA segments, In-vitro infection, Quantitative real-time PCR
Background Orthohantavirus genus in the hantaviridae family comprises more than 20 rodent borne viruses pathogenic to humans. Rodents, bats, moles and shrews have been recognised to carry distinct members of the Orthohantavirus genus [1]. The geographical distribution of the various strains of these viruses follow the distribution of the respective animal species [2]. Humans acquire infection via inhalation or direct contact with rodent faeces, urine and saliva. Globally, 150–200,000 persons are infected each year [3, 4]. The virus infects the endothelium * Correspondence: [email protected] 2 Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden Full list of author information is available at the end of the article
of various organs with symptoms of disease mainly from the kidneys and lungs [4]. Consequently, Orthohantavirus infections may lead to hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, or to the more lethal hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Th
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