The online version of this article (https://doi.org/10.1186/s12985-018-0932-8) contains supplementary material, which is available to authorized users.
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.
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.
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.
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.
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
Manigold T, Vial P. Human hantavirus infections: epidemiology, clinical features, pathogenesis and immunology. Swiss Med Wkly. 2014;144:w13937. PubMed
Ganaie SS, Mir MA. The role of viral genomic RNA and nucleocapsid protein in the autophagic clearance of hantavirus glycoprotein Gn. Virus Res. 2014;
Vapalahti O, Kallio-Kokko H, Narvanen A, Julkunen I, Lundkvist A, Plyusnin A, Lehvaslaiho H, Brummer-Korvenkontio M, Vaheri A, Lankinen H: Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response. J Med Virol 1995, 46:293-303.
Plyusnin A, Elliott RM. Bunyaviridae : molecular and cellular biology. Norfolk, UK: Caister Academic Press; 2011.
Lundkvist A, Cheng Y, Sjolander KB, Niklasson B, Vaheri A, Plyusnin A. Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys Glareolus, and leads to accumulation of mutants with altered genomic RNA S segment. J Virol. 1997;71:9515–23. PubMedPubMedCentral
- Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells
Julia Wigren Byström
Olivia Wesula Lwande
- BioMed Central
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