Advances in reverse genetics systems for rotavirus / 中华微生物学和免疫学杂志

Reverse genetics approaches can directly manipulate the genome of virus at the gene level, making it possible to quickly, directly and thoroughly study the mechanisms of virus replication and pathogenesis. At present, many viruses of the family Reoviridae, such as mammalian orthoreovirus ( MRV) and bluetongue virus ( BTV) , have made great progress in basic viral research using the powerful tool of re-verse genetics. However, for members of the genus Rotavirus in the family Reoviridae, progress in the con-struction of reverse genetic systems has been slow. The remarkable reverse genetics system based on helper-viruses was established in 2006, and it was not until 2017 that the entirely plasmid-based reverse genetics system was successfully established. This paper briefly reviewed the development of reverse genetics systems for rotavirus and prospected the direction for future research in order to provide technical support for acceler-ating the basic research on mechanisms of rotavirus infection.

Rescue of Synthetic Hantavirus-Like Foreign RNA by Hantavirus as a Helper Virus

A system for the expression of synthetic hantavirus-like luciferase RNA was developed using Hantaan (HTN) virus as a helper virus. The hantavirus-like luciferase RNA was constructed by the deletion of the coding region in HTN virus (76~118) S genome and by replacement of a luciferase gene. PCR was performed using primers designed to amplify the whole region of hantavirus-like foreign gene. The resulting PCR product was placed under the control of the T7 RNA polymerase promoter for in vitro transcription. The produced hantavirus-like luciferase RNA was transfected into Vero-E6 cell infected with HTN virus using lipofectin. The level of expression was measured using a luminometer. The hantavirus-like luciferase RNA was allowed to amplify and express. And also, the hantavirus-like luciferase RNA was packaged into HTN virions. The 5' terminal and 3' terminal conserved sequences of HTN virus genome were sufficient to provide the signals for RNA amplification and packaging. This suggests that the RNA promoter region for hantavirus RNA synthesis is located in 3' terminal region. The luciferase activity was analyzed from the progeny virus-infected cells in order to examine if the 5' and 3' terminal sequences play a role in regulating a packaging pathway while genome of HTN virus is packaged. The luciferase activity was detectable in every cell passage. However, the activity of luciferase was decreased gradually after each passage. The fact that the hantavirus-like luciferase RNA can be packaged into progeny virus suggests that the 5' and 3' terminal sequences of HTN virus genome play an important role in regulating a packaging pathway.