Generation of a set of SA11 expression plasmids for the development of a T7-RNA polymerase-dependent rotavirus reverse genetic system

Abstract

Rotavirus (RV) is a member of the family Reoviridae which contains a segmented double-stranded RNA genome comprised of 11 double-stranded RNA genome segments. Rotavirus is still the leading cause of severe diarrhoea worldwide in children less than five years of age and causes 215 000 deaths per year, most of which occur in Africa (Trask et al., 2016) Reverse genetic systems have been developed for members of the Reoviridae family, which include bluetongue virus (Boyce et al.,2008) orthoreovirus (Kobayshi et al., 2007) African horsesickness virus (Matsuo et al., 2010) and epizootic haemorrhagic disease virus (Yang et al., 2015). Reverse genetics allows for the manipulation of the viral genomes at cDNA level and also for the generation of information regarding the replication, pathogenesis and biological characterisation of these viruses. Until 2017, no reverse genetic system for rotavirus had been developed. Several helper virus dependent reverse genetic systems for rotaviruses have been described). However, they all depend on the presence of a helper virus and require strong selection. A true rotavirus reverse genetic system, which is free of any selection and allows manipulation of any genome segment, will enhance the understanding of the rotavirus replication cycle and elucidation of detailed host-pathogen interaction. This study was an attempt at developing a plasmid-based reverse genetic system for rotavirus with the use of cDNA expression plasmids based on the consensus SA11 sequence. The expression plasmids were constructed by cloning cDNA representing the consensus sequence of the 11 genome segment sequences of the rotavirus SA11 strain which were produced by PCR, into pSMART by means of FastCloning and In-Fusiong®HD cloning. The genome segments were flanked by a T7 promoter sequence on the 5’ end followed by a hepatitis delta virus (HDV) ribozyme sequence at the 3’ sequence to generate exact (+)ssRNA when transfected in mammalian cell cultures. The SA11 consensus sequence expression plasmids were transfected to BHK-T7 and BSR-T7 cells. Lysates of BHK-T7 and BSR-T7 cells were used to infect MA104 cells to generate viable virus indicating viral rescue. Viral rescue was evaluated with the use of immunofluorescent staining. Despite the indication of viral translation in one attempt of transfection, no viable virus was recovered following infection of MA104 cells with BHK-T7 and BSR-T7 cell lysates. The development of a reverse genetic system was unsuccessful in this study. Thus, the constructed set of SA11 CS expression plasmids will be the basis for further development towards a more robust rotavirus reverse genetic system.