Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics

Abstract

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

Recent advances in rotavirus reverse genetics and its utilization in basic research and vaccine development

“…Interestingly, either the VP4 or VP7 segment alone from one of the above three strains was reassorted into the genetic SA11 backbone successfully. However, efforts to generate recombinant RV with VP4 and VP7 segments in combination with one of these three strains in SA11 backbone were not successful [ 173 ].…”

Reverse genetics system for human rotaviruses

“…As well as being efficient, this procedure can be performed without the use of any helper expression plasmid encoding a harmful protein, which is preferable for clinical production of safe vaccines and viral vectors. Using the 11‐plasmid system and similar approaches based on the 11‐plasmid system, in which the concentration of T7 plasmids containing the NSP2 and NSP5 genes is threefold increased in relation to that of other plasmids, several novel SA11‐L2 mutants with modified segment(s) have already been successfully generated 29,31–36 . These SA11‐L2 mutants have helped us to determine viral protein roles in the virus multiplication cycle 34,36 .…”

“…In addition, the 11‐plasmid system and systems based on this are robust, allowing the generation of animal RVAs which grow well in cell culture, as well as HuRVAs which do not replicate well in cell culture. These systems have already been exploited widely in RVA laboratories throughout the world 29,31–36 …”

Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics

“…This system greatly improved our ability to manipulate the RVA genome. It also allowed the engineering of HuRVA VP7 genes into single-gene reassortants that have much higher infectivity titres in vitro than the parental HuRVAs [40]. This 11 plasmid-only system even allowed the rescue of recombinant HuRVAs with much lower infectivity compared to that of animal RVAs [37].…”