Recovery of bovine rotavirus infectious particles by gene transfection method in 3 cell lines

Rotaviruses, members of the family Reoviridae, are the principal agents of infectious diarrhea among wide range of animal species including mammalian. They cause many deaths per year. Rotavirus consists of three concentric layers of protein and 11 segments of double stranded RNA (dsRNA). Collectively, VP1, VP2, and VP3 and the dsRNA genome construct the core of the virion. VP1 is RNA dependent RNA polymerase and presence of VP2 is necessary for VP1 activity. Previously reported that VP1, VP2 and VP3 are necessary to replicate RNA genome of rotaviruses. Aim of this study is to illustrate only VP1 and VP2 and VP3 proteins are enough for the packaging. In this study, VP1 and VP2 and VP3 genes were cloned in a mammalian expression vector, pcDNA3.1(+). Then, cultured MA104 cell line transfected simultaneously by the segmented double stranded genome of rotaviruses and three resulted plasmids. Then, after 60 hours, transfected MA104 cells were fixed and observed under the transmission electron microscope (TEM) by positive staining. Finally, RT-PCR was performed by using standard primers of VP6 for confirmation of virus presence. In three sequentially passages, infection of MA104 cells was done and again the cells were fixed and observed under TEM by positive staining. Complete rotavirus particles were observed under TEM microscope in positive staining which shows the rotavirus genome could replicate in presence of VP1 and VP2 and VP3 proteins necessary for the replication and packaging. Also RT-PCR of particles proved that these particles are rotaviruses. TEM after third passage showed resulted particles are infectious particles of rotaviruses. This study illustrates that transfection of rotavirus genome with VP1 and VP2 and VP3 genes can trigger the replication process of rotaviruses. Furthermore, for isolation of rotavirus, usually filtered stool samples used to infect the host cells. But this new method provides the possibility of mass isolation of rotaviruses in an easier and less cost way. In addition, we can use this method for production and isolation of other viruses with similar structures and functions, and even new recombinant viruses.