Japanese encephalitis virus replication: A procedure for the selective isolation and characterization of viral RNA species

Replication of Flaviviruses

“…Small intracellular (8-15 S) RNAs have also been observed in actinomycin-D-treated flavivirus-infected cells (Zebovitz et al, 1972(Zebovitz et al, , 1974Takeda et al, 1978;Naeve and Trent, 1978). Wengler et al (1978) reported that three sizes of virus-specific low-molecular-weight flavivirus RNAs could be detected.…”

“…In addition to single-stranded 40 S genome RNA, RNaseresistant RNA (20-22 S) and RNA that is partially (50-70%) resistant to RNase (20-28 S) have been observed in cells infected with a number of different flaviviruses (Stollar et al, 1967;Trent et al, 1969;Zebovitz et al, 1972;Wengler et al, 1978;Cleaves et al, 1981;Chu and Westaway, 1985). Pulse-chase studies showed that [3H]uridine appeared first in the 20-28 S RNA and subsequently in the 40 S RNA (Cleaves et al, 1981).…”

“…Further, if a flavivirus infection induces a rapid cytopathic effect in these permissive cells, the possibility of multiple cycles of viral replication within the same host cell is eliminated. Small virus-specific RNAs (8-15 S) have often been observed in flavivirus-infected cells (Zebovitz et al, 1972(Zebovitz et al, , 1974Takeda et al, 1978;Naeve and Trent, 1978). Wengler et al (1978) reported that WN virus produced two low-molecular-weight RNAs (6.5 x 10 4 and 4.2 x 10 4 ) in BHK cells, but only the 6.5 x 10 dalton species in A. albopictus cells.…”

Flavivirus RNA synthesis in vitro

“…The genome-size RNA of 40–44S has type 1 cap at the 5′-end but no poly(A) at the 3′-end [1, 41–44]. In addition, double-stranded RNA of 20–22S, and heterogeneous 20–28S RNA presumably replicative form (RF) and replicative intermediate (RI) were also observed [45–48] as well as smaller RNAs of 8–12S RNA in flavivirus-infected cells [1]. It was concluded that this small RNA species were the degradation products of viral RNAs [49] as it was reported that there was no evidence for template activity of a small RNA in a previous study [1].…”

“…The replication complex consisted of viral RNA polymerase, ssRNA, the 20 S and 26 S replicative form (RF) and replicative intermediate (RI) form of viral RNA, respectively [53, 54]. In earlier studies, the endogenous membrane-bound replication complex has been characterized in viral RNA synthesis by Qureshi and Trent using Saint Louis encephalitis virus (SLE)-infected BHK-21 cells [55] and by Zebovitz et al using the JEV-infected porcine kidney cells, PS(Y-15) [47, 48]. To characterize the RNA species in the cytoplasmic membrane structures sedimenting at 250 S, the SLE-infected BHK-21 cells in the presence of actinomycin, were pulse-labeled with [ 3 H]labeled uridine for 30 min at 16 h post-infection [55].…”