Enterovirus 71 (EV-71) infections are generally manifested as mild hand, foot and mouth disease, but have been reported to cause severe neurological complications with high mortality rates. Treatment options remain limited due to the lack of antivirals. Octaguanidinium-conjugated morpholino oligomers (vivo-MOs) are single-stranded DNA-like antisense agents that can readily penetrate cells and reduce gene expression by steric blocking of complementary RNA sequences. In this study, inhibitory effects of three vivo-MOs that are complementary to the EV-71 internal ribosome entry site (IRES) and the RNA-dependent RNA polymerase (RdRP) were tested in RD cells. Vivo-MO-1 and vivo-MO-2 targeting the EV-71 IRES showed significant viral plaque reductions of 2.5 and 3.5 log10PFU/ml, respectively. Both vivo-MOs reduced viral RNA copies and viral capsid expression in RD cells in a dose-dependent manner. In contrast, vivo-MO-3 targeting the EV-71 RdRP exhibited less antiviral activity. Both vivo-MO-1 and 2 remained active when administered either 4h before or within 6h after EV-71 infection. Vivo-MO-2 exhibited antiviral activities against poliovirus (PV) and coxsackievirus A16 but vivo-MO-1 showed no antiviral activities against PV. Both the IRES-targeting vivo-MO-1 and vivo-MO-2 inhibit EV-71 RNA translation. Resistant mutants arose after serial passages in the presence of vivo-MO-1, but none were isolated against vivo-MO-2. A single T to C substitution at nucleotide position 533 was sufficient to confer resistance to vivo-MO-1. Our findings suggest that IRES-targeting vivo-MOs are good antiviral candidates for treating early EV-71 infection, and vivo-MO-2 is a more favorable candidate with broader antiviral spectrum against enteroviruses and are refractory to antiviral resistance.
This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments.
Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.
The complete genome sequence of the ALL-03 strain of rat cytomegalovirus (RCMV) has been determined. The RCMV genome has a length of 197,958 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. This strain is closely related to the English strain of RCMV in terms of genetic arrangement but differs slightly in size.