Establishment of infectious HCV virion-producing cells with newly designed full-genome replicon RNA

Hepatitis C virus (HCV) replicon systems enable in-depth analysis of the life cycle of HCV. However, the previously reported full-genome replicon system is unable to produce authentic virions. On the basis of these results, we constructed newly designed full-genomic replicon RNA, which is composed of the intact 5′-terminal-half RNA extending to the NS2 region flanked by an extra selection marker gene. Huh-7 cells harboring this full-genomic RNA proliferated well under G418 selection and secreted virion-like particles into the supernatant. These particles, which were round and 50 nm in diameter when analyzed by electron microscopy, had a buoyant density of 1.08 g/mL that shifted to 1.19 g/mL after NP-40 treatment; these figures match the putative densities of intact virions and nucleocapsids without envelope. The particles also showed infectivity in a colony-forming assay. This system may offer another option for investigating the life cycle of HCV

Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny

A Sabin 1 poliovirus-based vaccine vector transfects Vero cells with high efficiency

Over the past 40 years, live oral poliovirus (PV) vaccines have contributed to the eradication of wild PV in most countries. These live vaccine strains have a high safety record and can stimulate both cellular and humoral immune responses. As both of these factors are critical characteristics of a good vaccine, we aimed to modify the oral PV vaccines to create a powerful vaccine vector for extraneous antigen expression. In this study, we amplified three separate fragments from the Sabin 1 virus genome by RT-PCR and cloned them into the pGEM-TEasy vector. A cassette containing engineered protease cleavage sites and a polylinker was introduced into one of these fragments (f1) in front of the translation start site. This construction facilitated the insertion of foreign genes into the vector and the subsequent release of their co-translated antigens after digestion by endogenous protease. We also placed a ribozyme (Rz) sequence between the T7 promoter and viral genomic DNA so that in vitro transcription and Rz cleavage recreated the authentic 5′ end of the PV genome RNA. Poly(A)40 tails were added to the 3′ end of the genome to stabilize the transcribed RNA. The three PV genome fragments and their derivatives were cloned into various types of vectors that were transfected into Vero cells. Virus rescue experiments demonstrated that both the Rz and poly(A)40 elements were required for high transfection efficiency of the vector-derived RNAs