Development of stable, cold-adapted, temperature-sensitive/conditional lethal chimeric enterovirus A71 and coxsackievirus A16

Figures

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  Fig. 1. Schematic diagrams representing the genome structures and respective translated proteins of EV71:eTLLβP20 (A), EV71:TLLeC5 (B) and TLLeCA16 (C). The thicker bars denote the coding regions (P1, P2 and P3) and thinner bars denote the 5′ and 3′ non-coding (NC) regions of the virus genomes. The lighter colour denotes translated polyproteins and respective viral proteins after cleavage. The black arrows indicate the positions of cleavage sites separating P1, P2 and P3 component of the viral polyproteins. The red arrows indicate the sites of introduction of the restriction enzymes (MluI and EagI). The red lettering and black lettering respectively indicate the type and position of non-synonymous and synonymous mutations that occurred after 20 successive passages in Vero cells incubated at 28 ​°C.

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  Fig. 2. A–C Virus growth kinetics of EV71:TLLβP20, EV71:eTLLβP20, EV71:TLLeC5 and TLLeCA16 in comparison with the original wild-type ST strain by total viral RNA quantity in the culture supernatant of infected Vero cells incubated at 28 ​°C (A), 37 ​°C (B) and 39.5 ​°C (C). D–E Assessment of temperature reversion growth characteristics of EV71:eTLLβP20 (black bar), EV71:TLLeC5 (red bar) and TLLeCA16 (blue bar) following six successive passages in Vero cells incubated at 37 ​°C. Growth characteristics of the virus derived from each successive culture at 37 ​°C in terms of virus growth kinetics by cell death (D) and virus titre in CCID50/mL at full cytopathic effect (CPE) (E) at incubation temperature of 28 ​°C (solid bar) and 37 ​°C (dotted bar). F A composite photograph showing Vero cells incubated at 39.5 ​°C and infected with EV71:TLLβP20 (F-c), EV71:eTLLβP20 (F-d), EV71:TLLeC5 (F-e) and TLLeCA16 (F-f) after six successive passages at incubation temperature of 37 ​°C. EV71:TLLβP20-infected Vero cells incubated at 37 ​°C served as positive control (F-a). Non-infected Vero cells served as negative control (F-b). The infected cells were stained with fluorescein-tagged monoclonal antibody…