Table 1.

Mutations in EV-A71 genome that influence fitness and virulence.

		Phenotype/Mechanism
Region	Mutation	In vitro		In vivo	References
VP1	31G	DLD-1, RD and SK-N-SH cells ➢Reduced virion stability at 35°C, 36°C, 37°C, and 39°C ➢Reduced viral titer in DLD-1 cells ➢Increased growth in SK-N-SH cells	–		[35]
	L97R	SH-SY5Y cells ➢Replicative advantage ➢Improved binding to host cells ➢Associated with E167G/A (Entry, or assembly)	–		[66]
	E98K	L929 and Neuro-2a cells ➢Improved growth kinetic	Two-week-old (BALB/c mice) ➢Non-fatal ➢No visible damage to liver and lungs and were comparable to control mice infected with PBS		[67]
	107A	Vero cells ➢Reduced viral growth and titre ➢Regulates the cleavage of precursor VP0 and potentially affects virus uncoating and maturation	–		[50]
	145G	RD cells overexpressing hSCARB2 ➢Improved replication ➢Enhanced HS binding compared to 145E	Neonatal mice (ICR mice) ➢Lower viral loads in organs ➢Non-fatal  hSCARB2 transgenic mice ➢High adsorption to non-susceptible cells and rapid decrease in plasma of hSCARB2 transgenic mice ➢Lowered viral load in organs ➢Non-fatal		[44]
	145E	RD cells overexpressing hSCARB2 ➢Lower virus titer produced in mutants compared to 145G	Neonatal mice (ICR) ➢Higher viral load in organs ➢Lethal in mice hSCARB2 transgenic mice ➢More efficient replication and greater neuroinvasion compared to 145G
		RD cells ➢Mouse-adapted strain ➢Lower titers compared to parental strain ➢Decreased binding to cell	1-week old mice (BALB/c) ➢Caused 100% mortality		[45,46]
		Jurkat T cells ➢Molecular switch that changes virus from PSGL-binding (145G/Q) to non-PSGL-binding	–		[68]
		–	5-day-old BALB/c mice Caused 100% mortality		[69]
		–	Cynomolgous monkeys ➢CNS inflammation and damage ➢Neurological manifestations (tremors) ➢145E mutants recovered from monkeys inoculated with 145G mutant, while those inoculated with 145E maintained the same mutation, suggesting a selection pressure for 145E. ➢Innate immune system response with rise in IFNα, TNFα and IL-6 ➢More resistant to neutralizing antibodies		[47]
	Double mutant VP1 145E & VP2 149M	Neuro-2a cells ➢Higher viral titers ➢Higher viral protein expression	1-day old mice (ICR) ➢149M increased effect of 145E for mouse lethality		[48]
	Double mutant 145E & 98E	Murine NIH/3T3 and Neuro-2a cells ➢Productive infection ➢Addition of 169F mutation further enhanced infectivity	1-week old mice (BALB/c) ➢No significant signs of disease and non-lethal in mice		[51]
	K162A	Computer simulated Heparin ➢Reduced strength of interaction with heparin	–		[28]
	192M	Vero cells ➢Resistance to EV-A71 inhibitor pyridyl imidazolidinone	–		[27]
	K215A	Vero cells Increased thermostability where infectivity of mutant is higher than wild type after treatment at 420C	–		[70]
	K242A	Computer simulated Heparin ➢Reduced strength of interaction with heparin RD cells ➢Reduced binding on cell surface	–		[28]
	K244A
	K244E	–	1-day-old (BALB/c mice) ➢Increased virulence 5-day-old (BALB/c mice) ➢Caused 100% mortality at virus doses greater than 8.7 × 101 TCID50 6-week-old (AG129 mice) ➢Increased virulence		[71] [72]
	A289T	Human Brain Microvascular Endothelial Cells (HBMECs) ➢Decreased viral attachment, replication, protein synthesis, and virus particle secretion.	1-week-old (BALB/c&Sv129) ➢Decreased morbidity; decreased CNS infectivity		[73]
	Q172A	RD cells ➢Loss in both binding and infection	–		[49]
	Q152A R166A W171A T173A T175A N176A S178A F180A R236A	RD cells ➢Loss in binding and infection ➢No impairment of viral assembly except for N176A	–
VP2	K149I	Chinese Hamster Ovary cells ➢More efficient replication with higher virus titre	–		[45]
5’ UTR	C158U	–	Three-day-old ICR mice ➢Prolonged survival of infected mice ➢Reduced viral replication and virulence		[53]
	C115T	–	1-day old BALB/c mice ➢Reduced virulence		[60]
	A158T C475T A486G G487A	RD cells ➢Reduced CPE ➢Lower viral titers	–		[65]
Helicase 2C	K135A D176N C270A C281A C286A I141R S282R E325A L327K F328A F328Y	Vero cells ➢Reduced viral production ➢Reduced ATPase activity of protein 2C	–		[74]
Protease 3A	A5262G	RD cells ➢Absence of CPE ➢Reduced viral titer	–		[65]
Protease 3C	N69D	RD cells ➢Decreased enzymatic/catalytic activity ➢Decreased viral production	–		[75]
	T79V	RD cells ➢Increased viral replication Immunoprecipitation ➢Increased interaction with TRIM21	–		[58]
	R84Q	Vero cells ➢Loss of RNA-binding activity ➢No plaque formation at 8 d.p.i.	–		[76]
	I86A	Vero cells ➢Loss of proteolytic activity ➢No plaque formation at 8 d.p.i.	–		[76]
3D polymerase	I251T	SK-N-SH cells ➢Viral replication rate reduced by up to 100-fold at 39.5°C	ICR (newborn) ➢Reduced virulence		[77]
	L123F	RD cells ➢Increased replication fidelity ➢In vitro growth not altered	Ten-day-old (AG129 mice) ➢Reduced virulence ➢Reduced viral titers		[31]
	Double mutation G64R & L123F	RD cells ➢Increased replication fidelity ➢Attenuated in vitro growth	Ten-day-old (AG129 mice) ➢Reduced virulence ➢Reduced viral titer compared to L123F single mutation.		[31]