Viral determinants that drive Enterovirus-A71 fitness and virulence

Figures & data

Figure 1. Schematic illustration of EV-A71 genome organization.

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 42^0C	–		[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 × 10^1 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]

Table 2. Comparative studies of EV-A71 genome sequences and their phenotypic differences.

	Phenotypic Differences
Mutations	In vitro	In vivo	References
Protease 2A from a severe strain → protease 2A from a mild strain Protease 2A Position 64 (Y64H) Position 68 (M68R) Position 85 (D85E)	RD cells, Vero cells and SH-SY5Y cells ➢Slower replication compared to the severe strain at 37 °C and 39.5 °C ➢Less viral RNA compared to the severe strain at 37 °C and 39.5 °C	1-day-old ICR mice ➢Less severe pathological changes compared to severe strain ➢Lower viral load compared to severe strain	[78]
3CD region from severe strain → 3CD region from mild strain Protease 3C Position 138 (V138I) 3D polymerase Position 37 (S37N) Position 143 (R143K) Position 176 (I176V) Position 261 (E261G) Position 360 (G360A)	RD cells and Vero cells ➢Milder CPE as compared to severe strain at 37 °C and 39.5 °C ➢Decreased replication rate compared to severe strain at 39.5 °C	1-day-old ICR mice ➢Mice showed no paralysis, no death, less body weight loss, and milder symptoms as compared to the mice injected with the severe strain ➢Lower viral load as compared to mice infected with the severe strain	[38]
Weakly pathogenic strain → Highly pathogenic strain 5’ UTR Position 115 (115C>T) Position 132 (132T>C) Position 811 (811T>C) VP1 Position 639 (G639S) VP3 Position 485 (Q485R) Position 504 (A504V) Position 537 (V537I) Protease 2A Position 937 (G937S) Protease 2B Position 1014 (A1014V) Position 1045 (I1045V) Position 1062 (T1062I) Protease 2C Position 1135 (K1135R) Position 1145 (E1145D) Position 1155 (V1155A) Position 1361 (V1361M) Protease 3C Position 1597 (I1597V) Position 1617 (D1617N) 3D Polymerase Position 1746 (L1746F) Position 2146 (A2146V) 3’ UTR Position 7347 (7347A>G)	Nucleotide differences when a weakly pathogenic strain (JN200803) is compared to a highly pathogenic strain (JN200804) MA104 cells ➢Both strains (weakly and highly pathogenic strain) exhibited similar CPE ➢No difference in virulence as determined by TCID50 titration	1-day-old BALB/c mice ➢Mice infected with the highly pathogenic strain exhibited hind limb paralysis by day 4 post-infection. ➢Mice infected with the weakly pathogenic strain had no evident neurological symptoms and remained healthy.	[79]