A Rapid Phenotypic Assay for Detection of Acyclovir-Resistant Varicella-Zoster Virus with Mutations in the Thymidine Kinase Open Reading Frame

DOI: 10.1128/AAC.44.4.873-878.2000

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Fig. 1.
pkk-vzv-tk expression vector. The VZV TK ORF (VZV TK) is between the EcoRI and HindIII restriction sites downstream of the Tac promoter (Tac). The ampicillin (amp) resistance gene and the origin of replication (ori) are also indicated.
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Fig. 2.
Relative number of CFU on FUdR-containing plates expressed as the percentage of that on nucleoside analog-free plates. After PCR amplification, the TK ORF was digested with EcoRI and HindIII and was then inserted into pKK223-3 and subjected to the colony reduction assay as described in Materials and Methods. The number of colonies growing in the absence and in the presence of FUdR was recorded from at least three independent experiments. The relative number of CFU on FUdR-containing plates is expressed as the percentage of that on FUdR-free plates. The error bars correspond to the standard deviations. Sample names are indicated below each histogram. LY, LY-6625-resistant control; WT, a wild-type VZV TK control.

Table 1.

Colony reduction assay of control VZV strains

Strain	Origin^a	No. of colonies on plates:		%^b
Strain	Origin^a	Without FUdR	With FUdR	%^b
WT1	Infected cell culture	3,200	360	8
WT2	Infected cell culture	2,000	450	17
WT3	Clinical sample	1,800	330	12
LYON-6625	Infected cell culture	1,000	950	95
LYON-6625	Infected cell culture	3,000	2,600	86

↵a Template DNA was purified from the indicated source.
↵b Relative number of colonies on FUdR-containing plates expressed as the percentage of the number of colonies on plates not containing FUdR.

Table 2.

Correlation of clinical resistance, colony reduction assay with bacteria, and susceptibility assay with cells in culture^a

VZV strain (origin)	Source^b	Clinical resistance	Phenotype (CRA)	IC₅₀(μg/ml)^c		Size of the TK PCR fragment^d	TK genotype^e
VZV strain (origin)	Source^b	Clinical resistance	Phenotype (CRA)	ACV	BVDU	Size of the TK PCR fragment^d	TK genotype^e
A (NCLU II, CSF)	CC	No	S	0.28 ± 0.28 (S)	0.0018 ± 0.0013 (S)	WT	WT
B (NCLU III, CSF)	CC	Yes	R	8.8 ± 2.0 (R)	>50 (R)	Del	ND
C (NCLU IV, CSF)	CC	Yes	R	12.5 ± 7.2 (R)	>50 (R)	Del	ND
D (OKA)	CC	NA	S	0.22 ± 0.14 (S)	0.0008 ± 0.0003 (S)	WT	ND
E (OKA, ACV^r in vitro)	CC	NA	R	20.7 ± 4.7 (R)	>20^* (R)	WT	ND
F (OKA, BVDU^r in vitro)	CC	NA	R	27.4 ± 6.5 (R)	>20^* (R)	WT	ND
G (OKA, PFA^r in vitro)	CC	NA	S	4.7 ± 3.7 (R)	0.0032 ± 0.0014 (S)	WT	WT
H (CI-22)	CC	No	S	0.33 ± 0.27 (S)	0.0009 ± 0.0001 (S)	WT	ND
I (CI-13)	CC	No	S	0.24 ± 0.35 (S)	0.0014 ± 0.0012 (S)	WT	ND
J (CI-16)	CC	No	S	0.32 ± 0.36 (S)	0.0008 ± 0.0006 (S)	WT	ND
K (O7-1)	CC	NA	R	13.5 ± 4.4 (R)	24.8 ± 19.4 (R)	WT	ND
106	CSF	No	S	ND	ND	WT	WT
4.2	CSF	Yes	R	ND	ND	WT	Mut

↵a Abbreviations and symbols: CRA, colony reduction assay; CC, cell culture; S, susceptible; R, resistant; NA, not applicable; ND, not determined; WT, wild type; Del, deletion; Mut, mutant; ∗, only one determination.
↵b DNA was purified from the indicated source of material, either cell culture or CSF.
↵c Drug concentration (mean ± standard deviation of at least two independent experiments) required to reduce the viral cytopathic effect by 50% in the susceptibility assay.
↵d The size of the PCR product either was that of the wild type or was shorter owing to an internal deletion, as determined by gel electrophoresis.
↵e TK was either that of the wild type or that of the mutant as determined from the sequence of its corresponding PCR product.