Arch Virol (2015) 160:1385–1395 DOI 10.1007/s00705-015-2386-2
ORIGINAL ARTICLE
A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance Shouli Wu1,2,3 • Pingping Yan1,2,3 • Yansheng Yan1,2,3 • Lijun Qiu1,2 Meirong Xie1,2
•
Received: 11 November 2014 / Accepted: 27 February 2015 / Published online: 22 March 2015 Ó Springer-Verlag Wien 2015
Abstract HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotypedvirus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1–280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug
susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination (R2) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 9 104 cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents.
Introduction & Yansheng Yan
[email protected] 1
Fujian Provincial Center for Disease Control and Prevention, No. 76 Jintai Road, Fuzhou, Fujian Province 350001, China
2
Fujian Province Key Laboratory of Zoonosis Research, No. 76 Jintai Road, Fuzhou, Fujian Province 350001, China
3
School of Public Health, Fujian Medical University, No. 88 Jiaotong Road, Fuzhou, Fujian Province 350004, China
Since China’s first AIDS case was reported in a traveler from abroad in Beijing in 1985, the HIV/AIDS epidemic has expanded rapidly [1]. Currently, over 780,000 people living in China are estimated to be infected with HIV/AIDS [2], and the disease has been identified as a leading cause of death from infectious diseases [3]. Results from China’s public health surveillance system for HIV show that there were 167,837 HIV/AIDS patients receiving highly active
123
1386
antiretroviral therapy (HAART) until 2013 [4]. Effective ART, which has been considered one of the most costeffective actions in medical sciences [5], has been shown to improve health status, survival, and quality of life for people living with HIV/AIDS [6–8]. As a preventive intervention, it is also effective to reduce the risk of transmission of HIV infection [9–11]. However, the extremely high levels of virus production and the high mutation rate of the virus, as well as lack of alternative drug combinations that completely inhibit viral replication, result in increasing emergence of HIV-1 strains that are resistant to antiretroviral drugs, making ART less effective over time [12–15]. Intensive monitoring of HIV-1 drug resistance is therefore of great importance for evaluating the sensitivity of current strains to antiretroviral agents and is urgently needed. Antiretroviral drug resistance testing not only provides key information for the selection of an optimal ART but is also essential for the formulation of public-health policies to control HIV/AIDS pandemics. The International AIDS Society USA panel has recommended that HIV drug resistance testing be included in the clinical management of AIDS [16]. Drug resistance testing may be performed by direct detection of drug sensitivity of viruses (phenotyping) or through sequencing of genes encoding antiretroviral targets (genotyping). Genotypic resistance testing is a relatively simple, fast, and low-cost approach that is commonly used in clinical resistance testing; however, genotypic data are often not sufficient to predict the effect of complex mutations. Phenotypic resistance testing is relatively slow and expensive in relative to genotypic analysis, but it detects drug resistance directly and is very helpful when a patient harbors a virus with complex genetic patterns or the mutational resistance profile of a specific agent is not well characterized [17]. Therefore, phenotypic resistance testing is considered crucial for providing optimal care, particularly for antiretroviral-experienced patients [18]. Currently, commercial assays for phenotypic resistance testing mainly include PhenoSenseTM (Virologic), AntivirogramTM (VIRCO), and PhenoScriptTM (Viralliance) kits [19]. Although the commercial kits greatly simplify testing procedures and improve testing repeatability, the range of drug resistance detection is restricted to a narrow spectrum. In addition, assays for testing phenotypic resistance to a wide range of drugs have been developed to target different fragments separately, which not only increases the cost but also fails to provide adequate information about drug resistance. Based on a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B0 isolate [20], a single-loop recombinant pseudotyped-virus-based assay was developed to detect HIV-1 phenotypic resistance.
123
S. Wu et al.
Materials and methods PCR amplification of a target fragment Two sets of universal primers (Table 1) were designed according to sequences of different HIV-1 strains; the amplified regions consisted of gag p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (aa 1–280) genes. An Age I or Apa I restriction site was introduced at the 50 terminus of the primer, while an Aar I restriction site was introduced at the 30 -terminus of the primer. HIV-1 RNA was extracted from human plasma samples, and an approximately 3-kb fragment was amplified by nested RTPCR. PCR amplification was performed at room temperature for 10 min and then at 50 °C for 30 min, followed by 30 cycles of 94 °C for 30 s, 57 °C for 45 s, and 72 °C for 4 min, and finally, 72 °C for 7 min. The second round of PCR amplification was done at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 57 °C for 45 s, and 72 °C for 4 min, and finally, 72 °C for 7 min. PCR products were analyzed by electrophoresis on a 1 % agarose gel and purified using a QIAquick Gel Extraction Kit (QIAGEN; Valencia, CA, USA) following the manufacturer’s protocol. Construction of a resistance test vector (RTV) A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc was used as an indicator gene viral vector. This construct contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene. RTVs were constructed by incorporating amplified target genes into pLWJ-SV40-Luc using ApaI or AgeI and AarI restriction sites and conventional cloning methods. Monoclonal colonies were harvested and inoculated. Plasmids were extracted and digested with XbaI or KpnI. Positive clones were sequenced for further identification. The flow chart for construction of RTV is shown in Fig. 1. Detection of RTV infection activity 293T cells were co-transfected with the RTV and the membrane-protein-expressing plasmid VSV-G (2:1, mass ratio), and cells were then incubated at 37 °C in a humidified atmosphere containing 5 % CO2 for 6 h. The medium was replaced, followed by further incubation for 48–72 h. The medium, which contained HIV-1 pseudotyped virus, was filtered through a 0.45-lm filter and used to infect fresh 293T cells. Viral replication was monitored
Assay for HIV-1 drug resistance Table 1 Universal primers used for detection of HIV-1 phenotypic resistance
1387
Gene
Sequence (50 -30 )
Position (bp)
Note
AgeI
TAGACCGGTTCTWTAAAACTYTMAGAG
1678–1705
Outer primer: AgeI/Vif-HB
Vif-HB
CCTTTTCTCCTGTCTGCAGACCCCAATATG
5238–5266
AarI
ATCAKCACCTGCCATCTGTTTTC
5048–5071
Inner primer: AgeI/AarI
ApaI
TGCAGGGCCCCTAGRAAAAARGG
1997–2020
Inner primer: ApaI/AarI
Fig. 1 Flow chart for the construction of the RTV
to detect luciferase activity in infected target cells at approximately 48 h postinfection. Establishment of experimental conditions for phenotypic resistance testing Since pLWJ, which was used to construct the HIV backbone plasmid vector, was derived from a drug-sensitive strain and the HIV-1 pseudotyped virus derived from pLWJ was susceptible to antiretroviral agents, the pseudotyped virus served as a wild-type strain for phenotypic resistance testing in this study. To evaluate the association of luciferase activity with pseudotyped virus load and concentration of infected cells,
HIV-1 pseudotyped virus was gradient-diluted at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128, with a final volume of 100 ll. 293T cells were transferred at 5 9 104, 1 9 105, and 2 9 105 cells/well with 100 ll in each well. Cells were then incubated at 37 °C in a humidified atmosphere containing 5 % CO2 for 48 h, and luciferase fluorescence was measured in relative fluorescence units (RFU). All treatments were performed in triplicate. To investigate the effect of cell density on the drug sensitivity test, drugs were serially diluted tenfold and then transferred to 96-well plates, with 50 ll in each well, while 50 ll of medium served as a control. HIV-1 pseudotyped virus was transferred to 96-well plates, with 100 ll per well, and 293T cells were then added at densities of
123
1388
5 9 104, 1 9 105, and 2 9 105 cells/well, with 50 ll per well. Plates were then placed at 37 °C in a humidified atmosphere containing 5 % CO2 for 48 h, and luciferase RLU was measured. All treatments were performed in triplicate. To evaluate the repeatability of the test, the sensitivity of HIV-1 pseudotyped virus to the nucleoside RT inhibitors (NRTIs) zidovudine (AZT), lamivudine (3TC) and stavudine (d4T), the non-nucleoside RT inhibitors (NNRTIs) nevirapine (NVP) and favirenz (EFV), and a protease inhibitor (PI), indinavir (IDV), was determined at four different time points. Drug resistance testing of clinical samples Based on the aforementioned results, drug resistance testing of clinical samples was performed using the newly developed pseudotyped-virus-based assay. NRTIs and NNRTIs were used to inhibit pseudotyped virus infection, while the PI was used to inhibit the formation of pseudotyped viruses. All treatments were performed in triplicate. The target sequences obtained were uploaded to the Stanford HIV Drug Resistance Database, and the genotypic resistance analysis was performed using the HIVdb program. Data analysis Viral inhibition was calculated using the following formula: Viral inhibition (%) = (1-luciferase RLU in the drugtreated sample/ luciferase RLU in the control) 9 100 %. All analyses were done with the software GraphPad Prism version 2.01. Nonlinear regression analysis was used to estimate the half-maximal inhibitory concentration (IC50) of the drug on different HIV-1 strains. The final phenotypic resistance was expressed as a ratio of the IC50 of the tested strains to the IC50 of wild-type strains. A ratio of \4 indicated drug sensitivity, while a ratio of 4 or greater was considered drug resistance.
Results
S. Wu et al.
Experimental conditions for phenotypic resistance testing A clear-cut dose-dependent relationship was observed between the amount of pseudotyped virus used for inoculation and luciferase activity at 293T cell densities of 5 9 104, 1 9 105, and 2 9 105 cells/well. At the same virus dilution, the highest luciferase activity was found in infected cells at a density of 5 9 104 cells per well (Fig. 2). The coefficients of determination (R2) were 0.995, 0.9446, and 0.9948 between AZT concentration and viral inhibition when HIV-1 pseudotyped virus was tested at 5 9 104, 1 9 105, and 2 9 105 viruses per well, respectively, indicating a clear-cut dose-dependent inhibition of HIV-1 pseudotyped virus by AZT, with an IC50 of 0.02199 (95 % CI: 0.01116 to 0.04335), 0.07487 (95 % CI: 0.00831 to 0.6729), and 0.03861 lmol/L (95 % CI: 0.01911 to 0.07801), respectively. The highest R2 was observed with 5 9 104 viruses per well, while luciferase activity was much higher than at the other two viral concentrations. At 1 9 105 viruses per well, AZT inhibited HIV-1 pseudotyped virus in an irregular manner, notably at a low AZT concentration (Fig. 3). The results demonstrate that the concentration of HIV-1 pseudotyped virus affects the drug sensitivity test, and 5 9 104 viruses per well is the optimal concentration of HIV-1 pseudotyped virus inoculated into 96-well plates. In addition, the assay was found to have high stability for detecting HIV-1 phenotypic drug resistance (Table 3). In this study, six commonly used antiretroviral agents including 3TC, AZT, d4T, NVP, EFV and IDV, were selected for the drug sensitivity test. The IC50 values obtained with most of these drugs were similar to those reported previously [21, 22] (Table 4; Fig. 4). HIV-1 genotypic resistance-associated mutant locus The mutant loci associated with drug resistance in the 29 strains of recombinant HIV-1 pseudotyped virus to six antiretroviral agents, including 3TC, AZT, d4T, NVP, EFV and IDV, are described in Table 5.
RTV construction and testing of its infection activity HIV-1 phenotypic resistance of clinical samples Universal primers were used to amplify 35 clinical samples, and 34 samples were successfully amplified, with a successful amplification rate of 97.1 %; 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. The pseudotyped viruses derived from 29 RTVs were able to infect cells. Detailed characteristics of the clinical samples are described in Table 2.
123
Drug sensitivity tests were conducted in 29 HIV-1 pseudotyped viruses, while the wild-type HIV-1 strain served as a control. A comparison of phenotypic and genotypic resistance testing results is shown in Tables 6 and 7. There was no difference between genotypic and phenotypic resistance testing results, except that one HIV-1 strain was found to be AZT resistant by genotypic resistance testing
Assay for HIV-1 drug resistance
1389
Table 2 Characteristics of clinical samples and RT-nested PCR and phenotypic resistance testing results Patient ID
PCR results
Phenotypic resistance test
Treatment time (months)
CD4 (cells/mm3)
Viral load (copies/ml)
Drugs
CHQ08
?
?
16.5
228
22066
AZT?3TC?NVP
CJW09
?
?
52
219
2727
D4T?3TC?NVP
CMC09
?
?
45
324
17604
D4T?3TC?NVP
CSG05
?
?
12
253
15654
D4T?3TC?NVP
CWL09
?
-
52
117
1079
AZT?3TC?NVP
HXX09
?
?*
24
359
52195
D4T?3TC?NVP
KXC09 LJH11
? -
? /
17.5 40
121 151
29983 2563
AZT?3TC?NVP IDV?DDI?AZT
LXL11
?
?*
18
260
143011
AZT?3TC?NVP
LZY11
?
?
15
262
5538
AZT?3TC?NVP
MXZ09
?
?
24
41
89598
AZT?3TC?NVP
WJS11
?
?
43
49
/
EFV?3TC?D4T
WJT11
?
?
17
58
1500000
AZT?3TC?NVP
WQM11
?
?
14
254
4390
EFV?3TC?AZT
WZF07
?
?
28
107
170107
D4T?3TC?NVP
XSQ11
?
?
19
139
73454
AZT?3TC?NVP
XX11
?
?
29
181
15800
NVP?3TC?D4T
YYX08
?
?
13.5
710
31229
AZT?3TC?NVP
ZFL06
?
?
11
/
/
AZT?3TC?NVP
ZFL09
?
?
47
137
21161
AZT?3TC?NVP
ZGX07
?
?
8
/
/
D4T?3TC?EFV
ZGX09 ZJH11
? ?
? ?*
38 26
228 80
34649 25436
D4T?3TC?EFV AZT?3TC?NVP
ZJX09
?
?
47.5
115
26214
AZT?DDI?IDV
ZJX11
?
?
73
55
22066
3TC?LPV/r?TDF
ZYK09
?
?*
47.5
546
6369
D4T?3TC?NVP
DXS05
?
?
0
9
50898
-
JPD11
?
?
0
/
/
-
KXC08
?
?
0
/
/
-
LZY10
?
?
0
/
/
-
XSQ10
?
?
0
/
/
-
WQMU07
?
?
0
142
/
-
ZCS11
?
?
0
336
/
-
ZJH09
?
?
0
/
/
-
ZZM11
?
?
0
12
/
-
‘‘/’’ indicates no detection; ‘‘?’’ indicates positive PCR amplification or successful construction; ‘‘-’’ indicates negative PCR amplification or unsuccessful construction; ‘‘?*’’ indicates successful construction without infectivity
but AZT sensitive by phenotypic resistance testing. Nonlinear regression analysis revealed no significant difference between genotypic and phenotypic resistance testing results (P [ 0.05); however, an increase in IC50 observed in phenotypic resistance testing was found to be partly inconsistent with that detected in genotypic resistance testing.
Discussion The enzymes RT, PR, and integrase are critical for HIV replication, and all three of these enzymes are major targets of currently available anti-HIV agents [23, 24]. Resistance mutations have been identified in these drug targets, and a resistance-related mutation locus has been detected in the
123
1390
S. Wu et al.
Fig. 2 Dose-dependent relationship between the amount of pseudotyped virus used for inoculation and the concentration of cells and luciferase activity in infected cells
Fig. 3 Effect of cell concentration on AZT drug sensitivity
Table 3 Repeatability of phenotypic resistance test
Drug
IC50 (lmol/l)
Mean (lmol/l)
SD
CV (%)
1
2
3
4
AZT
0.0387
0.0225
0.0301
0.0341
0.0314
0.0069
22.02
NVP
0.0181
0.0168
0.0200
0.0154
0.0176
0.0019
11.10
IDV
0.0028
0.0028
0.0025
0.0027
0.0027
0.0001
4.87
Table 4 Sensitivity test for six antiviral drugs using the established single-loop recombinant pseudotyped-virus-based assay Method and statistics
NRTI (lM) 3TC
Recombinant pseudotypedvirus-based assay
NNRTI (nM) AZT
d4T
PRI (nM)
NVP
EFV
IDV 2.8
IC50
3.74
0.04
1.83
18.2
0.2
95 % CI
2.292–6.102
0.0170–0.0884
0.403–8.268
12.3–26.8
0.124–0.285
0.78–9.68
R2
0.9960
0.9887
0.9607
0.9972
0.9967
0.9741
IC50 in reference [21]
1.77
0.03
0.66
78
2
6
IC50 in reference [22]
0.53
0.01
0.64
170
0.8
–
123
Assay for HIV-1 drug resistance
Fig. 4 Inhibition curves for different drugs measured in the phenotypic resistance testing system
gag cleavage site (p7/p1/p6) [25]. It is well known that HIV-1 protease is responsible for the post-translational processing of gag and gag-pol polyprotein; therefore, mutations in the gag region are likely to cause resistance to PIs [26]. Although the cleavage site of HIV-1 gag and gag-pol has been included in drug sensitivity phenotypic testing [27], most currently available drug resistance tests still depend on detection of RT and PR. The commercial assay AntivirogramTM only detects PRs and most RTs (codons 1482) [28] while PhenoSenseTM targets part of gag, all PRs, and codon 313 of RTs [21]. The assay developed in this study detected full-length PR, RT, RNase, and integrase (1-280aa), as well as p7-p1-p6 protease cleavage sites in the gag region. The size of the construct that was used was approximately 3000 bp. Theoretically, such a drug resistance testing assay should be able to detect resistance to RT inhibitors, PIs and integrase inhibitors in HIV-1 strains. However, first-line antiviral agents remain the major choice for treatment of HIV infections, and PI-based second-line antiretroviral therapy is not widely used in China currently. Commercial integrase inhibitors have not yet been introduced for clinical treatment of HIV/AIDS in China. Therefore, in the present study, we used phenotypic resistance testing to detect resistance to commonly used antiviral drugs – NRTI (AZT, 3TC, d4T), NNRTI (NVP, EFV), and PI (IDV) – and compared these results with those of drug sensitivity tests and genotypic resistance tests. Further studies are required to validate the effectiveness of this single-loop recombinant pseudotypedvirus-based assay to detect phenotypic resistance to other PIs and integrase inhibitors. Our findings showed the highest luciferase activity at a cell concentration of 5 9 104 per well, while the highest R2 value was obtained when comparing drug concentration and viral inhibition. This may be explained by a high cell density, which may inhibit the growth of virus-infected cells, thereby resulting in a low infection rate. In addition, drugs inhibited HIV-1 pseudotyped virus in an irregular
1391
manner at a high cell density. These findings demonstrate that cell inoculation affects the experimental results, and the optimal cell inoculation volume should be determined for each experiment. In this study, we found that the optimal cell concentration was 5 9 104 cells/well, and a repeatability test showed that the recombinant pseudotypedvirus-based phenotypic resistance testing assay developed in this study was stable. The drug IC50 values estimated in this study were not completely consistent with previous findings [21, 22]; however, the differences, which may be associated with the HIV-1 strains used for construction of the resistance testing assay and development principles and methods, was still within the allowable range. It is therefore considered that the detection of drug resistance in clinical HIV-1 samples cannot only depend on IC50 values. We suggest that a comparison of the estimated drug IC50 values with those determined for wild-type HIV-1 strains in parallel experiments should be used to identify the resistance of tested HIV-1 strains based on an increase in IC50. However, various criteria have been employed to identify phenotypic resistance. For example, a reduction of drug sensitivity by 2.5, 4, or more than 10 times has been identified as indications for drug resistance [29, 30]. In this study, a IC50 increase of \4-fold relative to wild-type HIV-1 strains was defined as drug sensitivity, while a increase of 4-fold or more was identified as drug resistance. However, this criterion was defined based on the drug resistance testing results of 29 currently available clinical samples. Further experiments with a larger number of samples are required to adjust the diagnostic criteria for HIV-1 phenotypic resistance with the assay developed in this study, so as to develop targeted criteria of HIV-1 resistance specific to each antiretroviral drug. Our findings showed no significant difference between genotypic and phenotypic resistance testing results, which was in agreement with previous studies [31, 32]. However, partial divergence was observed between the IC50 increase detected in phenotypic resistance testing and genotypic resistance testing, which may be explained by the following causes. (1) Presence of new or atypical resistance mutations. Novel or atypical resistance-associated mutations are not detected using available genotypic resistance testing assays, which, however, can be detected in a phenotypic resistance testing assay. (2) Presence of antagonistic or complex mutations. For example, the M184V mutation is reported to partly reverse the resistance to tamoxifen (TAM), zidovudine, stavudine and tenofovir, and to increase the sensitivity of HIV-1 to these antiviral agents [33]. Therefore, HIV-1 strains carrying these mutations may be identified as sensitive by phenotypic resistance testing but resistant by genotypic resistance testing. In this study, we screened HIV-1 drug-resistance-associated
123
123
CRF01_AE
B
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
C
CRF01_AE
C
CRF01_AE
C
ZFL06
CSG05
YYX08
WQM11
LZY11
CHQ08
WJT11
KXC09
XSQ11
MXZ09
WZF07
XX11
ZGX09
WJS11
CMC09
ZFL09
ZJX09
CJW09
ZJX11
CRF01_AE
XSQ10
CRF01_AE CRF01_AE
CRF01_AE
WQMU07
ZZM11 ZGX07
CRF01_AE
LZY10
CRF01_AE
CRF01_AE
KXC08
CRF01_AE
CRF01_AE
JPD11
ZJH09
CRF01_AE
DXS05
ZCS11
Subtype
Patient ID
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99 1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
1-99
M46I, I54V, V82A
None
V82A, N88S
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None None
None
None
None
None
None
None
None
None
L10I, L24I, K43T
None
T74S
None
None
None
None
None
L10I
None
None
None
None
None
None
None
None
None
None
None None
None
None
None
L10V
None
None
None
V11I
1-560
1-560
1-560
1-560
1-560
1-560
4-560
1-560
1-560
1-560
1-560
1-560
161560
1-560
1-560
1-560
1-560
1-560
1-560
1-560 1-560
1-560
1-560
1-560
1-560
1-560
1-560
1-560
1-560
D67N, T69N, K70R, M184V, K219Q
D67G, K70R,M184V, T215F, K219Q
D67N, T69N, K70R, K219Q
M41L, D67N, T69N, K70R,M184V,T215F, K219Q
None
L74V
D67N,T69D,K70R, M184V,T215I,K219Q
M184V
K65R, M184V
M41L,D67N,M184V, L210W, T215F
M184V
D67N,M184V,T215Y,
M184V,L210W, T215F
D67N, K70R, L210S
M184V
M184V, T215F
M184V
K65R, D67N
D67N,T69N,K70R, M184V, T215L
None M184V
None
None
None
K219R
None
None
None
None
NRTI
Codons
Minor
Codons
Major
RT mutations
PR mutations
Table 5 Analysis of drug resistant-associated mutant loci in 29 recombinant pseudotyped virus samples
Y181I, N348I
K101Q, K103N
Y181I, N348I
A98G, Y181V
Y181C
K103N,E138G, P225H, K238T
A98G,K103N,G190A
K101E,E138G,G190A
Y181I, Y188C
K101E, G190A
K103N, M230L
K101E,G190A,N348I
G190A
K101E, G190A
K103S, G190A
K103N, Y188H
K101E, G190A
K101Q,Y181C, G190A, H221Y
A98G, Y181V
None K103N, G190A
None
None
None
None
None
None
None
None
NNRTI
1-288
1-280
1-280
1-280
1-280
1-288
1-280
1-288
1-280
1-280
1-288
1-280
1-288
1-280
1-288
1-288
1-280
1-280
1-280
1-288 1-280
1-288
1-279
1-288
1-280
1-288
1-280
1-279
1-280
Codons
None
None
None
None
None
None
None
None
None
None
Y143H
None
None
None
None
None
None
None
None
None None
None
None
None
None
None
None
None
None
Major
IN mutations
None
A128T
None
None
None
None
None
None
H51P
None
L68I
E157Q
None
V151I
None
F121S
None
None
A128T
None None
None
None
None
I203M
None
None
None
None
Minor
1392 S. Wu et al.
Assay for HIV-1 drug resistance
1393
Table 6 Comparison of phenotypic resistance test results and genotypic resistance test results Patient ID
Phenotypic resistance (level: fold increase in IC50)
Genotypic resistance (level) AZT
3TC
D4T
NVP
EFV
IDV
AZT
DXS05
S
S
S
S
S
S
1.08
0.49
0.51
2.66
1.01
1.92
JPD11
S
S
S
S
S
S
1.37
1.16
0.68
0.87
1.24
1.14
KXC08
S
S
S
S
S
S
1.88
1.08
0.78
1.08
0.94
2.14
LZY10
S
S
S
S
S
S
0.94
1.04
0.94
0.83
1.6
1.94
WQMU07
P-L
S
S
S
S
S
1.49
0.97
0.91
0.58
1.72
2.76
XSQ10
S
S
S
S
S
S
0.58
0.77
0.83
0.84
1.04
0.64
ZCS11 ZJH09
S S
S S
S S
S S
S S
S S
1.44 1.12
0.6 1.05
0.81 1.14
0.97 0.87
0.75 1.23
0.79 0.64
ZZM11
S
S
S
S
S
S
1.36
1.31
0.74
1.07
1.47
1.61
ZGX07
S
H60
S
H120
H100
S
0.74
39.14
2.03
14245.59
353499.47
0.54
ZFL06
I30
H65
L27
H70
I35
S
3.18
65.16
6.56
693
0.58
CSG05
S
I30
L27
H125
H75
S
2.09
6.15
6.43
26976.87
330169.67
3.52
YYX08
S
H60
S
H90
I55
S
1.98
17.32
1.15
20093.61
8441.15
2.84
WQM11
L27
H64
L25
H120
H90
S
3.79
89.56
5.73
161
426.11
0.87
LZY11
S
H60
S
H120
H100
S
2.12
88.38
1.12
671.26
334.84
0.57
CHQ08
I33
S
L22
H90
I55
S
15.98
0.27
5.77
607.93
22030.75
WJT11
I42
H68
I40
H60
I40
S
19.61
97.19
23.15
100.91
84.01
KXC09
I42
H64
I37
H90
I55
S
118.29
9.05
8981.28
14432.66
2.78
XSQ11
S
H60
S
H120
H90
S
1.86
77.01
0.87
417.21
320.32
0.65
MXZ09
H72
H72
H67
H90
I55
S
174.99
78.37
12846.92
31028.63
2.41
WZF07
S
H90
P-L10
H120
H60
S
1.95
330.48
3.12
1635.46
58695.65
0.54
XX11 ZGX09
S H65
H60 H65
S I57
H95 H130
H60 H105
S S
59.81 48.72
1.31 11.57
125.77 7461.45
241.14 410021.21
1.41 0.83
WJS11
S
S
S
H110
H110
S
7.5
1.25
336.39
497.06
0.72
CMC09
S
S
S
H60
I30
S
1.46
1.25
0.98
92.57
3630.43
0.51
ZFL09
H95
H73
H82
H70
I35
S
77.96
69.57
17.18
10897.58
2639.98
0.45
ZJX09
I53
S
I42
H60
I30
I45
33.32
1.58
14.92
17389.87
1510.6
41.16
CJW09
H75
S
I57
H65
H65
S
58.91
2.36
10.96
26998.9
386532.34
0.49
ZJX11
I45
H90
I37
H75
I30
H90
18.75
417.91
15.72
12.51
35.87
125.3
3.1 52.3
3TC
1.63
D4T
16
NVP
EFV
7913
116.16
IDV
0.13 1.1
S indicates sensitivity; L indicates low-level resistance; P-L indicates potential low-level resistance; I indicates medium-level resistance; H indicates high-level resistance
Table 7 Comparison between phenotypic and genotypic resistance testing results
Resistance pattern
Antiretroviral drug
Genotype
Phenotype
AZT
3TC
d4T
NVP
EFV
IDV
Resistance
Resistance
10
16
13
21
20
2
Sensitivity
Sensitivity
17
12
16
8
9
27
Resistance
Sensitivity
2
0
0
0
0
0
Sensitivity
Resistance
0
1
0
0
0
0
1.000
1.000
1.000
1.000
1.000
1.000
P-value (McNemar’s test)
P [ 0.05 indicates no significant difference between genotypic and phenotypic resistance testing results
mutations and used the newly developed phenotypic resistance testing assay to validate the finding through directional mutations.
In conclusion, the single-loop recombinant pseudotyped-virus-based HIV-1 phenotypic resistance testing assay appears to be applicable for testing phenotypic
123
1394
resistance to currently commonly used antiviral agents in clinical HIV-1 strains, but it is also effective for identification of novel resistance-associated mutant loci, which may further improve the HIV drug resistance database.
References 1. Zhang KL, Detels R, Liao S, Cohen M, Yu DB (2008) China’s HIV/AIDS epidemic: continuing challenges. Lancet 372:1791–1793 2. Zheng H, Wang L, Huang P, Norris J, Wang Q, Peng Z, Yu R, Wang N (2014) Incidence and risk factors for AIDS-related mortality in HIV patients in China: a cross-sectional study. BMC Public Health 14:831 3. Jiang ZS, Jiang JN (2012) Research progress in death risk factors of HIV infector and AIDS patients. Inter J Epidemiol Infect Dis 39:63–67 4. Jin Y, Liu Z, Wang X, Liu H, Ding G, Su Y, Zhu L, Wang N (2014) A systematic review of cohort studies of the quality of life in HIV/AIDS patients after antiretroviral therapy. Int J STD AIDS (in press) 5. Piacenti FJ (2006) An update and review of antiretroviral therapy. Pharmacotherapy 26:1111–1133 6. Brechtl JR, Breitbart W, Galietta M, Krivo S, Rosenfeld B (2001) The use of highly active antiretroviral therapy (HAART) in patients with advanced HIV infection: impact on medical, palliative care, and quality of life outcomes. J Pain Symptom Manag 21:41–51 7. Malteˆz F, Doroana M, Branco T, Valente C (2001) Recent advances in antiretroviral treatment and prevention in HIV-infected patients. Curr Opin HIV AIDS 6:S21–S30 8. Burgoyne RW, Tan DH (2008) Prolongation and quality of life for HIV-infected adults treated with highly active antiretroviral therapy (HAART): a balancing act. J Antimicrob Chemother 61:469–473 9. Granich R, Crowley S, Vitoria M, Smyth C, Kahn JG, Bennett R, Lo YR, Souteyrand Y, Williams B (2010) Highly active antiretroviral treatment as prevention of HIV transmission: review of scientific evidence and update. Curr Opin HIV AIDS 5:298–304 10. Granich R, Crowley S, Vitoria M, Lo YR, Souteyrand Y, Dye C, Gilks C, Guerma T, De Cock KM, Williams B (2010) Highly active antiretroviral treatment for the prevention of HIV transmission. J Int AIDS Soc 13:1 11. Auvert B, Males S, Puren A, Taljaard D, Carae¨l M, Williams B (2004) Can highly active antiretroviral therapy reduce the spread of HIV?: A study in a township of South Africa. J Acquir Immune Defic Syndr 36:613–621 12. Gupta R, Hill A, Sawyer AW, Pillay D (2008) Emergence of drug resistance in HIV type 1-infected patients after receipt of first-line highly active antiretroviral therapy: a systematic review of clinical trials. Clin Infect Dis 47:712–722 13. Shafer RW, Schapiro JM (2008) HIV-1 drug resistance mutations: an updated framework for the second decade of HAART. AIDS Rev 10:67–84 14. Little SJ, Daar ES, D’Aquila RT, Keiser PH, Connick E, Whitcomb JM, Hellmann NS, Petropoulos CJ, Sutton L, Pitt JA, Rosenberg ES, Koup RA, Walker BD, Richman DD (1999) Reduced antiretroviral drug susceptibility among patients with primary HIV infection. JAMA 282:1142–1149
123
S. Wu et al. 15. Clavel F, Hance AJ (2004) HIV drug resistance. N Engl J Med 350:1023–1035 16. Carpenter CC, Cooper DA, Fischl MA, Gatell JM, Gazzard BG, Hammer SM, Hirsch MS, Jacobsen DM, Katzenstein DA, Montaner JS, Richman DD, Saag MS, Schechter M, Schooley RT, Thompson MA, Vella S, Yeni PG, Volberding PA (2000) Antiretroviral therapy in adults: updated recommendations of the International AIDS Society-USA Panel. JAMA 283:381–390 17. Hirsch HH, Drechsler H, Holbro A, Hamy F, Sendi P, Petrovic K, Klimkait T, Battegay M (2005) Genotypic and phenotypic resistance testing of HIV-1 in routine clinical care. Eur J Clin Microbiol Infect Dis 24:733–738 18. MacArthur RD (2009) Understanding HIV phenotypic resistance testing: usefulness in managing treatment-experienced patients. AIDS Rev 11:223–230 19. Qari SH, Respess R, Weinstock H, Beltrami EM, Hertogs K, Larder BA, Petropoulos CJ, Hellmann N, Heneine W (2002) Comparative analysis of two commercial phenotypic assays for drug susceptibility testing of human immunodeficiency virus type 1. J Clin Microbiol 40:31–35 20. Wu SL, Yan YS, Yan PP, Huang HL, Wang HR (2010) Construction and characterization of a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B’ isolate. Arch Virol 155:1923–1931 21. Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin T, Huang W, Tian H, Smith D, Winslow GA, Capon DJ, Whitcomb JM (2000) A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 44:920–928 22. Paolucci S, Baldanti F, Zavattoni M, Gerna G (2004) Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants. J Antimicrob Chemother 53:766–771 23. Arts EJ, Hazuda DJ (2012) HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect Med 2:a007161 24. Hazuda DJ (2012) HIV integrase as a target for antiretroviral therapy. Curr Opin HIV AIDS 7:383–389 25. Zhang YM, Imamichi H, Imamichi T, Lane HC, Falloon J, Vasudevachari MB, Salzman NP (1997) Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites. J Virol 71:6662–6670 26. Banke S, Lillemark MR, Gerstoft J, Obel N, Jørgensen LB (2009) Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations. J Virol 83:8916–8924 27. Robinson LH, Gale CV, Kleim JP (2002) Inclusion of full length human immunodeficiency virus type 1 (HIV-1) gag sequences in viral recombinants applied to drug susceptibility phenotyping. J Virol Methods 104:147–160 28. Hertogs K, de Be´thune MP, Miller V, Ivens T, Schel P, Van Cauwenberge A, Van Den Eynde C, Van Gerwen V, Azijn H, Van Houtte M, Peeters F, Staszewski S, Conant M, Bloor S, Kemp S, Larder B, Pauwels R (1998) A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother 42:269–276 29. Pe´rez-Elı´as MJ, Lanier R, Mun˜oz V, Garcia-Arata I, Casado JL, Marti-Belda P, Moreno A, Dronda F, Antela A, Marco S, Moreno S (2000) Phenotypic testing predicts virological response in successive protease inhibitor-based regimens. AIDS 14:F95– F101 30. Condra JH (1998) Resistance to HIV protease inhibitors. Haemophilia 4:610–615
Assay for HIV-1 drug resistance 31. Dunne AL, Mitchell FM, Coberly SK, Hellmann NS, Hoy J, Mijch A, Petropoulos CJ, Mills J, Crowe SM (2001) Comparison of genotyping and phenotyping methods for determining susceptibility of HIV-1 to antiretroviral drugs. AIDS 15:1471–1475 32. Hirsch HH, Drechsler H, Holbro A, Hamy F, Sendi P, Petrovic K, Klimkait T, Battegay M (2005) Genotypic and phenotypic
1395 resistance testing of HIV-1 in routine clinical care. Eur J Clin Microbiol Infect Dis 24:733–738 33. Ring A, Dowsett M (2004) Mechanisms of tamoxifen resistance. Endocr Relat Cancer 11:643–658
123