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

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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.

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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

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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.

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A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance.

HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral thera...
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