AIDS RESEARCH AND HUMAN Volume 16, Number 8, 2000, pp. 807–813 Mary Ann Liebert, Inc.

Sequence Note

Analysis of HIV Type 1 Protease and Reverse Transcriptase in Antiretroviral Drug-Naive Ugandan Adults

G. BECKER-PERGOLA, 1 P. KATAAHA, 2 L. JOHNSTON-DOW, 3 S. FUNG,3 J.B. JACKSON, 1 and S.H. ESHLEMAN 1

ABSTRACT

We analyzed plasma HIV-1 from 27 antiretroviral drug-naive Ugandan adults. Previous subtype analysis of and gag sequences from these samples identified subtypes A, C, D, and recombinant HIV-1. Sequences of HIV-1 protease and reverse transcriptase (RT) were obtained with a commercial HIV-1 genotyping system. Subtypes based on protease sequences differed from gag subtypes for 5 of 27 samples, demonstrating a high rate of recombination between the gag and pol regions. Protease and RT sequences were analyzed for the presence of amino acid polymorphisms at positions that are sites of previously characterized drug resistance mutations. At those sites, frequent polymorphisms were detected at positions 36 and 69 in protease and posi- tions 179, 211, and 214 in RT. Subtype-specific amino acid motifs were identified in protease. Most of the sub- type A sequences had the amino acids DKKM at positions 35, 57, 69, and 89, whereas most subtype D se- quences had the amino acids ERHL at those positions. Detection of those polymorphisms may provide a useful approach for rapid identification of subtype A and D isolates in Uganda. This analysis significantly increases the number of Ugandan protease and RT sequences characterized to date and demonstrates successful use of a commercial HIV-1 genotyping system for analysis of diverse non-B HIV-1 subtypes.

URRENTLYLICENSED antiretroviral drugs target HIV-1 pro- prevalence of HIV-1 infection is high. While general use of an- Ctease and reverse transcriptase (RT). Resistance to anti- tiretroviral drugs in Uganda is limited, Uganda has been the site retroviral drugs is frequently associated with selection of HIV- of several clinical trials using antiretroviral drugs to treat and 1 variants with mutations in these enzymes. To date, most prevent HIV-1 infection. Most HIV-1 infections in Uganda are studies of HIV-1 protease and RT have been performed for sub- caused by subtype A and D HIV-1, 1,2 although infection with type (clade) B HIV-1, which is the most common subtype in subtypes C1,2 and G3,4 has also been reported. These subtypes the United States and . In contrast, there are few stud- have been found in diverse regions throughout the world. In the ies characterizing these enzymes in non-B HIV-1, which ac- United States, individuals have been identified with subtype A, counts for the majority of HIV-1 infections worldwide. Char- C, and D HIV-1. 5–9 One study found subtype A HIV-1 in 2 of acterization of these enzymes in non-B HIV-1 is becoming 22 individuals, 8 and another found 3 of 91 individuals to have increasingly important, as the prevalence of non-B HIV-1 in- non-B HIV-1, including 1 individual with subtype A who had creases in countries where antiretroviral drugs are widely used, no history of foreign travel or contact. 9 An important step in and the availability of antiretroviral drugs increases in devel- understanding the genetic correlates of drug resistance in non- oping countries. B HIV-1 is to characterize the baseline sequences of HIV-1 pro- Diverse HIV-1 subtypes are found in Uganda, where the tease and RT from individuals who have never received anti-

1Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205. 2Nakasero Blood Bank, Kampala, Uganda. 3PE Biosystems, Foster City, California 94404.

807 808 BECKER-PERGOLA ET AL. retroviral therapy. In this sequence note we analyze HIV-1 pro- tems) HIV genotyping system (HIV genotyping system tease and RT sequences from 27 antiretroviral drug-naive Ugan- Prt/59 RT; PE Biosystems, Foster City, CA). In this system, dan adults. HIV-1 RNA is isolated from plasma and reverse transcribed Plasma samples used for analysis were obtained from asymp- with random hexamer primers and Moloney murine leukemia tomatic volunteer blood donors at the Nakasero Blood Bank in RT. An HIV-1 DNA fragment including the regions en- Kampala, Uganda in 1993 and 1994. 1 Units from donors that coding protease (amino acids 1–99) and RT (amino acids were repeated reactive for HIV in donor-screening tests were 1–310) is amplified by polymerase chain reaction (PCR), using not used for transfusion. Plasma samples from 27 of those units TaqGold in a single 40-cycle reaction. The amplified DNA is have been analyzed in this article. HIV-1 from the same sam- then purified and sequenced with six or seven primers and ples was subtyped in a previous report by phylogenetic analy- BigDye terminator reagents. Sequences are edited, aligned, sis of env gp41 and gag p24 sequences. 1 Most of the samples translated into amino acids, and analyzed for the presence of (24 of 27) had the same subtype in both the env and gag re- amino acid polymorphisms. Controls to monitor for and pre- gions. Those included 15 subtype A, 2 subtype C, and 7 sub- vent DNA contamination in sample preparation and DNA am- type D samples. Three samples contained recombinant HIV-1, plification have been described previously. 10 one with subtype A in env and D in gag (A/D) and two with Protease sequences from the 27 Ugandan isolates are shown subtype D in env and subtype A in gag (D/A) (Table 1). in Fig. 1. Phylogenetic methods were used to determine the sub- Analysis of HIV-1 protease and RT sequences was per- type of each sample in the protease-coding region (Fig. 2). Of formed with the Perkin-Elmer/Applied Biosystems (PE Biosys- the 27 samples, 17 had subtype A protease regions, and 10 had

FIG. 1. Ugandan HIV-1 protease sequences. Ugandan plasma samples were analyzed with the PE Biosystems HIV genotyp- ing system as described. Protease sequences were aligned by the CLUSTAL method (MegAlign, DNASTAR, Lasergene). HIV- 1 subtypes were defined for the protease region by using the corresponding nucleotide sequences (see Fig. 2); the protease sub- type of each isolate is indicated in parentheses. Dashes indicate identity with the consensus sequence (Majority, top). Because the majority of samples are subtype A in protease, the consensus sequence resembles the Ugandan subtype A protease sequences. Dashes indicate identity with the consensus sequence. X indicates the presence of an amino acid mixture. Amino acid mixtures in the protease sequences are as follows: 135.544: 63L/P; 161.287: 57K/R; 194.793: 14K/R; 225.710: 63P/S; 326.675: 14K/R,15I/V; 108.448: 36I/M; 134.463: 13I/V; 326.570: 33L/V; 326.662: 37D/N; 501.045: 15I/V, 69H/Y, 72I/M: 503.083: 14K/R, 60D/E, 63P/S, 64I/V. The GenBank accession numbers of the corresponding protease nucleotide sequences are as follows: 129.733, 135.544, 140.223, 161.287, 171.005, 184.574, 190.574, 194.793, 204.987, 225.706, 230.580, 326.636, 326.642, 326.662, 326.675: GenBank numbers AF177347–AF177361, respectively. 108.448, 225.745: GenBank numbers AF176037–AF225.745, respectively. 134.463, 151.940, 42.877, 99.237, 326.570, 501.045, 503.083: GenBank numbers AF216993, AF216994, AF216996, AF216999, AF216995, AF216997, AF216998, respectively. 225.710, 602.174, 230.298: GenBank numbers AF216992, AF216991, AF216990, respectively. UGANDAN HIV-1 PROTEASE AND REVERSE TRANSCRIPTASE 809 subtype D protease regions. In 22 of 27 samples, the subtype those included both samples that subtyped as C in the env and of the gag and protease regions was the same (Table 1). How- gag regions (225.745 and 108.448). One of those samples was ever, 5 of 27 samples had different gag and protease subtypes, a C/A recombinant and the other was a C/D recombinant. Com- suggesting recombination between these regions. Interestingly, parison of the subtypes in all three regions ( env, gag, and pro-

FIG. 2. Phylogenetic analysis of HIV-1 protease sequences. Protease sequences (297 nucleotides) from the 27 Ugandan iso- lates were aligned with a set of 27 reference sequences from subtypes A–J recommended for HIV-1 subtyping. 11 Alignments were performed by the CLUSTAL method in the program Seqpup (Don Gilbert/ftp.bio.indiana.ed u). A neighbor-joining tree was constructed with 500 bootstrap replications of a Kimura two-parameter matrix to evaluate the robustness of the phylogenetic re- lationship between the sequences. The programs DNAdist and NEIGHBOR were used for this analysis. Bootstrap values were obtained from a consensus tree, using the program CONSENSE of the software package PHYLIP v3.572.0. The tree was rooted with the strain CPZGAB. Branch lengths are proportional to the amount of sequence divergence between taxa in the tree. The Ugandan isolates are indicated in boldface. 810 BECKER-PERGOLA ET AL.

TABLE 1. Env, Gag, AN D PROTEA SE tease) revealed a high rate of recombination, involving 7 of 27 SUBTY PES OF UG AND AN ISO LATE Sa (26%) of the samples; one sample (602.174) subtyped as D/A/D in env/gag/protease, suggesting that this variant was generated Sample Env Gag Pro by multiple recombination events. 99.237 D D A Protease sequences shown in Fig. 1 were analyzed for the 129.733 A A A presence of amino acid polymorphisms at positions of previ- 135.544 A A A ously characterized drug resistance mutations. 11 Polymor- 140.223 A A A phisms were detected at nine of those positions (Table 2). In 161.287 A A A subtype A, the polymorphisms M36I and H69K were present 171.005 A A A in all isolates. Comparison of the protease sequences in Fig. 1 184.574 A A A also revealed polymorphisms at four positions that were dif- 190.574 A A A ferent in subtype A versus D HIV-1. Most subtype A sequences 194.793 A A A 204.987 A A A had the amino acids D, K, K, and M at positions 35, 57, 69, 225.706 A A A and 89, whereas most subtype D sequences had the amino acids 225.710 D A A E, R, H, and L at those positions. For comparison, we analyzed 225.745 C C A the amino acid sequences at those positions in other HIV-1 sub- 230.580 A A A types (B, C, F, G, H, and J). 11 The DKKM pattern seen in Ugan- 326.636 A A A dan subtype A isolates was not seen in sequences from other 326.642 A A A subtypes. The ERHL pattern seen in Ugandan subtype D iso- 326.675 A A A lates was also prevalent among subtype B sequences, but was 42.877 D D D not seen in other subtypes. Rayfield et al. described a rapid 108.448 C C D method for identifying subtype A and D HIV-1 in Ugandan 134.463 D D D 2 151.940 D D D samples. That method is based on nucleotide differences in the 230.298 A D D C2–V3 region of these subtypes. Nucleotide differences that 326.570 D D D encode amino acid polymorphisms in protease may also be ex- 326.662 A A D ploited for rapid screening of Ugandan HIV-1 isolates to iden- 501.045 D D D tify those that are likely to have subtype A or D protease-cod- 503.083 D D D ing regions. Direct analysis of polymorphisms in protease may 602.174 D A D be desirable for studies of HIV-1 drug resistance, since our analysis reveals a high rate of intersubtype recombination be- aHIV-1 from 27 Ugandan plasma samples was amplified, tween pol and other regions, such as env and gag. sequenced, and subtyped by phylogenetic analysis. Subtyping of the env gp41 region and the gag p24 region was performed RT sequences from the 27 Ugandan isolates were also ana- in a previous study. 1 Subtyping of the protease region is lyzed (Fig. 3). The corresponding nucleotide sequences were described in Fig. 2. The subtype assignments for each region are shown.

TABLE 2. AM INO ACID POLYMORPHISMSIN UGA NDA N HIV-1 PROTEA SE AND RT SE QUEN CESa

Polymorphisms detected Previously characterized Subtype A Subtype D resistance Enzyme Position (n 5 17) (n 5 10) mutation

Protease 20 I (1), R (2) R (1) K20RM 33 F (1) V/L (1) L33F 36 I (17) I/M (1), I (5) M36I 45 R (1) K45I 60 E/D (1), E (2) D60E 63 V (1), I (1), L/ P (1), P/S (1) T (2) P/S (1) L63P 69 K (17) H/Y (1), Y (2) H69Y 77 I (2) V77I 82 I (1) V82AFIST RT 139 M (1) T139I 141 E (1) G141E 179 I (13) I/V (1), I (1) V179DE 211 N (1), K (3), S (12) S (1), K/R (1), K (5) R211K 214 F/L (1), F (14) F (10) L214F

aSubtype A and D protease sequences were analyzed for the presence of amino acid polymorphisms at positions of previously characterized drug resistance mutations (see text). The polymorphisms detected at each position are shown for subtype A and D isolates. Polymorphisms corresponding to known drug resistance mutations are shown in boldface. The number of isolates with each polymorphism is shown in parentheses. Amino acid mixtures are indicated (e.g., a mixture of proline [P] and leucine [L] at position 63 is indicated as L/P). UGANDAN HIV-1 PROTEASE AND REVERSE TRANSCRIPTASE 811 , , : ; : - 0 7 c 7 T V / a 8 3 3 /

I P 5 2 2 . . . k 2 7 0 9 n 9 9 2

a 3 9 9 2 :

2 , B : : 8

3 , s n 0 9 8 6 e 8 2 w . 0 0 5 . o G . 0 7 l

3 . l . 0 3 0 5 o 3 2 V f 5 2 / 2 ;

I , 2

s

; T 2 5 , a / E 4 9 7 / A e 0 2 8 . r

5 D , 9 1 a . 6 1

E 0 4 1 s 9 / 5 0 e , 2 D , r 2

: R 0 7 u , / t 5 4 7 3

x 4 K 8 : 9 i . 7 9 4 7 . 2 . m 4 7 5

4

4 ,

1 2 , d . 9 i 2 K 0 2 1 / c

; 7 0 , a E 5

6 4 Q . 6

/ o 7 ; 6 : n E 5 2 i 3 . K 0 3 / 6 0

m 0 , E 4 9 . 3 0 2 A 1

4

, 4 2 , . 3 9 . E 1 4 . n / 1

y , 7 1 l ; w D 5 5 e H . o 6 V / 1 v 4 h / i : , I t s D 8 0 3 c 2 1 1 e e 1 6 9 2 , r p 7 4 2 1 a . 5 .

s

,

, 0 4 5 e s S r 3 2 0 T e / . / , 1 2 c 1

A P 4 , ; n 7 8 9 4 0 e 1 V 8 3 1 5

u /

, 2 I 7 7 q

. 3 7 , 2 7 e 5 8 8 s 9 S 1 2 2 / 0 2 . . F 2

T A 1 , 3 , A 3 6 0 R L 4 – / 7 1 5 . 7 I 3

1 , ; 1 1 8 4 . , 3 E 7 . 5 2 / 2 V 1 g 7 0 / 2 i D . 7 , I 6 F

4 0 1 0 ,

N 2 4 / 5 F 8 n

2 i 1 : 9 D A

, , 8

2 6 d . s 4 4 e r : 0 M 4 4 / e b . 3 6 5 I i . b 8 2 0 r 8

5 c 7 0 . 7 m . s 3 1 y 1 u

5 l e 1

;

e n 2 d , 5

R v 2 3 i 4 / k s

t ; 3 0 a n c K .

7 S a e . 1 / 1 d p 9 B 1 0 L e s 2 n 2 5 z 3 e

e 1 : r y 7 ;

l : 7 , G 1 a V s

7 6 / : : n I 8 7 w 5 4 a . 2 3

o 7 4 2 l 9 e 7 l 5 7 4 r . 2 . 7

o

e ; 0 5 1 , f

2 9 w K F T s /

/ 2 1 a A s E ; A e – d e 2 l 6 L r 2 n 2 / p 1 a 6 a 1 F

2

3 m s

: 4 8 , 7 a e 5 1 4 s 7 c V 7 2 4 1 / n .

a 6 I , e . F 8 9 S u m 6 0 / A 7 s q 2

1 a 1 e s N 3 l . r

s , 3 ;

p y e l 2

e V b V e / 1 n d /

i v I a m : t i M t d 2 u 4 o 8 c 4 n n 4 e

e 7 l a 1 5

k p 1 c . g : s n 5 u : 2 e U a n 3 2 r 4

. B 1 6 , 6 s

T . n 6 ; 3 e . 6 e R 0 S c 6

2 / 5 G n 2 g 3 8 e N : 3 n ; 8 u i 1 5 ; 1 d q 5 G 7 / e F n G 2 6 . / s E o , A

6 E 7 p S – 2 T s 3 0 / 4 3 e 2 2 R

A r 9

,

1 , r 4 8

1 2 : o T 4 8 - 6 / c

0 8 , 6 V 7 A . 1 e I R 5 0 6 h F / . H t 0 2 6

A K 2 3 f

2

n

3 , s o , a 3 r 4

2

d s Q e ; : / 4 r n b 3 e Y 6 a K . / 3 b m g 4 6 7 D u . 7 m 2 U 1 n 9 1 3 u

2

2 : n , k 1

. 1 6 6 n

, n 3 a 3 ; 3

o S . 6 6 i B / N . . s / n G 6 L 6 s e I D 2 8 2 e F 6 3 4 c 3 G 812 BECKER-PERGOLA ET AL. used for phylogenetic subtyping, using the reference sequences in enhancing our understanding of drug resistance in non-sub- and methods described in Fig. 2 (data not shown). The subtype type B HIV-1, and monitoring the emergence of drug-resistant assignments based on protease sequences and RT sequences HIV-1 worldwide. were the same for 25 of 27 samples. For two of the samples (225.710 and 108.448), subtypes could not be definitively as- signed on the basis of sequences from the RT region. ACKNOWLEDGMENTS The Ugandan RT sequences in Fig. 3 were examined for the presence of amino acid polymorphisms at positions in RT that The authors thank Catherine Brennan (Abbott Laboratories) are sites of previously characterized drug resistance muta- for helpful discussions regarding sample analysis. The authors tions.11 Amino acid polymorphisms were detected at five of also thank Vernell Moutlon for technical assistance, and PE those positions (Table 2). Polymorphisms at the three positions Biosystems for providing reagents for this study. (179, 211, and 214) were observed in multiple isolates. The This work was supported in part by the Pediatric AIDS Clin- polymorphisms V179I, R211S, and L214F were present in the ical Trials Group (PACTG), the Adult AIDS Clinical Trials majority of subtype A isolates. In contrast, most of the subtype Group (AACTG), and NIH-CH/HD34348. D isolates had valine (V) at position 179 and lysine (K) at po- sition 211; all the subtype D isolates had the L214F polymor- phism. The significance of these polymorphisms in non-B HIV- REFERENCES 1 is not known. The low prevalence of primary drug resistance mutations in 1. Brennan CA, Lund JK, Golden A, Yamaguchi J, Vallari AS, this population is consistent with the lack of widespread use of Phillips JF, Kataaha PK, Jackson JB, and Devare SG: Serologic antiretroviral drugs in Uganda. It also demonstrates that pri- and phylogenetic characterization of HIV-1 subtypes in Uganda. mary drug resistance mutations identified in subtype B HIV-1 AIDS 1997;11:1823–1832. do not occur as common polymorphisms in HIV-1 from 2. Rayfield MA, Downing RG, Baggs J, Hu DJ, Pieniazek D, Luo CC, Biryahwaho B, Otten RA, Sempala SDK, and Dondero TJ: A Uganda. It is interesting to note that many of the isolates con- molecular epidemiologic survey of HIV in Uganda. HIV Variant tained polymorphisms at positions of previously characterized Working Group. AIDS 1998;12:521–527. drug resistance mutations. Furthermore, many of the isolates 3. Kaleebu P, Bobkov A, Cheingsong-Popov R, Bieniasz P, Garaev contained amino acid mixtures at those positions (e.g., protease M, and Weber J: Identification of HIV-1 subtype G from Uganda. 60D/E, 63L/P, 63P/S, and 69H/Y and RT 179I/V, 211K/R, and AIDS Res Hum Retroviruses 1995;11:657. 214F/L). This suggests that HIV-1 variants with polymorphisms 4. Mellquist JL, Bowman B, Kasturi L, Guay L, Kataaha P, Mmiro at those positions arise frequently in viral populations without F, Ndguwa C, Jackson JB, and Eshleman SH: Characterization of drug selection. HIV type 1 gp120 V3 region sequences from Ugandan infants. It is not known whether drug resistance mutations charac- AIDS Res Hum Retroviruses 1998;14:1391–1395. terized in subtype B HIV-1 have the same phenotypic effects 5. Brodine SK, Mascola JR, Weiss PJ, Ito SI, Porter KR, Artenstein AW, Garland FC, McCutchan FE, and Burke DS: Detection of in other HIV-1 subtypes. In theory, polymorphisms in HIV-1 diverse HIV-1 genetic subtypes in the USA. Lancet 1995;346: protease and RT that vary among subtypes could influence the 1198–1199. natural resistance of isolates to antiretroviral drugs, modulate 6. Gao F, Yue L, Hill SC, Robertson DL, Graves AH, Saag MS, Shaw the fitness of HIV-1 variants that acquire drug resistance mu- GM, Sharp PM, and Hahn BH: HIV-1 sequence subtype D in the tations, or influence the probability that other mutations caus- United States. AIDS Res Hum Retroviruses 1994;10:625–627. ing drug resistance would arise at those positions during drug 7. Beatrice ST, Oleszko WR, Punsalang A, Chaisson MA, Torian LV, selection. Studies comparing the genetic correlates of drug re- Schable CA, and Weisfuse IB: HIV-2 and non-B subtypes of HIV- sistance in different HIV-1 subtypes are clearly needed to ad- 1 group M in specimens submitted to the New York City (NYC) dress these issues. Department of Health Retrovirology Laboratory from 1993–1997. Genetic polymorphisms can complicate drug resistance In: 12th World AIDS Conference , Geneva, Switzerland, 1998. [Ab- stract 42116] analysis of HIV-1. Studies illustrate problems that can arise by 12–14 8. Irwin KL, Pau CP, Lupo D, Pienazek D, Luo CC, Olivo N, Ray- using genotypic assays based on hybridization. The PE field M, Hu DJ, Weber JT, Respess RA, Janssen R, Minor P, and Biosystems HIV genotyping system used in this article was de- Ernst J: Presence of human immunodeficiency virus (HIV) type 1 veloped for analysis of subtype B HIV-1. In this system, se- subtype A infection in a New York community with high HIV quence heterogeneity among HIV-1 subtypes can potentially in- prevalence: A sentinel site for monitoring HIV in terfere with reverse transcription, PCR amplification, or DNA North America. Centers for Disease Control and Prevention— sequencing, if primers fail to bind to the HIV-1 template. An Bronx Lebanon HIV Serosurvey Team. J Infect Dis 1997; abstract has described successful use of this system for analy- 176:1629–1633. sis of cultured non-B HIV-1 reference isolates, using both kit 9. Weidle PJ, Ganea CE, Pienniazek D, Ramos A, Schable CA, Ernst and nonkit primers. 15 Here, we demonstrate successful use of J, and McGowan J: Prevalence of HIV-1 group M, non-B subtypes in a Bronx New York community: A sentinel site for monitoring this system for analysis of diverse HIV-1 subtypes in plasma, HIV genetic diversity in the United States. In: 12th World AIDS without the use of culture, and using only primers supplied by Conference , Geneva, Switzerland, 1998. [Abstract 13225] the manufacturer. Continued development and optimization of 10. Mellquist JL, Becker-Pergola G, Gu J, Guay L, Himes L, Kataaha genotyping assays will be important for evaluation of HIV-1 P, Mmiro F, Ndugwa C, Jackson JB, and Eshleman SH: Dual trans- protease and RT in drug-naive and drug-experienced patients mission of subtype A and D HIV-1 from a Ugandan woman with non-B HIV-1 infection. Such assays will play a key role to her infant. AIDS Res Hum Retroviruses 1999;15:217–221. UGANDAN HIV-1 PROTEASE AND REVERSE TRANSCRIPTASE 813

11. Korber B, Kuiken C, Foley B, Hahn B, McCutchan F, Mellors J, deficiency virus type 1 clades and viral isolates with length poly- and Sodroski J: Human Retroviruses and AIDS: A Compilation and morphisms. J Clin Microbiol 1999;37:2533–2537. Analysis of Nucleic Acid and Amino Acid Sequences . Theoretical 15. Huang D, Polstra D, Geisler T, Scianna S, Bremer J, Robb M, Biology and Biophysics Group, Los Alamos National Laboratory, Michael N, Brun-Vezinet F, and Reichelderfer P: Characterization Los Alamos, New Mexico, 1998. of an international HIV-1 subtype panel for future use as standards 12. Koch N, Yahi N, Colson P, Fantini J, and Tamalet C: Genetic poly- and reference strains. In: 6th Conf. on Retroviruses and Oppor- morphism near HIV-1 reverse transcriptase resistance-associated tunistic Infections , Chicago, Illinois, 1999. [Abstract 719] codons is a major obstacle for the line probe assay as an alterna- tive method to sequence analysis. J Virol Methods 1999;80:25–31. 13. Puchhammer-Stockl E, Schmied B, Mandl CW, Vetter N, and Address reprint requests to: Heinz FX: Comparison of line probe assay (LIPA) and sequence Susan Eshleman analysis for detection of HIV-1 drug resistance. J Med Virol 1999;57:283–289. Department of Pathology 14. Vahey M, Nau ME, Barrick S, Cooley JD, Sawyer R, Sleeker AA, Johns Hopkins University School of Medicine Vickerman P, Bloor S, Larder B, Michael NL, and Wegner SA: Ross Building 646 Performance of the affymetrix GeneChip HIV PRT 440 platform 720 Rutland Ave. for antiretroviral drug resistance genotyping of human immuno- Baltimore, Maryland 21205