UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

HIV-1 subtype C in Ethiopia: genotypic and phenotypic variation

Abebe, A.

Link to publication

Citation for published version (APA):Abebe, A. (2000). HIV-1 subtype C in Ethiopia: genotypic and phenotypic variation

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 08 Jun 2018

CHAPTER II

HIV type 1 subtype C in Addis Ababa, Ethiopia

Almaz Abebe, Carla L. Kuiken, Jaap Goudsmit, Margreeth Valk, Tsehaynesh Messele, Tefera Sahlu, Hailu Yeneneh, Arnaud Fontanet, Frank De Wolf, and Tobias F. Rinke De Wit

AIDS Res Hum Retrov 1997J3 (12): 1071-1075

HIV Type 1 Subtype C in Addis Ababa, Ethiopia

ALMAZ ABEBE,1 CARLA L. KUIKEN,2 JAAP GOUDSMIT,2 MARGREETH VALK,2

TSEHAYNESH MESSELE,1 TEFERA SAHLU,' HAILU YENENEH,' ARNAUD FONTANET,1

FRANK DE WOLF,2 and TOBIAS F. RINKE DE WIT'

HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 variants in dif­

ferent geographic regions have been phylogenetically clas­

sified into several distinct genetic subtypes (A-I and 0) on the

basis of sequence differences in the V3 regions of their gpl20

envelope genes.'J In Africa the existence of all genetic subtypes

except subtype I has been confirmed. The presence of this mul­

titude of subtypes indicates the extensive divergence HIV-1 has

accumulated in the African continent.5'5 This study describes

the distribution of HIV-1 subtypes in Ethiopia, East Africa. The

first Ethiopian AIDS case was reported in 1986 and the AIDS

epidemic is a rapidly growing problem in Addis Ababa, the

capital city with more than 2 million inhabitants.6 In Addis Ab­

aba the HIV-1 seroprevalence is estimated to be 10-27% in

pregnant women (PW), 47-59% in commercial sex workers

(CSWs) (ENARP sentinel surveys 1995 and 1996) and 7%

among blood donors (BDs) (Ethiopian Red Cross Society, Na­

tional Blood Transfusion Service [ERCS-NBTS], unpublished

data, 1994). In the countries neighboring Ethiopia subtypes A

(Djibouti, Kenya). C (Kenya, Djibouti. Somalia), and D (Ken­

ya) are present.17 Preliminary sequence data on a limited number

of samples have indicated the presence of subtype C in Addis

Ababa in 1988.' To assess the distribution of HIV-1 subtypes

in Addis Ababa in more detail, 94 sera were analyzed, collected

from 3 different risk groups over 1989-1995 (Table I).

HIV-1 RNA was isolated from the collected sera according

lo a standard procedure.' Viral RNA was converted to cDNA

and then subjected to a nested polymerase chain reaction (PCR)

amplifying a 284-bp fragment covering the V3 region of the

gpl20 gene (positions 785-1069, HIV-1 subtype B consensus

database, Los Alamos, 1993). First and second PCR conditions

were as follows: 35 and 25 cycles of, respectively, 1 min at

95°C, 1 min at 55°C, and 2 min at 72°C, followed by a final

incubation for 10 min at 72°C. The amplified products were

sequenced directly by automated cycle sequencing with dye ter­

minators, using an ABI (Foster City, CA) 370 A system. Se­

quence alignments and comparisons were performed by using

CLUSTAL and the neighbor-joining algorithm.10" Figure 1

demonstrates the results of neighbor-joining sequence compar­

isons, clearly indicating that the vast majority of sequenced

samples (93 of 94) are of the C subtype. The prevalences of C

and A subtypes among the 57 samples analyzed from 1995

were, respectively, 56 of 57 (98.2%) and 1 of 57 (1.8%). In

addition, our data show that the C subtype is highly abundant

in samples collected before 1995.

The Ethiopian subtype C sequences tend to differ slightly

from the consensus C sequence (HTV-1 subtype C consensus

database, Los Alamos, 1995). Within the Ethiopian sequences,

a subcluster could be identified that is fairly homogeneous. Il­

lustrating this homogeneity, pairwise nucleotide comparisons

revealed a 7.4% difference within this subcluster, as compared

to an 11.5% difference within the remaining sequences. The

latter are more related to HIV-1 subtype C sequences from dif­

ferent countries (Fig. 1). Statistical comparisons, using a pre­

viously published method,'2 revealed significant sequence dif­

ferences between the main group and the subcluster at positions

270, 272, 278, 286, and 294 (Fig. 2). The presence of a sub-

cluster of the C subtype may suggest an independent introduc­

tion of the HIV subtype C virus in Addis Ababa. However, the

subcluster could not be detected with significant preference in

TABLE 1. STUDY SUBJECTS, NUMBER OF SERA SEQUENCED,

AND YEAR OF SAMPLE COLLECTION

Year Subjects Samples tested

1989 Commercial sex workers 1 1990 Commercial sex workers 6 1992 Commercial sex workers 24 1995 Commercial sex workers 19 1995 Pregnant women 9 1995 Blood donors 29

Total: 94

'Ethiopian-Netherlands AIDS Research Project (ENARP), Ethiopian Health and Nutntion Research Institute (EHNRI), Addis Ababa, Ethiopia. :Human Retrovirus Laboratory, Department of Human Retrovirology, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

49

88-i

S IA

^ ^ • • M If)

•OU

• - u w IS1-. ,a >.» •O Ä » »'S « E S£ « y u U 3 c • | | -

•g J *

= 18:

lil ! ! •

s j ï E °U c.% ..

a«! « « >

: = « - § OU .*s "PIS • «l •C Mi U 5 H. S = M ^ £ >> 3 2 -

•Cc13

nis v F t/J c

J 3 ^ > > 5 c £• I = O Î t l « ; c u v i . ï ' L ;

flSl

•ï E u £ ï ^ -< r.

« -o N jo

o ga«

c xi . . g U 3 O U

ä> L" §

C £ o B

50

C o m e n » u i

A_TP9S001

C_ÎSÎ91S5)

C _ : S H Î 3 S

C_3SB92"7B1

CJUJOlBCl

C~Ö9S)1

C~B9Slt

C_BS512

C_B9i)

C B 9 S H

C'BSSSI C_B9S90

C_KAZS9J*S

C_KP9S0H

C_Ki95034

C_XP9ÏO<0

C_KS9500*

C_KS95029

C_KS9SOSO

C_TV9SD91

C_TS9 5002

C^TS95Q10

C_TS9jO0*

C TS9SOCÏ

C B 9 5 3 9 1

C_TP9jOS9

C _ T I C ; 9 : 0 4 ï

C_KS9S077

C V3950ÏO

C_B9510O

C_B950St

C_B9S<MS

C B 9 5 0 Î 0

c3:sB92i-ii C 2S891B9S

:_B95S04

C^KA2S92Î9

C_KAZS92S5

C_KA:SWIOS

C_KPSSQ36

C_K£9Ü01Ï

C_K59SOiB

C_7KS920S5

:~TKS92:P'>

C2'KS9JÛ9! C_Tf9ÏD7S CTKS93O90 C .'KS^OS -1

C_TKS9202* C KAZS9J17

:_KAii9:sj C_KJVZS9206

rr~f'Azss:93 -, KAZSSIB*

_ ^j ' .r:B7

: " i J S 9 « 3

: _ L 9 i : i 9

C~b3SS33

IVIRS?^*TNKAXIIIVOLKSPVïTVCT*PNNyTRr.SMRIGP<OT .R I N- - -FJD- K-T -V - - -V-RSA Q-VIQ-KT-VG

TI D- O-N E-I F- G RAD I- -KO--KGL-K--N-

- H - - D H - - - I KDI-'

-V X-KNSX-

- V - T - - - H - N - S - -

-H R-I ALFKAT-

- S I I - R - -

- - Q - - K -

-n -o--rcrr-I I - X

GK R

R - I R - - - X

OK--A-- -K-IK

KM--D---K L-Y-

RQQ D K - W - - K —

- T - H - T - - K H - - - K

- 0 -K - - K - a - - G - L -

I - - - K - - H - T - I - ' - -FM-T-- -M-K-'

- -H-S-A-N

K -H-TK

K- - Q - N - - -

-K-HD---TT-,

•H-H-S-

-H-N-S-

---NHT-

RKH-

- E - I G - '

-R- -G-

RI - -1 I - - -K D

DI-GI G - K - - A - - - R - S I - - 0 R--

- - L K V - G - I - - V - - B l - i a i - - T - Y - l l - S I K

Y-H-MGT I--KQ--RJC--L

Jt RO R

A

- - - F X O D Q - X T - - I - - X 1 - - A - - - . - R - -

- ^ - F - - - G 3QK E D - R - - - K - - - .

K A - - D - - - K - - I - - K

-A T - - - Y G - I - - I - - H R - - I - - I R L - S K N I T

R V Y RTK Q

G-T K - S - -V L HT

OOQ G - a - - K

h Y GWDAT. . - -K-KCI-AX R--

- - K - - - D - - R D - - I - - 0 - T

-G-K D - - - P I - R X - S

- G - R - - T - - - R - R R - - I -

-RAQ--T- - IR-RI- -RK

- -OK-STV-RX-H

-R-K-KH---K-KIR-KX--

-R-K--N--IR-KXR-RX--

Y-IXX

IKH1CC

H-R--

M-T-T

LMKSM

•RUK--

- a - K - '

--HRQ---

-V-RQA--

KA-

-EK-K1--KS--I

- - K - K - - - I - L - 1

-N-SR

- - -A-O-RX-

-K- -R- -

-H-T-Q-H-

- H K 5 - - - S -

N G - K - - A - - - X - R I - - I K -

• l K GK-RTV--K-I

- - - V - H K X--

-0 A-G- -O- - -K-TV- -A- -

G Y - - - H - S K - - X 1 - 0 1 -

K H D - S - - - R - -•-A R - K - T R - - - R - M - - R - -

• GR--KA--BK-KD--KX-V-Q-K--A--KX-VN--KJC-

NTTM1

- - G I -

•D--RP

F1G. 2. Predicted amino acid sequences of the V3 region of gpl20 of the 94 Ethiopian HIV-1 envelope protein sequences, js compared to a denved Ethiopian consensus sequence. The subtype A sequence is shown first, followed by the main group (61 sequences) and the subcluster (32 sequences). Dashes indicate amino acid identity with the consensus sequence; dots indicate gaps introduced for the purpose of alignment; X, unreadable nucleotides (N); asterisks indicate nucleotide positions with signifi­cant differences between the main group and the subcluster. Sequence names have been indicated by codes as described in Fig 1.

samples collected in a certain year, risk group, or site, giving no information regarding possible independent introductions of these sequences.

Figure 2 presents the amino acid sequence alignment of die Ethiopian samples, subdivided into main group and subcluster. All V3 loops are 35 amino acids long and show the character­istic C-subtype pattern of GPGQT at the apex. The single Ethi­opian A-subtype sample shows at the apex a C-like GPGQT motif, instead of the consensus A subtype apex (GPGQA). The Ethiopian C-subtype sequences show a high frequency of me­

thionine at position 306, as compared to other C isolates.15 This is particularly true for the subcluster (30 of 32 sequences, 94%) as compared to the main group (26 of 62 sequences, 42%). The biological consequences of this mutation remain to be eluci­dated. On the basis of previous studies the amino acid positions 305 and 319 determine the non-syncytium-inducing (NSI) ver­sus syncytium-inducing (SI) phenotype of HIV-1 strains.'4-' According to the amino acids predicted at these positions in the sequenced Ethiopian isolates, 93 of them would be NSI and possibly 1 would be SI (B95032). Because the majority of sam-

51

C o n t a n i u i .

C_KSSS01«

C_*S90129O

C_TS9S007

C_KS9S0£I

C_TKS3Ï0T»

C~TXS920H C_B9S0<2

CB9S9S

C_B9S0I<

CJCP9S04Ï

C~KAZS»2*2

C_KA259ÎSe

C_KS9S0*1

C_BSS«2

C_B950J2

C_i»S4S

C_TP9S04(

C_TKS92102

C_K595020

C_XS9012(J

C^AS9012S2

C_TKS92DS9

C_AS90Ï01

C_:SB9213J

C_BS5077

C_KX2S9204

C^XS90S19

C_2SB91J«1

C_B9S9S

C BSSBS

C [ T K S 9 2 0 2 1

C TS95ÛC9

iviUBiLTmuaizvQLKipinivcTitnQamucsatiGpaQTfYATra

- I - K - - - .- l.-QSNKSIR

K V GXI L

J D K *

_ 1 Y T

K R T 1

K K L J 1

R--

K „ * L 11 R--

K

_ G Y K L U R--

K

T X IC

-!-• . . Q . X

K GK*. -!-• . . Q . S

D V a 1 L L I

I V K S

T K _ L Jl • G • G

FIG. 2. Continued.

pies collected in this study are from asymptomatic HIV-I-

infected individuals, this would be expected.

ACKNOWLEDGMENTS

We thank the National AIDS Control Program Referral Lab­

oratory at the Ethiopian Health and Nutrition Research Institute

(NACP. EHNRI) for sera collected before 1995. and the Region

14 Health Bureau and Ethiopian Red Cross Society, National

Blood Transfusion Service (ERCS-NBTS), Addis Ababa, for

collection of sera in 1995. This work was financially supported

by The Netherlands Ministry of Development Cooperation

through the Ethiopian-Netherlands AIDS Research Project

[ENARP). The Ethiopian-Netherlands AIDS Research Project

( EN ARP) is a collaborative effort of the Ethiopian Health and

Nutrition Research Institute (EHNRI), the Municipal Health

Service of Amsterdam, the Department of Human Retrovirol-

ogy of the Academic Medical Centre (AMC), and the Central

Laboratory of the Blood Transfusion Service (CLB), Amster­

dam, The Netherlands.

REFERENCES

Louwagie J. Janssens W, Mascola J, Heyndriclu L, Hegcrich P, van der Groen G, McCutchan FE, and Burke DS: Genetic diversity of the envelope glycoprotein from human immunodeficiency virus type 1 isolates of African origin. J Virol 1995;69:263-271. Myers G. Korber B. Wain-Hobson S. Jeang K-T. Henderson LE, and Pavlalus GN (eds.): Human Retroviruses and AIDS. Theoret­ical Biology and Biophysics Group, Los Alamos National Labo­ratory, Los Alamos, New Mexico, 1994.

Kostrikis LG, Bagdades E, Cao Y, Zhang L, Dimitriou D. and Ho DD: Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: Identificadon of new subtype des­ignated subtype I. J Virol 1995:69:6122-6130. Murphy E. Korber B, Georges-Courbot M, You B, Pinter A, Cook D, Kjeny M, Geogres A. Mathiot C, Barre-Sinoussi F, and Girard M: Diversity of V3 region sequences of human immunodeficiency viruses type 1 from the Central African Republic. AIDS Res Hum Retroviruses 1993;9:997-1006.

Nkengasong JN, Janssens W, Heyndrickx L. Fransen K, Ndumbe PM, Mone J, Leonaers A, Ngolle M, Ayuk J. Piot P, and van der Groen G: Genetic subtypes of HJV-1 in Cameroon. AIDS 1994; 8:1405-1412.

Eshete H and Sahlu T: Review article: The progression of HIV/ AIDS in Ethiopia. Ethiop J Health Dev 1996;10:179-190. Zachar V, Goustin AS. Zacharova V, Hager H, Koppclhus U, Womble DD. Lm X, Bambra C, Nyongo A. and Ebbesen P: Genetic polymorphism of envelope V3 region of HIV type I subtypes A, C. and D from Nairobi, Kenya AIDS Res Hum Retroviruses 19%; 12:75-78.

Ayehunie S, Johansson B, Sonnerborg A, Zewdie DW, Britton S. and Strannegard O: Sequence analysis of selected regions of the env (V3 loop and gp41) and gag (p7) reading frames of Ethiopian human immunodeficiency virus type 1 strains. Arch Virol 1993; 128:229-239.

Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, and van der Noordaa J: A rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495-503.

Higgins DC, Bleasby A, and Fuchs R: CLUSTAL V: Improved software for multiple sequence alignment. Comput Appl Biosci 1992;8:189-191.

Saitou N and Nei M: The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-425. Kuiken LC. Comelissen TEM, Zorgdrager F, Hartman S, Gibbs

52

JA, and Goudsmit J: Consistent nsk group-associated differences

in human immunodeficiency virus type I vpr, vpu and V3 se­

quences despite independent evolution. J Gen Virol 1996;

77:783-792. 13. Foley B and Korber B: Global variation in the HTV-1 V3 region.

In: Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory. Los Alamos, New Mexico. 1995.

14. De Wolf F, Hogervorst E. Goudsmit J, Fenyo EM, Rubsamen-Waigmann H, Holmers H, Galvao-Castro B, Karita E, Wasi C, Sempala SDK, Baan E, Zorgdrager F, Lukashov V, Osmanov S, Kuiken C. Comelissen M, and the WHO Network for FfTV Isolation and Characterization: Syncytium-inducing and nonsyncytium-inducing capacity of human immunodeficiency vims type 1 sub­types other than B: Phenolypic and genotypic characteristics. AIDS Res Hum Retroviruses 1994;10:1387-1400.

Fouchier RAM, Groemnk M. Kootstra NA, Tersmette M, Huisman HG, Miedema F, and Schuitemaker H: Phenotype-asscciated se­quence variation in the third variable domain of the human im­munodeficiency virus type I gpl20 molecule. J Virol 1992; 66:3183-3187.

Address reprint requests to:

Tobias F. Rinke de Wit

Ethiopian-Netherlands AIDS Research Project (ENARP)

Ethiopian Health and Nutrition Research Institute (EHNRI)

P.O. Box 1242

Addis Ababa, Ethiopia

53

54