AIDS RESEARCH AND HUMAN Volume 18, Number 15, 2002, pp. 1163–1166 ©Mary Ann Liebert, Inc.

Sequence Note

HIV Type 1 Strains from East and West Africa Are Intermixed in Sudan

MATTHEW HIERHOLZER, 1 R. ROSS GRAHAM, 2 I. EL KHIDIR, 3 SYBIL TASKER, 4 MAGDI DARWISH, 2 GAIL D. CHAPMAN, 2 ADEMOLA H. FAGBAMI, 2 ATEF SOLIMAN, 2 DEBORAH L. BIRX, 5 FRANCINE MCCUTCHAN,1 and JEAN K. CARR 1

ABSTRACT

The genetic subtypes of HIV-1 in the Sudan epidemic have not been characterized. Here we report the par- tial sequencing and analysis of 30 strains collected from HIV-1-positive patients and blood donors in Khar- toum in 1998 and 1999. From analysis of partial pol and sequences, it was determined that 50% were sub- type D and 30% were subtype C. Of interest, some subtype D clustered with those from East Africa whereas others joined subtype D from West Africa. Subtype A, subtype B, and three unique recombinants were also found, some partially unclassifiable. One unclassified strain matched another reported previously from the

Democratic Republic of Congo. Sudan borders nine other African countries, and has suffered more than 20 years of civil strife with large population displacements. The intermixing of HIV-1 subtypes previously sepa- rated in Africa may be occurring there, with the potential to generate novel new strains by recombination.

UDANISTHELARGESTCOUNTRY in Africa, with a land mass 5% in the 1996-to-1998 6 time frame. This represents the most Sapproximately the size of , and a population of about recent prevalence data available and there are no data about 28 million. Most of the population is in the north, comprised HIV-1 subtypes in Sudan. principally of Arabic-speaking Muslims, but about 20% live in HIV-positive patients and blood donors attending clinics and the south and are ethnically similar to sub-Saharan Africans. hospitals in Khartoum between 1998 and 1999 were recruited Sudan is located between countries with low HIV prevalence for study. Thirty study subjects gave written informed consent to the north 1 and those, to the east and south, with some of the and blood was collected, peripheral blood mononuclear cells highest HIV-1 infection rates in the world. 2–4 It has been en- (PBMCs) were purified, and DNA was extracted and used for gaged in a prolonged civil war for 20 years, which is still on- PCR amplification. A nested strategy was used to amplify about going; as a result, an estimated 4 million people are refugees, 1.1 kb of the pol gene, encoding protease and part of RT (nt many of whom travel in and out of Sudan to help relatives left 2167–3338 in HXB-2) 7 and 0.7 kb of the env gene, C2–V5. 8 behind. The amplified products were directly sequenced and phyloge- Little is known about the HIV epidemic in Sudan. In the netically analyzed. Sequences were submitted to GenBank un- southern town of Juba, close to the border with Uganda, the der the accession numbers in Table 1. HIV-1 prevalence among outpatients was 3% and among com- The information available about the study subjects is shown, mercial sex workers was 14% by 1995. 5 In Khartoum, the along with the subtype, in Table 1. Most of the subjects were prevalence among antenatal clinic patients was between 1 and males with late-stage HIV-1 infection (CDC stage 4). One of

1Henry M. Jackson Foundation, Rockville, Maryland 20850. 2Virology Research Program, U.S. Navy Medical Research Unit No. 3, Cairo, Egypt. 3Ministry of Health, Khartoum, Sudan. 4U.S. Naval Medical Research Center, Silver Spring, Maryland 20850. 5Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, Maryland 20850.

1163 1164 HIERHOLZER ET AL.

TABLE 1. INFORMATIONABOUT STUDY SUBJECTS

GenBank Age CDC Subtype Subtype I.D. Year accession no. (years) Sex stage (pol) (env)

98SU478 1998 AY102482 25 M A 98SU480 1998 AY102483 23 M 4 D 98SU488 1998 AY102484 31 M 4 D 98SU502 1998 AY102485 32 M 4 D 98SU504 1998 AY102486 41 M 4 C 98SU510 1998 AY102487 30 F 4 D 99SU005 1999 AY102488 30 D 99SU009 1999 AY102489 31 M 4 C 99SU015 1999 AY102490 23 M 4 B 99SU045 1999 AY102491 24 M 1 C C 99SU049 1999 AY102492 26 F D 98SU053 1999 AY102493 40 M 4 D D 99SU057 1999 AY102494 32 M 4 Ua A 99SU224 1999 AY102495 26 M 4 D 99SU227 1999 AY102496 32 M C 99SU231 1999 AY102497 24 M 1 C 99SU235 1999 AY102498 29 M 1 D D 99SU238 1999 AY102499 35 M A 99SU243 1999 AY102500 32 F D D 99SU245 1999 AY102501 32 M 4 D D 99SU247 1999 AY102502 35 F 1 C D 99SU249 1999 AY102503 24 F D D 99SU250 1999 AY102504 34 M D 99SU252 1999 AY102505 24 M 4 C 99SU254 1999 AY102506 26 M C 99SU255 1999 AY102507 33 M 4 C 99SU256 1999 AY102508 32 F 4 Ua/C

99SU259 1999 AY102509 40 F 1 D D 99SU272 1999 AY102510 39 M C 99SU273 1999 AY102511 29 F 4 D D

aU, Unclassifiable.

the study subjects reported foreign travel to Saudi Arabia, but fall into the existing HIV-1 classification. The pol gene of the rest had not left the country. Ten of the 30 samples se- SU256 was recombinant between subtype C and an unknown quenced in pol were also sequenced in env. Seven were con- subtype, explaining its unique position in the cordant D, one was concordant C, and three were recombinant. (Fig. 1). The 600 nt at the 5 9 end of the amplicon were un- Phylogenetic analysis of the viral sequences from the 30 classifiable, while the remaining 500 nt were clearly derived study subjects and from reference sequences of relevant sub- from subtype C (bootstrap 5 96%, data not shown). SU057 was types is shown in Fig. 1. Subtype D was the most common, entirely unclassifiable in pol (Fig. 1). A search of GenBank re- consisting of 50% of samples, and subtype C was the next most vealed one sequence that resembled it, another unclassified common, found in 30% of the samples (Fig. 2). There were two , 97DRC-MBCD, collected in 1997 from the Democratic subtype A, one subtype B, and three recombinants. Globally, Republic of Congo. 9 The two are clearly related phy- subtype D consists of two different lineages, one circulating in logenetically in pol (100% bootstrap; Fig. 1). The partial env East Africa and another in West Central Africa. Genetically, gene sequence for SU057 was subtype A, but did not cluster they are distinguishable as two significant subclusters within with the East African subtype A viruses (data not shown). subtype D (Fig. 1). Most of the Sudanese viruses were of East On the basis of this small sample, the HIV epidemic in Su- African origin, similar to those from Uganda, but two SU259 dan appears to be dominated by East African viruses (Fig. 2; and SU273, clustered with those from West Central Africa. The subtypes A, C, and D). Three of the viruses (10%), however, subtype C viruses probably originated in Ethiopia, Sudan’ s were genetically related to viruses from West Central Africa, neighbor to the east. There were also two subtype A viruses, including two subtype D and one unclassified recombinant. Su- common in Kenya, and one subtype B. dan, with millions of refugees both inside and outside the coun- Listed in Table 1, three viruses were recombinant either try, sharing borders with nine other African countries and the within pol or between pol and env. One, SU247, was C in pol Red Sea, is clearly a candidate for generating new genetic forms and D in env. Two other recombinants had regions that did not of HIV-1. HIV-1 SUBTYPES IN SUDAN 1165

FIG. 1. Phylogenetic analysis of 1.1 kb of the pol genes from Sudan. Partial pol sequences from Sudan were phylogenetically analyzed in a neighbor-joining tree along with reference sequences from the major subtypes of HIV-1. A genetic distance of 10% is shown by the scale bar. The significance of the nodes defining the clusters was assessed by maximum parsimony with the bootstrap. Significant bootstrap values are indicated at the appropriate nodes.

FIG. 2. Proportions of different genetic forms. The proportion of the population that was genetically classified as either sub- types or recombinants is shown. 1166 HIERHOLZER ET AL.

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