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Journal of Virology, July 2005, p. 8249-8261, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8249-8261.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In-Depth, Longitudinal Analysis of Viral Quasispecies from an Individual Triply Infected with Late-Stage Human Immunodeficiency Virus Type 1, Using a Multiple PCR Primer Approach

M. Gerhardt,1* D. Mloka,1,2 S. Tovanabutra,3 E. Sanders-Buell,3 O. Hoffmann,1,4 L. Maboko,5 D. Mmbando,6 D. L. Birx,3 F. E. McCutchan,3 and M. Hoelscher1

Department of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany,1 Muhimbili University College of Health Sciences, Dar-es-Salaam, Tanzania,2 U.S. Military HIV Research Program, Rockville, Maryland,3 Centre for Population Studies, London School of Hygiene and Tropical Medicine, London, United Kingdom,4 Mbeya Medical Research Programme, Mbeya, Tanzania,5 Mbeya Regional Medical Office, Ministry of Health, Mbeya, Tanzania6

Received 11 November 2004/ Accepted 15 February 2005

Coinfections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of coinfections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.

* Corresponding author. Mailing address: Department of Infectious Diseases and Tropical Medicine, Ludwig-Maximilians-University, Leopoldstr. 5, 80802 Munich, Germany. Phone: 49 89 2180-3925. Fax: 49 89 33 60 38. E-mail: martina.gerhardt{at}lrz.uni-muenchen.de.

Journal of Virology, July 2005, p. 8249-8261, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8249-8261.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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