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Journal of Clinical Microbiology, June 2004, p. 2742-2751, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2742-2751.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Subtype C Human Immunodeficiency Virus Type 1 by Subtype-Specific PCR and Its Use in the Characterization of Viruses Circulating in the Southern Parts of India

Nagadenahalli B. Siddappa,^1,2 Prashanta K. Dash,¹ Anita Mahadevan,³ Narayana Jayasuryan,⁴ Fen Hu,⁵ Bethany Dice,⁵ Randy Keefe,⁵ Kadappa S. Satish,⁶ Bhuthiah Satish,⁶ Kuttan Sreekanthan,⁷ Ramdas Chatterjee,⁸ Kandala Venu,⁹ Parthasarathy Satishchandra,¹⁰ Vasanthapuram Ravi,² Susarla K. Shankar,³ Raj Shankarappa,^5,11,12* and Udaykumar Ranga^1*

Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research,¹ Department of Neuropathology,³ Department of Neurology,¹⁰ Department of Neurovirology,² National Institute of Mental Health and NeurosciencesMicrotest Innovations Pvt. Ltd.,⁴ Seva Free Clinic, Bangalore, Karnataka,⁶ Department of Infectious Diseases, Medical College Hospital, Thiruvananthapuram, Kerala,⁷ Department of Virology, Chittaranjan National Cancer Institute, Kolkata, West Bengal,⁸ Government General Chest Hospital, Erragadda, Osmania Medical College, Hyderabad, Andhra PradeshIndia,⁹ Center for Genomic Sciences, Allegheny-Singer Research Institute,⁵ Thomas E. Starzl Transplantation Institute, Department of Surgery,¹¹ Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania,¹²

Received 25 September 2003/ Returned for modification 29 October 2003/ Accepted 21 February 2004

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-B sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

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* Corresponding author. Mailing address for Udaykumar Ranga: Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur (PO), Bangalore 560 064, India. Phone: 91-80-2362 2750, ext. 2241. Fax: 91-80-2362 2766 or 91-80-2362 6581. E-mail: udaykumar{at}jncasr.ac.in. Mailing address for Raj Shankarappa: Thomas E. Starzl Transplantation Institute, CNBIO, Department of Surgery, 300 Technology Dr., University of Pittsburgh, Pittsburgh, PA 15219. Phone: (412) 383-9704. Fax: (801) 605-6821. E-mail: gpgr{at}yahoo.com.

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Journal of Clinical Microbiology, June 2004, p. 2742-2751, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2742-2751.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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