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Nucleic Acids Research Advance Access published online on May 29, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp455
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences

Luiz Carlos Junior Alcantara1, Sharon Cassol2, Pieter Libin3, Koen Deforche4, Oliver G. Pybus5, Marc Van Ranst3, Bernardo Galvão-Castro1, Anne-Mieke Vandamme3 and Tulio de Oliveira6,*

1Laboratório Avançado de Saúde Publica, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Brazil, 2MRC Unit for Inflammation and Immunity, Department of Immunology, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, Pretoria, South Africa, 3Laboratory for Clinical and Epidemiological Virology, Katholieke Universiteit Leuven, BG, 4MyBioData bvba, Rotselaar, Belgium, 5Department of Zoology, University of Oxford, UK and 6Africa Centre for Health and Population Studies, University of KwaZulu-Natal, South Africa

*To whom correspondence should be addressed. Tel: +27 35 5650 7531; Fax: +27 35 550 7565; Email: tuliodna{at}gmail.com; tdeoliveira{at}africacentre.ac.za

Received February 11, 2009. Revised May 13, 2009. Accepted May 14, 2009.

Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including:

http://bioafrica.mrc.ac.za/rega-genotype/html/

http://lasp.cpqgm.fiocruz.br/virus-genotype/html/

http://jose.med.kuleuven.be/genotypetool/html/.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.


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