TY - GEN T1 - Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach AU - Prepens, Sandra AU - Kreuzer, Karl-Anton AU - Leendertz, Fabian AU - Nitsche, Andreas AU - Ehlers, Bernhard AB - Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized. KW - Animals KW - DNA KW - Molecular Sequence Data KW - Phylogeny KW - Cluster Analysis KW - Herpesviridae Infections/veterinary KW - Viral/genetics KW - Sequence Analysis KW - Viral Proteins/genetics KW - Polymerase Chain Reaction/methods KW - Primate Diseases/virology KW - DNA Primers/genetics KW - Glycoproteins/genetics KW - Virology/methods KW - Herpesviridae Infections/virology KW - Viral/chemistry KW - Herpesviridae/genetics KW - Herpesviridae/isolation & purification KW - Herpesviridae/classification KW - DNA-Directed DNA KW - Polymerase/genetics KW - Exodeoxyribonucleases/genetic KW - Primates KW - Viral Structural Proteins/genetics KW - 610 Medizin PY - 2007 LA - eng PB - Robert Koch-Institut, Infektionskrankheiten / Erreger; Robert Koch-Institut, Biologische Sicherheit VL - 4 IS - 84 DO - 10.1186/1743-422X-4-84 ER -