Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach

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

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach-4

<p><b>Copyright information:</b></p><p>Taken from "Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach"</p><p>http://www.virologyj.com/content/4/1/84</p><p>Virology Journal 2007;4():84-84.</p><p>Published online 6 Sep 2007</p><p>PMCID:PMC2014757.</p><p></p>0copy numbers). Real-time PCR was carried out in the presence or absence of LNA-PtroLCV1 or LNA-EBV

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach-1

<p><b>Copyright information:</b></p><p>Taken from "Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach"</p><p>http://www.virologyj.com/content/4/1/84</p><p>Virology Journal 2007;4():84-84.</p><p>Published online 6 Sep 2007</p><p>PMCID:PMC2014757.</p><p></p>esence (+) of LNA-PtroLCV1 and/or LNA-PtroRHV1. The electropherogram is shown. Below, the amplified DPOL sequences are indicated. Marker: 100 bpladder (lanes 1 and 8)

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach-3

<p><b>Copyright information:</b></p><p>Taken from "Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach"</p><p>http://www.virologyj.com/content/4/1/84</p><p>Virology Journal 2007;4():84-84.</p><p>Published online 6 Sep 2007</p><p>PMCID:PMC2014757.</p><p></p>el primate herpesviruses and of known human and non-human primate herpesviruses, available in GenBank. A multiple alignment of concatenated 1100 aa was analysed with the neighbor-joining method. A midpoint-rooted phylogram is shown. The branch length is proportional to evolutionary distance (scale bar). Results of bootstrap analysis (100-fold) are indicated at the nodes of the tree, to the left of the first vertical divider. In addition, the alignment was analysed with Tree-Puzzle 5.0. Support values, estimated by the quartet puzzling (QP) tree search and expressing the QP reliability in percent, are indicated at the nodes of the tree to the left of the vertical divider. Nodes with values below 70% in both analyses were depicted as pat of a multifurcation (black bar). Viruses, which are entirely novel or viruses for which additional sequence information was generated, are highlighted with black arrows. Herpesvirus genera and families are indicated. Full names of known viruses and their nucleotide sequence accession numbers are listed in the section

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach-0

<p><b>Copyright information:</b></p><p>Taken from "Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach"</p><p>http://www.virologyj.com/content/4/1/84</p><p>Virology Journal 2007;4():84-84.</p><p>Published online 6 Sep 2007</p><p>PMCID:PMC2014757.</p><p></p>0copy numbers). Real-time PCR was carried out in the presence or absence of LNA-PtroLCV1 or LNA-EBV

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach-2

<p><b>Copyright information:</b></p><p>Taken from "Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach"</p><p>http://www.virologyj.com/content/4/1/84</p><p>Virology Journal 2007;4():84-84.</p><p>Published online 6 Sep 2007</p><p>PMCID:PMC2014757.</p><p></p>tial sequences of the ORFs UL57, UL55 and UL54, obtained through PCR with deg/dI primers, are depicted by thin solid lines, and the type of gB sequence (gB1 or gB2) is indicated. LD amplimers are depicted by dashed lines. The solid line represents the final 8 kbp contiguous sequences of CgueCMV-1.1 and CgueCMV-1.2. Pairwise alignments of the UL56 and gB proteins and the partial DPOL proteins of CgueCMV-1.1 and CgueCMV-1.2 are shown. Dots represent identical amino acids, dashes indicate regions of non-colinearity

Nonredundant function of soluble LTα3 produced by innate lymphoid cells in intestinal homeostasis

Immunoglobulin A (IgA) production at mucosal surfaces contributes to protection against pathogens and controls intestinal microbiota composition. However, mechanisms regulating IgA induction are not completely defined. We show that soluble lymphotoxin α (sLTα3) produced by RORγt(+) innate lymphoid cells (ILCs) controls T cell-dependent IgA induction in the lamina propria via regulation of T cell homing to the gut. By contrast, membrane-bound lymphotoxin β (LTα1β2) produced by RORγt(+) ILCs is critical for T cell-independent IgA induction in the lamina propria via control of dendritic cell functions. Ablation of LTα in RORγt(+) cells abrogated IgA production in the gut and altered microbiota composition. Thus, soluble and membrane-bound lymphotoxins produced by ILCs distinctly organize adaptive immune responses in the gut and control commensal microbiota composition