Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats

BACKGROUND: In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR). FINDINGS: In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences. CONCLUSIONS: Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family Rhabdoviridae

Organization of the catalytic center of <i>^Dm</i>cN-IIIB.

<p>The magnesium ion (green sphere) is octahedrally coordinated by several residues of <i>^Dm</i>cN-IIIB shown as stick model (carbon in yellow, nitrogen in blue, oxygen in red) as well as several water molecules (red). One equatorial coordination position of Mg²⁺ is occupied by a fluorine atom of the co-crystallized metal fluoride MgF₃^– (magnesium in green; fluorine in light blue). MgF₃^– is coordinated by Asp55, Asp57, Lys219, Ser171 as well as water molecules. Note that the coordination of MgF₃^– represents the pentavalent phosphate transition state in the nucleotidase reaction cycle. Contacts are represented by dashed lines and the model is defined by a representative 2 mF_o – DF_c electron density map (blue mesh) contoured at a sigma level of 2.0.</p

Kinetic properties of the cytosolic 5′-nucleotidases IIIA and IIIB.

<p>Kinetic properties of the cytosolic 5′-nucleotidases IIIA and IIIB.</p

X-ray diffraction data and structure refinement statistics of <i>Drosophila melanogaster</i> cN-IIIB bound to different reaction products.

<p>Values in parentheses indicate the specific values in the particular highest resolution shell.</p><p>* Twinned refinement: twin law = h,-k,-l; twin fraction = 0.10.</p

Structural comparison of <i>^Dm</i>cN-IIIB and <i>^Mm</i>cN-IIIA.

<p>Detail view on the substrate-binding pockets of <i>^Dm</i>cN-IIIB bound to m⁷guanosine in red (A) and <i>^Mm</i>cN-IIIA bound to UMP in dark blue (B). The left panel shows the binding pocket with labeled residues and substrates represented as sticks. Note that Phe75 of <i>^Dm</i>cN-IIIB is replaced by a histidine in <i>^Hs</i>cN-IIIA while the Trp121 of <i>^Dm</i>cN-IIIB is replaced by a tyrosine in <i>^Hs</i>cN-IIIA. In the right panel the substrate-binding pockets are shown as surfaces, which were calculated using a probe radius of 1.65 Å and for clarity reasons were clipped at the level of the nucleobases. Note that the substrate-binding pocket for m⁷G (cN-IIIB, red surface) is significantly larger than the UMP binding pocket of cN-IIIA (blue surface), which was thought to be the structural reason for its purine substrate exclusion. (C) Orientation of substrate binding residues and the corresponding pocket after docking m⁷GMP to <i>^Mm</i>cN-IIIA (light blue). Note that Asn69 (marked by an asterisk) rotates by 180 degrees in comparison to (B) and adopts another common rotamer thus enlarging the binding pocket and neatly accommodating the m⁷GMP.</p

Overall structural comparison of <i>^Dm</i>cN-IIIB bound to m⁷G with <i>^Mm</i>cN-IIIA bound to UMP.

<p>Superposition of <i>^Dm</i>cN-IIIB (HAD core in white; cap domain in red) and cN-IIIA (HAD core in grey; cap domain in blue) showing the overall structural similarity of both enzymes. Note that the cap domain of cN-IIIB is shifted by 4 Å when the structures are superposed <i>via</i> their the HAD core. Thus, the substrate-bound cN-IIIA adopts a closed conformation while the product-bound cN-IIIB represents an open conformation. A movie of the trajectory from the open-to-closed state is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090915#pone.0090915.s005" target="_blank">Movie S1</a></b>.</p

Detail view on the substrate-binding pocket of <i>^Dm</i>cN-IIIB.

<p>(A) The co-crystallized reaction product 7-methylguanosine is shown in ball-and-stick mode (carbon in grey, nitrogen in blue, oxygen in red) and is defined by an mF_o – DF_c omit-electron density map (blue mesh) contoured at a sigma level of 3.0. m⁷G is stacked in a coplanar but off-centered fashion by Phe75 and Trp120 (colors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090915#pone-0090915-g003" target="_blank"><b>Figure 3</b></a>). Trp121 packs by T-shaped edge-to-face stacking against the bound product and binding is additionally supported by numerous direct or water-mediated hydrogen bonds (dashed lines with distances) to various protein residues of <i>^Dm</i>cN-IIIB. (B) Binding of the reaction product cytidine by <i>^Dm</i>cN-IIIB. In contrast to m⁷G, the nucleobase moiety of cytidine makes only a hydrogen bond to Ser124.</p

Kinetic properties of the cytosolic 5′-nucleotidases IIIA, IIIB and variants of <i>^Dm</i>cN-IIIB.

<p>Kinetic properties of the cytosolic 5′-nucleotidases IIIA, IIIB and variants of <i>^Dm</i>cN-IIIB.</p