Dendrobium Swarz.(ラン科)の類縁に関する研究 : I. Eugenanthe Schlechter節内での交配親和性

1.ノビルタイプのデンドロビウム品種に.新しい遺伝子を導入する可能性を調べるため, Eugenanthe節内の22種と、D. moniliforme(セッコク), D. nobileとの交配を行なった.2.交配稔性からみて, 本節内にはD. moniliforme, D. nobileとは遠縁と思われる種が含まれていた.3.D. moniliformeは, D. nobileに比べ, 多くの種と交雑可能で, 今後の育種のために有用な種と考えられた.In order to check the possibility of introducing new genes into the modern nobile-type cultivars of Dendrobium, D. nobile Lindl. and D. moniliforme (L.) Swarz. were crossed with selected species of section Eugenanthe Schlechter. D. moniliforme showed a wider range of crossability with Eugenanthe species compared to D. nobile. Eugenanthe species were divided into two groups according to their crossability with D. moniliforme

HA154–156 glycosylation site in Egyptian H5N1 influenza viruses.

<p>The H5N1 sequences evaluated were obtained from the Global Initiative on Sharing Avian Influenza Data (GISAID; <a href="http://platform.gisaid.org/epi3/frontend" target="_blank">http://platform.gisaid.org/epi3/frontend</a>).</p

Quantitative analysis of vRNA, cRNA, and mRNA levels for wild-type and mutant PB1 proteins.

<p>293T cells were transfected with plasmids expressing PB2, PA, NP, and wild-type or mutant PB1 protein, along with a plasmid transcribing a viral RNA. The levels of vRNA, cRNA, and mRNA were assessed by quantitative RT-PCR and compared to those obtained with wild-type PB1 protein. The results shown represent the average levels of RNAs ± standard deviations from a representative experiment carried out in triplicate. As a negative control (Mock), the PB1 protein expression plasmid was omitted.</p

Effects of mutations in the four conserved motifs of PB1 on influenza polymerase activity.

<p>(<b>A</b>) Motif I; (<b>B</b>) Motif II; (<b>C</b>) Motif III; (<b>D</b>) Motif IV. Consensus sequences are listed by motif with numbers at each end indicating the position of the first and last amino acid residue. Invariant amino acids are underlined. Selected mutations found in the four conserved motifs of natural isolates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036113#pone-0036113-t003" target="_blank"><b>Table 3</b></a>) were introduced into the PB1 protein of A/bar-headed goose/Qinghai/1/2005 (H5N1) virus. The polymerase activity of each PB1 mutant was assessed in human 293T cells by quantifying luciferase activity in the cell lysates of cells transfected with PB1- (wild type or mutant), PB2-, PA- and NP-protein expression plasmids along with a reporter plasmid expressing an influenza virus-like RNA construct for luciferase. Luciferase activity levels were normalized by the <i>Renilla</i> internal control and expressed as a percentage relative to wild-type PB1 activity set as 100%. Data values are averaged over three independent transfection experiments where each experiment was run in triplicate. Error bars represent one standard deviation.</p

Mutations in the conserved motifs I – IV of the PB1 protein and their effects on replicative ability in human and chicken cells.

1<p>The indicated mutations were introduced into the PB1 protein of A/bar-headed goose/Qinghai/1/2005 (H5N1; BHG) virus. Polymerase activity in human 293T and avian DF-1 cells is indicated as percentage relative to wild-type PB1 activity.</p>2<p>For some of these isolates, different sequences are reported in the Los Alamos National Laboratory and NIH NCBI databases.</p

Growth characteristics of wild-type virus and virus possessing the PB1 K480R mutation.

<p>Wild-type A/California/04/09 virus and recombinant virus possessing the K480R mutation were used for infection of human Calu-3 cells (MOI = 0.001). Supernatants were collected at specified time points following infection, and virus titers were quantified by plaque assays in MDCK cells. Results shown are averages of three plaque assays.</p

Avian virus PB1 proteins with mutations in the conserved motifs I – IV.

*<p>Percentage of total changes.</p

Influenza A virus PB1 proteins with mutations in the conserve motifs I – IV.

*<p>Percentage of total changes.</p

Effects of PB1 mutation K480R on influenza polymerase activity in human and avian cells.

<p>The effects of PB1 mutation K480R were evaluated in the background of the A/California/04/09 (H1N1) virus replication complex in human 293T and chicken DF-1 cells. The experiments were carried out as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036113#pone-0036113-g001" target="_blank">Figure 1</a>. Luciferase activity levels were normalized by the <i>Renilla</i> internal control and expressed as a percentage relative to wild-type PB1 activity in the particular cell line. *P<0.07 by Student’s t-test.</p

Molecular Differences among ZEBOV Mouse-Adapted Variants

<div><p>(A) Comparison of ZEBOV variants. Pre–MA-ZEBOV differs from wild-type ZEBOV (WT-ZEBOV) by three amino acids in the GP (red triangles) and a silent mutation in the ORF of the VP40 gene (gray triangle). Serial passage of pre–MA-ZEBOV in progressively older mice yielded MA-ZEBOV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020073#ppat-0020073-b014" target="_blank">14</a>], which contains coding changes in the NP, VP35, VP24, and polymerase (L) (all shown in red triangles). Two nucleotide changes localize to the NCRs at the 3′ end of the VP30 and the 5′ end of the VP24 gene (red triangles); the remaining three modifications in the NP, VP40, and L ORFs are silent (gray triangles).</p><p>(B) Nucleotide and amino acid differences among WT-ZEBOV, pre–MA-ZEBOV, MA-ZEBOV, and MA-RG. The nucleotide changes in GP of pre–MA-ZEBOV, compared to WT-ZEBOV, as well as the changes acquired during adaptation of pre–MA-ZEBOV in mice are shown in red.</p><p>−, no change.</p></div