Primers used for quantitative reverse transcription PCR.

<p>Primers used for quantitative reverse transcription PCR.</p

Virus titer determination and qPCR analysis of BV genomic DNA copy number in the supernatant.

<p>Sf9 cells were infected with vAcΔ<i>cc</i> or vAcΔ<i>cc</i>Δ<i>fp25k</i> at an MOI of 5. The supernatants from infected cells were collected at 0, 18, 24, 48, 72 and 96 h. p.i. Virus titers were determined by EPDA (A), genomic DNA was detected by qPCR (B), and the results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.</p

BV structural proteins incorporation and genome integrity analysis.

<p>(A) Western blot analysis of structural proteins incorporated into recombinant BVs. Equal copy numbers of vAcΔ<i>cc</i> and vAcΔ<i>cc</i>Δ<i>fp25k</i> virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibodies against GP64, Ac23, and VP39. (B) Transformation assay. Genomic DNA isolated from vAcΔ<i>cc</i> or vAcΔ<i>cc</i>Δ<i>fp25k</i> BVs harvested at 36 h and 48 h p.i. was quantified by qPCR. Equal copy numbers (5×10^<i>8</i>) of genomic DNA was used to transform competent <i>E</i>. <i>coli</i> DH10B cells, and the number of CFUs was calculated. (C) Transfection assay. Equal copy numbers of genomic DNA isolated from vAcΔ<i>cc</i> and vAcΔ<i>cc</i>Δ<i>fp25k</i> BVs were transfected into Sf9 cell. Transfection efficiency was calculated as the number of cells expressing EGFP. The mean number of cells transfected by BV genomic DNA of control virus was set as 100%. Data are representative of three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student’s <i>t</i>-test. <b>*</b><i>P</i><0.05.</p

Characterization of recombinant bacmids and viruses.

<p>(A) Construction of <i>fp25k</i> knockout bacmid. The <i>fp25k</i> gene in AcBacΔ<i>cc</i> bacmid was deleted and replaced with the zeocin resistance gene (<i>zeocin</i>^<i>r</i>) through homologous recombination. (B) Strategy for construction of recombinant viruses, vAcΔ<i>cc</i>, vAcΔ<i>cc</i>Δ<i>fp25k</i> and vAcΔ<i>cc</i>Δ<i>fp25k-rfp25k</i>, the e<i>gfp</i> gene was inserted into the <i>polyhedrin</i> locus by transposition. (C) Transfection-infection assay of recombinant bacmids for viral propagation. At 48 h p.t., GFP-expressing cells were observed by fluorescence microscopy (upper panel). The fluorescent signals of GFP in cells infected by transfection supernatant were visualized at 72 h p.i. (lower panel). (D) One step growth curves of vAcΔ<i>cc</i>, vAcΔ<i>cc</i>Δ<i>fp25k</i> and vAcΔ<i>cc</i>Δ<i>fp25k-rfp25k</i>. Sf9 cells were infected with each virus at an MOI of 5, the supernatants were harvested at 0, 24, 48 and 96 h p.i. and determined for the production of infectious virus by EPDA. The results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.</p

Schematic diagram of our hypothesis for parental and <i>fp25k</i>-negative AcMNPV infection.

<p>(A) In parental AcMNPV-infected cells, normal ODVs are formed in the nucleus, and most of the BVs that bud through the plasma membrane are non-infectious. (B) In <i>fp25k</i>-negative virus-infected cells, incompletely enveloped ODVs are formed, and incompletely enveloped ODVs are retained in the nucleus, while a larger number of infectious BVs are produced.</p

Hydrazine-Assisted Syntheses and Properties of Mercury Tellurides Containing Transition-Metal Complexes

With assistance of reactive and coordinative hydrazine, transition-metal telluromercurates [Mn­(trien)­(N₂H₄)₂]₂­[Hg₂Te₄]₂ (<b>A</b>), [Zn­(trien)­(N₂H₄)₂]­Hg₂Te₄ (<b>B</b>), [Mn­(tepa)­(N₂H₄)]₂­Hg₄Te₁₂ (<b>C</b>), [TM­(trien)­(Hg₂Te₄)] (TM = Mn (<b>D</b>), Zn (<b>E</b>)), and [Zn­(atep)]₂Hg₅Te₁₂ (atep = 4-(2-aminoethyl)­triethylenetetramine) (<b>F</b>) were solvothermally prepared in triethylenetetramine (trien) or tetraethylenepentamine (tepa) solvents using elemental Te as precursor in lower temperature range. Compounds <b>A</b> and <b>B</b> consist of mixed coordination cations [TM­(trien)­(N₂H₄)₂]²⁺ (TM = Mn, Zn) and one-dimensional polyanion [Hg₂Te₄]^2– with the five-membered Hg₂Te₃ rings being coplanar. Compound <b>C</b> is composed of two [Mn­(tepa)­(N₂H₄)]²⁺ cations and a [Hg₄Te₁₂]^4– cluster with a centrosymmetric structure. Compounds <b>D</b> and <b>E</b> consist of coordination polymer [TM­(trien) (Hg₂Te₄)] containing novel doubled [Hg₂Te₄]_<i>n</i> chain with tetrahedrally coordinated Hg­(II) centers, which is quite different from the common single chain with the same composition of [Hg₂Te₄]_<i>n</i>. <b>D</b> and <b>E</b> are the first examples of telluromercurates incorporated with TM complex units via TM–Te bonds. Compound <b>F</b> contains fivefold coordinated [Zn­(atep)]²⁺ cations and zigzag [Hg₅Te₁₂^4–]_<i>n</i> polymeric anion. The [Hg₅Te₁₂^4–]_<i>n</i> anion is a new species of the binary telluromercurates. It is built from [Hg₄Te₆] and [HgTe₂(Te₄)] subunits via interconnectivity, which generates Hg₃Te₃ and Hg₄Te₄ rings in the structure. Compounds <b>A</b>–<b>F</b> are potential semiconductors with narrow band gaps in the range of 0.96–1.09 eV. Photocatalytic investigation of Mn­(II) complexes show that they are photocatalytically active in the degradation of CV under visible-light irradiation with the highest catalytic effective of cluster compound <b>C</b>

Expression analyses of viral structural proteins in infected cells.

<p>(A) Sf9 cells were infected with vAcΔ<i>cc</i> or vAcΔ<i>cc</i>Δ<i>fp25k</i> at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting using the corresponding antibodies, the results were repeated twice. (B) Sf9 cells infected with vAcΔ<i>cc</i> or vAcΔ<i>cc</i>Δ<i>fp25k</i> (MOI = 5) were collected at 48 h p.i. and analyzed by qRT-PCR. The transcriptional levels of viral genes were normalized to the internal control <i>28S</i> rRNA, and the transcription difference between vAcΔ<i>cc</i>- and vAcΔ<i>cc</i>Δ<i>fp25k</i>-infected cells was analyzed by the 2^{<i>-ΔΔC</i>}T method. The results of corresponding genes in vAcΔ<i>cc</i>-infected cells were set as 100%. The data are from three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student's <i>t</i>-test. <b>*</b><i>P</i><0.05.</p

Overexpression of GP64.

<p>(A) Genomic organization of AcBacΔ<i>cc</i>-<i>gp64</i>. (B) Western blot analysis of cells infected with vAcΔ<i>cc</i> or vAcΔ<i>cc</i>-<i>gp64</i> and virions of vAcΔ<i>cc</i> or vAcΔ<i>cc</i>-<i>gp64</i>. For expression of GP64 in infected cells, Sf9 cells were infected with vAcΔ<i>cc</i> or vAcΔ<i>cc</i>-<i>gp64</i> at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting. For GP64 incorporation into virions, equal copy numbers of vAcΔ<i>cc</i> and vAcΔ<i>cc</i>-<i>gp64</i> virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibody against GP64, experiments were repeated twice. (C) One-step growth curve of vAcΔ<i>cc</i> and vAcΔ<i>cc</i>-<i>gp64</i>, virus titers were determined by EPDA. Data are representative of three independent experiments. Error bars represent standard deviation.</p

Morphologic and Molecular Characterization of a Strain of Zika Virus Imported into Guangdong, China

<div><p>The recent outbreaks of Zika virus (ZIKV) disease have caused worldwide concerns. Guangdong province is one of the commercial centers in China and communicates frequently with the epidemic areas. To date, 65.2% of the ZIKV infection cases in China were imported via port of entry in Guangdong. The continuous surveillance of imported cases is crucial for the prevention and control of potential ZIKV infection outbreak in China. In this study, a strain of ZIKV was isolated from the serum of a 6-year-old child returning from Venezuela. The morphology of the ZIKV was analyzed <i>in vivo</i> and <i>in vitro</i> by electron microscopy, and clusters of virus particles were found in the loose cytoplasmic membrane structures. The genomic sequence of the isolated ZIKV was determined, and the alignment and phylogenetic analysis identified one unique amino acid substitution occurring in the non-structural protein 4B (NS4B), and the isolated virus belonged to the Asian lineage.</p></div

The isolation and amplification of the ZIKV GZ02 strain.

<p>(A) The amplification of ZIKV GZ02 strain in BHK-21 and Vero cells infected with the homogenized supernatant of brain. Typical cytopathic effects were observed at 5 days post-infection. (B) The viral loads in tissue samples from ZIKV-infected mice were quantified by qRT-PCR and expressed on a log₁₀ scale as viral RNA copies per gram after comparison with a standard curve. The experiments were repeated twice. Error bars represent standard deviation. Data were analyzed by a two-tails student t test. * and ** represent <i>P</i> < 0.05 or 0.01, respectively.</p