The correlation between the mutual deletions of amino acids within porcine circovirus rep protein and the discrepancy of replication.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.

Immunogenicity of porcine circovirus type 2 nucleic acid vaccine containing CpG motif for mice.

BACKGROUND: This study aimed at reseaching the immune effect of porcine circovirus type 2 (PCV2) DNA vaccine containing CpG motif on mice. METHODS: A total of 40 6-week-old female BALB/c mice were randomly divided into four groups which were immunized by 18CpG-pVAX1-ORF2, pVAX1-ORF2, pVAX1 and PBS, respectively, and immunized again 2 weeks later. All mice were challenged with 0.2 mL PCV2 cells virulent strain SD (106.0 TCID50/mL) after 4 weeks. Average daily gain, blood antibody levels, microscopic changes and viremia were detected to estimate the effect of DNA vaccine. RESULTS AND DISCUSSION: The results showed that compared to those of the control mice, groups immunized with pVAX1-ORF2 and 18CpG-pVAX1-ORF2 could induce PCV2-specific antibodies. The PCV2-specific antibodies level of 18 CpG-pVAX1-ORF2 groups was higher significantly than other groups and decreased slowly along with time. There was no distinct pathological damage and viremia occurring in mice that inoculated with CpG motif DNA vaccines. The results demonstrated that the DNA vaccine containing 18 CpG could build up resistibility immunity and reduce immune organ damage on mice.

Reactivation of Multidrug-Resistant HSV-1 in a Post-Allogenic Hematopoietic Stem Cell Transplant Patient: Dynamic Detection of the Rare A605V Mutation by Next-Generation Sequencing.

We present an immunocompromised patient with a multiresistant herpes simplex virus-1 reactivation with a rare mutation (A605V) in the viral DNA polymerase gene. Next-generation sequencing suggests the presence of multiple drug-resistant strains before treatment and altered ratios during treatment, affecting the clinical response to aciclovir and foscarnet.

Genome-wide in silico identification of glutathione S-transferase (GST) gene family members in fig (Ficus carica L.) and expression characteristics during fruit color development.

Glutathione S-transferase (GSTs), a large and diverse group of multi-functional enzymes (EC 2.5.1.18), are associated with cellular detoxification, various biotic and abiotic stress responses, as well as secondary metabolites transportation. Here, 53 members of the FcGST gene family were screened from the genome database of fig (Ficus carica), which were further classified into five subfamilies, and the tau and phi were the major subfamilies. These genes were unevenly distributed over all the 13 chromosomes, and 12 tandem and one segmental duplication may contribute to this family expansion. Syntenic analysis revealed that FcGST shared closer genetic evolutionary origin relationship with species from the Ficus genus of the Moraceae family, such as F. microcarpa and F. hispida. The FcGST members of the same subfamily shared similar gene structure and motif distribution. The α helices were the chief structure element in predicted secondary and tertiary structure of FcGSTs proteins. GO and KEGG indicated that FcGSTs play multiple roles in glutathione metabolism and stress reactions as well as flavonoid metabolism. Predictive promoter analysis indicated that FcGSTs gene may be responsive to light, hormone, stress stimulation, development signaling, and regulated by MYB or WRKY. RNA-seq analysis showed that several FcGSTs that mainly expressed in the female flower tissue and peel during 'Purple-Peel' fig fruit development. Compared with 'Green Peel', FcGSTF1, and FcGSTU5/6/7 exhibited high expression abundance in the mature fruit purple peel. Additionally, results of phylogenetic sequences analysis, multiple sequences alignment, and anthocyanin content together showed that the expression changes of FcGSTF1, and FcGSTU5/6/7 may play crucial roles in fruit peel color alteration during fruit ripening. Our study provides a comprehensive overview of the GST gene family in fig, thus facilitating the further clarification of the molecular function and breeding utilization.

Synchronous removal of tetracycline and copper (II) over Z­scheme BiVO4/rGO/g-C3N4 photocatalyst under visible-light irradiation.

The combined pollution of heavy metals and organic pollutants in water body has become one of vital environmental issues. Herein, a series of BiVO4/rGO/g-C3N4 nanocomposites were synthesized for concurrent removals of organic pollutant and heavy metal. Results showed that using the optimized photocatalyst BiVO4/rGO/g-C3N4-28, tetracycline (TC) removal of 87.3% and copper (Cu (II)) removal of 90.6% were achieved under visible-light irradiation within 3 h, respectively; much higher than those using BiVO4 and g-C3N4. More importantly, synergistic effect of TC and Cu (II) removals occurred on the surface of BiVO4/rGO/g-C3N4 in the TC-Cu (II) coexistence condition. Additionally, the ·OH and ·O2- were the most important active species for TC oxidation, while photogenerated electrons were the most responsible for Cu (II) reduction. Results of various characterizations and electron spin resonance test demonstrated that BiVO4/rGO/g-C3N4 was a Z-scheme photocatalyst. Based on the identified intermediates, possible degradation pathways and mechanisms for photocatalytic degradation of TC were proposed. This study advances the development and mechanism of photocatalysts for collaborative removal of pollutants.

Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome.

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.