Isolation, Genome Phylogenetic Analysis and In vitro Rescue of a Newly Emerging Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) is the major causative agent of post-weaning multisystemic wasting syndrome (PMWS). Infection by PCV2 may cause heavy losses in pig industry. In this study, we report the isolation of a newly emerging PCV2 from northeastern China. The complete genome of the PCV2 isolate named PCV2-LJR contains 1766 nucleotides and was compared with reference sequences published in GenBank followed by topology analysis of the resulting phylogenetic tree. The data indicated that the prevalent PCV2 isolates in the northeastern China had close relationship, although various genotypes of PCV2 existed. In addition, by gene recombination and transfection techniques, the PCV2 infectious clone was achieved and was able to rescue virus in vitro determined by indirect immunofluorescence assay and PCR. The obtained biological materials may be used for biological characterization of PCV2

Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p \u3c 0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment

Phages Bearing Affinity Peptides to Bovine Rotavirus Differentiate the Virus from Other Viruses

The aim of this study was to identify potential ligands and develop a novel diagnostic test to pathogenic bovine rotavirus (BRV) using phage display technology. The viruses were used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After five rounds of biopanning, phages had a specific binding activity to BRV were isolated. DNA sequencing indicated that phage displayed peptides HVHPPLRPHSDK, HATNHLPTPHNR or YPTHHAHTTPVR were potential ligands to BRV. Using the specific peptide-expressing phages, we developed a phage-based ELISA to differentiate BRV from other viruses. Compared with quantitative real-time PCR (qPCR), the phage-mediated ELISA was more suitable for the capture of BRV and the detection limitation of this approach was 0.1 µg/ml of samples. The high sensitivity, specificity and low cross-reactivity for the phage-based ELISA were confirmed in receiver operating characteristics (ROC) analysis

A phage-displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluorescence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pre-treatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells. These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells

Template-dependent multiple displacement amplification for profiling human circulating RNA

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material. </jats:p

Crustal and mantle velocity models of southern Tibet from finite frequency tomography

Using traveltimes of teleseismic body waves recorded by several temporary local seismic arrays, we carried out finite‐frequency tomographic inversions to image the three‐dimensional velocity structure beneath southern Tibet to examine the roles of the upper mantle in the formation of the Tibetan Plateau. The results reveal a region of relatively high P and S wave velocity anomalies extending from the uppermost mantle to at least 200 km depth beneath the Higher Himalaya. We interpret this high‐velocity anomaly as the underthrusting Indian mantle lithosphere. There is a strong low P and S wave velocity anomaly that extends from the lower crust to at least 200 km depth beneath the Yadong‐Gulu rift, suggesting that rifting in southern Tibet is probably a process that involves the entire lithosphere. Intermediate‐depth earthquakes in southern Tibet are located at the top of an anomalous feature in the mantle with a low Vp, a high Vs, and a low Vp/Vs ratio. One possible explanation for this unusual velocity anomaly is the ongoing granulite‐eclogite transformation. Together with the compressional stress from the collision, eclogitization and the associated negative buoyancy force offer a plausible mechanism that causes the subduction of the Indian mantle lithosphere beneath the Higher Himalaya. Our tomographic model and the observation of north‐dipping lineations in the upper mantle suggest that the Indian mantle lithosphere has been broken laterally in the direction perpendicular to the convergence beneath the north‐south trending rifts and subducted in a progressive, piecewise and subparallel fashion with the current one beneath the Higher Himalaya