Red recombination enables a wide variety of markerless manipulation of porcine epidemic diarrhea virus genome to generate recombinant virus

Porcine epidemic diarrhea virus (PEDV) is a member of the genera Alphacoronavirus that has been associated with acute watery diarrhea and vomiting in swine. Unfortunately, no effective vaccines and antiviral drugs for PEDV are currently available. Reverse genetics systems are crucial tools for these researches. Here, a PEDV full-length cDNA clone was constructed. Furtherly, three PEDV reporter virus plasmids containing red fluorescent protein (RFP), Nano luciferase (Nluc), or green fluorescence protein (GFP) were generated using Red recombination with the GS1783 E. coli strain. These reporter-expressing recombinant (r) PEDVs showed similar growth properties to the rPEDV, and the foreign genes were stable to culture up to P9 in Vero cells. Using the Nluc-expressing rPEDV, the replication of PEDV was easily quantified, and a platform for rapid anti-PEDV drug screening was constructed. Among the three drugs, Bergenin, Umifenovir hydrochloride (Arbidol), and Ganoderma lucidum triterpenoids (GLTs), we found that GLTs inhibited PEDV replication mainly after the stage of virus “Entry”. Overall, this study will broaden insight into the method for manipulating the PEDV genome and provide a powerful tool for screening anti-PEDV agents

Molecular dynamics study of the interactions of incident N or Ti atoms with the TiN(001) surface

The interaction processes between incident N or Ti atoms and the TiN(001) surface are simulated by classical molecular dynamics based on the second nearest-neighbor modified embedded-atom method potentials. The simulations are carried out for substrate temperatures between 300–700 K and kinetic energies of the incident atoms within the range of 0.5–10 eV. When N atoms impact against the surface, adsorption, resputtering and reflection of particles are observed; several unique atomic mechanisms are identified to account for these interactions, in which the adsorption could occur due to the atomic exchange process while the resputtering and reflection may simultaneously occur. The impact position of incident N atoms on the surface plays an important role in determining the interaction modes. Their occurrence probabilities are dependent on the kinetic energy of incident N atoms but independent on the substrate temperature. When Ti atoms are the incident particles, adsorption is the predominant interaction mode between particles and the surface. This results in the much smaller initial sticking coefficient of N atoms on the TiN(001) surface compared with that of Ti atoms. Stoichiometric TiN is promoted by N/Ti flux ratios larger than one

The compensational boundary method to calculate the projected contact area of nanoindentation in atomistic simulations

The atomistic simulation of nanoidentation has become a powerful method to probe the mechanical behaviour and properties of small volumes of materials. It is crucial to calculate the projected contact area (PCA) accurately in order to obtain a reliable value of nanoindentation hardness. In this work, atomistic simulations of nanoindentation were performed on the Cu(111) and Ag(111) surfaces, and a new compensational boundary method is proposed to calculate the PCA. Compared with other available methods, this method provides a clear physical implication, and works well independently of the contact depth and the deformation behaviour of the material. It is also concluded that the widely-used experimental Oliver–Pharr (O–P) method significantly underestimates the PCA in atomistic simulations, and does not work for shallow nanoindentation at the nanoscale

Distinctive Genes Determine Different Intramuscular Fat and Muscle Fiber Ratios of the longissimus dorsi Muscles in Jinhua and Landrace Pigs

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF

Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

AbstractHuman respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses – one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection