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

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

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

Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN- and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1)

Phylogeny and evolution of porcine parvovirus.

International audiencePorcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. Previous phylogenetic studies based on the NS1 or VP2 genes indicated that current PPV strains diverged 30 years ago and that VP2 was under neutral or positive selection. Our analysis of NS1, VP2 and complete ORFs indicated that the most recent common ancestor of PPV strains existed about 250 years ago and that the 127-nt repeat in the 3'NTR was present in viruses of some subclades that evolved about 80 years ago. Nucleotide substitution rates of NS1 and VP2 genes were 3.03 × 10(-5) and 1.07 × 10(-4), respectively. Both the NS1 and VP2 proteins were under purifying selection and recombination did not contribute to the genetic diversity of PPV. As expected, surface amino acids are hydrophilic and make up the majority of mutations in the VP2 protein; residues in VP2 interfaces were substituted gradually, often in conjunction with complementary substitutions in the neighboring VP2

Phylogeny and expression of the nucleocapsid gene of porcine epidemic diarrhoea virus

Porcine epidemic diarrhoea virus (PEDV) is one of the important pathogens that may cause severe diarrhoea in piglets. In this study, the nucleocapsid (N) gene of a Chinese PEDV isolate designated HLJBY was cloned. The phylogeny of PEDV strains was investigated by constructing a phylogenetic tree based on the N protein sequences. The results indicate that there are two major groups of Chinese PEDVs, a Japanese PEDV group and a Korean PEDV group. High-level expression of the N protein was achieved in Escherichia coli. The immunoreactivity between PEDV particles or the bacterially expressed N protein and rabbit anti-PEDV serum was confirmed by immunofluorescence assays and Western blot. Both PEDV N protein and the polyclonal antibody generated in this study are valuable diagnostic reagents for PEDV surveillance

Incidence rates and deaths of tuberculosis in HIV-negative patients in the United States and Germany as analyzed by new predictive model for infection.

Incidence and mortality due to tuberculosis (TB) have been decreasing worldwide. Given that TB is a cosmopolitan disease, proper surveillance and evaluation are critical for controlling dissemination. Herein, mathematical modeling was performed in order to: 1) demonstrate a correlation between the incidence of TB in HIV-free patients in the US and Germany, and their corresponding mortality rates; 2) show the utility of the newly developed D-R algorithm for analyzing and predicting the incidence of TB in both countries; and 3) inform us on population death rates due to TB in HIV-negative patients. Using data published by the World Health Organization between 1990 and 2009, the relationship between incidence and mortality that could not be ascribed to HIV infection was evaluated. Using linear, quadratic and cubic curves, we found that a cubic function provided the best fit with the data in both the US (Y = 2.3588+2.2459X+61.1639X(2)-60.104X(3)) and Germany (Y = 1.9271+9.4967X+18.3824X(2)-10.350X(3)) where the correlation coefficient (R) between incidence and mortality was 0.995 and 0.993, respectively. Second, we demonstrated that fitted curves using the D-R model were equal to or better than those generated using the GM(1,1) algorithm as exemplified in the relative values for Sum of Squares of Error, Relative Standard Error, Mean Absolute Deviation, Average Relative Error, and Mean Absolute Percentage Error. Finally, future trends using both the D-R and the classic GM(1,1) models predicted a continued decline in infection and mortality rates of TB in HIV-negative patients rates extending to 2015 assuming no changes to diagnosis or treatment regimens are enacted

The correlation (R) and variance (F) between incidence (I) and death (D) in TB patients in the US (U) and Germany (G) using linear, quadratic, and cubic parametric equations.

<p>For F, p<0.01.</p