Porcine Deltacoronavirus Nucleocapsid Protein Suppressed IFN-ß Production by Interfering Porcine RIG-I dsRNA-Binding and K63-Linked Polyubiquitination.

Porcine deltacoronavirus (PDCoV) is a newly detected porcine coronavirus causing serious vomiting and diarrhea in piglets, especially newborn piglets. There has been an outbreak of PDCoV in worldwide since 2014, causing significant economic losses in the pig industry. The interferon (IFN)-mediated antiviral response is an important component of virus-host interactions and plays an essential role in inhibiting virus infection. However, the mechanism of PDCoV escaping the porcine immune surveillance is unclear. In the present study, we demonstrated that the PDCoV nucleocapsid (N) protein antagonizes porcine IFN-ß production after vesicular stomatitis virus (VSV) infection or poly(I:C) stimulation. PDCoV N protein also suppressed the activation of porcine IFN-ß promoter when it was stimulated by porcine RLR signaling molecules. PDCoV N protein targeted porcine retinoic acid-inducible gene I (pRIG-I) and porcine TNF receptor associated factor 3 (pTRAF3) by directly interacting with them. The N-terminal region (1-246 aa) of PDCoV N protein was important for interacting with pRIG-I and interfere its function. We confirmed that PDCoV N antagonizes IFN-ß production by associating with pRIG-I to impede it from binding double-stranded RNA. Furthermore, porcine Riplet (pRiplet) was an important activator for pRIG-I by mediating the K63-linked polyubiquitination. However, PDCoV N protein restrained the pRiplet binding pRIG-I to inhibit pRIG-I K63-linked polyubiquitination. Taken together, our results revealed a novel mechanism by which PDCoV N protein interferes with the early activation of pRIG-I in the host antiviral response. The novel findings provide a new insight into PDCoV on evading the host innate immune response and may provide new therapeutic targets and more efficacious vaccines strategies for PDCoV infections.

Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.

[Construction of a stx2 deletion mutant of Shiga toxin 2 phage phiMin27 and its infectious properties].

OBJECTIVE: To construct an stx2 gene mutant phage phi Min27(delta stx::cat) and to observe its infectiousness of various serotypes Escherichia coli strains. METHODS: With the help of Red recombinant system, the stx2 gene of the E. coli O157:H7 Min27 strain isolated from intestinal feces of piglet with diarrhea at a swine farm of Shanghai, was replaced by the chloramphenicol acetyltrasferase (cat) gene from plasmid pLacI. Phage phi Min27(delta stx::cat) was isolated after induction of E. coli Min27(delta stx::cat) strain with mitomycin C. Twenty-one E. coli strains with various serotypes were infected with phi Min27(delta stx::cat), and plaque formation and lysogenic conversion of them were investigated. RESULTS: Of the 21 E. coli isolates, 2 with the serotypes of O60 and O138 integrated the phi Min27(delta stx::cat) in their chromosomes and expressed resistance to chloramphenicol. With the exception of one laboratory E. coli strain MG1655, none of the tested E. coli strains supported the formation of plaques and lysogenization when used as indicators for phi Min27(delta stx::cat). Following induction with mitomycin C, these lysogenic strains released infectious particles of phi Min27(delta stx::cat) that formed plaques on a lawn of E.coli laboratory strain MC1061. CONCLUSION: These results demonstrated that phi Min27(delta stx::cat) was able to infect and lysogenize particular E. coli strains and that the lysogens could produce infectious phage progeny. It could be inferred that Stx bacteriophages were able to spread exogenous genes among E. coli strains. The work provided a basis for further study on mechanisms of Stx phages infection and control of Stx expression.

Short-tailed stx phages exploit the conserved YaeT protein to disseminate Shiga toxin genes among enterobacteria.

Infection of Escherichia coli by Shiga toxin-encoding bacteriophages (Stx phages) was the pivotal event in the evolution of the deadly Shiga toxin-encoding E. coli (STEC), of which serotype O157:H7 is the most notorious. The number of different bacterial species and strains reported to produce Shiga toxin is now more than 500, since the first reported STEC infection outbreak in 1982. Clearly, Stx phages are spreading rapidly, but the underlying mechanism for this dissemination has not been explained. Here we show that an essential and highly conserved gene product, YaeT, which has an essential role in the insertion of proteins in the gram-negative bacterial outer membrane, is the surface molecule recognized by the majority (ca. 70%) of Stx phages via conserved tail spike proteins associated with a short-tailed morphology. The yaeT gene was initially identified through complementation, and its role was confirmed in phage binding assays with and without anti-YaeT antiserum. Heterologous cloning of E. coli yaeT to enable Stx phage adsorption to Erwinia carotovora and the phage adsorption patterns of bacterial species possessing natural yaeT variants further supported this conclusion. The use of an essential and highly conserved protein by the majority of Stx phages is a strategy that has enabled and promoted the rapid spread of shigatoxigenic potential throughout multiple E. coli serogroups and related bacterial species. Infection of commensal bacteria in the mammalian gut has been shown to amplify Shiga toxin production in vivo, and the data from this study provide a platform for the development of a therapeutic strategy to limit this YaeT-mediated infection of the commensal flora.