Transcriptome Analysis in Spleen Reveals Differential Regulation of Response to Newcastle Disease Virus in Two Chicken Lines.

Enhancing genetic resistance of chickens to Newcastle Disease Virus (NDV) provides a promising way to improve poultry health, and to alleviate poverty and food insecurity in developing countries. In this study, two inbred chicken lines with different responses to NDV, Fayoumi and Leghorn, were challenged with LaSota NDV strain at 21 days of age. Through transcriptome analysis, gene expression in spleen at 2 and 6 days post-inoculation was compared between NDV-infected and control groups, as well as between chicken lines. At a false discovery rate <0.05, Fayoumi chickens, which are relatively more resistant to NDV, showed fewer differentially expressed genes (DEGs) than Leghorn chickens. Several interferon-stimulated genes were identified as important DEGs regulating immune response to NDV in chicken. Pathways predicted by IPA analysis, such as "EIF-signaling", "actin cytoskeleton organization nitric oxide production" and "coagulation system" may contribute to resistance to NDV in Fayoumi chickens. The identified DEGs and predicted pathways may contribute to differential responses to NDV between the two chicken lines and provide potential targets for breeding chickens that are more resistant to NDV

\u3cem\u3eRhizobium phaseoli\u3c/em\u3e Symbiotic Mutants with Transposon Tn5 Insertions

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained

Parenteral vaccination of mammalian livestock with Newcastle disease virus-based vector vaccines offers optimal efficacy and safety

Newcastle disease virus (NDV) is an avian virus that is being evaluated as a vaccine vector for the delivery of foreign genes in mammals. The use of NDV as a vaccine vector in these species offers two major advantages. First, NDV is highly attenuated in mammals, rendering its use inherently safe. Second, mammals lack pre-existing NDV immunity, which minimizes the risk of vaccination failure. NDV-vector vaccines are generally administered to mammals via the respiratory route. We recently showed that intramuscular vaccination with NDV-based Rift Valley fever virus (RVFV) vaccines provides complete protection in mice and induces neutralizing antibodies in sheep and cattle, the main target species of RVFV. Here, we discuss the use of NDV as a vaccine vector for applications in mammalian livestock with an emphasis on the vaccination route. We also report the results of novel experiments that underscore our notion that vaccination via a parenteral route is more effective than immunization via the respiratory route

Sharp Moser-Trudinger inequalities on Riemannian manifolds with Negative curvature

Let $M$ be a complete, simply connected Riemannian manifold with negative curvature. We obtain some Moser-Trudinger inequalities with sharp constants on $M$.Comment: 13 page

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex

Synthesis of IFN-β by Virus-Infected Chicken Embryo Cells Demonstrated with Specific Antisera and a New Bioassay

Transcripts of interferon-α(IFN-α) and IFN-β genes are present in virus-infected chicken cells, but because of a lack of appropriate assays and reagents, it was unclear if biologically active IFN-β is secreted. We have established a nonviral bioassay for the sensitive detection of chicken IFN (ChIFN). This assay is based on a quail cell line that carries a luciferase gene that is controlled by the IFN-responsive chicken Mx promoter. Luciferase activity was strongly stimulated when the indicator cells were incubated with ChIFN-α, ChIFN-β, or ChIFN-γ but not with chicken interleukin-1β (ChIL-1β). Unlike the classic antiviral assay that preferentially detects ChIFN-α, the Mx-luciferase assay detected ChIFN-α and ChIFN-β with similar sensitivity. With the help of this novel assay and with rabbit antisera specific for either IFN-α or IFN-β, we analyzed the composition of IFN in supernatants of virus-infected chicken embryo cells. Virtually all IFN produced in response to Newcastle disease virus (NDV) was IFN-α. However, IFN produced in response to influenza A or vaccinia virus (VV) was a mixture of usually more than 80% IFN-α and up to 20% IFN-β. Thus, IFN-α and IFN-β both contribute to the cytokine activity in supernatants of virus-infected chicken cells. Furthermore, the infecting virus appears to determine the IFN subtype composition

Syndromic surveillance and pathogen detection using multiplex assays for respiratory infections in small ruminants

Background Several bacterial and viruses can infect the respiratory tract of small ruminants causing similar clinical signs. The differential diagnosis of respiratory diseases in small ruminants can be achieved using multi-plex assays for an accurate identification of the causative agents. Objective The present study was aimed at developing molecular multiplex as-says using different methods, such as real time PCR and micro fluidics bead-based technology, applicable for the syndromic surveillance of respiratory infection in small ruminants. The targeted infections were those caused by Capripoxviruses (CaPVs), Peste-des-petits ru-minants' virus (PPRV), Parapoxvirus, Mycoplasma Capricolum subsp. Capripneumoniae (MCCP) and Pasteurella multocida (PM). An inter-nal control was included in order to determine the quality of sam-ples being tested. Methodology Primers and probes were designed for the conserved regions of the genomes of all the targeted pathogens. The probes for real time PCR were labelled with compatible fluorescent dyes and quenchers, whereas for microfluidics bead based method, primers and probes were biotinylated, phosphorylated and C12 amino-modified accor-dingly. Total nucleic acid extraction procedures were evaluated to extract both DNA or RNA. The amplification protocols were opti-mized and the procedures were validated for the amplification of the above-mentioned pathogens in a single test (or tube). Results A one-step multiplex real time PCR method was developed to ampli-fy four targets, CaPVs, PPRV, MCCP and PM in order to accommodate real time PCR platforms from different manufacturers and reduce complexity in performing the assay. This real time PCR method was highly specific and sensitive in detecting the targeted pathogens as well as co-coinfections. Out of 314 samples tested from different African countries, 80 samples were positive for PPRV, 50 for PM, 2 for CaPV and 8 were mixed infections of PPRV and PM. The same patho-gens were included, and the panel was expanded with Parapoxvirus, an internal control, and tested in microfluidics bead-based method. The validated microfluidics bead-based method displayed a similar analytical sensitivity and specificity to the real time PCR based assay. Conclusion The real time PCR method is being implemented in routine diag-nostics and surveillance of different veterinary laboratories in Africa and Asia for differential diagnosis of PPR. Microfluidics bead-based assays will extend the scope by allowing the screening of more pathogens. These two multiplex approaches facilitate the syndromic surveillance of respiratory infection in small ruminants in regions where several pathogens with similar clinical symptoms are present