Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.

We report the isolation and characterization of a highly pathogenic avian influenza A/H5N1 virus from Crested Hawk-Eagles smuggled into Europe by air travel. A screening performed in human and avian contacts indicated no dissemination occurred. Illegal movements of birds are a major threat for the introduction of highly pathogenic avian influenza.

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The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

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The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-g (ChIFN-g) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-g production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-g could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-g production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-g ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-g after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-g production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-g.

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