VERY few veterinary surgeons have thought fit to write a book on diseases of poultry. Mr. Ernest Gray has done justice to the subject and is to be congratulated on his effort. A book of this size, written by one with specialized knowledge, will add to the value of any library or private bookshelf. Diseases of Poultry Their Aetiology, Diagnosis, Treatment and Control; with a Section on the Normal Anatomy and Physiology of the Fowl. By Ernest Gray. (Lockwood's Agricultural and Horticultural Handbooks.) Pp. x + 198 + 16 plates. (London: Crosby Lockwood and Son, Ltd., 1940.) 9s. 6d. net.

Diseases of Poultry

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.

Rescue of Influenza A Virus from Recombinant DNA

Regulation of translation via mRNA structure in prokaryotes and eukaryotes.

Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications

Newcastle disease: Evolution of genotypes and the related diagnostic challenges

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence surrounding the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. Nevertheless, comparison of NDV strains that carry exactly the same F protein cleavage site shows that significant differences in virulence still exist. For instance, virulent field strain Herts/33 with the F cleavage site 112RRQRRF117 had an intracerebral pathogenicity index of 1·88 compared with 1·28 for strain NDFLtag, which has the same cleavage site. This implies that additional factors contribute to virulence. After generating an infectious clone of Herts/33 (FL-Herts), we were able to map the location of additional virulence factors by exchanging sequences between FL-Herts and NDFLtag. The results showed that, in addition to the F protein cleavage site, the haemagglutinin–neuraminidase (HN) protein also contributed to virulence. The effect of the HN protein on virulence was most prominent after intravenous inoculation. Interestingly, both the stem region and the globular head of the HN protein seem to be involved in determining virulence.

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain

A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was produced in high amounts by tumor cells infected with rec(IL-2). Tumor vaccine cells infected by rec(IL-2) stimulated human T cells to exert anti-tumor activity in vitro in a tumor neutralization assay. These effects were significantly increased when compared to vaccine infected by rec(-) virus without IL-2 gene. After incubation with rec(IL-2) infected tumor cells, T cells showed increased expression of the activation marker CD69 and produced increased amounts of IFNgamma when compared to T cells co-incubated with rec(-) infected tumor cells. CD8 T cells incubated with rec(IL-2) infected tumor cells showed upregulation of perforin, cell surface exposure of the degranulation marker CD107a and increased anti-tumor cytotoxic activity. Purified T cells from lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients could be stimulated to secrete IFNgamma in an ELISPOT assay upon 40 h of stimulation with rec(IL-2) infected autologous tumor cells [ATV-rec(IL-2)] but not upon stimulation with rec(IL-2) infected allogeneic U937 tumor cells. This suggests direct activation of patient derived tumor antigen-specific memory T cells by ATV-rec(IL-2). In conclusion, the already inherent immunostimulatory properties of NDV could be further augmented by the introduction of the therapeutic gene IL-2. Active specific immunization of patients with ATV-rec(IL-2) should provide the microenvironment at the vaccination site with IL-2 and avoid side effects as seen after systemic IL-2 application.

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Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

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Olav S. de Leeuw

Papers

Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain

Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice