Nucleotide sequence and vaccinia expression of the nucleoprotein of a highly virulent, neurotropic strain of Newcastle disease virus.

The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB.

Functional characterization of mouse granulocytes and macrophages produced in vitro from bone marrow progenitors stimulated with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF).

Bone marrow from C3H/ouj mice was depleted to < 1% of CD11b+ granulocytes and macrophages using paramagnetic beads coated with sheep anti-rat antibodies. CD11b- cells, enriched three- to fourfold in colony-forming cells, were stimulated in liquid culture with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Cultures stimulated with IL-3 or GM-CSF increased cell numbers fourfold at 7 days, with the CD11b+ population increasing to 63% +/- 9% (n = 5) with IL-3 or 96% +/- 1% (n = 4) cells with GM-CSF. Functional responsiveness of the granulocytes and macrophages was assessed by flow cytometry in an oxidative burst assay using dichlorofluorescein (DCF) and a quantitative phagocytosis assay using opsonized fluorescent beads. Granulocytes and macrophages, identified by light scatter characteristics and allophycocyanine staining of CD11b, were assayed simultaneously with granulocytes from fresh mouse bone marrow and peripheral blood. GM-CSF-generated CD11b+ cells had higher oxidative responses than similar populations produced in response to IL-3. The oxidative burst of these in vitro generated CD11b+ populations was similar to the equivalent fresh bone marrow population. Oxidative burst responses of peripheral blood phagocytic cells could not be adequately measured in this system. Peripheral blood CD11b+ cells were the most phagocytic, followed by GM-CSF-stimulated CD11b+ cells; IL-3-stimulated and bone marrow CD11b+ cells were the least phagocytic. These data demonstrate that functional granulocytes can be produced in vitro using growth factors and that GM-CSF produces a more responsive cell than IL-3.

Bronchoalveolar lavage study in victims of toxic gas leak at Bhopal.

Bronchoalveolar lavage using flexible fibreoptic bronchoscope was carried out in 50 patients 1-2 1/2 yr after exposure to the 'toxic gas' at Bhopal. Thirty six patients in the analysis were categorised into 3 groups (viz., mild, moderate and severe), depending upon the severity of exposure. There was an increase in cellularity in the lower respiratory tract (alveolitis) of the severely exposed patients (in both smokers and non-smokers), compared to normals (P less than 0.05). The increase in cellularity in severely exposed non-smokers was due to abnormal accumulation of macrophages (P less than 0.01), and in severely exposed smokers, to macrophages (P less than 0.01) and neutrophils (P less than 0.05). Mild and moderately exposed patients did not show significant change in cellularity in lower respiratory tract, compared to normal individuals (P greater than 0.2). There was a trend towards increasing cellularity, as the severity increased (P less than 0.0001) and higher numbers of total cells were seen in severely exposed smokers, suggesting that smoking is a risk factor. It appears, therefore, that subjects severely exposed to the toxic gas at Bhopal may have a subclinical alveolitis characterised by accumulation and possibly activation of macrophages in the lower respiratory tract. Smokers, who were exposed to the gas had in addition, accumulation of neutrophils.

Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus.

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strain's neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.