Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics.

Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.

Design and intracellular activity of a human single-chain antibody to human immunodeficiency virus type 1 conserved gp41 epitope.

A human lymphoid cell line (F172-D8) excreting a human immunodeficiency virus type 1 (HIV-1) anti-gp41 monoclonal antibody was used to construct a plasmid containing the cDNA of the single-chain variable fragment (scFvD8) corresponding to this antibody. A stable human osteosarcoma cell line was obtained which expressed the scFvD8 protein in the cytoplasm. Whereas a cell line transfected with a control construct (pCI-neo) was readily and productively infected with laboratory (Ba-L) or primary HIV-1 isolates, the scFvD8 cell line did not support productive infection. Binding of the virus, internalization, and reverse transcription were not altered by scFvD8 expression, but gp160 expression was dramatically reduced. These data suggest that cytoplasmic expression of this artificial single-chain antibody can interfere with gp160 expression, thereby reducing the production of mature viral envelope proteins.

Visna-maedi virus induces interleukin-8 in sheep alveolar macrophages through a tyrosine-kinase signaling pathway.

The mechanisms leading to the severe lung damage seen in some sheep naturally infected with the visna-maedi virus, and to pulmonary lesions in other lentiviral diseases, appear to involve the recruitment of large numbers of uninfected inflammatory cells. Only a few alveolar macrophages from experimentally infected lambs express virus, but high levels of interleukin (IL)-8 mRNA are present in the macrophage population. In vitro infection with visna-maedi virus at low multiplicity of alveolar macrophages from uninfected sheep also strongly induced the expression of IL-8 mRNA and the accumulation of IL-8 in the extracellular medium. An initial peak of IL-8 mRNA expression at 3 or 6 h after infection was followed by a fall, then a more persistent expression lasting at least 48 h after infection. The early peak was accompanied by expression of mRNA for IL-1beta, and a possible rise in tumor necrosis factor alpha (TNFalpha) mRNA, although this was frequently elevated in uninfected ovine alveolar macrophages. Interestingly, these events occurred identically in cells treated with non-infectious heat-treated virus, suggesting that interaction between viral components and cellular membrane receptors could suffice for both early and late IL-8 induction. The level of IL-8 mRNA induced by treatment with live or inactivated virus could be severely reduced by pretreatment of the macrophages with genistein but not with staurosporine, suggesting the involvement of a tyrosine-kinase signaling pathway. The early induction of IL-1beta and possibly of TNFalpha may explain the occurrence of a later persistent expression of IL-8 mRNA through an autocrine mechanism.

Alveolar macrophages from sheep naturally infected by visna-maedi virus contribute to IL-8 production in the lung.

Sheep naturally infected by visna-maedi virus often develop a chronic interstitial lung disease characterized by an alveolitis comprising lymphocytes, neutrophils and macrophages. The alpha chemokine interleukin-8 (IL-8) was detected in cell free bronchoalveolar lavage fluid from naturally infected animals, confirmed by RT-PCR, presenting typical lesions of maedi and elevated total alveolar cell counts. No detectable IL-8 was found in the fluid obtained from uninfected animals. IL-8 concentration in alveolar fluid is correlated with alveolar neutrophil counts. Bronchoalveolar lavage cells from infected animals were found to contain a large amount of IL-8 mRNA and may contribute to IL-8 production. In situ hybridization showed that macrophages were the predominant cell type expressing IL-8 mRNA. Sustained production of IL-8 by alveolar macrophages during visna-maedi infection could suffice for neutrophil attraction to the alveoli, and may contribute to the development of lesions.

Visna-maedi virus-induced expression of interleukin-8 gene in sheep alveolar cells following experimental in vitro and in vivo infection.

Visna-maedi virus is a lentivirus which causes inflammatory disorders in sheep, including a chronic interstitial lung disease resembling that observed in human immunodeficiency virus type 1 (HIV 1) infection. In view of our previous demonstration of the production of neutrophil chemotactic activity by alveolar macrophages, and given the lymphocytic and neutrophilic nature of the alveolar cell infiltrate in both naturally and experimentally infected animals, we hypothesized that interleukin-8 (IL8) could be a candidate for at least part of the chemotactic activity we described. In this study, we investigated IL8 mRNA expression following visna-maedi virus infection. Northern analysis of total RNA using an ovine IL8-specific probe demonstrated that the IL8 gene is upregulated in alveolar macrophages as a consequence of in vitro infection and in alveolar cells from experimentally infected animals. Using a semi-quantitative RT-PCR method, we showed that various levels of IL8 mRNA are expressed by alveolar cells from infected animals and that they correlate with the intensity of the lesions. In conclusion, visna-maedi virus is able to induce IL8 mRNA expression in sheep alveolar cells. Results from in vivo infected animals suggest that IL8 could play a role in the early build-up of visna-maedi virus-induced lesions.

Sequencing of the ovine interleukin-8-encoding cDNA using the polymerase chain reaction.

The nucleotide (nt) sequence encoding the ovine homologue of interleukin-8 (IL-8) was determined. The mRNA is 1494-nt long with an ORF of 101 codons. The long 3' non-coding element contains several ATTTA repeats implicated in the swift turnover of other chemokine mRNAs. The encoded protein of 11 kDa before processing, and 9 kDa as mature protein, contains the Cys-Xaa-Cys motif common to alpha-chemokines, and has conserved amino acids (aa) at positions identified as receptor contact sites for IL-8. Identities with other published IL-8 aa sequences are: dog, 91%; pig, 87%; rabbit, 84%; human, 78%; guinea pig, 69%. A 49% aa identity is also found with a chicken embryo fibroblast protein.