Cytologic diagnosis of respiratory syncytial virus infection in a bronchoalveolar lavage specimen from a bone marrow transplant recipient.

Cytologic examination of a bronchoalveolar lavage specimen from a 6-year-old bone marrow transplant recipient revealed pulmonary infiltrates and occasional cells containing discrete pink cytoplasmic inclusions on a May-Grunwald-Giemsa stain. Direct immunofluorescence stains of cytospins prepared from the same specimen were positive for respiratory syncytial virus (RSV). Electron microscopy revealed occasional epithelial cells with cytoplasmic inclusions composed of filamentous virions. The patient died 6 months after the specimen was taken. An autopsy showed ongoing bronchiolitis with diffuse alveolar damage. Occasional bronchiolar epithelial cells contained discrete eosinophilic cytoplasmic inclusions, which on ultrastructural examination proved to be compatible with RSV. Examination of bronchoalveolar lavage fluid from bone marrow transplant recipients should include a search for cytopathic changes compatible with RSV infection. Electron microscopy can be helpful in confirming this diagnosis.

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells.

OBJECTIVES: To generate non-haematopoietic tissues from mobilized haematopoietic CD133(+) stem cells. MATERIALS AND METHODS: Mobilized peripheral blood CD133(+) cells from adult healthy donors were used. In vitro ability of highly enriched CD133(+) cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. RESULTS: We found that a recently identified population of CD45(+) adherent cells generated in vitro after culture of highly purified CD133(+) cells for 3-5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. CONCLUSION: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.

Irreversible binding of phage phi X174 to cell-bound lipopolysaccharide receptors and release of virus-receptor complexes.

At 37 degrees C, binding of phi X174 to the lipopolysaccharide receptors in the outer membrane of Escherichia coli C is followed by an irreversible ejection of its DNA. DNA ejection marks the beginning of the eclipse period in the infection cycle. Binding data with a phi X mutant Fcs70 at 15 degrees C, where the DNA ejection, or eclipse, rate is essentially zero, do not follow the law of mass action. This rules out a simple mechanism of reversible binding followed by irreversible DNA ejection. A more complex reaction model was devised to fit the data [Incardona, N. L. (1983) J. Theor. Biol. 105, 631-645]. It takes into account the fact that lipopolysaccharide-containing outer membrane fragments are continually released from infected E. coli cells, some of which have phi X bound to them. In this paper the model is shown to fit the binding data for wild-type virus at 15 degrees C and to account for the nonlinearity observed at 37 degrees C in the pseudo-first-order binding kinetics and first-order eclipse kinetics for both mutant and wild-type virus. This leads to the conclusion that phi X174 binding to cell-bound receptors is irreversible but binding to released receptors is reversible. The release of virus-receptor complexes from infected cells and the dissociation of these complexes were confirmed by electron microscopy. We propose that initially a single phi X174 vertex interacts reversibly with E. coli lipopolysaccharide but dissociation from the cell is prevented by the subsequent interaction of additional vertices with adjacent receptor molecules.

The Sendai virus nucleocapsid exists in at least four different helical states.

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3- and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because of the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.

Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin.

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.