The differentiation state of monocytic cells affects their susceptibility to infection and the effects of infection by dengue virus.

This study describes the susceptibility to dengue virus infection of a monocytic cell line at different states of differentiation. Infectious virus titres increased in undifferentiated U937 cells following infection with clinical isolates but only when the cells were infected via their Fc receptors. No. c.p.e. was observed and virus was not secreted into supernatant fluid. Once differentiated, cells were susceptible to infection either with virus alone or with virus-antibody complexes. Infection was cytolytic and virus was released into the supernatant fluid. Similar results were obtained with freshly isolated peripheral blood monocytes. Increased blood vessel permeability, which occurs in dengue haemorrhagic fever and dengue shock syndrome patients, has been correlated with secondary heterotypic infections and has been postulated to arise from antibody-enhanced infection of monocytes. The data presented suggest a possible mechanism whereby infected monocytes undergoing diapedesis through blood vessel walls might differentiate sufficiently during the process to release virus and cytokines at localized sites on blood vessels.

A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity.

The virulent strain SFV4 of Semliki Forest virus (SFV), produced from the infectious clone pSP6-SFV4, is lethal after intranasal (i.n.) infection of adult mice and for pregnant mice after intraperitoneal (i.p.) infection. In contrast, the A7 strain of SFV is avirulent when given i.n. to adult mice, but induces fetal death in pregnant mice after i.p. infection. The nucleotide and deduced amino acid sequences of part of the core and all of the envelope region of A7-SFV were determined and compared to those of SFV4. A7 differed from SFV4 at 80 nucleotides (nt) in the coding sequence, 15 of which were associated with amino acid differences and seven of which (two in the E2 protein and five in E1) were non-conservative. The 3' non-coding sequence of A7 was longer (415 nt) than that of SFV4 (263 nt) and a divergent sequence of 181 nt was present adjacent to the end of the E1 coding region. The effects on virulence of two mutations in the E2 gene of SFV4, resulting in the non-conservative amino acid substitutions present in A7, were analysed. One mutation (mut 8729 a/c) resulted in only slight attenuation, whereas the other (mut 8902 a/g) resulted in avirulence for pregnant mice. However, mut 8902 a/g was lethal for the majority of developing fetuses after i.p. infection of the mother.

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

The effects of tritium on embryo development: the embryotoxic effects of [3H]tryptophan.

The effects of tritiated amino acids on the development of the early mouse embryo in vitro are described. [3H]tryptophan was extremely toxic, compared with [3H]glutamic acid, and inhibited growth of the two-cell embryo to the blastocyst stage. The LD50 values for the two amino acids were, respectively, 1 and 296 kBq/ml of medium. The two-cell embryo was more sensitive to the irradiation from incorporated [3H]tryptophan is selectively incorporated into two chromosomal proteins in the two-cell embryo, and that those proteins were stage-specific markers of development.

Identification of a neutralization-specific antigen of a calf rotavirus.

Monospecific polyclonal antisera were raised in guinea-pigs against the calf rotavirus polypeptides VP1, VP2, VP3 + 4, VP4.2, VP6, VP7.1, VP7.2 and VP10. All of the antisera gave a similar pattern of cytoplasmic immunofluorescence in rotavirus-infected cells, but spots of fluorescence of varying intensity with different sera were seen over the nucleus. Immune precipitation, using Staphylococcus aureus to collect immune complexes, showed that VP2 was precipitated by antiserum to VP2 (alpha-VP2) and VP6 by alpha-VP6, alpha-VP7.1 and alpha-VP7.2 both precipitated the same range of proteins from infected cells (VP7, VP7.1 and VP7.2) or from virions (VP7.1 and VP7.2). VP10, either from virions or infected cells, was not precipitated by alpha-VP10. The only antiserum which efficiently neutralized infectivity was alpha-VP7.2. There were low levels of neutralization with alpha-VP10 (but the results varied from experiment to experiment) and traces with alpha-VP6. alpha-VP7.1 and the other antisera did not neutralize even though alpha-VP7.1 agglutinated double-shelled particles as seen in immune electron microscopy to a greater extent than alpha-VP7.2. Both VP7.1 and VP7.2 were shown to be glycoproteins by tunicamycin treatment of infected cells. Core particles only were agglutinated by alpha-VP10. All the evidence leads us to conclude that there were major neutralizing antigenic determinants present on VP7.2, a minor component of the outer shell of the virion.