The Semliki Forest virus vector induces p53-independent apoptosis.

Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.

Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5' nontranslated region has two nucleotide changes, and the 3' nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and non-structural regions of the SFV genome.

Death mechanisms in cultured cells infected by Semliki Forest virus.

We have investigated the induction of cell death in cultured cells by the virulent SFV4 and avirulent A7 strains of Semliki Forest virus (SFV). In BHK cells, death occurred by a typical apoptotic mechanism, as did the death of oligodendrocytes in glial cell cultures. For cerebellar neuron cultures, virus-induced death was due to necrosis. Although the SFV4 and A7 strains did not differ in the mechanism of induction of cell death, the virulent SFV4 strain did multiply to a higher titre in cultured neurons than the avirulent A7 strain. This is consistent with previous animal studies which indicate that the virulence of SFV strains is controlled by rapidity of multiplication in the CNS, leading to a lethal threshold of damage, rather than differential cell tropism or cell death mechanisms. The immune-mediated demyelination induced by avirulent strains may be triggered by apoptosis of oligodendrocytes, the consequences of which are obscured by death for virulent strains.

Analysis of the molecular basis of neuropathogenesis of RNA viruses in experimental animals: relevance for human disease?

RNA viruses with segmented genomes were the first model used for molecular analysis of viral neuropathogenesis, since they could be analysed genetically by reassortment. Four viruses with non-segmented genomes have been used as models of neurovirulence and demyelinating disease: JHM coronavirus, Theiler's virus, Sindbis virus and Semliki Forest virus (SFV). Virus gene expression in the central nervous system of infected animals has been measured by in situ hybridization and immunocytochemistry. Cell tropism has been analysed by neural cell culture. Infectious clones have been constructed for Theiler's virus, Sindbis virus and SFV, and these allow analysis of the sequences involved in the determination of neuropathogenesis, through the construction of chimeric viruses and site-specific mutagenesis. Measles and rubella viruses have been studied in animal systems because of their importance for human disease. The importance of two recently discovered mechanisms of neuropathogenesis, antibody-induced modulation of virus multiplication, and persistence of virus in the absence of multiplication, remains to be assessed.

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.

Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice.

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.

Quality of care in occupational health through nursing diagnosis.

Nursing diagnosis as a part of nursing is not a new activity, but rather one that has been more clearly delineated and structured. Nursing diagnosis is the natural outcome following nursing assessment, and directs the establishment of care goals. Nursing diagnosis promotes quality of care by providing a common framework for the description of client needs, by delineating the areas of nursing responsibility, and by providing criteria to measure the comprehensiveness of the care provided. Consistency in terminology and structure is important for the effectiveness of nursing diagnosis. The use of a widely accepted diagnostic label promotes a clear understanding of the nurse's intended statement of the client's condition. Although consistency and standardization are features of the taxonomy of nursing diagnosis, the concept is flexible enough to be used in a variety of care settings. In occupational health nursing, this flexibility is provided through use of group data, through the diagnostic category of health maintenance, and by the identification of potential nursing diagnoses.

Teratogenicity of the Semliki Forest virus mutant ts22 for the foetal mouse: induction of skeletal and skin defects.

The maximum proportion of skeletal and/or skin defects induced by the Semliki Forest virus (SFV) mutant ts22 in the 17-day-old foetal mouse occurred following infection of the mother at day 10 of pregnancy. The skeletal defects were detected using a combination of Alcian blue staining for cartilage and Alizarin red staining for bone. Using immunogold-silver staining with anti-SFV IgG and in situ hybridization with a cDNA probe to SFV non-structural sequences, we have shown that mesenchymal cells in the dermis and surrounding developing cartilaginous plates were heavily infected in most foetuses at day 17 of pregnancy, following infection of the mother at day 10. Other infected foetal tissues contained less viral antigen and nucleic acid; they included the liver, muscle (including myocardium), lung and kidney. The central nervous system contained only small amounts of viral antigen and nucleic acid. It is proposed that the skeletal and skin defects induced in mouse foetuses by ts22 infection result from the tropism of the virus for mesenchymal cells involved in the development of such tissue.