Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in vero cells or human liver cells and attenuated in mice.

A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2ADelta30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2ADelta30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Delta30) in the 3' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2ADelta30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5' and 3' UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Delta30 deletion, thereby creating rDEN4Delta30-4995, a recombinant virus which is ts and more attenuated than rDEN4Delta30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.

Electromyographic response of the elbow flexors to a changing, dislocating force.

The electromyographic (EMG) outputs of the biceps brachii, brachialis, and brachioradialis muscles of 14 subjects were monitored during the application of an externally generated, dislocating force across the elbow joint. Subjects were seated in an apparatus designed to maintain the right upper extremity at 90 degrees shoulder flexion and 180 degrees elbow extension. The dislocating force was increased from 0-22 Newtons (N) at an average rate of 2N/sec to determine if a minimum load were necessary before muscles would be activated. Although no muscle was found to be significantly more active than any other over the entire load range, post hoc analysis showed that both the brachialis and brachioradialis were significantly more active at loads above 18N than below. The biceps brachii showed no significant increase in activity. The practice of strengthening muscles as a means of protecting joint integrity can be questioned by these results since the muscles under study did little to resist elbow joint dislocation at loads up to 18N.