Introduction of Ebola virus into a remote border district of Sierra Leone, 2014: use of field epidemiology and RNA sequencing to describe chains of transmission.

In early October 2014, 7 months after the 2014-2015 Ebola epidemic in West Africa began, a cluster of reported deaths in Koinadugu, a remote district of Sierra Leone, was the first evidence of Ebola virus disease (Ebola) in the district. Prior to this event, geographic isolation was thought to have prevented the introduction of Ebola to this area. We describe our initial investigation of this cluster of deaths and subsequent public health actions after Ebola was confirmed, and present challenges to our investigation and methods of overcoming them. We present a transmission tree and results of whole genome sequencing of selected isolates to identify the source of infection in Koinadugu and demonstrate transmission between its villages. Koinadugu's experience highlights the danger of assuming that remote location and geographic isolation can prevent the spread of Ebola, but also demonstrates how deployment of rapid field response teams can help limit spread once Ebola is detected.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.