Persistence Of Dengue Virus In Stored Platelets and Red Blood Cells

Dengue virus (DENV), a single stranded RNA member of the Flavivirus family, is responsible for morbidity and mortality, causes ∼50 million infections annually, exists at a relatively high asymptomatic number in blood and is transfusion-transmitted. It is therefore a significant risk to blood systems globally. Universal pathogen inactivation (PI) of platelets and plasma promises to eventually eradicate DENV from blood systems, however, approval of PI in North America has been complicated as some patients may develop respiratory distress and clinical efficacy is currently being further evaluated. Moreover, the technology for PI red cells and whole blood is only in early development with a phase III clinical trial underway. While these studies will ultimately protect the blood system, it is currently unknown whether DENV can maintain its viability or even replicate under the various conditions of stored cellular blood products. Therefore, we investigated the persistence of DENV in platelet (PC) and red blood cell concentrates (RCC) throughout storage under standard blood bank conditions. Purified DENV particles (serotypes 1-4), were produced by sucrose gradient ultracentrifugation of infected Vero cell supernatants. To mimic the approximate DENV concentration known to circulate at an asymptomatic level, purified virus at 10⁵ plaque forming units/ml was spiked into donor-derived, leukoreduced PC or RCC obtained under institutionally approved informed consent and produced by Canadian Blood Services standard operating procedures at the Network Centre for Applied Development. All units were free of bacterial contamination after production as determined by the BacT/Alert microbial detection system. Following inoculation, the PCs were stored for up to 7 days on an industry-approved rocker at 22^oC, while RCC were stored for 42 days at 1-6^oC. Platelet and RBC were sampled antiseptically daily or weekly (respectively) through side couplers added to the product bags. Each DENV serotype was detected using cytolytic plaque assays immediately following inoculation of platelets or RBC demonstrating the presence of infectious virus. However, possibly due to the 22^oC storage conditions, viable DENV underwent logarithmic inactivation in platelet units by Day 1 post inoculation. Nevertheless, between 1-5% of the initial infectious DENV added was detected up to Day 7. This was in contrast to purified DENV in the absence of cells, suggesting virus replication in or protection by the donor platelets. The presence of DENV1-4 was confirmed by quantitative rtPCR, which showed that the DENV RNA genome was replicated with a peak at Day 3 by as much as 7-fold, with continued detection throughout the 7 day platelet storage period. These observations are consistent with platelets having known translational machinery to produce protein from mRNA. Analysis of the RBCs by plaque assays showed viable DENV throughout the 42 day storage period, consistent with stabilization of the virus at 1-6^oC. Interestingly, even though RBCs do not have the ability to translate mRNA, evidence of virus replication was observed, possibly due to the presence of residual white blood cells in the leukoreduced RBC products. The possibility that Dengue viruses or other RNA viruses may persist and replicate in cellular blood products highlights the need to hasten the development and implementation of universal pathogen inactivation.