The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes.

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50) values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development.

Experience with uncrossmatched blood refrigerator in emergency department.

BACKGROUND: Uncrossmatched packed red blood cell (PRBC) transfusion is fundamental in resuscitation of hemorrhagic shock. Ready availability of uncrossmatched blood can be achieved by storing uncrossmatched blood in a blood bank refrigerator in the emergency department (ED), but could theoretically lead to inappropriate uncrossmatched use. METHODS: This retrospective study was performed at a level I trauma center from January 2013 to March 2014. Possibly inappropriate transfusion was defined as patients who received at least one unit of blood from the ED refrigerator and no more than two units of PRBC in the first 24 hours. Deaths within the first 24 hours were excluded. Patients who received blood from the ED refrigerator who received ≤2 units total in 24 hours were compared with those who received >2 units. RESULTS: 158 adults received blood from the ED refrigerator. 140 (88.6%) were trauma patients. 37 (23.4%) received massive transfusion (MT). 42 (26.6%) deaths were excluded. 29 patients received ≤2 units and 87 received >2 units in the first 24 hours. The ≤2 units group had a higher systolic blood pressure (116 mm Hg vs. 102 mm Hg, p=0.042), lower base deficit (6.4 mEq/L vs. 9.4 mEq/L, p=0.032), higher hematocrit (34% vs. 30%, p=0.024), lower rate of MT protocol activation (27.6% vs. 58.6%, p=0.005), and lower rates of transfusion of fresh frozen plasma (17.2% vs. 54.0%, p=0.001) and platelets (13.8% vs. 39.1%, p=0.012). Appropriately transfused patients were more likely to have evidence of shock with active, non-compressible hemorrhage. Potentially inappropriate uses were more likely in patients either without evidence of hemorrhage or without signs of shock. DISCUSSION: Storing uncrossmatched blood in the ED is an effective way to get PRBCs transfused quickly in hemorrhaging patients and is associated with a low rate of unnecessary uncrossmatched transfusion. Provider education and good clinical judgment are imperative to prevent unnecessary use. LEVEL OF EVIDENCE: Level III, therapeutic.