The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA14-14-2 and its parental virulent strain SA14, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5' and 3' untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5'- and 3'-terminal untranslated regions contain one- and two- point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.

Recombinant, human, erythropoietin (rhEPO) is a glycoprotein hormone which is prescribed throughout the world to treat anaemia caused by chronic kidney disease or chemotherapy. rhEPO is at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Due to its complex glycosylation profile, which has a crucial influence upon biological activity, therapeutic rhEPO preparations must be closely monitored to ensure consistency, safety and efficacy. Here, we have compared twelve rhEPO preparations from eleven manufacturers in China and one in Japan, measuring in vivo biological activity and exploring its relationship with glycosylation through sialic acid content determination, isoform distribution via capillary electrophoresis (CE), O-glycan profiling, and N-glycan mapping using a novel anion-exchange/hydrophilic interaction chromatography-mass spectrometry (AEX/HILIC-MS) approach. We observed differences between glycosylation profiles, including the varying occurrence of sialic acid O-acetylation, extension of N-glycan antennae with N-acetyllactosamine units, and the distribution of sialic acids across multi-antennary structures. The presence of unusually high levels of suspected penta- and hexa-anionic N-glycans in several samples is consistent with elevated rhEPO isoform acidity, which is reflected by slightly elevated in vivo bioactivities. This aside, the observed differences in glycosylation profile do not appear to have a significant influence upon biological activity in mice. Nonetheless, with the continued emergence of biosimilars, the study highlights the importance of monitoring glycosylation profiles in biological medicines, in order to detect and account for divergence between products, as well as the presence of unusual or unexpected glycans.

ObjectiveA recombinant antitumor/antiviral protein (Novaferon, Nova) is a new type of interferon, which is produced by artificial design technology combining DNA-shuffling and High Throughput Screening (HTS).MethodsThe in vitro biological activities, such as anti-tumor activity and antiviral activity of Nova and recombinant human interferon alpha-2b (rhIFN-α2b) was performed; in vivo anti-tumor activity in nude mice was also tested. Flow cytometry, histo-pathological analysis including HE staining and immunohistochemistry, and surface plasmon resonance assay were performed to investigate the underlying mechanisms analysis.ResultsNova exhibited stronger anti-cancer effects compared to rhIFN-α2b in vitro and in vivo. The antitumor mechanisms of Nova may be related to S phase arrest, pro-apoptosis, and inhibition of tumor angiogenesis. Moreover, Nova exhibited a higher binding affinity for IFN receptor 2 (IFNR2) than rhIFN-α2b, which is one of the possible reasons accounting for its stronger actions against tumor cells compared with rhIFN-α2b.ConclusionNova has strong antitumor activity and could be a potentially effective therapeutic drug for cancer.

Aim: M 2 ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M 2 ES in rats. Methods: 125 I-radiolabeled M 2 ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M 2 ES were investigated using the trichloroacetic acid (TCA) precipitation method. Results: The serum M 2 ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a noncompartment model. The pharmacokinetic parameters were evaluated as follows: C max =28.3 μg·equ/mL, t 1/2 =71.5 h, AUC (0-∞) =174.6 μg·equ·h/mL, Cl=17.2 mL·h -1 ·kg -1 , MRT=57.6 h, and V ss =989.8 mL/kg for the total radioactivity; C max =30.3 μg·equ/mL, t 1/2 =60.1 h, AUC (0-∞) =146.2 μg·equ·h/mL, Cl=20.6 mL·h -1 ·kg -1 , MRT=47.4 h, and V ss =974.6 mL/kg for the TCA precipitate radioactivity. M 2 ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose. Conclusion: PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M 2 ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M 2 ES.