The 50?% lethal dose (LD50) of JEV in BALB/c mice was determined by the method of Reed and Munech [19]

The 50?% lethal dose (LD50) of JEV in BALB/c mice was determined by the method of Reed and Munech [19]. synthesis, construction, expression and purification of recombinant GRFT Five pairs of primers were designed (Table?1) based on the GRFT sequence (GenBank ID: “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ594069.1″,”term_id”:”222090410″,”term_text”:”FJ594069.1″FJ594069.1) to synthesize the GRFT gene by the splicing by overlap extension PCR (SOE-PCR) method [7]. JEV or other flavivirus infections. or in the expression system does not alter its potent anti-HIV activity and its safety profile compared to native GRFT [16, 17]. GRFT is a dimeric lectin with six principal binding sites that can bind to the SARS-CoV spike glycoprotein (S) and prevent virus entry into target cells [27]. Because of the presence of glycans on the JEV virion, we investigated whether GRFT displays antiviral activity against JEV infection. Here, we demonstrate that GRFT inhibits JEV entry into host cells at nanomolar concentrations. Furthermore, we show that the inhibition was due to the binding ability of GRFT to the glycans on JEV virions. Finally, we show that GRFT protects BALB/c mice from challenge with a lethal dose of JEV. In summary, our data establish that GRFT is an antiviral agent that is potentially applicable in the development of therapeutics against JEV or other flavivirus infections. Materials and methods Cell, virus and animal Baby hamster kidney (BHK)-21 cells were cultured in Dulbeccos modified Eagles medium (DMEM) (Invitrogen) containing 10?% heat-inactivated fetal calf serum (FCS), penicillin (100?U/ml) and streptomycin (100 g/ml) at 37?C and 5?% CO2. The Japanese encephalitis virus (JEV) strain NJ2008 (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”GQ918133″,”term_id”:”296802975″,”term_text”:”GQ918133″GQ918133) was propagated and titrated by plaque assay in BHK-21 cells. BALB/c mice (2?weeks old) Lerociclib (G1T38) were purchased from the Animal Center of Nanjing Army Hospital (Nanjing, China) and handled according to the ethical guidelines of Nanjing Agricultural University, China. The 50?% lethal dose (LD50) of JEV in BALB/c mice was determined by the method of Reed and Munech [19]. synthesis, construction, expression and purification of recombinant GRFT Five pairs of primers were designed (Table?1) based Lerociclib (G1T38) on the GRFT sequence (GenBank ID: “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ594069.1″,”term_id”:”222090410″,”term_text”:”FJ594069.1″FJ594069.1) to synthesize the GRFT gene by the splicing by overlap extension PCR (SOE-PCR) method [7]. The full-length GRFT PCR product (375?bp) was digested with and restriction enzymes and ligated into pCold-I vector (Takara Bio Inc.) that had been digested with the same enzymes. The construct was confirmed by restriction enzyme digestion and DNA sequencing analysis. Table?1 List of primers used for the synthesis of GRFT by splicing by overlap extension PCR (SOE-PCR) and restriction enzyme sequences are underlined and were introduced in P5-forward and P5-reverse, respectively The N-terminal 6-His-tagged GRFT was expressed and purified as a dimer as described previously [11] with minor modifications. Briefly, Rosetta 2 cells (Novagen) were transformed with pCold-I-GRFT plasmid. A single colony was used to inoculate 10?ml of Luria-Bertani (LB) medium containing ampicillin (100?g/ml), and the culture was grown at 37?C overnight. The cultures were diluted in 1 liter of LB medium containing ampicillin (100?g/ml) and grown to an expression system. The monoclonal antibody was generated as described previously [8] and produced as ascites in BALB/c mice by injecting them with the hybridoma. Antibody was purified by protein A chromatography and characterized by western blot analysis and ELISA. Cytotoxicity assay BHK-21 cells grown in a 96-well plate at a density of 1 1??104 cells/well were incubated in the presence of GRFT (1.0-500?g/ml) for 72?h. The cytotoxicity was assessed by measuring the activity of lactate dehydrogenase (LDH) in the culture medium using an LDH diagnostic kit (Promega) according to manufacturers instructions and analyzed by regression analysis. Plaque assay BHK-21 cells at a density of 2??105 Lerociclib (G1T38) cells/well in a 24-well plate were incubated at 37 C for 1.5?h in the presence of GRFT/JEV mixture or supernatant of homogenized mouse brain tissue. The cells were washed and incubated in DMEM containing 2?% FCS (maintenance medium). Forty-eight hours later, the cell supernatant was diluted and used to inoculate BHK-21 cells at a density of 1 1. 2??106 cells/well in a 6-well plate for 1.5 hrs. The medium was then removed and replaced with agar overlay medium containing 2?% agar and 4?% FCS. Lerociclib (G1T38) The plates were incubated for 3-4?days at 37 C, and the cells were stained with 0.1?% methylene blue to observe plaque formation. The 50?% inhibitory concentration (IC50) was determined. The antiviral activity of GRFT assayed by quantitative real-time reverse transcription PCR (qRT-PCR) JEV genomic RNA was extracted Lerociclib (G1T38) from infected cells Rabbit polyclonal to MICALL2 using TRIzol (Takara) and used to synthesize cDNA by reverse transcription.