Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells.

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus-host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Detection of Neutralizing Antibodies to Tembusu Virus: Implications for Infection and Immunity.

Neutralizing antibodies are the key mediators of protective immune response to flaviviruses after both infection and vaccination. Plaque reduction neutralization test (PRNT) is considered the "gold standard" for measurement of the immunity. To date, little is known regarding neutralizing antibody response to Tembusu virus (TMUV), a novel flavivirus emerging in ducks in 2010. Here, we developed a PRNT for detection of TMUV neutralizing antibodies. Following optimization and validation, the PRNT was applied to test serum samples from different flocks of ducks. Using sera prepared in experimental conditions, the levels of 50% end point titer (neutralizing dose, ND50) generated from positive sera (5,012-79,433) were significantly higher than those from mock-infected sera (10 to 126), indicating that the test can be used in the detection of TMUV-specific neutralizing antibodies. Dose-dependent efficacy test of a cell-derived 180th passage of a plaque-purified virus of the PS TMUV isolate (PS180) in combined with immunization-challenge experiments revealed that ND50 titer of ~1,258 is the minimum capable of providing adequate protection against challenge with virulent TMUV. In the investigation of serum samples collected from three flocks infected by TMUV and four flocks vaccinated with a licensed attenuated vaccine (the 120th passage virus), ND50 titers peaked at 1 week after both disease onset (7,943-125,893) and vaccination (3,612-79,432), and high levels of ND50 titer were detected in sera collected at 15 weeks after disease onset (5,012-63,095) and 17 weeks after vaccination (3,981-25,119). Together these findings demonstrated that spontaneous and experimental infections by TMUV and vaccination with the licensed TMUV attenuated vaccine elicit high, long-lasting neutralizing antibodies. The highest ND50 titer of neutralizing antibodies elicited by PS180 was determined to be 3,162, suggesting that attenuation of TMUV by more passages has a dramatic impact on the neutralizing antibody response of the virus.