Abstract
BackgroundSince April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV.Methodology/Principal FindingsA neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ±5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples.Conclusions/SignificanceThe blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV.
Highlights
Since April 2010, an outbreak of an infectious disease has spread widely throughout most of the domestic duck population in China, resulting in retarded growth, high fever, loss of appetite, decline in egg production, and death of the birds [1]
The monoclonal antibodies secreted by hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using 96-well polystyrene flat-bottomed microtiter plates coated with the inactive duck Tembusu virus (DTMUV) purified by ultra-centrifugation through 40% w/v sucrose
Characterization of monoclonal antibody (mAb) A total of 6 antibodies against DTMUV secreted by the monoclonal hybridomas were selected by indirect ELISA using 96well polystyrene flat-bottomed microtiter plates coated with the purified DTMUV
Summary
Since April 2010, an outbreak of an infectious disease has spread widely throughout most of the domestic duck population in China, resulting in retarded growth, high fever, loss of appetite, decline in egg production, and death of the birds [1]. The causative agent of the disease was identified as a newly emerged duck Tembusu virus (DTMUV). Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The disease remains a constant threat to the duck industry; it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world.
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