Abstract
Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.
Highlights
A new infectious disease has been causing a sudden fall in the egg production of ducks from Jiangsu, Fujian, Anhui, Shandong, Hebei and other provinces in China since April 2010 [1,2,3]
The pathogen was identified by the NS5 gene sequence as Tembusu virus (TMUV), which belongs to the Ntaya virus (NTAV) group in the genus Flavivirus of the family Flaviviridae [1]
Preparation and characterization of monoclonal antibodies (mAbs) The purified recombinant E protein was used as the immunogen
Summary
A new infectious disease has been causing a sudden fall in the egg production of ducks from Jiangsu, Fujian, Anhui, Shandong, Hebei and other provinces in China since April 2010 [1,2,3]. The pathogen was identified by the NS5 gene sequence as TMUV, which belongs to the Ntaya virus (NTAV) group in the genus Flavivirus of the family Flaviviridae [1]. This duck TMUV disease has since caused an enormous loss in many layers and meat-type duck farms in China [2,3,4,5]. The translation product of the genome is a polyprotein that contains three structural proteins (capsid, prM and envelope) and eight nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B and NS5) [5,6,7,8]. The E protein is the major surface protein of flaviviruses and is the dominant antigen that induces immunological responses in infected hosts and elicits virus-neutralizing antibodies [11,12,13]
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