| Muscovy duck (Cairna moschata) belongs to the class of cairinini. Its wild populations are identified as three-level protected species by Washington Convention (CITES). Wild ducks inhabits tropical regions. At present, its wild populations exist in Mexico, Brazil and Paraguay. Muscovy duck parvovirus (MDPV) infection can cause a highly contagious, acute and lethal disease in young ducklings of 1-3 week old. The growth and development of recovered ducklings will be inhibited and loss economic value. The desease is widespread in all major Muscovy duck farming countries, leading to a huge economic loss. All the MDPV strains are similar to each other and share the same serological type. Humoral immune responses play a major role in MDPV immune protection. B-cell epitope is the basic unit of humoral immune. But, up to now, the localization and immunological characteristics of B-cell epitopes on MDPV protein are unknown. In inactivated vaccine production process, most of the non-structural proteins can be removed after virus purification. Inactivated vaccine immuned animals will not have the live virus to proliferate, so there is no non-structural protein of virus expression and non-structural protein antibodies. And naturally infected animals have the live virus in vivo. In the assembly process of the virus there has non-structural protein expression and can stimulate the body to produce the corresponding antibody. So we can use the non structural protein antibody to differentiate infected animals and immune animals. This provides a theoretical basis for the establishment of a way to detection hidden infection and distinction of the immune animals and infected animals.To map the antigenic epitopes of MDPV YL08 strain NS1 protein, a set of 10aa partially overlapping fragments of 30aa spanning the protein were designed. The totals of 31 pairs of primers were synthesized. Each pair of primers were annealed and cloned into expression vector, pET-32a and the map of these fragments confirmed by sequencing. Each fragment was expressed in Escherichia coli Rosetta(DE3) and purified by elution from sodium dodecyl sulphate (SDS) polyacrylamide gels. Anti-His tag MAb was used to identify the purified protein. Then western blot reactivity of these short peptide fused protein to viral infected sera were surveyed. Linear immunodominant B-cell epitopes were primarily found in seven fragments:NS(481-510), NS(501-530), NS(521-550), NS(541-570), NS(561-590), NS(581-610) and NS(601-627). Thus, the non-structural protein linear B-cell epitopes are located on the C-terminal, (481-627aa). Fragments NS(501-530) showed stronger positive than the other fragments in Dot-ELISA assays.An indirect enzyme-linked immunosorbent assay (i-ELISA) based on epitope NS(501-530) was developed. Distributions of true positive vs. true postive+false positive (T/P) ratios were carried out in ELISA for recombinant protein from examination of 100 sera (30 negative sera and 70 vaccinated sera) and 240 infected sera. There was a significant difference between the positive and negative populations (P< 0.05). For the negative sera and vaccinated sera, the mean value was 0.228± 0.024. On this basis, T/P value was set at 0.300 (mean±3 SD). There were none of false negatives in this ELISA assay. The detection value of two negative sera or vaccinated sera was up to the mean value (one in naive sera and the other one in vaccinated sera). There were 100 sera (30 negative sera and 70 vaccinated sera) in total. Therefore, the adoption of this positive-negative threshold value for this assay resulted in specificity of 98.0 %. Further more, the ELISA tests developed were used to detect antibodies against other Muscovy Duck viruses, including duck plague virus (DPV), duck hepatitis virus (DHV) and duck reovirus (DRV). The results showed that there were no cross-reactivity between the epitope NS(501-530) of MDPV and antibodies against other Muscovy Duck viruses. This proved that the epitope NS(501-530) was specific for antibody against MDPV. Seven infected sera (positive sera) were used to analyse the detection limit of serum neutralization (SN) and the ELISA developed in this study. The data indicate that the ELISA is a more sensitive method for antibody detection. The coefficient of variation (CVs) of intro-batch and inter-batch repetition tests were less than 10%, respectively. The reproducibility was reliable. The earliest antibody against epitope NS(501-530) in experimentally infected Muscovy Ducks could be detected at the first week. The reactivity to NS epitope peaked at the third week, and then declined slightly over time untile 12th week. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy Ducks from Muscovy Ducks vaccinated with inactivated virus.The ELISA detection method based on epitope of NS protein was established, which provides technical support for the rapid diagnosis of MDPV, epidemic surveillance, epidemiological investigation and immune antibody titer assay. In particular, it is a technical reserve for the identification of natural infection and vaccination in the future by using inactivated vaccine or recombinant protein subunit vaccine. |
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