Background Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. Results In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R 2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. Conclusions A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses. Electronic supplementary material The online version of this article (10.1186/s12951-019-0468-0) contains supplementary material, which is available to authorized users.
The zoonotic transmission of hepatitis E virus (HEV) is mainly mediated by HEV genotypes 3 and 4, with domestic pigs serving as an important reservoir for both genotypes. In China, genotype 4 HEV is the primary prevalent genotype on pig farms. In this study, the prevalence of HEV infection in pig herds of Shaanxi Province was investigated. Serological testing detected anti-HEV antibody-positive pigs in five selected cities, with 13 of 17 farms harbouring at least one positive pig (76.47%). Within positive farms, the proportion of positive pigs ranged from 1.6% to 37.5%. Genetic detection analyses of faecal samples revealed that pigs in four cities and on nine of 17 farms were positive for sequences homologous to a partial ORF2-coding region of HEV (306 bp), as were 6 of 53 bile and 1 of 26 semen samples. Meanwhile, DNA coding for partial HEV ORF1 (1,080 bp) and a longer gene segment coding for partial ORF2 (1,594 bp) were successfully amplified from RNA isolated from pig semen from one HEV-positive pig. Sequence comparisons of partial ORF2 gene sequences showed that HEV isolates from Shaanxi Province shared the highest identity (81.4%-96.1%) with genotype 4 HEV. Phylogenetic tree analysis grouped these isolates into three subgenotypes (4d, 4h and 4i), with subgenotype 4i the predominant subgenotype. In addition, the HEV isolate from pig semen belonged to subgenotype 4i HEV based on phylogenetic trees constructed both using partial ORF1 and ORF2 gene sequences. In conclusion, HEV infection is endemic on pig farms of Shaanxi Province, China, and 4i is the predominant HEV subgenotype. More important, this is the first study demonstrating detection of HEV RNA in pig semen, suggesting that artificial insemination can transmit HEV in pigs.
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