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Background African swine fever virus (ASFV) is the etiologic agent of African swine fever (ASF), a disease of highly contagious and significant threat to pork production. At present, the sensitive detection methods are the keys to the disease control. Objective Full-length p72 is produced by a eukaryotic system, and its monoclonal antibody (mAb) 34C10 is subsequently recovered. A blocking ELISA kit for detection of ASFV antibody is developed based on p72 trimers and 34C10. Methods Full-length p72 is expressed and is used as an immunogen to prepare a panel of monoclonal antibodies. The mAb 34C10 is verified by immunofluorescence and tested by ELISAs with positive serums. The constant affinity of 34C10 is then confirmed. A blocking ELISA kit is further developed and is compared with two commercial kits. Results The mAb 34C10 is specifically bound to p72 protein, and it exhibits a blocking effect to positive serum. The immunofluorescence assay experiment shows that 34C10 could bind to p72 expressed by baculoviruses, and the binding affinity of 34C10 is found to be as high as 1.85 × 1011 L/mol. The blocking ELISA kit shows high coincidence with a commercial ELISA kit. The sensitivity between these two kits is 97.6% (95%, CI: 90.65–99.58), and the specificity between them is 100% (95%, CI: 98.34–100). Conclusions The blocking ELISA developed in this study may have great potential for diagnosis of ASF. The structure of the antigen p72 is found to be a key factor for the performance of the kit. Highlights For the first time, the eukaryotic expressed full-length p72 protein is used to recover the monoclonal antibody, and it is coated as antigen during the development of the blocking ELISA kit. This study sheds new light on the development of the blocking ELISA kits, especially for the development of a diagnostic kit for the contagious virus with bio-safety problems.

Additional file 1: Figure S1. Sequence aligment of rAKAV and wtAKAV. Figure S2. The sequence of rAKAV S segment. Figure S3. The plague of rAKAV and wtAKAV. Figure S4. The PCR result of rAKAV and wtAKAV.

Metabolites of serum and milk from genetically modified (GM) cows and contrast check (CK) cows were comparatively investigated. Serum and milk were collected from genetically modified (GM) cows and contrast check (CK) cows, and then, they were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Although the level of some blood biochemical indexes for GM cows was shifted up or down, they were generally in normal physiological condition. Serum samples from lactoferrin GM cows exhibited reduced levels of amino acids and elevated levels of indoleacetate, α-keto acids, long-chain fatty acids, etc. GM milk possessed elevated levels of pentose and amino sugar metabolites, including arabitol, xylulose, glucuronate, and N-acetylgalactosamine. Interestingly, some essential nutrients, such as certain unsaturated fatty acids (e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA)), and some necessary rare sugars were significantly upregulated. Compared to the CK group, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted based on the increased or decreased metabolites identified in the serum and milk samples of the GM group. The results showed that the GM cows were in healthy condition and their milk has improved benefits for customers. The milk from genetically modified cows was found to be a promising milk source for producing recombinant human lactoferrin (rhLF) for human beings.

Carp edema virus (CEV) is a poxvirus associated with outbreaks of clinical disease that may lead to high mortality in both koi and common carp populations, potentially causing severe economic losses for carp aquaculture and the global koi trade. Therefore, the establishment of a sensitive and specific assay for detection of CEV is an essential requirement for the prevention of further spread of this emerging viral disease. In this study, a novel droplet-digital PCR (ddPCR) assay, targeting the p4a gene of CEV was developed. The assay exhibits good linearity, repeatability and reproducibility. Linearity was maintained at extremely low concentrations of CEV nucleic acid templates. The detection limit of ddPCR was 2.2 ± 0.26 copies of CEV DNA per reaction (n = 8), which represents a greater sensitivity than the quantitative PCR (qPCR) assay. Specificity analysis showed that the ddPCR assay for CEV had no cross-reactivity with other important aquatic pathogens. In clinical diagnosis of 151 koi and common-carp samples, 58 and 45 tested positively by ddPCR and qPCR assays, respectively. The overall agreement between the two assays was 91.39% (138/151). Linear regression analysis demonstrated a significant correlation between ddPCR and qPCR results with an R2 value of 0.9627. Our findings indicate that the ddPCR assay could provide a robust diagnostic tool for the sensitive detection of CEV, even in samples with a low viral load.

Molecular characterization of transgenic animals is crucial for the phenotype analysis, unintended effects prediction, commercial and economic requirements. In this study, a comprehensive molecular characterization of a transgenic pig, which expressed hCD46 gene, was verified using next-generation sequencing (NGS). One complete and one incomplete sequence of pBSCD46-neo and multiple backbone fragments were inserted into the host genome. The whole insertion sequence was 28,243bp, and 12bp sequence on the genome was deleted during the transgene. The cost of NGS is higher than other molecular methods, in order to reduce the cost and to make the sequencing strategy more economically effective, we carried out a computer simulation to identify the smallest amount of data that could meet the requirements of the molecular characterization analysis. 15× depth of coverage was shown to be sufficient with good accuracy, while 3× might be effective, 20× was recommended. The obtained molecular characterization information would provide a stronger foundation for the safety evaluation of the GM animals. Meanwhile, the gene integrity of the porcine pathogen PERV was analyzed using the same procedure. No complete sequence of the PERV subtype was observed in the porcine genome, which was the same as the previous research. In consequence, NGS combined bioinformatics analysis is a reliable and accurate approach for the molecular characterization of GMOs, even for the safety evaluation.

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0 TCID50/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.

To separate and concentrate koi herpesvirus (KHV) from large-volume samples, a separation method based on immunomagnetic beads (IMBs) coated with polyclonal antibody directed against KHV was developed. After treatment with IMBs, viral DNA was extracted from samples and used as a template for quantitative PCR (qPCR). The results showed that the concentration of the template DNA extracted from the virus that had been separated using IMBs was 9.65-fold higher than that from virus not treated with IMBs. The detection limit of the IMBs/qPCR method was found to be at least 10 times lower than that of qPCR alone.

Three monoclonal antibodies (MAbs) (27E4, 17H2, 8B6) against channel catfish virus (CCV) were developed by immunizing Balb/C mice. Using indirect ELISA, these MAbs reacted only with CCV and not with three other fish viruses or nine fish cell lines. During western blotting analysis, MAb 27E4 recognized 170 kDa and 47 kDa proteins, while MAb 17H2 and MAb 8B6 recognized 47 kDa and 56 kDa proteins, respectively. Furthermore, a sandwich ELISA was developed for detection of CCV. The detection limit of the test was 105 TCID50/mL.

Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 105 TCID50 of GCRV per mL.