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Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. To develop an effective diagnostic assay for the detection of AKAV, we generated and characterized one AKAV N-reactive monoclonal antibody (mAb) which was designated as 2D3. Western blot and indirect immunofluorescence assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168-182 of AKAV N protein. Then, mAb 2D3 was applied to establish a double antibody sandwich ELISA (DAS-ELISA), which was able to detect both the purified AKAV N protein (with detection limit of 6.25 ng/ml ) and AKAV-infected cell culture supernatant (with detection limit of 250 TCID /ml). Taken together, the successfully prepared mAb and the preliminarily established DAS-ELISA provide valuable materials and a promising method for the serological diagnosis of AKAV.

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

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.

On August 14th, 2018, a Beijing resident living in Xicheng District found a female H. longicornis tick attached to the skin at the front of his upper shin. On examination, the patient was afebrile and appeared well. The species of the tick was identified through morphological characteristics and phylogenetic analysis based on cytochrome C oxidase subunit I. This H. longicornis tick was screened for tick-borne pathogens such as viruses, bacteria and parasites. RNA pathogens were screened by PCR and sequencing, while DNA pathogens were screened by metagenomic analyses. It was found that the tick was positive for the DNA sequences of zoonotic and animal pathogens such as A. phagocytophilum, Ehrlichia minasensis and C. burnetii. Considering the good health condition of the patient, we hypothesized that the pathogens originated from the tick specimen itself rather than host blood meal. For the first time, our study reveals the possible risk of transmission of tick-borne pathogens to human beings through tick bit in downtown Beijing. Further research is needed to screen for tick-borne pathogens among unfed ticks collected from central Beijing.

Advances in molecular testing and microfluidic technologies have opened new avenues for rapid detection of animal viruses. We used a centrifugal microfluidic disk (CMFD) to detect 6 important swine viruses, including foot-and-mouth disease virus, classical swine fever virus, porcine reproductive and respiratory swine virus–North American genotype, porcine circovirus 2, pseudorabies virus, and porcine parvovirus. Through integrating the loop-mediated isothermal amplification (LAMP) method and microfluidic chip technology, the CMFD could be successfully performed at 62℃ in 60 min. The detection limit of the CMFD was 3.2 × 102 copies per reaction, close to the sensitivity of tube-type LAMP turbidity methods (1 × 102 copies per reaction). In addition, the CMFD was highly specific in detecting the targeted viruses with no cross-reaction with other viruses, including porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine rotavirus. The coincidence rate of CMFD and conventional PCR was ~94%; the CMFD was more sensitive than conventional PCR for detecting mixed viral infections. The positive detection rate of 6 viruses in clinical samples by CMFD was 44.0% (102 of 232), whereas PCR was 40.1% (93 of 232). Thirty-six clinical samples were determined to be coinfected with 2 or more viruses. CMFD can be used for rapid, sensitive, and accurate detection of 6 swine viruses, offering a reliable assay for monitoring these pathogens, especially for detecting viruses in widespread mixed-infection clinical samples.

Porcine circovirus 3 (PCV3), a newly emerged circovirus, is associated with porcine dermatitis and nephropathy syndrome, reproductive failure and multi-systemic inflammation disease, and is widely distributed in pig populations worldwide. Therefore, developing specific diagnostic assays will be important for controlling this emerging pathogen. In this study, we developed a novel droplet digital PCR (ddPCR) assay targeting the PCV3 cap gene to improve the sensitivity of PCV3 detection. The established assay is highly specific to PCV3, and does not cross react with other important swine pathogens. The assay's detection limit was 1.68 ± 0.29 copies of PCV3 DNA per reaction (n = 8), an approximately 10-fold greater sensitivity than that of our previously developed quantitative real-time PCR (qPCR) assay for the same virus. The ddPCR assay results were highly reproducible, with intra- and inter-assay coefficient of variation values of9.0%. Of the 239 archived pig tissue and serum samples, 42 tested positive for PCV3 by the ddPCR assay. Among the 42 positive samples, 31 tested positive by the qPCR assay. Notably, PCV3 was detected in the serum samples collected from commercially imported healthy boars from the US, France and the UK during 2011-2017. The overall agreement between the two assays was 95.39% (228/239). Furthermore, the linear regression analysis showed that the ddPCR and the qPCR results were significantly correlated with an R

Porcine circovirus type 3 (PCV3) is a novel pathogen first identified in the United States in 2016. As there is a high possibility that no clinical signs of infection are observed in the host, an accurate and sensitive method is needed for quarantine on numerous live pigs especially for international pig trade. In this study, a TaqMan‐based real‐time PCR assay specifically for PCV3 was established without cross‐reactions with other non‐targeted pig viruses. The sensitivity of the current approach is about 1.5 × 101 copies μL−1 plasmid DNA while the sensitivity of the conventional PCR is about 1.5 × 102 copies μL−1 plasmid DNA. Further, this assay was applied in the retrospective quarantine on serum samples of 601 commercial live boars imported to China from the United States, France and the United Kingdom from 2011 to 2017. The results revealed that PCV3 could be detected positive in the commercial boars imported from the United States and the above‐mentioned western European countries and phylogenetic study also revealed that viral isolates were grouped with some isolates from Korea and the United States. Our study suggested that PCV3 may be prevalent globally since 2011.

In pigs, Senecavirus A (SVA) causes a vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. Sensitive and specific detection of SVA is critical for controlling this emerging disease. In this study, a novel reverse transcription droplet digital PCR (RT-ddPCR) assay, targeting the conserved viral polymerase 3D gene, was established for the detection of SVA. This assay exhibited good linearity, repeatability and reproducibility, and maintained linearity at extremely low concentrations of SVA nucleic acid templates. The detection limit of RT-ddPCR was 1.53 ± 0.22 copies of SVA RNA per reaction (n = 8), and the assay showed approximately 10-fold greater sensitivity than a reverse transcription real-time PCR (RT-rPCR) assay. Moreover, specificity analysis showed that the RT-ddPCR for SVA had no cross-reactivity with other important swine pathogens. In clinical diagnosis of 134 pig serum and tissue samples, 26 and 21 samples were identified as positive by RT-ddPCR and RT-rPCR, respectively. The overall agreement between the two assays was 96.27% (129/134). Further linear regression analysis showed a significant correlation between the RT-ddPCR and RT-rPCR assays with an R2 value of 0.9761. Our results indicate that the RT-ddPCR assay is a robust diagnostic tool for the sensitive detection of SVA, even in samples with a low viral load.

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.

Background Channel catfish virus disease (CCVD) has resulted in great economic losses and has restricted the development of fisheries. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of CCVD. Objective A colloidal gold immunochromatographic strip has been developed for the detection of CCVD. Methods In this study, a colloidal gold immunochromatographic strip for channel catfish virus (CCV) detection was developed using the monoclonal antibody 8B6 conjugated with colloidal gold as the detector antibody. A rabbit anti-CCV antibody was used as the capture complex at the test line, and a goat anti-mouse IgG antibody was used as the capture antibody at the control line. The strip was characterized in its specificity, sensitivity, and stability. In addition, an infection experiment was performed to test the applicability of the test strip. Result The strip was able to detect concentrations of the virus (104 tissue culture infective dose (TCID50)/mL) and showed analytical specificity when tested against other viral pathogens. The strips were still usable after 30 days of storage at 60°C. It was possible to detect CCV experimentally in infected fish within 10–15 min of using the strip. Conclusions The strip can be used as a rapid and convenient tool for on-site diagnosis to control outbreaks and the spread of CCVD. Highlights The immunochromatographic strip was the first to be developed and applied for the detection of CCVD.

Background Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. Methods Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 μl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg’s disease virus, and 3000 clinical samples were tested for practicability. Results The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. Conclusions This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies. Electronic supplementary material The online version of this article (10.1186/s13071-018-2996-0) contains supplementary material, which is available to authorized users.

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.

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.

Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni–NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.

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.