The well-being and subsistence of giant pandas, an endangered species with a limited distribution, are currently threatened by a number of viruses, including canine parvovirus (CPV-2), canine distemper virus (CDV), and giant panda rotavirus (GPRV). To allow for timely intervention upon viral infection, it is necessary to execute rapid and accurate diagnosis of potential mixed viral infections. In the present study, we developed and validated a multiplex PCR (mPCR) approach for the detection of CPV-2, CDV, and GPRV infections. The results indicate that the method could selectively amplify the three viruses with high sensitivity and specificity, which are necessary attributes in clinical settings. Utilizing the established method, (sub)clinical giant panda samples were examined, and CPV-2, CDV, and GPRV were found in 19.72% (43 out of 218), 7.34% (16 out of 218), and 6.42% (14 out of 218) of the samples, respectively. Noticeably, mixed infections of two or three viruses were common, and this was generally observed in CDV- or GPRV-positive samples. Meanwhile, mPCR results were further validated with sequencing and the phylogenetic analysis of full-length sequences of viral genes. Taken together, our study provides an approachable assay which enables the quick detection of the three viruses mentioned above, which will benefit clinical diagnosis and laboratory epidemiological-based investigations of the giant panda population.

Porcine epidemic diarrhea virus (PEDV) is a major pathogen that causes serious economic losses to the swine industry. To aid PEDV clinical diagnosis and vaccine development, sensitive and precise serological methods are demanded for rapid detection of (neutralizing) antibodies. Aiming for the development of a novel virus-free hemagglutination inhibition (HI) assay, the N-terminal region of the PEDV S1 subunit, encompassing the sialic acid-binding motif, was first expressed as an Fc-fusion protein with a C-terminal Spy Tag (S1^0A-Spy). The S1^0A-Spy protein was then presented on SpyCatcher-mi3 nanoparticles, forming virus-like particles designated S1^0A-NPs. Electron microscopy and dynamic light scattering analysis confirmed its topology, and the hemagglutination assay showed that S1^0A-NPs can efficiently agglutinate red blood cells. The HI assay based on S1^0A-NPs was then validated with PEDV-positive and -negative samples. The results showed that the HI assay had high specificity for the detection of PEDV antibodies. Next, a total of 253 clinical serum samples were subjected to the HI testing along with virus neutralization (VN) assay. The area under the receiver operating characteristic curve with VN was 0.959, and the kappa value was 0.759. Statistical analysis of the results indicated that the HI titers of the samples tested exhibited high consistency with the VN titers. Taken together, a novel virus-free HI assay based on the multivalent display of a chimeric PEDV spike protein upon self-assembling nanoparticles was established, providing a new approach for PEDV serological diagnosis.

Porcine epidemic diarrhea virus (PEDV) is a major viral pathogen responsible for severe diarrhea and substantial economic losses in the swine industry. Characterized by its high infectivity, pathogenicity and mortality rates in neonatal piglets, PEDV has caused devastating epidemics globally over the past decade. Therefore, development of rapid, sensitive, and specific serological tests is therefore critical for effective disease surveillance and immunity evaluation. In this study, we developed and validated an indirect ELISA using the immunodominant S1^0AB region of the PEDV spike protein. The assay exhibited acceptable sensitivity, strong reproducibility (CV < 10%) and excellent specificity with no cross-reactivity against other common swine viruses. The test results also exhibited high concordance with virus neutralization tests (AUC = 0.913; kappa = 0.797). In addition, the ELISA was utilized to analyze 1629 field serum samples collected from southwestern China. The results indicated significant regional differences occur regarding PEDV seroprevalence and proved its potential for large-scale seroepidemiological studies. In conclusion, the S1^0AB-based indirect ELISA developed in the present study provides a reliable, specific, and practical tool for PEDV serodiagnosis and control.