Feline coronavirus (FCoV), as one of the important pathogens of feline viral gastroenteritis, has been attracting great attention. A total of 1869 rectal and nasal swabs, feces, and ascites samples were collected from eight regions in Guangxi province during 2021–2024. The multiplex RT-qPCR established in our laboratory was used to test these samples for FCoV, and 17.66% (330/1869) of the samples were positive for FCoV. The S, M, and N genes of 63 FCoV-positive samples were amplified and sequenced, and the genetic and evolutionary characteristics were analyzed. Similarity analysis showed that the nucleotide and amino acid homologies of S, M, and N genes were 81.2–99.6% and 70.2–99.5%, 89.9–100% and 91.6–100%, and 90.1–100% and 91.5–100%, respectively. Phylogenetic analysis revealed that all 63 FCoV strains, based on S gene sequences, belonged to type I FCoV (FCoV-I), and were clustered with Chinese strains and the Netherlands UU strains. Recombinant signals were detected in the S gene of strains GXLZ03-2022, GXLZ08-2022, and CCoV GD/2020/X9. The results suggest that FCoV is still prevalent in the Guangxi province of southern China, and the prevalent FCoV strains show high genetic diversity and novel epidemic characteristics.

Background and Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. Materials and Methods: We designed three pairs of specific primers and probes for the detection of FCoV 5′ untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. Results: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%–0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. Conclusion: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis. Keywords: detection method, feline coronavirus, feline leukemia virus, feline panleukopenia virus, multiplex reverse transcription-quantitative polymerase chain reaction.

Background: Canine coronavirus (CCoV), canine rotavirus (CRV), canine parvovirus (CPV), and canine distemper virus (CDV) cause gastroenteritis in dogs, and co-infections of these pathogens are common in China. In particular, CCoV and CRV are confirmed to have important zoonotic potential and cause public health issues. It is difficult to diagnose these diseases based only on clinical manifestations and pathological damage. Methods: In this study, four pairs of specific primers and probes targeting the CCoV M, CRV VP7, CPV VP2, and CDV N genes were designed. The reaction conditions, including the primer and probe concentrations, annealing temperatures, and reaction cycles, were optimized for the development of a quadruplex RT-qPCR for the detection of CCoV, CRV, CPV, and CDV. The assay was used to test 1028 clinical samples to validate its application. Results: A quadruplex RT-qPCR was successfully established for the differential detection of CCoV, CRV, CPV, and CDV, with good specificity, high sensitivity, and excellent repeatability. The assay could specifically detect CCoV, CRV, CPV, and CDV without cross-reactivity with the other canine viruses tested. It showed high sensitivity with limits of detection (LOD) of 1.1 × 10² copies/reaction for all four plasmid constructs. It showed excellent repeatability, with 0.05–0.90% intra-assay variation and 0.02–0.94% inter-assay variation. The 1028 clinical samples were tested using the quadruplex RT-qPCR and a reported reference RT-qPCR. The positivity rates of CCoV, CRV, CPV, and CDV were 9.53%, 0.97%, 25.68%, and 5.06% using the developed assay, and 9.05%, 0.88%, 25.68%, and 4.86% using the reference assay, with agreements higher than 99.32%. Conclusion: The results indicated that a rapid and accurate quadruplex RT-qPCR was developed for the detection and differentiation of CCoV, CRV, CPV, and CDV.

Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022–2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 10² copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19–1.31% and inter-assay variability of 0.10–0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.

Canine coronavirus (CCoV) is an important gastrointestinal pathogen that causes serious harm to pet dogs worldwide. Here, 1791 clinical samples were collected from pet dogs in different pet hospitals in Guangxi Province, southern China, for the 2021–2024 period and detected for CCoV by a multiplex RT-qPCR. The results showed that 8.43% (151/1791) of samples were positive for CCoV. Sixty-five positive samples were selected to amplify, sequence, and analyze S, M, and N genes. A sequence comparison revealed that the nucleotide and amino acid similarities of the S, M, and N genes were 94.86% and 94.62%, 96.85% and 97.80%, and 96.85% and 97.80%, respectively. Phylogenetic analysis indicated that 65 CCoV strains obtained in this study belonged to the CCoV-II genotype, of which 56 CCoV strains belonged to the CCoV-IIa subtype and 9 CCoV strains belonged to the CCoV-IIb subtype. A potential recombination event analysis of S gene sequences indicated that two CCoV strains, i.e., GXBSHM0328-34 and GXYLAC0318-35, have recombination signals. A Bayesian analysis indicated that the evolutionary rates of the S, M, and N genes were 1.791 × 10⁻³, 6.529 × 10⁻⁴, and 4.775 × 10⁻⁴ substitutions/site/year, respectively. The population size grew slowly before 1980 and then began to shrink slowly; it then shrank rapidly in 2005 and expanded sharply in 2020, leveling off thereafter. These results indicated the CCoV strains prevalent in Guangxi Province, southern China, showed a high level of genetic diversity and maintained continuous variation among clinical epidemic strains.