Rotaviruses (RVs) are important pathogens which induce gastroenteritis in different kinds of animals, including mammals and birds. Rotaviruses are divided into nine species (RVA-RVD and RVF-RVJ), and RVA-RVC and RVH can infect both humans and pigs. It is vital to understand the genetic diversity and evolution of porcine rotavirus (PoRV) for effective prevention and control of this disease. In this study, 5320 intestinal tissue samples and fecal swabs were collected from different pig farms in Guangxi Province, southern China, from 2022 to 2025. These samples were tested for PoRV species A (PoRVA), PoRVB, PoRVC, and PoRVH using the multiplex RT-qPCR. The positive samples of PoRVA were further selected to amplify and analyze the VP4, VP6, and VP7 gene sequences. The phylogenetic trees were constructed based on the PoRVA VP4, VP6, and VP7 gene sequences. Bayesian time-dynamic analysis and recombination analysis were performed for the PoRVA VP4 gene. The results indicated that the PoRVA, PoRVB, PoRVC, and PoRVH positivity rates were 16.92% (900/5320), 0.51% (27/5320), 12.71% (676/5320), and 6.22% (331/5320), respectively. Fifty-two VP4, VP6, and VP7 gene sequences were obtained from the 52 selected PoRVA-positive clinical samples. The nucleotide and amino acid identity analysis of the obtained PoRVA VP4, VP6, and VP7 genes indicated that the genetic diversity of the VP4 gene was higher than that of the VP6 and VP7 genes. The phylogenetic trees based on the VP4, VP6, and VP7 genes revealed that the predominant strains of PoRVA in Guangxi Province were the G9P[13]I5 genotype. Bayesian analysis indicated that the population size of PoRVA kept steady with no significant expansion from its discovery in the 1970s to approximately 2016, then exhibited gradual growth. Sequence analysis of the PoRVA VP4 gene revealed substitutions and recombination in the PoRVA strains, and one strain was derived from recombination of a porcine-originating strain and a human-originating strain. This study provided useful information on the molecular characteristics and genetic diversity of PoRVA and supplied important clues for in-depth research on the cross-species transmission of PoRVA.

Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are highly contagious waterfowl viruses causing significant harm to the poultry industry. This study designed primers and probes targeting the non-structural genomic region of MDPV and GPV, optimizing amplification for both quantitative polymerase chain reaction (qPCR) and duplex crystal digital polymerase chain reaction (dPCR). The resulting duplex crystal dPCR method showed strong specificity, detecting MDPV and GPV without cross-reactivity with other waterfowl viruses. Sensitivity tests revealed that the lower detection limit for qPCR was 11.5 copies/μL, while duplex crystal dPCR achieved a significantly lower detection limit of 0.3 copies/μL. This indicates that dPCR is approximately 38.3-fold more sensitive than qPCR in detecting MDPV and GPV The method demonstrated good repeatability with variation coefficients below 8.0%. In 52 clinical samples from Guangxi, the positive rates for MDPV and GPV were 15.38% and 23.08%, respectively, with one co-infection. This study confirms that duplex crystal dPCR is a specific, sensitive, and efficient technique for detecting MDPV and GPV, especially in the detection of samples containing low concentrations of viruses.

Avian leukosis virus (ALV), chicken infectious anemia virus (CIAV), avian reovirus (ARV), and fowl adenovirus (FAdV) are important viral pathogens that can transmitted horizontally and vertically, and induce immunosuppression to the poultry flocks. They exhibit diverse pathogenic characteristics in clinical settings, and pose continuous threat to the health of poultry flocks. Here, the specific primers and probes were designed for the env gene of ALV, the VP1 gene of CIAV, the M1 gene of ARV, and the ORF1 gene of FAdV. The RNA standards for ALV, and ARV, and the plasmid DNA standards for CIAV, and FAdV were constructed. To establish a quadruplex real-time quantitative PCR (RT-qPCR) for detecting these four viruses, the reaction conditions (primer and probe concentrations, annealing temperature, and reaction cycles) were optimized, and the specificity, sensitivity, and repeatability were analyzed. The results indicated that the developed assay could specifically detect ALV, CIAV, ARV, and FAdV, and had no cross-reaction with other chicken viruses; the limits of detection (LODs) of them were 136.66, 129.59, 133.20, and 139.79 copies/reaction, respectively, demonstrating high specificity and sensitivity. In addition, this assay had excellent repeatability, with coefficients of variation (CVs) of 0.29-0.93% for the intra-assay and of 0.29-0.99% for the inter-assay. The developed assay was validated via testing 1,575 clinical samples from Guangxi province, China. The positivity rates of ALV, CIAV, ARV, and FAdV were 36.89% (581/1,575), 17.65% (278/1,575), 2.16% (34/1,575), and 7.05% (111/1,575), respectively. These 1,575 clinical samples were also tested using the reported reference methods, and the results were compared with those of the established method. The coincidence rate of the developed and the reference assays exceeded 99.31%. In conclusion, a quadruplex RT-qPCR was successfully developed for the efficient and precise detection and differentiation of ALV, CIAV, ARV, and FAdV.

Avian leukosis virus (ALV), chicken infectious anemia virus (CIAV), and fowl adenovirus (FAdV) are significant immunosuppressive agents that cause huge financial losses to the poultry industry. In this study, specific primers and probes for ALV, CIAV, and FAdV were designed, and the RNA standard for ALV and plasmid standards for CIAV and FAdV were constructed. A triplex reverse transcription-crystal digital PCR (RT-cdPCR) assay was developed for the detection of ALV, CIAV, and FAdV after the optimization of key reaction parameters, including primer and probe concentrations, annealing temperature, and reaction cycle. Subsequently, the assay was assessed for its specificity, sensitivity, and repeatability. The results showed that the assay specifically detected ALV, CIAV, and FAdV, without cross-reactivity with other avian pathogens. The limits of detection (LODs) for sensitivity to ALV, CIAV, and FAdV were 3.75, 3.75, and 5.50 copies/reaction, respectively. Repeatability analysis showed the coefficients of variation (CVs) with 0.38-1.77% for intra-assay and 0.31-3.02% for inter-assay. The assay was applied to evaluate 1211 clinical samples from Guangxi Zhuang Autonomous Region in China and yielded positivity rates of 45.58% (552/1211) for ALV, 10.73% (130/1211) for CIAV, and 5.20% (63/1211) for FAdV. Additionally, these 1211 clinical samples were tested using the reference methods, and the results were compared with those of the established method, showing coincidence rates ≥ 98.18%. In conclusion, a triplex RT-cdPCR was successfully established for the simultaneous and accurate detection of ALV, CIAV, and FAdV.

Background Porcine reproductive and respiratory syndrome (PRRS) is a widely prevalent disease of reproductive failure of pregnant pigs and respiratory syndromes in pigs of different ages, especially in piglets. The etiological agents include PRRS virus (PRRSV) genotypes 1 (PRRSV-1) and PRRSV-2, whereas their clinical symptoms are similar and hard to differentiate. It is necessary to establish accurate and reliable methods for differential detection of PRRSV-1 and PRRSV-2.Methods Two pairs of specific primers and probes were designed basing on the PRRSV-1 and PRRSV-2 ORF6 gene. The reaction conditions and procedures of the duplex crystal digital PCR (cdPCR) were optimized. The specificity, sensitivity, and repeatability of the developed assay were evaluated. The application of the developed assay was assessed by testing 2,185 clinical tissue samples.Results The results indicated that the concentration of the templates and their Ct values had good linear relationship with R2 of 0.998. This method could specifically detect PRRSV-1 and PRRSV-2, without cross-reaction with other swine viruses. The limits of detection (LODs) of the assay were 4.507 copies/reaction and 5.607 copies/reaction for PRRSV-1 and PRRSV-2, respectively, which was approximately 30 times more sensitive than that of the duplex real-time quantitative PCR (qPCR). The repeatability test showed that the intra- and inter-assay coefficients of variation (CVs) were 0.74%-0.93% and 0.63%-1.62%, respectively. This method was validated by testing 2,185 clinical samples from Guangxi Province in South China, and the positivity rates of PRRSV-1 and PRRSV-2 were 2.20% (48/2,185) and 23.43% (512/2,185), respectively. The coincidence rates of the developed assay with the qPCR assay recommended by the World Organisation of Animal Health (WOAH) were 99.73% and 99.73%, respectively, while with the duplex qPCR developed in this study were 99.82% and 99.77%, respectively.Conclusions These results indicated that a rapid and accurate duplex cdPCR method with high sensitivity and excellent specificity had been successfully developed for the differential detection of PRRSV-1 and PRRSV-2.

Kobuvirus is a new genus of viruses in the Picornaviridae family causing diarrhea in animals. Porcine kobuvirus (PKV) is an important pathogen with a high rate of infection in pig herds. In this study, a total of 10,990 fecal swabs and tissue samples were collected from different areas of Guangxi province in southern China during 2021–2025 and then tested for PKV using RT-qPCR. The results showed a 19.19% (2109/10,990) PKV positivity rate. Sixty-two PKV-positive samples, which were selected according to sampling regions, sampling seasons, and detection Ct values, were used for PCR amplification and gene sequencing. A sequence comparison showed that the nucleotide and amino acid identities of VP1, 2B, and 3D genes were 78.6–99.5% and 83.5–100%, 77.7–99.8% and 80.9–100%, and 90.9–99.8% and 94.9–99.9%, respectively, indicating that the 3D gene was more conserved than the VP1 and 2B genes. The phylogenetic trees based on these three genes revealed that the PKV VP1 gene sequences from different countries could be classified into two groups (Groups I and II), and the PKV VP1 gene sequences obtained from Guangxi province were distributed in Groups I and II and formed independent clades. The 2B and 3D gene sequences could also be classified into two groups (Groups I and II). Bayesian analysis indicated a state of population growth for PKV strains from the time of their discovery until 2009, at which point it began to decline. Amino acid sequence analysis of the VP1 gene identified mutations and insertions in the obtained PKV strains. Recombinant analysis showed that no recombinant event was found in the VP1, 2B, and 3D genes of the obtained strains. The results indicated the geographically specific inheritance and variation in PKV, provided more information on the prevalence and genetic evolution of PKV in Guangxi province, Southern China, and emphasized the importance of regularly monitoring genetic variation in PKV for better comprehension of PKV.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus (SIV) are important pathogens of significant infectious diseases. They cause similar clinical respiratory symptoms, including fever, cough, runny nose, and respiratory distress, which makes these diseases difficult to distinguish from each other. In this study, three pairs of specific primers and TaqMan probes were designed for the conserved regions of the PRCoV S gene, PRRSV N gene, and SIV M gene, respectively. The annealing temperature, primer and probe concentrations, and reaction cycle were optimized, and a triplex crystal digital PCR (cdPCR) assay was established for the detection of PRCoV, PRRSV, and SIV. According to the test results, the assay was capable of specifically detecting PRCoV, PRRSV, and SIV, and there was no cross-reaction with other control swine viruses. Based on the Poisson distribution analysis, the limits of detection (LODs) for PRCoV, PRRSV, and SIV were 6.00, 5.75 and 6.00 copies/reaction, respectively, and the sensitivity was 26 times higher than those of the corresponding multiplex RT-qPCR. The coefficients of variation (CVs) of the intra-assay and inter-assay ranged from 0.19 to 1.84%. The assay was used to test 1,657 clinical samples, and the positivity rates of PRCoV, PRRSV, and SIV were 1.15, 12.79, and 2.05%, respectively. It showed diagnostic sensitivity and specificity of 100 and 99.82% for PRCoV, 100 and 99.24% for PRRSV, and 100 and 99.69% for SIV, respectively. These results indicated that the triplex cdPCR assay has strong specificity, high sensitivity, and excellent repeatability, which provides a valuable tool for the detection and differentiation of PRCoV, PRRSV, and SIV.

Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5' untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28-1.58% and the inter-assay CV of 0.20-1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses.

Porcine respiratory coronavirus (PRCV) is an important coronavirus to cause respiratory syndrome in pigs. To analyze the genetic and evolutional characteristics of PRCV in Guangxi province, southern China, a total of 6,267 clinical samples were collected from different pig farms, harmless treatment plants and abattoirs in Guangxi province during 2022-2024. Seventeen positive samples of PRCV were selected to amplify and analyze the S, M, and N gene sequences. The results showed that the positivity rate of PRCV was 1.13% (71/6,267) using RT-qPCR. The homology analysis revealed that the nucleotide (amino acid) identity were 98.2-100% (96.6-100%) among the obtained 17 S, M, and N gene sequences, and 91.3-98.1% (88.8-98.0%) among the obtained strains and the reference strains from different countries. The phylogenetic analysis indicated that all PRCV strains could be divided into two groups, tentatively designated group I and group II, and each group was further divided into different clades. The strains obtained from Guangxi province distributed in group I, and formed an independent clade. They were most closely related to PRCV 137 isolate 86/135308 strain from the United Kingdom basing on the S, M, and N gene sequences. The sequence analysis revealed that all PRCV strains from Guangxi province obtained in this study showed 672 nt/224 aa deletion in the 5' region of S gene, and there existed 37 amino acid mutations in different regions in S gene of different PCRV strains. Recombination analysis of S gene sequences did not find any recombination event. The Bayesian analysis indicated that all PRCV strains could be divided into Group I and II, and the obtained Guangxi strains belonged to group I. The population size of circulating PRCV strains kept slightly upward trend since its discovery in 1984 until 2010, showed a steady downward trend until 2020, and then a slight increase. The results suggested that the PRCV strains circulating in Guangxi province originated from Europe, and have evolved independently with genetic diversity. These findings enriched the epidemiological data of PRCV, and provided new information on the prevalence and genetic evolution of PRCV in Guangxi province of China.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV) are currently prevalent worldwide and cause similar neurological symptoms in infected pigs. It is very important to establish a detection method that can rapidly and accurately detect and differentiate these three viruses. Targeting the PHEV N gene, PRV gB gene, and CSFV 5 ' untranslated region (5 ' UTR), three pairs of specific primers and probes were designed, and a triplex crystal digital reverse transcription-PCR (cdRT-PCR) was developed to detect PHEV, PRV, and CSFV. The results indicated that this assay had high sensitivity, and the limitation of detection (LODs) for PHEV, PRV, and CSFV were 4.812, 4.047, and 5.243 copies/reaction, respectively, which was about 50 times higher than that of multiplex real-time quantitative RT-PCR (RT-qPCR). This assay showed good specificity, without cross-reaction with other important swine pathogens, i.e., FMDV, PRRSV, PEDV, SIV, TGEV, PoRV, and PCV2. This assay had high repeatability, with intra-assay coefficients of variation (CVs) of 0.73-1.87%, and inter-assay CVs of 0.57-2.95%. The developed assay was used to test 1,367 clinical tissue samples from Guangxi province in China, and the positive rates of PHEV, PRV, and CSFV were 3.44% (47/1,367), 1.24% (17/1,367), and 1.90% (26/1,367), respectively, with a coincidence rate of 98.98% and a Kappa value of 0.94 to the reference multiplex RT-qPCR. The established triplex cdRT-PCR was a highly rapid, sensitive, and accurate assay to detect and differentiate PHEV, PRV, and CSFV.

Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09–1.24% for intra-assay and 0.08–1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases.

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.

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.

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.

The variant porcine epidemic diarrhea virus (PEDV) has caused considerable economic losses to the global pig industry since 2010. In this study, a total of 5859 diarrhea samples were collected from different pig farms in China’s Guangxi province during January 2020 and March 2024 and tested for PEDV using RT-qPCR. The positivity rate of PEDV was 11.90% (697/5859). Ninety-two PEDV-positive samples were selected based on sampling time, and the sampling region for amplification, sequencing, and analysis of the S1, M, and N genes. Phylogenetic analysis of the S1 gene revealed that all strains from Guangxi province were distributed in three subgroups, i.e., 81.5% (75/92) in the G2a subgroup, 4.3% (4/92) in the G2b subgroup, and 14.1% (13/92) in the G2c subgroup. The sequence analysis revealed that the S1 gene sequences from Guangxi province had higher homology with the variant strains than with the classical strains, showing as high as 99.2% with the variant strain AJ1102 and only 94.3% with the classical strain CV777. Recombination analysis revealed that the GX-BS08-2023 strain (G2c) from Guangxi province originated from inter-lineage recombination between the GX-BS09-2023 (G2a) and CH-JN547228-2011 (G1a) strains. In addition, the S1 gene of the G2a and G2b subgroup strains shared many mutations and insertions. There were common mutations of N143D and P235L in the G2a subgroup. Evolutionary analysis revealed that all Guangxi strains belonged to the G2 genotype. These strains have spread rapidly since the PEDV variant strains that emerged in 2010, weakened until 2021, and then remained stable. In conclusion, the results revealed the latest genetic evolution of circulating PEDV strains in Guangxi province in recent years, providing important information for preventing and controlling PEDV infection. Currently, the G2a subgroup strains are the predominant strains circulating in pig herds in Guangxi province, southern China.

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is the only known porcine neurotropic coronavirus, which is prevalent worldwide at present. It is of great significance to understand the genetic and evolutionary characteristics of PHEV in order to perform effective measures for prevention and control of this disease. In this study, a total of 6,986 tissue samples and nasopharyngeal swabs were collected from different regions of Guangxi province in southern China during 2021-2024, and were tested for PHEV using a quadruplex RT-qPCR. The positivity rate of PHEV was 2.81% (196/6,986), of which tissue samples and nasopharyngeal swabs had 2.05% (87/4,246) and 3.98% (109/2,740) positivity rates, respectively. Fifty PHEV positive samples were selected for PCR amplification and gene sequencing. Sequence analysis revealed that the nucleotide homology and amino acid similarities of S, M, and N genes were 94.3%-99.3% and 92.3%-99.2%, 95.0%-99.7% and 94.7%-100.0%, 94.0%-99.5% and 93.5%-99.3%, respectively, indicating M and N genes were more conservative than S gene. Phylogenetic trees based on these three genes revealed that PHEV strains from different countries could be divided into two groups G1 and G2, and the PHEV strains from Guangxi province obtained in this study distributed in subgroups G1c and G2b. Bayesian analysis revealed that the population size of PHEV has been in a relatively stable state since its discovery until it expanded sharply around 2015, and still on the slow rise thereafter. S gene sequences analysis indicated that PHEV strains existed variation of mutation, and recombination. The results indicated that the prevalent PHEV strains in Guangxi province had complex evolutionary trajectories and high genetic diversity. To the best of our knowledge, this is the first report on the genetic and evolutionary characteristics of PHEV in southern China.

African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV.

African swine fever (ASF) is a highly contagious and lethal viral disease that causes severe hemorrhagic fever in pigs. It keeps spreading around the world, posing a severe socioeconomic risk and endangering biodiversity and domestic food security. ASF first outbroke in China in 2018, and has spread to most provinces nationwide. Genotypes I and II ASF virus (ASFV) as the etiological pathogens have been found in China. In this study, three pairs of specific primers and probes targeting the ASFV B646L gene, F1055L gene, and E183L gene were designed to detect universal, genotype I, and genotype II strains, respectively. A triplex crystal digital PCR (cdPCR) was established on the basis of optimizing various reaction conditions. The assay demonstrated remarkably sensitive with low limits of detection (LODs) of 5.120, 4.218, 4.588 copies/reaction for B646L, F1055L, and E183L gene, respectively; excellent repeatability with 1.24-2.01% intra-assay coefficients of variation (CVs) and 1.32-2.53% inter-assay CVs; good specificity for only detection of genotypes I and II ASFV, without cross-reactivity with PCV2, PRV, SIV, PRRSV, PEDV, FMDV, and CSFV. The triplex cdPCR was used to test 1,275 clinical samples from Guangxi province of China, and the positivity rates were 5.05, 3.22, and 1.02% for genotype I, genotype II, and co-infection of genotypes I and II, respectively. These 1,275 clinical samples were also detected using a reported reference triplex real-time quantitative PCR (qPCR), and the agreements of detection results between these two methods were more than 98.98%. In conclusion, the developed triplex cdPCR could be used as a rapid, sensitive, and accurate method to detect and differentiate genotypes I and II strains of ASFV.

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.