One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus.

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.

Genetic and Evolutionary Analysis of Porcine Deltacoronavirus in Guangxi Province, Southern China, from 2020 to 2023.

Porcine deltacoronavirus (PDCoV) has shown large-scale global spread since its discovery in Hong Kong in 2012. In this study, a total of 4897 diarrheal fecal samples were collected from the Guangxi province of China from 2020 to 2023 and tested using RT-qPCR. In total, 362 (362/4897, 7.39%) of samples were positive for PDCoV. The S, M, and N gene sequences were obtained from 34 positive samples after amplification and sequencing. These PDCoV gene sequences, together with other PDCoV S gene reference sequences from China and other countries, were analyzed. Phylogenetic analysis revealed that the Chinese PDCoV strains have diverged in recent years. Bayesian analysis revealed that the new China 1.3 lineage began to diverge in 2012. Comparing the amino acids of the China 1.3 lineage with those of other lineages, the China 1.3 lineage showed variations of mutations, deletions, and insertions, and some variations demonstrated the same as or similar to those of the China 1.2 lineage. In addition, recombination analysis revealed interlineage recombination in CHGX-MT505459-2019 and CHGX-MT505449-2017 strains from Guangxi province. In summary, the results provide new information on the prevalence and evolution of PDCoV in Guangxi province in southern China, which will facilitate better comprehension and prevention of PDCoV.

Seroprevalence of Toxoplasma gondii in water buffaloes and cats in Guangxi, China.

Toxoplasma gondii is a pathogen that poses a serious threat to human health and causes significant economic losses to the global livestock industry. The prevalence of toxoplasmosis infection has been reported to be high in humans and animals around the world, but the occurrence of the disease has not yet been reported in water buffaloes in Guangxi Zhuang Autonomous Region, southern China. To understand the overall seroprevalence of T. gondii infection in Guangxi, a total of 1041 water buffalo and 114 cat serum samples were examined using an indirect enzyme-linked immunosorbent assay (I-ELISA). Of the 1041 water buffaloes analyzed, an overall seroprevalence of 52.9% (551/1041) was obtained, with year, season, and city location being significant factors affecting the rate of T. gondii infection in water buffaloes (P < 0.001). The results also revealed a high seroprevalence of 57% (65/114) in cats. Given that buffalo milk and meat products are vital food sources, these findings suggest that toxoplasmosis in water buffaloes may be a public health threat. This study provides the first T. gondii seroprevalence data in Guangxi, which could contribute to the prevention and control of toxoplasmosis in the region.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/µL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15-1.14% and inter-assay CVs of 0.07-0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus.

Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. APPV distributes throughout the world and causes certain economic losses to the swine industry. The specific primers and probe were designed targeting the 5' untranslated region (UTR) of APPV to amplify a 90 bp fragment, and the recombinant standard plasmid was constructed. After optimizing the concentrations of primers and probe, annealing temperature, and reaction cycles, a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) were successfully established. The results showed that the standard curves of the qRT-PCR and the cdRT-PCR had R2 values of 0.999 and 0.9998, respectively. Both methods could specifically detect APPV, and no amplification signal was obtained from other swine viruses. The limit of detection (LOD) of the cdRT-PCR was 0.1 copies/µL, and that of the qRT-PCR was 10 copies/µL. The intra-assay and inter-assay coefficients of variation of repeatability and reproducibility were less than 0.90% for the qRT-PCR and less than 5.27% for the cdRT-PCR. The 60 clinical tissue samples were analyzed using both methods, and the positivity rates of APPV were 23.33% by the qRT-PCR and 25% by the cdRT-PCR, with a coincidence rate of 98.33%. The results indicated that the cdRT-PCR and the qRT-PCR developed here are highly specific, sensitive methods for the rapid and accurate detection of APPV.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/µL for the multiplex qRT-PCR and 3.20 × 10-1 copies/µL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

Neospora caninum seroprevalence in water buffaloes in Guangxi, China.

Neospora caninum is an important obligate intracellular apicomplexan parasite that causes spontaneous abortions in cattle and leads to huge economic losses to the farming industry. Although a high prevalence of N. caninum infection has been reported in Asia, data on the prevalence of water buffaloes in China remain unclear. To understand the seroprevalence of N. caninum infection in water buffaloes and its definitive host dogs in China, a total of 987 water buffalo sera from Guangxi Province were tested using an indirect enzyme-linked immunosorbent assay. We obtained an overall seroprevalence of 50.9% (502/987) for water buffalo samples. And the positive rate was higher in border cities (56.8%, 425/748) than in central cities (32.3%, 77/239). We further tested 240 serum samples from dogs in Guangxi and found an overall prevalence of 57.9% (139/240). The high prevalence of N. caninum infection in both dogs and water buffaloes was first reported in southern China, and these data will surely contribute to the prevention and control of the disease.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

Molecular Characterization of African Swine Fever Virus From 2019-2020 Outbreaks in Guangxi Province, Southern China.

African swine fever virus (ASFV) causes contagious hemorrhagic disease of pigs with high morbidity and mortality. To identify the molecular characteristics of ASFV strains circulating in Guangxi province, southern China, a total of 336 tissue samples collected from 336 domestic pigs that died as a result of severe hemorrhagic disease during 2019-2020 were tested for ASFV. Furthermore, 66 ASFV strains were genetically characterized by sequence analysis of the C-terminal region of B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v) gene, the central variable region (CVR) of B602L gene, the full MGF505-2R gene, and the tandem repeat sequence (TRS) within intergenic region (IGR) between the I73R and I329L (I73R/I329L) genes. Phylogenetic analysis revealed that the ASFV strains from Guangxi province belonged to genotypes I and II based on the B646L (p72) and E183L (p54) genes, and there were eight different tetrameric TRS variants based on the CVR of B602L gene. Phylogenetic analysis of the EP402R (CD2v) gene revealed that these ASFV strains belonged to serogroups 4 and 8. Eight of the 66 strains belonged to genotype I and serogroup 4, and showed deletion of whole MGF505-2R gene. The sequence analysis of the IGR between the I73R/I329L genes showed that IGR II and III variants were co-circulating in Guangxi province. The results indicated that ASFV strains circulating in Guangxi province during 2019-2020 outbreaks showed high genetic diversity, of which genotypes I and II, as well as serogroups 4 and 8, were simultaneously circulating in Guangxi province, and there existed wild-type and naturally gene-deleted strains in the field. This is the first detailed report on the molecular characterization of the ASFV strains circulating in southern China, and serogroup 4 in China.

A Multiplex Crystal Digital PCR for Detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus.

African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) are highly infectious diseases of domestic pigs and wild boars. The co-infections of ASF virus (ASFV), CSF virus (CSFV), and PRRS virus (PRRSV) have been reported in different pig farms. Early differential detection and diagnosis of ASFV, CSFV, and PRRSV in the clinical samples is very important for the effective prevention and control of these diseases. A multiplex crystal digital PCR (dPCR) was developed for differential detection of ASFV, CSFV, and PRRSV in this study, targeting p72, 5' untranslated region (UTR), and ORF7 genes, respectively. The different reaction conditions were optimized, and the specificity, sensitivity, and repeatability of the assay were evaluated. The results showed that the multiplex crystal dPCR was able to accurately and differentially detect ASFV, CSFV, and PRRSV with a limit of detection of 4.69 × 10-1 copies/µl, respectively, and could not detect other porcine viruses, i.e., foot-and-mouth disease virus (FMDV), Senecavirus A (SVA), atypical porcine pestivirus (APPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV). The assay showed excellent repeatability and reproducibility, with coefficients of variation (CV) of the intra- and inter-assay from 0.09 to 1.40%, and from 0.64 to 2.26%, respectively. The 289 clinical samples from different pig herds in Guangxi province, China, were tested by the multiplex crystal dPCR and a reference multiplex real-time quantitative RT-PCR (qRT-PCR) established previously in our laboratory. The positive rates of ASFV, CSFV, and PRRSV were 30.10, 13.49, and 22.49% by the multiplex crystal dPCR, and 24.57, 8.65, and 18.34% by the multiplex qRT-PCR, with coincidence rates of 94.66, 95.16, and 95.84%, respectively. The results indicated that the established multiplex crystal dPCR was a specific, sensitive, and accurate method for the detection and quantification of ASFV, CSFV, and PRRSV. This is the first report on the multiplex dPCR for detecting ASFV, CSFV, and PRRSV.

Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. RESULTS: The one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/µL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. CONCLUSION: A one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

Development of a multiplex qRT-PCR assay for detection of African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. OBJECTIVES: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. METHODS: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. RESULTS: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. CONCLUSIONS: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

Evolution and genetic diversity of atypical porcine pestivirus (APPV) from piglets with congenital tremor in Guangxi Province, Southern China.

Atypical porcine pestivirus (APPV) was identified and associated with congenital tremor (CT) type A-II in new born piglets and has been reported in many countries. In China, the first APPV identification in swine herds was reported in Guangdong province in 2016. To investigate the genetic characteristics of APPV in Guangxi province, 53 tissue samples from neonatal piglets with CT were collected and detected from October 2017 to May 2019. Five APPV strains which were named as GX04/2017, GX01-2018, GX02-2018, GX01-2019 and GX02-2019 were obtained. Sequence analysis revealed that all six APPV strains from Guangxi province, including five strains from this study and one from a previous report, shared 83.3%-97.5% nucleotide identity of complete genome and 91.7%-99.1% amino acid identity of the open reading frame (ORF), and shared 77.7%-97.7% nucleotide identity of complete genome and 90.6%-99.3% amino acid identity of ORF with reference strains. Phylogenetic analysis indicated that all APPV strains could be divided into three clades based on the complete genome, Npro , Erns and E2 gene sequences, respectively; and the APPV strains from Guangxi province distributed in two clades (clades I and II). No sign of recombination was observed from Guangxi strains. Evolution analysis performed on the complete genome of 58 APPV strains showed that America, Europe and Asia strains during 2006-2019 evolved at a mean rate of 1.37 × 10-4 substitutions/site/year, and the most recent common ancestor (tMRCA) of them was estimated as 1,700.5 years ago. The findings of this study indicated that there existed a high degree of genetic diversity of APPV from Guangxi province, Southern China, which provided important information on the epidemiological features and evolutionary relationships of APPV.

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 102 copies/µL for ASFV and 6.34 × 101 copies/µL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018-2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

Emergence of Thailand-like strains of porcine deltacoronavirus in Guangxi Province, China.

Porcine deltacoronavirus (PDCoV) has been detected sporadically in China since its first description in 2012. In our study, 62 faecal and intestinal samples from pigs with diarrhoea were collected in Guangxi Province, China, during 2017 and 2018. Twelve samples (19.4%, 12/62) were positive for PDCoV. Five complete genomes of PDCoV were then determined, and sequence alignment revealed that the five strains had discontinuous deletions at 400-401 aa in non-structural protein 2 (NSP2) and 758-760 aa in non-structural protein 3 (NSP3) compared with the respective proteins in the HKU15-44 strain. Notably, the CHN-GX81-2018 strain contained two insertions in the S gene and 3'-UTR. Multiple sequence alignment and phylogenetic analysis showed that four strains shared 98.2%-98.4% nucleotide identity with CHN-AH-2004 and were classified into a new cluster of China lineage strains, whereas the CHN-GX81-2018 strain shared 98.7% nucleotide identity with Vietnam/Binh21/2015 and belonged to the Vietnam/Laos/Thailand lineage. Recombination analyses revealed that four strains were the result of recombination between CHN-HB-2014 and Vietnam/Binh21/2015 strains. This study demonstrated the co-existence of multiple lineages of PDCoV in China, and our findings will aid the reorganization and evolution of the virus.

First identification of a novel parvovirus distantly related to human bufavirus from diarrheal dogs in China.

Bufaviruses are small, nonenveloped, single-stranded DNA viruses belonging to the subfamily Parvovirinae. Human bufaviruses were first identified in 2012 in fecal samples from children with diarrhea. A new parvovirus of canines that was first detected in various samples from dogs with enteric and respiratory symptoms in Italy between 2014 and 2018 is closely related to the newly described human bufavirus. To explore the prevalence and genetic diversity of CBuV in Chinese dogs, 540 canine parvovirus (CPV)-positive serum and diarrhea samples were collected in Guangxi Province between 2016 and 2018. Among the samples, 6.25% (5/80) of rectal swabs and 2.5% (5/200) of CPV PCR-positive samples were positive for CBuV. However, the virus was not detected in CPV PCR-negative samples or nasal swabs. Two CBuV isolates were identified from CPV-positive fecal and serum samples by complete sequence analysis, with 99.8%-99.9% NS1 and VP2 protein identity to each another. Sequence analysis indicated that the CBuV GXNN01-2018 isolate VP2 protein shares 99.6% identity with the Italian CBuV ITA/2015/297 isolate and 62.3%-65.5% identity with human bufavirus. Phylogenetic analysis showed that CBuV was significantly distinct from other known bufaviruses and was most closely related to CBuV ITA/2015/297. This is the first report of the existence of CBuV in China, and our findings will strengthen the understanding of the epidemiology of bufaviruses in different animals.

Complete Genome Sequence of an Atypical Porcine Pestivirus Strain, GX01-2018, from Guangxi Province, China.

We report the complete genome sequence of an atypical porcine pestivirus (APPV) strain named GX01-2018 that was isolated in Guangxi Province, China, from a suckling piglet showing congenital tremor. The whole genome consisted of 11,565 bp and shared 83.4% to 98.2% nucleotide identities and 91.9% to 99.1% amino acid identities with other APPV strains from different countries.