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Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT–PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay’s limit of detection (LOD) was 2.89 × 102 copies/μL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay’s applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study’s multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Pigs are the main host of Seneca Valley virus (SVV), previously known as Senecavirus A (SVA). Pigs affected by SVV have vesicles in the nose, hooves, and limp and may cause death in some severe cases. Occasionally, SVV has also been detected in mice, houseflies, environmental equipment, and corridors in pig farms. Moreover, it was successfully isolated from mouse tissue samples. In this study, an SVV strain (SVA/GD/China/2018) was isolated from a buffalo with mouth ulcers in the Guangdong province of China using seven mammalian cell lines (including BHK-21, NA, PK-15, ST, Vero, Marc-145, and MDBK). The genome of SVA/GD/China/2018 consists of 7,276 nucleotides. Multiple-sequence alignment showed that SVA/GD/China/2018 shared the highest nucleotide similarity (99.1%) with one wild boar-origin SVV strain (Sichuan HS-01) from the Sichuan province of China. Genetic analysis revealed that SVA/GD/China/2018 clustered with those porcine-origin SVV strains. To the best of our knowledge, this is the first report of SVV infection in buffalo, which might expand the host range of the virus. Surveillance should be expanded, and clinical significance of SVV needs to be further evaluated in cattle.

Bovine rhinitis B virus (BRBV) is an emerging viral species in the genus Aphthovirus, family Picornaviridae. Studies suggested that BRBV was considered a potential etiological agent of bovine respiratory disease complex (BRDC). BRBV has been reported in the United States, Sweden, Canada, Japan, and Mexico. However, little information of BRBV was available in China. In this study, we performed viral metagenomic analysis in a calf with respiratory disease. The results showed high abundance (3.85) of BRBV nucleotide and 248 mapped reads in calf samples. Online BLASTn analysis showed that three contigs of those had the highest nucleotide similarity (95%) with one Swedish BRBV isolate (BRBV_SWE1, GenBank accession no. KY432299). To identify the genome characterization of the Chinese BRBV isolate (designated CHN1), six couples of overlapping RT-PCR primers were designed according to genome sequences of BRBV_SWE1. Through gene cloning and splicing, we obtained the genome information of CHN1, possessing 7,465 nucleotides (46.6% G+C). Although CHN1 had the highest nucleotide similarity (95.1%) with BRBV_SWE1, one 11-nucleotide (ACATTTGTTGT) deletion occurred in the 5′ untranslated region compared to SWE1. Phylogenetic analysis showed that CHN1 clustered together with BRBV_SWE1, and far from other BRBV isolates. This study recorded the first discovery of BRBV infection in China. Further investigation should be made in order to evaluate the infection status and epidemiological significance of BRBV in China.

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/μl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

Porcine circovirus (PCV) is one of the smallest known DNA viruses in mammals. At present, PCVs are divided into three species, PCV1, PCV2, and PCV3. PCV1 and PCV2 were found in the 1970s and the 1990s, respectively, whereas PCV3 was discovered recently in 2016. PCV1 does not cause diseases in pigs. However, PCV3, similar to PCV2, is reported to be associated with several swine diseases, including porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCVs are very common in domestic pigs as well as wild boars. However, PCVs have been occasionally isolated from non-porcine animals, including ruminants (such as cattle, goats, wild chamois, and roe deers), rodents (such as NMRI mice, BALB/c mice, Black C57 mice, ICR mice, Mus musculus, and Rattus rattus), canines (such as dogs, minks, foxes, and raccoon dogs), insects (such as flies, mosquitoes, and ticks), and shellfish. Moreover, PCVs are frequently reported in biological products, including human vaccines, animal vaccines, porcine-derived commercial pepsin products, and many cell lines. PCVs are also abundant in the environment, including water samples and air samples. Interestingly, PCV1 and/or PCV2 antibody or antigen has also been detected in sera, stool samples and respiratory swab samples of human, revealing zoonotic potential of PCVs. Thus, PCVs inhabit many types of reservoirs. In this review, we summarize the reservoirs of PCVs, and this information would be helpful in understanding the natural circulating status and possible cross-species transmission of PCVs.