Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae.

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.

Buffalo-Origin Seneca Valley Virus in China: First Report, Isolation, Genome Characterization, and Evolution Analysis.

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.

Genome Characterization and Phylogenetic Analysis of the First Bovine Rhinitis B Virus Isolate in China.

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.

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

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.

Reservoirs of Porcine Circoviruses: A Mini Review.

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.

A Novel Enzootic Nasal Tumor Virus Circulating in Goats from Southern China.

Enzootic nasal tumor virus (ENTV) has two types, ENTV-1 in sheep and ENTV-2 in goats, respectively. In China, the incidence of ENTV-2 related diseases has increased year by year. In this study, we reported an outbreak of ENTV-2 in a commercial goat farm in Qingyuan city, Guangdong province, southern China. A full-length genome of ENTV-2 (designated GDQY2017), with 7479 base pairs, was sequenced. Although GDQY2017 shared the highest nucleotide identity with a Chinese ENTV-2 isolate (ENTV-2CHN4, GenBank accession number KU258873), it possesses distinct genome characteristics undescribed, including a non-continuous 21-nucleotide insertion in the gag gene and a non-continuous 12-nucleotide deletion in the env gene. Notably, most of these indel nucleotide sequences were originated from a Chinese jaagsiekte sheep retrovirus (JSRV) isolate (GenBank accession number DQ838494). In the gag and env genes, GDQY2017 was phylogenetically related to those Chinese ENTV-2 isolates and a Chinese JSRV isolate (DQ838494). For GDQY2017-like viruses, more surveillance work should be made to explain their pathogenicity in goat herds. To our knowledge, this study represents the first to demonstrate the circulating pattern of ENTV-2 in Guangdong province, China, which will help to better understand the epidemiology and genetic diversity of ENTV-2.

Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Preliminary serological evidence for Schmallenberg virus infection in China.

Schmallenberg virus (SBV) is an emerging and rampant arbovirus in Europe, and even Africa and West Asia. Investigating whether SBV existed in new regions or countries, it was very helpful for the early warning and control of SBV. In this study, we collected 317 serum samples (n = 242 for dairy cattle, n = 13 for yellow cattle, n = 21 for buffalo, and n = 41 for goats) from Guangdong province of southern China, which is located in a subtropical region and is an important distribution area for arboviral diseases. A commercial competitive enzyme-linked immunosorbent assay (cELISA) kit and a previously established real-time PCR were used to detect SBV antibody and RNA in those serum samples. Via testing, serological evidence of SBV was confirmed, with total positive rates (57.4, 15.4, 19, and 9.8%) in dairy cattle, yellow cattle, buffalo, and goats, respectively, while no positive signal for SBV RNA was found. To summarize, this study for the first time provided preliminary serological evidence of SBV infection in China, East Asia. Further investigations on molecular evidence, origin, and pathogenesis of SBV in ruminants needed to be studied in China.

Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations.

BACKGROUND: Porcine circovirus type 3 (PCV3), as an emerging circovirus species, was reported to be widely circulating in the United States, China, South Korea and Poland. Previous studies revealed that PCV3 was mainly concentrated in sick animals with respiratory disease, skin disease, reproductive disorders and so on. However, the circulating status of PCV3 in pigs with other clinical presentations (especilly asymptomatic or diarrhea) was not well established. FINDINGS: In this study, to conduct a comparative epidemiological survey of PCV3, 80 weaned pig serum samples with severe respiratory disease (SRD), 175 weaned pig serum samples with mild respiratory disease (MRD), 216 asymptomatic weaned pig serum samples, 35 diarrheal weaned pig samples and 35 non-diarrheal weaned pig samples were collected from eight provinces of China. Via qPCR testing, PCV3 was circulating in all sampling provinces, with total positive rates varying from 1.04% to 100%. Interestingly, the PCV3-positive rate was significantly higher in weaned pigs with SRD (63.75%, 51/80) than in those weaned pigs with MRD (13.14%, 23/175) and asymptomatic pigs (1.85%, 4/216) (P < 0.01). Similarly, the PCV3-positive rate was significantly higher in diarrheal weaned pigs (17.14%, 6/35) than in non-diarrheal weaned pigs (2.86%, 1/35) (P < 0.05). Moreover, the lower Ct values of qPCR were frequently found in those weaned pigs or fattening pigs with respiratory disease and diarrhea rather than that in asymptomatic pigs. Sequence analysis showed that low genetic diversity existed among those PCV3 sequences collected from pigs with different clinical presentations. CONCLUSIONS: The present study further extends evidence that newly described PCV3 widely circulates in six additional provinces of Southern and Northern China and has high similarity to previously reported isolates. As an emerging virus of swine, although the present case-control study reveals that PCV3 has a potential association with swine respiratory disease and diarrhea, further investigations into the pathogenesis are needed to ascertain the role of PCV3 in swine health.

Influenza D Virus in Animal Species in Guangdong Province, Southern China.

Molecular tests revealed influenza D viruses of D/OK lineage widely circulating in farmed animal species in Guangdong Province, southern China. In particular, we found high levels of influenza D virus infection in goats and pigs. We also detected viral RNA in serum specimens and feces of animals with certain severe diseases.

Genome Sequence of a Novel Porcine Circovirus Type 2 Strain That Reemerged in Southern China.

Here, we describe a novel porcine circovirus type 2 (PCV2) variant (GD2014) found in the Guangdong province, southern China. Its complete genome is 1,766 nucleotides and contained a 708-nucleotide open reading frame 2 (ORF2). Sequence analysis suggested that GD2014 is closest to JS2015 originating from the Jiangsu province of China and belongs to the PCV2d genotype.

Occurrence and sequence analysis of porcine deltacoronaviruses in southern China.

BACKGROUND: Following the initial isolation of porcine deltacoronavirus (PDCoV) from pigs with diarrheal disease in the United States in 2014, the virus has been detected on swine farms in some provinces of China. To date, little is known about the molecular epidemiology of PDCoV in southern China where major swine production is operated. RESULTS: To investigate the prevalence of PDCoV in this region and compare its activity to other enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis coronavirus (TGEV), and porcine rotavirus group C (Rota C), 390 fecal samples were collected from swine of various ages from 15 swine farms with reported diarrhea. Fecal samples were tested by reverse transcription-PCR (RT-PCR) that targeted PDCoV, PEDV, TGEV, and Rota C, respectively. PDCoV was detected exclusively from nursing piglets with an overall prevalence of approximate 1.28 % (5/390), not in suckling and fattening piglets. Interestingly, all of PDCoV-positive samples were from 2015 rather than 2012-2014. Despite a low detection rate, PDCoV emerged in each province/region of southern China. In addition, compared to TGEV (1.54 %, 5/390) or Rota C (1.28 %, 6/390), there were highly detection rates of PEDV (22.6 %, 88/390) in those samples. Notably, all five PDCoV-positive piglets were co-infected by PEDV. Furthermore, phylogenetic analysis of spike (S) and nucleocapsid (N) gene sequences of PDCoVs revealed that currently circulating PDCoVs in southern China were more closely related to other Chinese strains of PDCoVs than to those reported in United States, South Korea and Thailand. CONCLUSIONS: This study demonstrated that PDCoV was present in southern China despite the low prevalence, and supported an evolutionary theory of geographical clustering of PDCoVs.

Molecular detection and genome characterization of porcine circovirus type 2 in rats captured on commercial swine farms.

Porcine circovirus type 2 (PCV2) is considered the major etiological pathogen of porcine circovirus-associated diseases (PCVADs) in pigs. Recently, PCV2 was also found in non-porcine animals such as cattle, rats, and mice. However, there was no record of PCV2 in rats in China. The goal of this study was to investigate whether PCV2 was present in rats (Rattus norvegicus, RN) on three swine farms, using molecular tools. PCR results showed that 30 of 95 (31.6 %) rat samples were positive for PCV2. Moreover, further genotype analysis suggested that 10 of 30 (33.3 %) were positive for PCV2a, 19 of 30 (63.3 %) were positive for PCV2b, and only one sample (1/30, 3.33 %) was co-infected by PCV2a and PCV2b. To determine the possible origin of PCV2, 60 serum samples were also collected from weaned pigs on those swine farms, and 23 out of 60 samples were positive for PCV2. In addition, two distinct RN-origin and two distinct porcine-origin PCV2 full-length nucleotide sequences were obtained from the farms. Sequence and phylogenetic analysis indicated that they had the highest nucleotide similarity and closest genetic relationships to each other. In this study, we report the infection and genome characterization of PCV2 in rats and compare RN-origin and porcine-origin PCV2 sequences obtained from the same pig farm, revealing possible cross-species transmission of PCV2.

Emergence of reticuloendotheliosis virus in pigeons in Guangdong Province, Southern China.

Reticuloendotheliosis virus (REV), an important immunosuppressive pathogen, has many hosts, including chickens, ducks, geese, turkeys, and wild birds. Clinically, REV may lead to increased susceptibility to other pathogens, resulting in serious tissue damage (especially tumors) and the death of its host. In this study, we encountered a disease outbreak resulting in a large number of deaths of pigeons in Guangdong Province, Southern China. Histopathological analysis revealed apparent tumor-like lesions in multiple organs of pigeons. PCR assays for detection of tumor-associated pathogens (REV, avian leukosis virus, and Marek's disease virus) in poultry revealed the presence of REV sequences only. Moreover, fowlpox virus (FPV) with an insertion of REV long terminal repeat (LTR) sequences was also considered, but it was excluded using a specific PCR assay. To gain more genetic information, two full-length REV genome sequences were determined and found to have the highest nucleotide sequence similarity (99.9 %) and the closest genetic relationship to a vaccine strain (MD-2) and had a more distant genetic relationship (94.3 %) to a duck-origin strain (ATCC-VR775). To confirm the presence of REVs in pigeons, specific-pathogen-free (SPF) chickens and healthy pigeons were inoculated with microfiltered tumor tissue homogenates and were found to be susceptible to infection with REV. To our knowledge, this is the first report of REV in pigeons, and the data suggest that pigeons may be the natural host of REV.

Genome characterization and phylogenetic analysis of a lineage IV peste des petits ruminants virus in southern China.

Since 2013, the second outbreak of peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) has spread over more than 20 provinces, municipalities, and autonomous regions in China, resulting in major economic losses for livestock industry. In 2014, we encountered a clinical PPR case on a goat farm in Guangdong province, southern China. The complete genome of this PPRV strain, named CH/GDDG/2014, was sequenced to determine its similarities and differences with other strains. The CH/GDDG/2014 genome comprised 15,954 nucleotides (six nucleotides more than classical PPRVs identified before 2013, but complying with the rule of six) with six open reading frames encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. The whole-genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus (99.8 %) to China/XJYL/2013 and the least consensus (87.2 %) to KN5/2011. The phylogenetic analysis showed that CH/GDDG/2014 was clustered in one branch (lineage IV) with other emerging strains during the second outbreak. This study is the first report describing the whole-genome sequence of PPRV in Guangdong province, southern China and also suggests the PPR outbreak may be closely related to illegal cross-regional importation of goats.