Genetic diversity of porcine circovirus type 2 in China between 1999–2017

Porcine circovirus type 2 (PCV‐2), is linked to PCV‐2 associated disease, which has caused considerable economic loss in the swine industry. Here, we report the genetic diversity of PCV‐2 in China. A total of 74 Chinese PCV‐2 strains sequenced between 1999 and 2017 were studied. Based on the ORF2 and complete genomes, we found that apart from the PCV‐2a, PCV‐2b, and PCV‐2d genotypes, two unstable recombination genotypes also exist, referred to as IM1 and IM2 genotypes. We found that the patterns of PCV‐2 genetic shift in China are similar to the patterns at the global level. Additionally, for the PCV‐2 ORF2 gene of Chinese isolates, we found a similar time to the most recent common ancestor and evolutionary rate to the global values. This indicates that PCV2 genetic diversity in China is driven by genetic drift/recombination of local strains and by the sporadic introduction of foreign genotypes from other countries. Overall, our study illustrates the genetic diversity and evolution dynamics of PCV‐2 in China.     

First detection and genetic analysis of fox‐origin porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is a causative agent of porcine circovirus‐associated disease (PCVAD), which is a serious problem in the swine industry worldwide. In recent years, nonporcine‐origin PCV2 has attracted more and more attention of the researchers. This study reported on the first identification of PCV2 in farmed foxes with reproductive failure. Three fox‐origin PCV2 strains were successfully isolated, sequenced, and designated as FoxHB1, FoxHB2, and FoxHB3 respectively. Pairwise‐sequence comparisons of the complete genomes revealed that three fox‐origin PCV2 strains had nucleotide identities varied from 91.9% to 99.7% with representative strains of PCV2 different genotypes. Meanwhile, phylogenetic analysis based on complete genomes of 44 PCV2 strains indicated that the fox‐origin PCV2 strains belonged to Chinese epidemic genotypes PCV2b and PCV2d. These results provided the first supported evidence that PCV2 could infect foxes, implying that the cross‐species transmission of PCV2 would be a big threat to Chinese fur animal‐bearing industry.     

Detection and genome sequencing of porcine circovirus 3 in neonatal pigs with congenital tremors in South China

Porcine circovirus 3 (PCV3) is a novel circovirus first discovered in the United States in piglets and sows with porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multisystemic inflammation. Here, seven PCV3 strains were identified for the first time from neonatal pigs with clinical signs of congenital tremors (CT) in South China. The tissue tropism of PCV3 in CT‐affected piglets was analysed by the real‐time quantitative PCR, and the result showed that high loads of viral genomes were detected in the brains and hearts. The complete genomes of seven new PCV3 revealed 96.8%–99.6% nucleotide identities with eleven other PCV3 strains previously reported from the United States and China. Phylogenetic analysis based on the complete genome sequences showed that all PCV3 strains clustered together and were clearly separated from other circovirus species. This study reports on the first identification of PCV3 in CT‐affected newborn piglets and provides the epidemiological information of neonatal piglets with CT in Guangdong and Guangxi Provinces of China.     

Emergence of a novel recombinant porcine circovirus type 2 in China: PCV2c and PCV2d recombinant

Porcine circovirus type 2 (PCV2) has been causing huge economic losses in Chinese swine herds since it was first identified in China in 1999. Genotypes of PCV2 except for PCV2c coexist in swine herds in China, which may facilitate virus recombination. In the current study, six novel PCV2 strains were detected in China, and these strains shared high nucleotide similarity of the Rep gene with the PCV2c strain DK1987PMWSfree and high homology of the Cap gene with PCV2d. Genome sequence analysis revealed that the complete genomes of these strains were 1767 nucleotides (nt) in length and shared 99.8%–99.9% nucleotide identity with each other and 91.7%–98.7% with representative strains. Phylogenetic analysis, sequencing analysis, base‐by‐base comparisons and comprehensive recombination analysis demonstrated that these six strains originated from recombination within the Rep gene between PCV2c and PCV2d strains. Surprisingly, further investigation through theoretical recombination analysis of Chinese PCV2 GenBank sequences showed that these novel patterns of recombinant PCV2 strains have been generated since 2010. Collectively, our findings provide additional evidence of inter‐genotypic recombination of PCV2.     

Genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from South Korea, 2017

Porcine epidemic diarrhoea virus (PEDV ) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. Since the re‐emergence of massive PEDV outbreaks in South Korea in 2013−2014, domestic pig farms have continued to experience PED epidemics or endemics. This study represents the molecular characterization of PEDV isolates identified in diarrhoeic animals collected across the country in 2017. Initial sequencing analysis of the full‐length S genes revealed that 70% of the 2017 isolates (7/10) belong to the G2b subgroup, while the remaining isolates were classified as G1b. The data indicated that both variant G1b and global epidemic G2b strains were responsible for current PED outbreaks in South Korea. The 2017 G1b and G2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the S gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding Korean prototype G1b and G2b strains, respectively. In an interesting manner, one G2b‐like KNU ‐1705 strain was found to possess a large 39‐nucleotide deletion in the ORF 1a region theoretically encoding nonstructural protein 3. Phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global G1b or G2b strains but formed different branches within the same genogroup. These results indicate that PEDV s undergo continuous evolution in the field. In addition, one 2017 PEDV strain, KOR /KNU ‐1705/2017, was successfully isolated and propagated in Vero cells. The antisera raised against the Korean prototype 2014 G2b strain efficiently neutralized KNU ‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 PEDV strains. Our data advance the understanding of the molecular epidemiology and antigenicity of PEDV circulating in South Korea.     

First detection of novel enterovirus G recombining a torovirus papain‐like protease gene associated with diarrhoea in swine in South Korea

Enterovirus species G (EV‐G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV‐G strain (KOR/KNU‐1811/2018) that resulted from cross‐order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV‐G genome possessed an exogenous 594‐nucleotide (198‐amino acid) sequence, flanked by two viral 3C^pro cleavage sites at the 5′ and 3′ ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain‐like cysteine protease (PLCP), which was recently found in the EV‐G1, ‐G2, and ‐G17 genomes. The complete KNU‐1811 genome shared 73.7% nucleotide identity with a prototype EV‐G1 strain, but had 83.9%–86.7% sequence homology with the global EV‐G1‐PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV‐G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1‐PLCP and G17‐PLCP strains respectively. These results implied that the torovirus‐derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV‐G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV‐G driven by infrequent viral recombination events, by which EV‐G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.     

Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan

Porcine circovirus (PCV) is a small non‐enveloped virus with a single‐stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2‐systemic disease (PCV2‐SD), previously known as post‐weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2‐SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.     

Phylogenetic and genetic variation analyses of porcine circovirus type 2 isolated from China

Porcine circovirus type 2 (PCV 2) is a causative agent of PCV 2‐associated disease, which is a growing problem in the swine industry worldwide. High nucleotide substitution occurs in the capsid (Cap) gene of PCV 2, which allows the continuous evolution and the emergence of novel PCV 2 strains. In this study, we sequenced 24 Chinese PCV 2 strains collected from healthy and diseased pigs between 2013 and 2015. Analyses of the genome, Cap and phylogeny classified the 24 Chinese PCV 2 strains as PCV ‐2a (four of 24), PCV ‐2b (five of 24) and PCV ‐2d (15 of 24). All strains shared 89.5%–100% and 87.2%–100% identities with the nucleotide and amino acid (aa) sequences of Cap, respectively. Selection pressure analysis showed that five sites at the epitope regions in Cap were under positive selection. Further analysis by Jameson–Wolf antigenic index indicated that aa substitutions occurring at the epitope regions contributed to the antigenic alterations of the different PCV 2 strains. High genetic variation and genotype shift to PCV 2d occurred in recent years, and different genotypes coexisted in Chinese pig herds. The data provide evidence for the increased genetic diversity and insights into the molecular epidemiology of PCV 2.     

Phylogenetic and Phylogeographic Analyses of Porcine Circovirus Type 2 Among Pig Farms in Vietnam

This study demonstrated the prevalence of Porcine circovirus type 2 (PCV2) among pig farms in Vietnam. Analyses of the genome, capsid protein and phylogeny classified all 30 Vietnamese PCV2 strains as the PCV2b genotype, belonging to the clusters of 1A, 1B, 1C and recombinant forms. Each viral genome was 1767 nucleotides long and shared 96.0–100% nucleotide sequence identity. The amino acid substitutions in the capsid protein of the Vietnamese PCV2 strains were in immunodominant regions, and the majority of strains (24/30) contained a lysine extension at the C‐terminus. Bayesian phylogeographic analysis revealed epidemic links of the PCV2 recombinant cluster within and among countries, which supports a circulating recombinant form of PCV2. Further analysis by the Jameson–Wolf antigenic index indicated antigenic alterations at important sites in the capsid protein (sites 131–133) among the recombinant cluster and the other clusters of PCV2b.     

Molecular characteristics and pathogenic assessment of porcine epidemic diarrhoea virus isolates from the 2018 endemic outbreaks on Jeju Island,South Korea

Since the 2013–2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate‐scale regional outbreaks have recurred. In particular, areas of Jeju Island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full‐length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non‐structural protein (nsp)‐ or spike (S) protein‐coding regions. The 2018 Jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the S gene and whole‐genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re‐emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU‐1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV‐induced syncytia formation. Genomic sequencing identified a unique 8‐nt DEL in the extreme C‐terminal region of the S gene at the 4th passage (KNU‐1807‐P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU‐1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.     

Retrospective study of porcine circovirus type 2 infection reveals a novel genotype PCV2f

Porcine postweaning multisystemic wasting syndrome (PMWS ) caused by porcine circovirus type 2 (PCV 2) is a disease causing severe economic losses annually worldwide to the pig industry. PCV 2 infection was first reported in China in 2000, and currently has three major genotypes, PCV 2a, b and d, circulating in this country. To further elucidate the origin and prevalence of PCV 2 in China, 123 clinical pig tissue samples collected in 25 provinces between 1990 and 1999 were analysed by PCV 2‐specific PCR , resulting in identification of 23 PCV 2 strains collected between 1996 and 1999. Phylogenetic analysis based on the nucleotide sequences of open reading frame 2 (ORF 2) showed that 20 of the 23 grouped within PCV 2a, while the remaining three strains formed an independent clade, so far unreported and therefore named PCV 2f. This genotype shared lower sequence identity with other known genotypes. This study provides further understanding of the genetic diversity and evolution of PCV 2 and has tracked PCV 2 infection in China back to 1996 rather than 2000.     

Development and Evaluation of Single‐tube Nested PCR (STNPCR) for the Detection of Porcine Circovirus type 2 (PCV2)

Porcine circovirus 2 (PCV2) is a common virus in pig population and is associated with the postweaning multisystemic wasting disease (PMWS). In this study, it was developed and evaluated the single‐tube nested PCR (STNPCR) method for the detection of PCV2 DNA. PCV2 reference controls and swine tissue samples were used, and primers were selected for targeting specific regions of the viral genome. In comparison of the methods, STNPCR was 10 times more sensitive than conventional PCR and showed the same sensitivity to nested PCR (NPCR), but with reduction in the risk of cross‐contamination. In clinical application, 55 tissue samples were analysed by conventional PCR and resulted in 67% (37/55) of positive reactions, while the NPCR and STNPCR were able to identify the presence of viral DNA in 100% (55/55) of the samples. The high sensitivity combined with the elimination of cross‐contamination makes the STNPCR method suitable for the epidemiological studies of PCV2 and can aid in the diagnosis of PMWS.     

Detection and genetic characterization of porcine circovirus 3 (PCV3) in pigs in India

Porcine circovirus type 3 (PCV3), a novel circovirus, has been reported recently from major swine growing countries globally, and the virus is associated with diseases like porcine dermatitis, nephropathy syndrome and reproductive failure. This report describes the identification of PCV3 associated with reproductive failure in sows and piglet mortality and circulation of the virus in healthy pigs in India. The pathological changes in various tissues from stillborn piglet and characterization of the virus genomes were reported. The genome sequences of Indian PCV3 strains showed 91.4%–99.8% nucleotide identity with other sequences of PCV3 strains circulating worldwide. The phylogenetic analysis showed clustering of Indian strains into a separate group with the isolate from USA (MN/2016) under PCV3a genotype. The results confirmed the circulation of PCV3 in Indian pigs and its association with clinical cases. This study speculates emergence of PCV3 as an important pig pathogen in the country, which warrants the thorough investigation on PCV3 epidemiology, pathogenesis and to implement the control measures.     

A novel NADC30‐like porcine reproductive and respiratory syndrome virus (PRRSV) plays a limited role in the pathogenicity of porcine circoviruses (PCV2 and PCV3) and PRRSV co‐infection

Co‐infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co‐infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17‐36) collected from a diseased pig was co‐infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17‐36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30‐like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17‐36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30‐like PRRSV SD17‐36 isolate is low pathogenic. Our results indicate that the co‐infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co‐infection.     

Prolonged Detection of PCV2 and Anti‐PCV2 Antibody in Oral Fluids Following Experimental Inoculation

The onset, level and duration of PCV2 and anti‐PCV2 antibody in oral fluid were evaluated using samples collected from experimentally inoculated pigs for 98 days post‐inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (i) negative control group; (ii) inoculated with PCV2a (strain ISU 40895) on DPI 0; (iii) inoculated with PCV2a (strain ISU‐40895) on DPI 0 and re‐challenged at DPIs 35 and 70; (iv) inoculated with PCV2a (ISU‐40895), PCV2b (PVG4072) and PCV2a (ISU‐4838) on DPIs 0, 35 and 70, respectively. Serum was collected from each animal, and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by quantitative polymerase chain reaction (qPCR) and anti‐PCV2 IgG, IgA and IgM antibody isotypes by enzyme‐linked immunosorbant assay (ELISA). Serum was assayed for anti‐PCV2 IgG by ELISA. Anti‐PCV2 antibodies (IgG, IgM and IgA) were detected in oral fluid from experimentally inoculated pigs from DPI 14 with IgG and IgA clearly present at 98 DPI. PCV2 was detected in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout 98 DPI. Overall, the data indicated that PCV2 infection in swine is prolonged, persists in the face of an active antibody response and can be efficiently monitored using oral fluid specimens.     

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV ‐2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV ‐3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV ‐3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV ‐3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12 months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV ‐3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV ‐3 infection. The high prevalence and the absence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations.     

The prevalence of novel porcine circovirus type 3 isolates in pig farms in China

The emerging porcine circovirus type 3 (PCV3) has been reported in Chinese swine herds since 2017. We performed a nationwide investigation on the prevalence of PCV3 in pig breeding farms and slaughterhouses in China. A total of 4,040 tonsil samples were collected from 89 farms in 25 provinces, and 1,419 lymph node samples were collected from 50 slaughterhouses in 27 provinces. The PCR results showed that in pig breeding farms, the positive rate was 41.6% (37/89) at the farm level and 5.0% (201/4040) at the individual level. In the slaughterhouses, the positive rate was 62.0% (31/50) at the farm level and 8.0% (114/1419) at the individual level. The PCR‐positive samples were further sequenced, and 19 new PCV3 isolates were identified. The complete genomes of the 19 virus isolates showed 97.4%–99.7% nucleotide identity with other PCV3 isolates. The phylogenetic analysis revealed that the 19 isolates were divided into PCV3a and PCV3b genotype clusters based on the PCV3 complete genome sequences. This study indicated that PCV3 has spread extensively in both pig breeding farms and slaughterhouses. The positive rate of PCV3 was higher in eastern China compared to other regions in China. Furthermore, this study will help us understand the prevalence and genetic variation of PCV3 in Chinese swine herds.     

The detection of porcine circovirus 3 in Guangxi,China

Porcine circovirus type 3 (PCV 3) is a novel circovirus that was firstly detected in the USA . PCV 3 is associated with porcine dermatitis and nephropathy syndrome (PDNS ), reproductive failure and cardiac and multisystemic inflammation. Latterly, PCV 3 was detected in Guangxi, China. Forty‐one of 108 (37.96%) samples and nine of 47 (19.14%) samples were PCV 3 positive in pig farms and pig slaughter houses, respectively. Three PCV 3 strains were sequenced and designated PCV 3‐China/GX 2016‐1, PCV 3‐China/GX 2016‐2 and PCV 3‐China/GX 2016‐3. The complete genome of PCV 3‐China/GX 2016‐2 and PCV 3‐China/GX 2016‐3 is both 2,000 bp in length, while PCV 3‐China/GX 2016‐1 is of 1,999 bp and has a G deletion at position of 1,155 in its genome. The complete genome and capsid nucleotide of the three PCV 3 strains identified in this study shared 97.5%–99.4% and 96.7%–99.1% identities with that of the other PCV 3 strains available in NCBI , respectively. Phylogenetic analysis based on complete genome and capsid gene of 35 PCV 3 strains showed that the three PCV 3 sequences from Guangxi Province were divided into two clusters. The results of this study contribute to the understanding of PCV 3 molecular epidemiology.     

Detection and phylogenetic analysis of porcine circovirus type 3 in central China

Porcine circovirus type 3 (PCV3) is the pathogen responsible for a new infectious disease that was first reported in 2016 in the United States. To further investigate the epidemic profile and genetic diversity of the virus, one hundred and seventy clinical samples (110 tissue samples and 60 serum samples) were collected from 41 different pig farms in 14 cities in central China, and a SYBR Green I‐based quantitative real‐time PCR method was developed to detect PCV3. The partial cap genes of four field strains from four different farms were sequenced and analysed. The results showed the detection limit was 2.19 × 10¹ genome copies/μl. Fifty‐three of 170 samples were detected as positive for PCV3, giving a PCV3‐positive rate of 31.18%, with 48.78% (20/41) of pig farms harbouring PCV3, which varied from 20% to 42.86% between 2013 and 2017. PCV3 could be detected in samples from pigs with different clinical presentations, and the PCV3‐positive rates varied for these different clinical presentations. The partial capsid genes of four PCV3 strains (designated YZ, LY‐03, NY and SP) shared 96.3%–99.4% nucleotide identity with those available in GenBank. Phylogenetic analysis based on the capsid gene of 32 PCV3 strains showed that the four PCV3 strains in this study were clustered with the China/GD2016 and South Korea Ku‐1606 strains. The results of this study will aid our understanding of the molecular epidemiology of PCV3.