Naturally Co‐Infected Boars with both Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

In this study, the humoral response against porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), the presence of the virus in semen and serum and the genetic characteristics of the virus detected in 15 boars from a commercial farm were analysed. The results showed that 53% of the boars presented anti‐PRRSV antibodies and 100% presented anti‐PCV2 antibodies. Porcine reproductive and respiratory syndrome virus was detected in 43% of the boars and 73% were positive to PCV2. The complete ORF5 gene of PRRSV of 14 samples and a fragment of the ORF2 gene of PCV2 of 22 samples were sequenced. Porcine reproductive and respiratory syndrome virus analysis revealed <92% identity in viruses from semen and serum of two boars, whereas in the rest of the boars the identity was >97.5%. As for PCV2, two boars presented an identity <95% in serum and semen and the rest had an identity >96%. The results showed that PRRSV‐ and PCV2‐naturally infected boars can be found, and at least two different strains of viruses from semen and serum can be detected.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Genome characterization of a porcine circovirus type 3 in South China

Porcine circovirus type 3 (PCV 3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. Recently, a PCV 3 strain was identified from pyretic and pneumonic piglets in Guangdong province, China. This virus strain was sequenced and designated PCV 3‐China/GD 2016. The complete genome of PCV 3‐China/GD 2016 is 2,000 bp in length and shared 99.1% and 99.1% nucleotide identities with PCV 3/29160 and PCV 3/2164, respectively. [Corrections added after initial online publication on 13 March 2017: The numbers ‘98.5%’ and ‘97.4%’ has been changed to ‘99.1%’ and ‘99.1%’ in the previous sentence.] Phylogenetic analysis based on the complete genome showed that PCV 3‐China/GD 2016 clustered with the emerging PCV 3 and separated with other virus in genus Circovirus . The results of this study suggest that PCV 3 has existed within the pigs of China. It is urgent to investigate the pathogenicity and epidemiology of this novel circovirus China.     

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.     

Molecular characterization of two novel atypical porcine pestivirus (APPV) strains from piglets with congenital tremor in China

As one of emerging porcine viruses, atypical porcine pestivirus (APPV) was found in three continents since it emerged in 2015. It is now thought as the causative agent for congenital tremor type A‐II in piglets. At the end of 2017, two APPV strains were identified from piglets with congenital tremor in Guangxi and Yunnan, China. The genome of APPV GX04/2017 strain was so far determined to be 11,534 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a polyprotein comprising 3,635 amino acids. Comparative analysis of ORF, N^pro, E2, and NS3 gene sequences revealed that the APPV GX04/2017 strain shares nucleotide sequence identities of 82.8%–92.8% with other APPV strains, while YN01/2017 strain is 79.4%–97.4% homology to the others. Phylogenetic analysis showed that the APPV GX04/2017 and YN01/2017 are two novel APPV strains with the highest homology to each other, and relative high similarity to the APPV 000515 and JX‐JM01 strains in genome sequence. The current findings provide updated information about APPV epidemiology and divergence in China, which would certainly help to establish reliable diagnosis and surveillance programs for APPV.     

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.     

Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV), which emerged in Taiwan in late 2013, have caused a high morbidity and mortality in neonatal piglets. To investigate the molecular characteristics of the spike (S) gene of the emerging Taiwan PEDV strains for a better understanding of the genetic diversity and relationship among the Taiwan new variants and the global PEDVs, full‐length S genes of PEDVs from nine 1–7 day‐old piglets from three pig farms in the central and southern Taiwan were sequenced and analysed. The result of phylogenetic analysis of the S gene showed that all the Taiwan PEDV strains were closely related to the non‐S INDEL strains from US, Canada and China, suggesting a common ancestor for these strains. As compared with the historic PEDVs and CV777‐based vaccine strains, the nine Taiwan PEDV variants shared almost the same genetic signatures as the global non‐S INDEL strains, including a series of insertions, deletions and mutations in the amino terminal as well as identical mutations in the neutralizing epitopes of the S gene. The high similarity of the S protein among the Taiwan and the globally emerged non‐S INDEL PEDV strains suggests that the Taiwan new variants may share similar pathogenesis and immunogenicity as the global outbreak variants. The development of a novel vaccine based on the Taiwan or the global non‐S INDEL strains may be contributive to the control of the current global porcine epidemic diarrhoea outbreaks.     

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.     

Identification and genetic characterization of porcine circovirus type 3 in China

A novel circovirus called porcine circovirus type 3 (PCV3) was recently reported to exist in the USA. This circovirus is associated with porcine dermatitis, nephropathy syndrome and reproductive failure. This study reports on the first identification, widely epidemic, different phylogenetic clusters, potential role in sow reproductive failure and possible origins of PCV3 in China.     

Genetic Characterization of African Swine Fever Viruses from a 2008 Outbreak in Tanzania

Outbreaks of African swine fever (ASF) have been reported in the past from several countries in sub‐Saharan Africa. The aim of this study was to genotype ASF viruses (ASFVs) from the 2008 outbreak in Morogoro and Dar es Salaam regions of Tanzania. Tissue samples from domestic pigs that died as a result of severe haemorrhagic disease were collected and analysed with PCR and genome sequencing methods using ASFV‐specific primer sets. Nucleotide sequence data were obtained for the B646L (p72), E183L (p54) and the variable region of the B602L gene sequences. Phylogenetic analyses based on DNA sequences showed that the 2008 Tanzanian isolates belonged to p72 genotype XV and clustered together with those derived from the 2001 outbreak in Tanzania. Analysis of the tetrameric amino acid repeat regions within the variable region of the B602L gene showed that the repeat signature of the 2008 Tanzanian ASFV was unique and contained three novel tetramers (U = NIDT/NTDT and X = NTDI). Epidemiological investigation suggested that transportation of live pigs continues to play an active role in the epidemiology of ASF in Tanzania. It is recommended that future control of ASF spread in Tanzania should focus on the early detection and confirmation of the disease, prompt institution of quarantine measures, culling and proper disposal of infected and in‐contact animals and decontamination of affected premises.     

Genetic characterization and diversity of porcine circovirus type 2 in non‐vaccinated South African swine herds

The porcine circovirus type 2 (PCV2) is a swine infectious viral pathogen of great significance in global swine herds. It was recently detected at another Province of South Africa sequel to the first detection of North American‐like strain (PCV2a) at Gauteng about two decades ago, but there is a dearth of information about the genomic features and diversity of the viral strains in circulation within the country and the entire sub‐Saharan Africa region. To date, only one complete genome of the virus from South Africa is available on global data base. This current effort is therefore geared towards the full‐genome characterization of the circulating PCV2 strains in the pigs of Eastern Cape Province. With the use of conventional polymerase chain reaction method, fifteen complete PCV2 genomes were successfully amplified, sequenced and assembled from field samples obtained from non‐vaccinated pigs in the region. Neighbor Joining and Maximum Likelihood phylogenetic analyses of the ORF2 gene and full genomes unanimously showed that most of the assembled genomes (11) belong to genotype PCV2b. Furthermore, three of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (i.e. PCV2d) strains from the USA, China and South Korea. The last sequence, however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2‐IM2), recently identified in a global PCV2 strains phylogenetic analysis. This study reports the first complete genome sequences of PCV2b, PCV2d and PCV2‐IM2 in pigs from South Africa, and it gives a possible insight into the genetic characteristics and variability of the viral strains presently in circulation within the country. It further emphasizes the need for more stringent measures in curtailing the introduction and spread of transboundary swine pathogens in the country and entire Southern African region.     

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.