The porcine circovirus type 1 in porcine kidney 15 cell line is not transferred to mice lymphoid cells after xenoimplantation into the peritoneal cavity

The porcine circovirus type 1 (PCV1) has been identified within lymphoid tissues of experimental infected pigs and suggested to induce an immunosuppressive stage in pigs. The virus does not induce a cytophatic effect in the pig-derived cell line PK-15. Because PCV1 is prevalent in many pig cells and tissues, the risk of inducing a viral xenozoonosis by PCV1 was raised for the xenoimplantation of pig cells into human hosts. The present work evaluated if PCV1 is able to replicate in mice tissues after xenoimplantation of PCV1-infected pig cells. Active growing PK-15 cells harboring PCV1 with or without microencapsulation in sodium alginate were implanted into the peritoneal cavity of mice. After 1 month postimplantation in mice, peritoneal macrophages, spleen, and lymph nodes were harvested and analyzed with the polymerase chain reaction technique (PCR). No evidence of circovirus type 1 DNA was detected within the mice tissues.     

Ultrastructural alterations in human blood leukocytes induced by porcine circovirus type 1 infection

BACKGROUND: Swine infectious pathogens, especially viruses, represent a potential public health risk associated with the use of pig tissues for xenotransplantation in humans. We hypothesized that porcine circovirus type I (PCV-1) may infect human mononuclear cells, resulting in ultrastructural alterations of the target cells. METHODS: Transmission electron microscopy was used for evaluating ultrastructural alterations of human cells exposed to a PCV-infected PK15 cell line. A polymerase chain reaction (PCR) assay and fluorescence in situ hybridization (FISH) were developed for detecting PCV-1 in human mononuclear cells. RESULTS: Morphological alterations of the human T cells exposed to PCV PK15 showed 'boomerang-shaped' intracytoplasmic inclusions. Nucleocapsids appeared free, close to the nucleus, or contained into cytoplasmic vacuoles. Virions were observed near the surface of the human cells. A considerable number of mature virions and immature forms could be observed in the human cells that had a completely intact nuclear membrane with no alteration in the disposition of chromatin. PCV-1 particles were identified budding into typical Golgi saccules and vacuoles. Virions sized up to 23 nm in diameter, and appeared in the nucleus and in the periphery of the cellular core. PCV-1 infection was detected on CD4+, CD8+, CD14+, CD19+, and CD56+ human cells by PCR assay and FISH. CONCLUSIONS: These results suggest that PCV has the capability of infecting human leukocytes in vitro, and should be considered a potential risk of viral transmission during xenotransplantation.     

Presence of Torque teno sus virus 1 and 2 in porcine circovirus 3‐positive pigs

In this study, the co‐infection of Torque teno sus virus (TTS uV) and porcine circovirus type 3 (PCV 3) was reported. One hundred and ten of 132 (83.3%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 1 (TTS uV1). Ninety‐four of 132 (71.2%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 2 (TTS uV2). Sixty‐six of 132 (50.0%) of PCV 3‐positive samples were co‐infected with both TTS uV1 and TTS uV2. There were no clinical signs of infection in pigs that were both PCV 3‐positive and PCV 2‐negative, in either multiparous sows or live‐born infants. The high co‐infection rate provides valuable information for the further study of the pathological correlation between PCV 3 and TTS uVs.     

Rapid specific and visible detection of porcine circovirus type 3 using loop‐mediated isothermal amplification (LAMP)

In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV 3) was established using loop‐mediated isothermal amplification (LAMP ). Four primers were specifically designed to amplify PCV 3. The LAMP assay was effectively optimized to amplify PCV 3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10¹ copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV 2), both gE and gD genes of pseudorabies virus (PRV ) and porcine parvovirus (PPV ), the LAMP assay showed a high specific detection of PCV 3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency.     

Identification of pig circovirus type 2 in New Zealand pigs

Interest in porcine circovirus has been stimulated by the recent emergence of postweaning multisystemic wasting syndrome (PMWS) in pigs and the potential use of pig organs for xenotransplantation in humans. Porcine circovirus type 1 (PCV1) is considered to be widespread in pigs but nonpathogenic. Circovirus type 2 (PCV2) is a similar virus but has been differentiated only recently as a separate type. High tissue concentrations of PCV2 are associated with lesions in PMWS cases, but the etiological role of this agent in the disease remains unclear. The presence of PCV1 in New Zealand pigs has been previously reported based on serological data. PMWS has been recently recorded in New Zealand pigs. The epidemiology of PCV2 in New Zealand pigs has not been examined. The purpose of the study was to look for evidence of circoviruses in New Zealand pig herds. Pig circovirus DNA was sought in various tissues using the polymerase chain reaction. Circovirus type 2 was found in New Zealand pig herds, without any evidence that PMWS has ever occurred in these herds. Newborn piglets were shown to have infection, suggesting vertical transmission of the virus.     

The retrospective identification and molecular epidemiology of porcine circovirus type 3 (PCV3) in swine in Thailand from 2006 to 2017

Porcine circovirus type 3 (PCV3) has recently been detected in pigs worldwide, with similar clinical manifestations to porcine circovirus‐associated disease (PCVAD) from porcine circovirus type 2 (PCV2) infection. Here, we report the identification and molecular epidemiology of PCV3 in swine in Thailand from clinical samples retrieved from 2006 to 2017. The epidemiological data revealed co‐infection with PCV2, PRRSV, and PCV2/PRRSV was common in our samples. Circulating PCV3 from this study shared a high similarity of nucleotide and deduced amino acid sequences of the partial capsid gene (96.7%–100% and 96.7%–100% respectively), indicated the genetic stability of PCV3 in Thailand. Phylogenetic analysis based on the capsid gene revealed scatter clustering with current PCV3 having no relation to the geographical origin of the virus strains. In this retrospective study, results have demonstrated that PCV3 has spread extensively within Thai swine from as early as 2006 and may also be involved in PRDC and PCVAD.     

Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus

BACKGROUND: An important issue with respect to virus safety in xenotransplantation is the search for human analogues of porcine viruses, because transmission of a porcine virus followed by recombination with a related human virus may lead to a new emerging virus of unknown pathogenicity, host range and virulence. In case of circoviruses, two types of porcine circovirus (PCV1 and PCV2) are described, but the existence of an analogous human circovirus has not yet been investigated. METHODS: This study describes the analysis of human samples with a consensus primer-PCR approach designed to amplify conserved regions from the rep gene of circoviruses from the genus Circovirus. DNA from human sera, lymph nodes, blood and urine was extracted and investigated with this method that has led previously to the identification of a new avian circovirus. RESULTS: By screening 1101 samples (there of 168 from immunocompromised patients), no evidence for the existence of a human circovirus related to the genus Circovirus was obtained. CONCLUSIONS: This result renders the existence of a human circovirus related to the porcine circoviruses more unlikely, nevertheless the presence of such a virus cannot be ruled out.     

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.     

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.     

First detection of porcine circovirus type 3 on commercial pig farms in Poland

Porcine circovirus type 3 (PCV3) is a novel circovirus species recently discovered in USA and China in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, respiratory disease and multisystemic inflammation. This study reports on the first identification of PCV3 in Europe, in serum from pigs from Polish farms. A total of 1,050 serum samples were collected between 2014 and 2017 from sows and 3–20 weeks old pigs from 14 commercial farms representing different regions of Poland, different size and health status. The samples were pooled by 4–6 and tested with real‐time PCR for PCV3. PCV3 DNA was detected in 12 of 14 farms (85.7%). On the PCV3‐positive farms, the virus was detected in 5.9% to 65% serum pools. PCV3 was most common among weaned pigs and finishers (26.1% and 28.0% of serum pools, respectively). Sequence analysis of 359 nucleotide fragment of ORF2 showed highest identity of 99.7% to PCV3‐US/SD2016 from USA. Our results indicate that PCV3 is a common virus among Polish pigs but no links to unexplained disease conditions were established.     

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.     

Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin

Millard A‐L, Häberli L, Sinzger C, Ghielmetti M, Schneider MKJ, Bossart W, Seebach JD, Mueller NJ. Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin.
Xenotransplantation 2010; 17: 274–287. © 2010 John Wiley & Sons A/S. Abstract: Background: Human cytomegalovirus (HCMV) infection or reactivation has been linked to allograft rejection resulting from endothelial injury and immune activation. In pig‐to‐human xenotransplantation, currently investigated to circumvent the shortage of human organs in transplantation medicine, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV. We investigated the susceptibility of porcine endothelial cells (pEC) to HCMV infection. Methods: Immortalized porcine aortic (PEDSV15) and porcine microvascular bone‐marrow derived EC (2A2) as well as a panel of primary pEC originated from different vascular beds were inoculated with the endotheliotropic (TB40/E) and the fibroblast propagated (TB40/F) HCMV strains at multiplicity of infection (MOI) ranging from 0.1 to 5. Viral replication kinetics, development of cytopathology and release of viral progeny were analyzed. Results: All viral strains infected pEC with differences in both infection efficiency and kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV‐infected pEC released viral particles but with a lower efficiency than infected HAEC. Conclusions: Our data demonstrate that HCMV productively infects pEC, therefore finding strategies to render pEC resistant to HCMV infection will be of interest to reduce the potential risk carried by HCMV reactivation in xenotransplantation.     

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.     

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.     

The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province

Porcine circovirus type 3 (PCV3) was detected in Shandong, China. One hundred and thirty‐two of 222 (59.46%) samples were PCV3 positive, while 52 of 132 (39.39%) samples were co‐infected with PCV2. There were no clinical signs of infection in either multiparous sows or live‐born infants. Two strains of PCV3 were indentified from natural stillborn foetuses. Phylogenetic analysis showed the two strains of PCV3 are 96% identical to the known PCV3/Pig/USA (KX778720.1, KX966193.1 and KX898030.1) and closely related to Barbel Circovirus. Further studies of the epidemiology of PCV3 and the co‐infection with PCV2 are needed.     

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.     

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.     

Insights into the epidemic characteristics and evolutionary history of the novel porcine circovirus type 3 in southern China

Porcine circovirus type 3 (PCV 3) is a newly identified circovirus from swine in the USA , China and Poland. This novel circovirus has been associated with porcine dermatitis and nephropathy syndrome (PDNS ), reproductive failure and multisystemic inflammation; moreover, PCV 3 poses a potential threat to the swine industry. In this retrospective study, a phylogenetic analysis was conducted to address the epidemiology and evolutionary dynamics of this novel circovirus. The total positive sample rate of PCV 3 was 26.7% (76/285) and has increased gradually over the past 3 years. Of these PCV 3‐positive samples, 22.3% (17/76) were coinfected with PCV 2. PCV 3 can be detected in multiple sample types with different positive rates, and the positive rate is highest among stillborn. We also divide PCV 3 into three clades (PCV 3a, PCV 3b and PCV 3c) based on two amino acid mutations (A24V and R27K) on the cap protein in this study. In addition, the origin of PCV 3 was approximately 1966 and may have originated from a bat‐associated circovirus. Our results suggested that PCV 3 is widely distributed in southern China and has been circulating in swine herds for nearly half a century. PCV 3 has evolved into different clades caused by mutations in cap proteins; thus, further research on PCV 3 epidemiology should be conducted.     

Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province,China

In pigs, three circovirus species within the genus Circovirus have been identified so far, including the non‐pathogenic Porcine circovirus 1 (PCV1), the pathogenic Porcine circovirus 2 (PCV2) and the recently identified Porcine circovirus 3 (PCV3). In April 2019, a new circovirus with a distinct relationship to other circoviruses was identified in several pigs with severe clinical disease in Hunan province, China. The size of the viral genome, tentatively designated as porcine circovirus type 4 (PCV4), is 1,770 nucleotides (nt). PCV4 shows the highest genomic identity to mink circovirus (66.9%) and has identities of 43.2%–51.5% to the other PCV genomes. Two major genes, a replicase (Rep) gene spanning 891 nt and a capsid (Cap) gene spanning 687 nt, were predicted. Furthermore, a TaqMan^® real‐time polymerase chain reaction (PCR) targeting the replicase gene was developed to investigate the prevalence of PCV4 in 187 clinical samples from Hunan province, China. The results revealed an overall PCV4 prevalence of 12.8%, with the highest positive rates in nasal swabs (28.5%, 6/21) followed by serum samples (13.4%, 11/82). The clinical significance and pathogenesis of this virus needs further investigation.