A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Molecular detection and genetic characteristics of porcine circovirus 3 and porcine circovirus 4 in central China.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies.

Porcine Circovirus Type 3 (PCV3) in Poland: Prevalence in Wild Boar Population in Connection with African Swine Fever (ASF).

Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78-99.80%.

Simultaneous detection and genetic characterization of porcine circovirus 2 and 4 in Henan province of China.

Porcine circovirus 4 (PCV4) was identified as a novel porcine circovirus in China in 2019. To investigate the prevalence and genetic characteristics of PCV2 and PCV4, 133 clinical samples (103 tissue samples and 30 serum samples) were collected from 30 different pig farms in Henan province of China, and a SYBR Green I-based duplex quantitative real-time polymerase chain reaction assay was established to detect PCV2 and PCV4 genomes simultaneously. The complete genome sequences of 20 PCV2 and 6 PCV4 strains from 19 and 6 clinical samples respectively were sequenced and analyzed. The results showed the detection limits of this assay were 80.2 copies/µL for PCV2 and 58.6 copies/µL for PCV4. The detection results of clinical samples revealed the PCV2 positive rate was 63.16% (84/133), the PCV4 positive rate was 33.33% (45/133), and the PCV2 and PCV4 co-infection positive rate was 21.05% (28/133). Among 20 PCV2 strains, 6 belonged to PCV2a, 6 belonged to PCV2b and 8 belonged to PCV2d. Co-infection with JZ1 (PCV2b) and JZ2 (PCV2d) strains was identified in one sample (JZ-1). Eleven putative recombination events were found through the recombination analysis, suggesting that the new PCV2 variant strains had circulated in Henan province, which contributes to our understanding of evolutionary characteristics of PCV2 in China. The possible genotypes of PCV4 strains were determined based on genomic sequences of 6 PCV4 strains in this study and 29 PCV4 reference strains available at GenBank. According to three different phylogenetic trees (ORF1, ORF2 and complete genome), all 35 PCV4 strains were clustered into two major genotypes (PCV4a and PCV4b), and 6 PCV4 strains in this study belonged to PCV4a. Additionally, the functional regions of PCV4 strains were predicted by comparison with other circoviruses, which are conducive to the further study of the biological functions of PCV4 genome.

Recent Progress on Epidemiology and Pathobiology of Porcine Circovirus 3.

The recently discovered porcine circovirus 3 (PCV3) belongs to the Circovirus genus of the Circoviridae family together with the other three PCVs, PCV1, PCV2, and PCV4. As reported, PCV3 can infect pig, wild boar, and several other intermediate hosts, resulting in single or multiple infections in the affected animal. The PCV3 infection can lead to respiratory diseases, digestive disorders, reproductive disorders, multisystemic inflammation, and immune responses. Up to now, PCV3 infection, as well as the disease caused by PCV3, has been reported in many swine farms worldwide with high positive rates, which indicates that the virus may be another important pathogen in the swine industry. Therefore, we reviewed the current progress on epidemiology and pathobiology of PCV3, which may provide the latest knowledge of the virus and PCV3-related diseases.

Identification of a novel circovirus in blood sample of giant pandas (Ailuropoda melanoleuca).

The members of the family Circoviridae are considered to be one of the smallest autonomously replicating viruses that are classified into two genera, Circovirus and Cyclovirus. Circoviruses have been found in a variety of vertebrates, but whether they infect endangered protected animals has not been studied in much detail. Here, viral metagenomics and PCR methods were used to detect and verify viral nucleic acid in the blood sample from giant pandas. According to these methods, the complete genome sequence of a novel circovirus, the giant panda associated circovirus (GPCV) from the blood sample of three giant pandas was identified. The GPCV genome is 2090 bp in size and reveals two putative ambisense open-reading frames, encoding the major structural capsid protein and the replication associated protein, respectively, the latter having two predicted introns. Pairwise sequence comparison and phylogenetic analyses indicated GPCV was a putative new species within genus Circovirus based on the species demarcation criteria of the International Committee on the Taxonomy of Viruses. It is the first time that circovirus has been identified from blood sample of giant pandas. These efforts will contribute to future analyses to illuminate the evolutionary relationships between classified and newly identified members of the family Circoviridae.

Circoviruses and cycloviruses identified in Weddell seal fecal samples from McMurdo Sound, Antarctica.

Circoviridae is a family of circular single-stranded DNA viruses whose members infect a wide variety of hosts. While well characterized in avian and mammalian hosts, little is known about circoviruses associated with Antarctic animals. From 48 Weddell seal (Leptonychotes weddellii) fecal samples collected on the sea ice in McMurdo between Nov 2014 and Dec 2014, we identified and determined the genomes of novel viruses that fall within two genera of the family Circoviridae, i.e. Circovirus (n = 7) and Cyclovirus (n = 45). We named these viruses as werosea circovirus (WerCV) and werosea cyclovirus (WerCyV). The genomes of WerCV and WerCyV share ~63-64% genome-wide pairwise identity with classified circoviruses and cycloviruses, respectively. Based on the species demarcation threshold of 80% for members of the Circoviridae, the genomes of WerCV and WerCyV represent new species in their respective genera. Evidence indicated recombination in five of the 45 WerCyV genomes identified in this study. These are the first circoviruses found associated with Antarctic pinnipeds, adding to those recently identified associated with Adélie (Pygoscelis adeliae) and chinstrap penguins (P. antarcticus).

First detection and phylogenetic analysis of porcine circovirus 3 in female donkeys with reproductive disorders.

BACKGROUND: PCV3 is a pathogen associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and cardiac and multiorgan inflammation, which was newly identified in 2016 in sows in USA. Recently, PCV3 has also been identified from several non-porcine species like (cattle, dog, wild boar, deer, mice and ticks). However, PCV3 infection in donkey is not well established. Since 2019, 300 blood samples were collected from female donkey, which was characterized by abortion and sterility, in Liaocheng city of China. RESULTS: In the present study, an investigation of PCV3 in donkey blood samples was undertaken employing by real time PCR. Positive rates of PCV3 in donkeys reach to 21.0 %. In addition, one full-length PCV3 genome sequence was obtained, and it had a highest identity with porcine circovirus 3 PCV3/CN/Nanjing2017 strain and is clustered to PCV3a genotype based on ORF2 sequences. CONCLUSIONS: This is the first report of detection of PCV3 from female donkeys presenting reproductive failure in large-scale donkey farms, China. In addition, the PCV3 strain identified in this study shared the closest relationship with those from porcine, suggesting that PCV3 may be transmitted from pigs to donkeys. Totally, PCV3 infection in donkey should be concerned although the association between it and reproductive failure are not better understood.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species.

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90-80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5'-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Detection and molecular characterization of two canine circovirus genotypes co-circulating in Vietnam.

BACKGROUND: Canine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2). AIM: To characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam. METHOD: PCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny. RESULTS: In total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0-87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%). CONCLUSION: The co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated. CLINICAL RELEVANCE: The mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.

Circovirus in Blood of a Febrile Horse with Hepatitis.

Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse's serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73-74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62-63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1's genome sequence.

Genetic changes and evolutionary analysis of canine circovirus.

Canine circovirus (canineCV) has been found to be associated with vasculitis, hemorrhage, hemorrhagic enteritis, and diarrhea of canines. CanineCV, like other circoviruses, may also be associated with lymphoid depletion and immunosuppression. This circovirus has been detected worldwide in different countries and species. Recombination and mutation events in the canineCV genome have been described, indicating that the virus is continuing to evolve. However, the origin, codon usage patterns, and host adaptation of canineCV remain to be studied. Here, the coding sequences of 93 canineCV sequences available in the GenBank database were used for analysis. The results showed that canineCV sequences could be classified into five genotypes, as confirmed by phylogenetic and principal component analysis (PCA). Maximum clade credibility (MCC) and maximum-likelihood (ML) trees suggested that canineCV originated from bat circovirus. G/T and A/C nucleotide biases were observed in ORF1 and ORF2, respectively, and a low codon usage bias (CUB) was found in canineCV using an effective number of codon (ENC) analysis. Correlation analysis, ENC plot analysis and neutrality plot analysis indicated that the codon usage pattern was mainly shaped by natural selection. Codon adaptation index (CAI) analysis, relative codon deoptimization index (RCDI) analysis, and similarity index (SiD) analysis revealed a better adaption to Vulpes vulpes than to Canis familiaris. Furthermore, a cross-species transmission hypothesis that canineCV may have evolved from bats (origin analysis) and subsequently adapted to wolves, arctic foxes, dogs, and red foxes, was proposed. This study contributes to our understanding of the factors related to canineCV evolution and host adaption.

Experimental efficacy of a trivalent vaccine containing porcine circovirus types 2a/b (PCV2a/b) and Mycoplasma hyopneumoniae against PCV2d and M. hyopneumoniae challenges.

The purpose of this experimental study was to evaluate the efficacy of a new trivalent vaccine containing porcine circovirus types 2a/b (PCV2a/b) and Mycoplasma hyopneumoniae. Pigs were administered the vaccine intramuscularly as either at 3 and 24 days of age with 1.0 mL or at 21 days of age with 2.0 mL according to the manufacturer's recommendations. The pigs were challenged at 42 days of age with either PCV2d (intranasal route) or M. hyopneumoniae (intratracheal route), or both. No statistical differences were observed between the one-dose and two-dose experiments based on clinical (growth performance), immunological (protective immunity), microbiological (viremia and laryngeal swab), and pathological (pulmonary and lymphoid lesion) outcomes. Pigs in vaccinated/challenged and unvaccinated/unchallenged groups showed significant difference in growth performance compared to pigs in the unvaccinated/challenged group in both dosage experiments. Vaccinated pigs elicited a significant amount of protective immunity for PCV2d-specific neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC) as well as M. hyopneumoniae-specific IFN-γ-SC significantly post-challenge compared to unvaccinated/challenged pigs. Vaccination and challenge reduced the viral load amount of PCV2d in the blood and reduced the M. hyopneumoniae load in laryngeal swab, while simultaneously reducing both pulmonary and lymphoid lesion severity when compared to unvaccinated/challenged pigs. Trivalent vaccination provided good protection against a single PCV2d challenge, single M. hyopneumoniae challenge, and a PCV2d/M. hyopneumoniae dual challenge.

Porcine circovirus type 2 (PCV2) infection in Hebei Province from 2016 to 2019: a retrospective study.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in swine, the most common of which are postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). To investigate the prevalence and genetic diversity of PCV2 in Hebei Province, Northern China, from 2016 to 2019, a total of 448 suspected cases of PCV2 infection were studied, and 179 samples were positive for PCV2. A pathological and histopathological examination suggested PCV2 to be cause of the observed lesions. Phylogenetic analysis showed that four genotypes were prevalent in Hebei Province: PCV2a, 2b, 2d, and 2e. Analysis of PCV2 strains using RDP4 and SimPlot showed that there were genetic recombination events among PCV2 strains in Hebei Province. A total of 3284 serum samples were screened by ELISA, and the positive rate of PCV2 antibodies was 73.9% (2428/3284). This study provides a scientific reference for the prevention and treatment of PCV2 in Hebei Province.

Field infection of a gilt and its litter demonstrates vertical transmission and effect on reproductive failure caused by porcine circovirus type 3 (PCV3).

BACKGROUND: PCV3 is a member of the Circovirus family, associated with disease and mortality in pigs. It is not clear whether PCV3 putatively causes clinical symptoms and disease. In the present case, we reported a gilt infected with PCV3 associated with reproductive failures, vertical transmission, tissue lesions, viral replication by in situ hybridization, and the hypothesis that some strains of PCV3 clade one are associated with reproductive failures at the field level. CASE PRESENTATION: In May 2019, a pig farm in Colombia reported increased reproductive failures, and the presence of PCV3 in gilts and sows was established in a single form or coinfections, mainly with PCV2 and PPV7. Ten sows with a single infection with PCV3 were found, and one gilt with a pre-farrowing serum viral load above 103 was studied. This gilt was followed up during the pre-farrowing, farrowing period and on her litter for 6 weeks. During dystocic farrowing, a mummy and ten piglets were released, including two weak-born piglets. The highest viral loads for PCV3 were found in the mummy and the placenta. In the weak-born piglets, there were viral loads both in serum and in tissues, mainly in the mesenteric ganglia and lung. Replication of PCV3 in these tissues was demonstrated by in situ hybridizations. PCV3 was also found in the precolostrum sera of piglets and colostrum, showing vertical transmission. The viral load in piglets decreased gradually until week six of life. The viral genome's complete sequencing was made from the mummy, and its analysis classified it as PCV3 clade one. CONCLUSIONS: This report confirms that PCV3 can cause disease at the field level, and putatively, in this case, we find the generation of reproductive failures. The ability of PCV3 to cause disease as a putative pathogen may be associated with the viral load present in the pig and the strain that is affecting the farm. For this case, we found that viral loads above 103 (4.93 log genomic copies / mL) in the gilt were associated with clinical manifestation and that some PCV3 strains belonging to clade one are more associated with the reproductive presentation.

Porcine Circovirus 3 Detection in Aborted Fetuses and Stillborn Piglets from Swine Reproductive Failure Cases.

Porcine circovirus 3 (PCV-3) has been widely detected in healthy and diseased pigs; among different pathologic conditions, the strongest evidence of association comes from reproductive disease cases. However, simple viral detection does not imply the causality of the clinical conditions. Detection of PCV-3 within lesions may provide stronger evidence of causality. Thus, this study aimed to assess the frequency of PCV-3 detection in tissues from fetuses/stillborn piglets in cases of reproductive problems in domestic swine, as well as the histopathologic assessment of fetal tissues. Fetuses or stillborn piglets from 53 cases of reproductive failure were collected and analyzed by PCV-3 qPCR. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV-2), and porcine parvovirus 1 (PPV1) was also checked. PCV-3 qPCR positive samples with a high viral load were tested by PCV-3 in situ hybridization (ISH), sequenced, and phylogenetically analyzed. PCV-3 DNA was detected in 18/53 (33.9%) reproductive failure cases and in 16 of them PCV-3 was the only pathogen found. PCV-2 DNA was found in 5/53 (9.4%), PRRSV RNA in 4/53 (7.5%) and PPV1 was not detected. Four out of the six PCV-3 qPCR-positive cases with Ct value <30 were positive when tested by ISH. In these samples, PCV-3 was detected within mild histopathologic lesions, such as arteritis and periarteritis in multiple tissues. The present work emphasizes the need to include PCV-3 as a potential causative agent of reproductive failure in swine.

Genetic and phylogenetic analysis of porcine circovirus type 2 on Jeju Island, South Korea, 2019-2020: evidence of a novel intergenotypic recombinant.

Porcine circovirus type 2 (PCV2) is the most ubiquitous viral pathogen of pigs and has persistently affected the global swine industry. Since first being identified in South Korea in 1999, the virus has undergone considerable genetic change and genotype shifts during the past two decades. These events have contributed to the coexistence of genotypes PCV2a, PCV2b, and PCV2d in Korean pig populations, which may promote viral recombination. The genotypic and phylogenetic characteristics of PCV2 strains circulating in pig herds on Jeju Island from 2019 to 2020 were the focus of this study. Genotype-specific PCR indicated that PCV2d is the dominant viral genotype and that coinfections with PCV2d and PCV2a (75%) or PCV2a and PCV2b (25%) are common in provincial pig herds. The complete genome sequences of 11 PCV2 strains, including three PCV2a, two PCV2b, and six PCV2d strains, were determined. A genomic comparison showed that all of the viruses had the highest nucleotide sequence identity to their corresponding genotypic reference strain. Notably, genetic and phylogenetic analysis revealed that one PCV2d strain, KNU-1931, exhibited nucleotide sequence variation in the ORF1 gene when compared to other PCV2d strains but showed a high degree of similarity to the PCV2b strains. Comprehensive recombination analysis suggested that KNU-1931 originated from natural recombination within ORF1 between PCV2b (the minor parent) and PCV2d (the major parent) strains. Our findings provide information about the frequency of genetic recombination between two different PCV2 genotypes circulating in the field domestically, illustrating the importance of continual intergenotypic recombination for viral fitness when multiple genotypes are present.

Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China.

Porcine circovirus 4 (PCV4), a new circovirus with a distinct relationship to other circoviruses, was identified in 2019 in several pigs with severe clinical disease in Hunan Province, China. To investigate the epidemic profile and genetic diversity of the virus, 63 clinical samples were collected from 24 different pig farms in 14 cities in Henan and Shanxi Provinces, China, between February 2018 and December 2019, and the partial Cap gene of PCV4 was amplified by PCR. Among the 63 samples, 16 (25.40%) were positive for PCV4, and 50% (12/24) of the pig farms were positive for PCV4. PCV4 was detected in samples from pigs with different clinical presentations. One PCV4 strain (Henan-LY1-2019) was sequenced in this study, and shared 98.4% genomic nucleotide identity with PCV4 strain HNU-AHG1-2019 (accession no. MK986820) detected on a pig farm in Hunan Province in 2019. A phylogenetic analysis based on the genomes of Henan-LY1-2019 and 31 reference strains showed that the Henan-LY1-2019 strain together with PCV4 strain HNU-AHG1-2019 was grouped in a relatively independent sub-branch, and separated from other viruses in the genus Circovirus. The results of this study extend our understanding of the molecular epidemiology of PCV4.