Novel Porcine Circoviruses in View of Lessons Learned from Porcine Circovirus Type 2-Epidemiology and Threat to Pigs and Other Species.

Porcine circovirus type 2 (PCV2) plays a key role in PCV2-associated disease (PCVAD) etiology and has yielded significant losses in the pig husbandry in the last 20 years. However, the impact of two recently described species of porcine circoviruses, PCV3 and PCV4, on the pork industry remains unknown. The presence of PCV3 has been associated with several clinical presentations in pigs. Reproductive failure and multisystemic inflammation have been reported most consistently. The clinical symptoms, anatomopathological changes and interaction with other pathogens during PCV3 infection in pigs indicate that PCV3 might be pathogenic for these animals and can cause economic losses in the swine industry similar to PCV2, which makes PCV3 worth including in the differential list as a cause of clinical disorders in reproductive swine herds. Moreover, subsequent studies indicate interspecies transmission and worldwide spreading of PCV3. To date, research related to PCV3 and PCV4 vaccine design is at early stage, and numerous aspects regarding immune response and virus characteristics remain unknown.

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.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.

First detection and genomic characterization of porcine circovirus 3 in mosquitoes from pig farms in China.

The porcine circovirus type 3 (PCV3) becomes an important causative agent of swine disease since its discovery in 2016. PCV3 infection exhibits a wide range of clinical syndromes causing substantial economic losses in swine industry. Previous studies have reported the detection of numerous known viruses including circovirus in mosquitoes. However, the transmission of PCV3 in field-caught mosquitoes remains largely unknown. This study aims to detect PCV3 infection in mosquitoes and analyze its genomic characteristics. Here, we performed a PCR to detect the PCV3 in 269 mosquito samples collected from pig farms located in Heilongjiang, Jilin, and Yunnan provinces. The proportion of PCV3-positive mosquitoes was 32.0 % (86/269), ranging from 21.4%-42.5% at farm level, which may imply that mosquito serves as a route of transmission for PCV3. To determine the possible origin of PCV3 in mosquitoes, 80 pig serum samples were collected from the pig farms where mosquito sampling was also performed. The proportion of PCV3-positive farms ranged from 15.0%-30.0 % in which infection of positive pigs positively correlated with mosquitoes carrying the virus. Additionally, we sequenced the entire genome of 6 strains of PCV3 in mosquitoes and 2 strains of PCV3 in pigs. Sequence analysis indicated a 100 % nucleotide similarity between mosquito and pig viral isolates that were all collected from similar farms. Phylogenetic analysis showed that PCV3 could be divided into two clades, PCV3a and PCV3b, and the PCV3 strains isolated in mosquitoes were distributed on the two clades. Our results demonstrate that mosquitoes may serve as a potential transmission vector in the life-cycle of PCV3, revealing possible transmission routes of PCV3.

Environmental distribution of Porcine Circovirus Type 2 (PCV2) in swine herds with natural infection.

Porcine circovirus type 2 (PCV2) is the aetiological agent of PCV2-Systemic Disease (PCV2-SD) and PCV2-Subclinical Infection (PCV2-SI). PCV2 is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance. The aim of this study was to identify, under field conditions, the possible critical points in the environment of non-vaccinated farrow-to-weaning swine farms where PCV2 could accumulate and persist. For that, environmental samples from five swine farms with PCV2-SD or PCV2-SI were taken and analysed by qPCR, including different farm areas, farm personnel and management implements. PCV2 DNA was detected in the environment of all farms (42.9% of positive samples). Overall, the PCV2-SD herd seemed to present more positive samples and higher viral loads than the PCV2-SI herds. At individual farm level, weaning areas appeared to be the most contaminated facilities. In addition, PCV2 was found at high levels in most samples from farm workers, especially work boots, suggesting that they may play a role in within-farm transmission. In addition, PCV2 was detected in areas without animals the like warehouses, offices and farm perimeter. Therefore, this study is helpful to improve measures to reduce within-farm PCV2 dissemination.

Transmission of Porcine Circovirus 3 (PCV3) by Xenotransplantation of Pig Hearts into Baboons.

Porcine circovirus 3 (PCV3) is a newly described member of the virus family Circoviridae. PCV3 is highly distributed among pigs and wild boars worldwide. A sudden introduction of PCV3 was recently observed in a herd of triple genetically modified pigs generated for xenotransplantation. These animals were used as donor pigs for orthotopic heart transplantation into baboons. In four cases, PCV3-positive hearts were transplanted, and transmission of PCV3 to the recipient was observed. PCV3 was found in all organs of the recipient baboons and a higher virus load was found in animals with a longer survival time of the transplant, indicating replication of the virus. This is the first report showing trans-species transmission of PCV3 to baboons by transplantation of a heart from a PCV3-positive donor pig. Sequence analysis showed that PCV3a and PCV3b were present in the infected pigs and were transmitted. Experiments to infect human 293 cells with PCV3 failed.

Phylogenetic analysis of porcine circovirus type 2 in Sardinia, Italy, shows genotype 2d circulation among domestic pigs and wild boars.

Porcine circovirus type 2 (PCV2) is associated with multi-factorial syndromes, commonly known as porcine-circovirus-associated diseases, which cause severe economic losses in the swine industry worldwide. Four genotypes (PCV2a, PCV2b, PCV2c, and PCV2d) have been identified. Lately, the prevalence of PCV2d has been increasing in many countries, thereby prefiguring a global replacement of PCV2b. Wild boars are also susceptible to PCV2 infection, with virus prevalence similar to that of domestic pigs. This work was aimed at expanding the knowledge about the molecular epidemiology of PCV2 in Italy. For this purpose, we analysed 40 complete ORF-2 sequences from PCV2 strains isolated from domestic pigs and wild boars in Sardinia (Italy) over a period of 5 years (2009-2013). Phylogenetic and Bayesian analyses were performed on three data sets compiled from DNA sequences over a large geographical area. PCV2b was found to be dominant in Sardinia, whereas no PCV2a and PCV2c were found. This study indicates the presence of genotype PCV2d-2 infecting both domestic and wild pigs, thus confirming its circulation in Italy. Sardinian sequences clustered mostly with Italian isolates and with strains from China, Belgium, Croatia, Taiwan, Korea, and Portugal. Genetic variability of PCV2 in Sardinia appears to be a result of both local viral evolution and different epidemic introduction events.

Porcine circovirus 2 (PCV-2) genetic variability under natural infection scenario reveals a complex network of viral quasispecies.

Porcine circovirus 2 (PCV-2) is a virus characterized by a high evolutionary rate, promoting the potential emergence of different genotypes and strains. Despite the likely relevance in the emergence of new PCV-2 variants, the subtle evolutionary patterns of PCV-2 at the individual-host level or over short transmission chains are still largely unknown. This study aimed to analyze the within-host genetic variability of PCV-2 subpopulations to unravel the forces driving PCV-2 evolution. A longitudinal weekly sampling was conducted on individual animals located in three farms after the first PCV-2 detection. The analysis of polymorphisms evaluated throughout the full PCV-2 genome demonstrated the presence of several single nucleotide polymorphisms (SNPs) especially in the genome region encoding for the capsid gene. The global haplotype reconstruction allowed inferring the virus transmission network over time, suggesting a relevant within-farm circulation. Evidences of co-infection and recombination involving multiple PCV-2 genotypes were found after mixing with pigs originating from other sources. The present study demonstrates the remarkable within-host genetic variability of PCV-2 quasispecies, suggesting the role of the natural selection induced by the host immune response in driving PCV-2 evolution. Moreover, the effect of pig management in multiple genotype coinfections occurrence and recombination likelihood was demonstrated.

Molecular assessment of visitor personal protective equipment contamination with the Aleutian mink disease virus and porcine circovirus-2 in mink and porcine farms.

Personal protective equipment (PPE) is an element of biosecurity intended to prevent the access or spread of diseases in farms. Nevertheless, to date no extensive reports exist about the effectiveness of different available PPE on farms. Thus, our aim was to estimate the degree of protection of PPE from viral contamination during farm visits. Two farms, infected with Aleutian mink disease virus and porcine circovirus-2 respectively, were visited by six visitors wearing different combinations of PPE: coveralls with hood and bootcovers, both with a certified barrier to infective agents (certified PPE group) and non-certified bootcover and coverall without hood (non-certified PPE group). Seventy-two swab samples from PPE and both hair and street clothes under PPE were taken after the visit and analysed by qPCR. Our results reveal viral exposure during visits, and the external protections of body and shoes were contaminated in all cases (24/24). In addition, protection from viral contamination varied noticeably according to the biosecurity elements used. A higher number of positives were detected in the non-certified PPE group than in the certified PPE group, both in elements under external protections (14/18 vs 3/18) and also in hair (4/6 vs 0/6). In fact, non-certified bootcovers broke during visits, resulting in viral contamination of the internal elements under them; these are consequently not suitable for using with wrinkled surfaces usually found in farm facilities. Thus, certified coveralls should be used in order to prevent contaminations, and workers and personnel of farms should be trained in their proper use. qPCR is a useful tool in the risk management of biosecurity programmes, and our results may serve as a model to evaluate different biosecurity measures.

High prevalence of PCV2d in Hunan province, China: a retrospective analysis of samples collected from 2006 to 2016.

Porcine circovirus 2 (PCV2) has been widely prevailing in China since the first report in 2001, causing huge economic losses to the pig industry. In the present study, 674 samples were collected from 2006 to 2016 in Hunan province, and 62% were positive for PCV2. An increase was observed from 2006 to 2011 (72.1%-89.1%), and a decrease was observed from 2012 to 2016 (78.9%-36.8%). The prevalence of genotype PCV2a, PCV2b, and PCV2d was 0, 44.7% and 67%, respectively. During 2006-2007, PCV2b was the main genotype circulating in Hunan, while, in 2008, PCV2d became the predominant one. Coinfection with PCV2b and PCV2d was observed frequently, and the positive rates of coinfection ranged from 6.3% to 18.9% during 2006-2016. The complete genome was sequenced for 54 positive samples, and four were identified as PCV2b-1, 22 as PCV2b-2, four as PCV2d-1 and 24 as PCV2d-2, based on phylogenetic analysis of the complete genome and ORF2 region. Recombination analysis using the complete genome sequences of these isolates revealed a high recombination rate of 27.7% (17/54), and showed that recombination occurred mainly in the ORF1 region. This shows that the prevalence of PCV2 has clearly decreased in recent years and that PCV2d has become a predominant genotype since 2008. In addition, frequent recombination events were observed in the PCV2 isolates from Hunan, China.

First findings of duck circovirus in migrating wild ducks in China.

To probe the epidemiology of duck circovirus (DuCV) in migrating wild ducks in China, 189 samples collected from 11 species of wild ducks from 2013 to 2016 were analyzed by PCR. Four positive samples were obtained from Mallard (2), Green-winged Teal (1), and Falcated Duck (1), and the positive rate was 2.12%. The homologous alignment of the complete genome shows that the homology of the wild duck strains is 81.6 to 95.1% to each other and 81.4 to 100% to the 32 strains from other origins. Amino acids alignment revealed that the mutable ORF2 gene has six major variable regions and many random mutation points. Phylogenetic analysis showed that the four sequences belong to two genotypes: wd2013017, wd2013046 and AY228555 (representative strain of genotype 1) belong to genotype 1, while wd2014012, wd2015028 and AY394721 (representative strain of genotype 2) belong to genotype 2. The results indicate that both genotypes of DuCV are distributed and common in migrating wild ducks. Further analysis shows that duck circovirus infection is mainly concentrated in the eastern coastal cities of China, which are part of the East Asian-Australian flyway. This suggests that wild ducks with circovirus may be an important factor in the epidemic and spread of DuCV.

Genome sequence characterization of pigeon circoviruses in China.

Pigeon circovirus (PiCV) was detected by PCR in pigeons from China. Altogether, 48 out of 244 pigeons tested positive for PiCV (positive rate, 19.67%), suggesting that the virus was prevalent in China. From the 48 PiCV-positive samples, about 2040bp complete genome fragments were obtained by full length genome amplification and sequenced with a next-generation sequencing platform. Characteristics of the ORFs from different PiCV strains tested in this study were analyzed. Several insertion, deletion or substitutions were discovered during the analysis of the nucleotide sequence compared with sequences reported previously. In phylogenetic tree analysis, 48 sequences isolated in this study could be further divided into five clades (A, B, C, D, and F), clade E includes reference sequences only. Two major groups were found in the six clades, distinguished by ATA and ATG initiation codons. Most of the viruses isolated in the study were in the ATG group, with fewer in the ATA branch.

Goose Parvovirus and Circovirus Coinfections in Ornamental Ducks.

Clinical observations and diagnostic procedures carried out to elucidate the cause of high mortality in 2-8-wk-old ornamental ducks (mandarin, wood, falcated, and silver teal ducks) are described. At necropsy, ducklings showed general pallor of skeletal and heart muscles, subcutaneous gelatinous transudates, pericarditis, ascites, and severe edema and hyperemia of lungs. Histopathologic examination revealed that the most important changes were located in the crop, bursa of Fabricius, and lungs with presence of amorphic basic intracytoplasmic inclusions. No bacteria or fungi could be detected from affected organs and ascitic fluid. Viral diagnosis included molecular detection for the presence of goose parvovirus (GPV), circovirus, avian influenza, herpesviruses, paramyxovirus, reovirus, and polyomavirus. Both GPV and circovirus could be detected by real-time PCR and nested broad-spectrum PCR, respectively. Phylogenetically, full-length nucleotide sequence of GPV showed a close similarity ranging from 95.6% to 97.9% with European and Asian pathogenic GPV. On the other hand, the detected circovirus showed nucleotide identity of 90% to 98% with goose circoviruses (GoCVs). This is the first report of GoCVs and GPV in ornamental ducks. The concurrence of GPV and GoCV infections is thought to contribute to the high mortality.

A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions.

Transmission characteristics of PCV2 have been compared between vaccinated and non-vaccinated pigs in experimental conditions. Twenty-four Specific Pathogen Free (SPF) piglets, vaccinated against PCV2 at 3weeks of age (PCV2a recombinant CAP protein-based vaccine), were inoculated at 15days post-vaccination with a PCV2b inoculum (6⋅10(5) TCID50), and put in contact with 24 vaccinated SPF piglets during 42days post-inoculation. Those piglets were shared in six replicates of a contact trial involving 4 inoculated piglets mingled with 4 susceptible SPF piglets. Two replicates of a similar contact trial were made with non-vaccinated pigs. Non vaccinated animals received a placebo at vaccination time and were inoculated the same way and at the same time as the vaccinated group. All the animals were monitored twice weekly using quantitative real-time PCR and ELISA for serology until 42days post-inoculation. The frequency of infection and the PCV2 genome load in sera of the vaccinated pigs were significantly reduced compared to the non-vaccinated animals. The duration of infectiousness was significantly different between vaccinated and non-vaccinated groups (16.6days [14.7;18.4] and 26.6days [22.9;30.4] respectively). The transmission rate was also considerably decreased in vaccinated pigs (ß=0.09 [0.05-0.14] compared to ß=0.19 [0.11-0.32] in non-vaccinated pigs). This led to an estimated reproduction ratio of 1.5 [95% CI 0.8 - 2.2] in vaccinated animals versus 5.1 [95% CI 2.5 - 8.2] in non-vaccinated pigs when merging data of this experiment with previous trials carried out in same conditions.

Preliminary evidence of age-dependent clinical signs associated with porcine circovirus 2b in experimentally infected CH3/Rockefeller mice.

Mice and rats are susceptible to porcine circovirus 2b (PCV2) infection under field and experimental conditions. However, whether PCV2 induces disease in rodents remains a matter of debate. The objectives of the present study were to determine whether PCV2-induced disease in mice is age-dependent and whether intranasally inoculated animals are able to infect animals they come into contact with. Twenty-five CH3/Rockefeller mice were divided into six groups and intranasally inoculated with 25µL of either PCV2b or PBS on days 0, 3 and 6. One group remained untreated. Two age groups were tested: 3-week-old mice and 6-week-old mice. The administration of three PCV2 intranasal inoculations at intervals of three days was able to induce infection and support virus transmission in susceptible mice, regardless of the age at inoculation. The clinical signs associated with PCV2 infection were more severe in younger mice, and PCV2-DNA load was higher in their faeces. In conclusion, PCV2 induced disease in mice.

Evidence of possible vertical transmission of duck circovirus.

To test the hypothesis that duck circovirus (DuCV) may be vertically transmitted from infected breeder ducks to their ducklings, we investigated 120 newly hatched ducklings, 30 dead duck embryos and 80 non-embryonated duck eggs with the duplex polymerase chain reaction (PCR). DuCV DNA was present in 15 newly hatched ducklings, 4 duck embryos and 3 non-embryonated eggs. Four ducklings from two flocks were co-infected by DuCV-1 and DuCV-2, three ducklings from three flocks were DuCV-1 single infection, and eight ducklings from six flocks were DuCV-2 single infection. One duck embryo and one non-embryonated egg were positive for both DuCV-1 and DuCV-2 DNAs, one embryo for DuCV-1 DNA, and two embryos and two non-embryonated eggs for DuCV-2 DNA. The findings provide evidence of possible vertical transmission of DuCV and simultaneous transmission of DuCV-1 and DuCV-2 from breeder ducks to ducklings.

Mutability dynamics of an emergent single stranded DNA virus in a naïve host.

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10(-3) subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.

Multiple routes of porcine circovirus type 2 transmission to piglets in the presence of maternal immunity.

Porcine circovirus 2 (PCV2), the cause of porcine circovirus-associated disease (PCVAD), is widespread in swine farms throughout the United States with vaccine controlling disease, but not eliminating infection. We examined the PCV2 virological and immunological status of sows, pre-suckling piglets, and the farrowing environment of sow farms to determine PCV2 exposure risks, transmission dynamics, and immunological impacts at the time of farrowing. PCV2 was widely distributed in animals and the farrowing environment of 6 midwestern US sow farms irrespective of sow vaccination status. High levels of PCV2 capsid-specific antibodies were observed in sow serum and colostrum and had no apparent effect on PCV2 transmission to and infection in piglets. In 281 pre-suckling piglets from 59 sows, PCV2 DNA was detected in 63% of serum samples and on 93% of axillary skin swabs. PCV2 was present in one or more samples from 58 of 59 sows and in the farrowing environment. Isolated infectious virus samples from sows, presuckling piglets, and the environment were shown by sequencing to be genetically similar from all farms. In conclusion, piglets are readily infected with PCV2 in utero and are under constant challenge by PCV2 through contact with infected sows and a contaminated farrowing environment. However, maternal immunity did not affect PCV2 transmission to piglets or the viral load in sows. These findings illustrate the importance of maternal infection, despite robust anti-PCV2 immunity, in early infection of newborn piglets, and the need to develop appropriate infection models for elucidation of mechanisms of protective immunity.