Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome.

A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.     

A review of porcine circovirus 2-associated syndromes and diseases

Clinical expression of porcine circovirus 2 (PCV2) infection in swine may result in several distinct syndromes and diseases including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, porcine respiratory disease complex, granulomatous enteritis, necrotizing lymphadenitis, and possibly exudative epidermitis. Association of PCV2 with congenital tremor in piglets is still controversial. The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. This review concentrates on PCV-2-associated syndromes and diseases other than PMWS.     

Transmission of different variants of PCV2 and viral dynamics in a research facility with pigs mingled from PMWS-affected herds and non-affected herds

Post-weaning Multisystemic Wasting Syndrome (PMWS) has been identified in most swine-producing countries worldwide. The disease has resulted in significant health challenges and economic damage to the swine industry. The aim of this study was to determine horizontal transmission of porcine circovirus type 2 (PCV2) and to examine viral dynamics in pigs in a controlled PMWS transmission study. In the study pigs from PMWS-affected herds and non-affected herds were permitted to have close contact (same pen), nose-to-nose contact (to pigs in neighbouring pens) or no physical contact (pen across the aisle and pens in other compartments). By DNA sequence analysis, eight variants of genotype PCV-2b were identified in the research facility. From the spread of these PCV2-variants it was concluded that PCV2 primarily infects through close contact and nose-to-nose contact. PCV2 genome sequences were obtained from selected pigs at arrival to the research facility and again when the same pigs developed PMWS. This analysis showed that pigs from PMWS-affected herds developed PMWS caused by the same variant of PCV2 as they carried when entering the research facility. In contrast, pigs from non-affected herds developed PMWS with PCV2-variants identified in pigs from PMWS-affected herds. This was probably connected to at least 10³ higher mean serum-titer of PCV2 in pigs from PMWS-affected herds as compared to pigs from non-affected herds at the beginning of the transmission study. The study further showed that pigs able to control the PCV2 infection, as measured by the PCV2-titer in serum, recovered clinically (pigs from PMWS-affected herds) or stayed healthy (pigs from non-affected herds). Like this, pigs with a PCV2 titer below 5 × 10⁸ copies/ml serum during the study period had a chance of recover from the PCV2 infection whereas pigs with PCV2 titers above 5 × 10⁸ copies/ml serum at any time point generally died from PMWS.     

Recent advances in the epidemiology,diagnosis and control of diseases caused by porcine circovirus type 2

Post-weaning multisystemic wasting syndrome (PMWS) emerged as a significant disease affecting pig production in the 1990s although the causal agent, porcine circovirus type 2 (PCV2), and the disease itself, had existed in swine for many years prior to this. The important multifactorial ‘triggers’ of PMWS include the immune and infection status of the sow, the timing of PCV2 infection, variations in the virulence of PCV2, co-infections, immune modulation as well as host genetics and management factors. In terms of diagnosis, histopathological examination and the detection of PCV2 within lymphoid tissue remains the ‘gold standard’ as quantitative PCR techniques are currently not specific or sensitive enough. The recent commercial availability of PCV2 vaccines provides an excellent tool for reducing the impact of PMWS and other porcine circovirus-related diseases. This review assesses recent advances in the epidemiology, diagnosis and control of PMWS.     

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.     

Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Recently, the disease has been reproduced with inocula containing a newly described porcine circovirus (PCV), designated PCV 2, and porcine parvovirus (PPV). In order to determine if these viruses interact in naturally acquired PMWS, affected tissues from field cases were examined by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for PCV 2 and PPV, as well as by PCR for the other recognized porcine circovirus, PCV 1. Porcine circovirus 2 was detected by PCR or IHC in affected fixed or frozen tissues from 69 of 69 cases of PMWS collected over 3 years from 25 farms. Porcine parvovirus was detected in 12 of the same cases, and PCV 1 was detected in 9 of 69; however, an apparent decrease was found in the sensitivity of the PCRs used to detect the latter 2 viruses when fixed tissue from the same cases were compared with the use of frozen tissues. Porcine circovirus 2 was not detected by PCR in affected tissues from 16 age-matched pigs that had Streptococcus suis-associated disease. Electron microscopic examination of plasma pooled from 15 pigs with PMWS revealed the presence of PCV and PPV, whereas these viruses were not observed in pooled plasma from 5 age-matched clinically normal pigs. These results confirm and extend previous findings documenting a consistent association of PCV 2 with PMWS. As well, infection by PPV or PCV 1 or both may be an important cofactor in the pathogenesis of some, but apparently not all, cases of PMWS.     

Porcine circovirus type 2 and associated diseases in Romania--short communication

Porcine circovirus type 2 (PCV2) has been demonstrated to be the causal agent for postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). This report describes the first detection of PCV2 and associated diseases in a Romanian swine herd located in Transylvania. The clinical signs, pathological and histopathological changes observed in affected pigs were similar to those previously described for PDNS and PMWS. Polymerase chain reaction and in situ hybridisation were used for the detection of PCV2 nucleic acids from tissues and serum samples. Complete PCV2 genomes of both PMWS and PDNS cases were sequenced and analysed, and by comparing them with each other no genomic differences could be detected. The sequence analysis showed that the Romanian PCV2 was closely related to PCV2 identified in France and in Hungary.     

Detection and molecular characterization of porcine circovirus type 2 (PCV2) from piglets with exudative epidermitis in Uruguay

Porcine circovirus type 2 (PCV2) is an economically important emerging pathogen associated with distinct syndromes and diseases in swine, collectively known as porcine circovirus associated diseases (PCVAD). The main purpose of this study was to investigate the presence of PCV2 in piglets affected with exudative epidermitis (EE) in Uruguay. In addition we aimed to analyze the phylogenetic relationships of the isolated strains. In June 2011 an outbreak of EE detected in a small herd was reported. Piglets presented skin lesions compatible with EE and symptoms associated with postweaning multisystemic wasting syndrome (PMWS) were also observed. Sera from affected and healthy animals were tested for the presence of viral DNA. Exclusively, diseased piglets were infected with PCV2. Phylogenetic analysis showed that PCV2 isolates belonged to PCV2b genotype. We report the detection and molecular characterization of PCV2 strains for the first time in Uruguay.     

Temporal distribution of porcine circovirus 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland.

Porcine circovirus type 2 (PCV2) is now recognized as the essential infectious component of porcine postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in high-status, specific pathogen-free pigs in Canada in 1991 and is now an economically important disease that affects the swine industry around the world. Recently, reports of genomic studies on PCV2 viruses indicated that 2 distinctive genogroups of PCV2 exist.4,10 This report involves the results of a study on the distribution of predominant PCV2 genogroups recovered from samples taken from PMWS-affected and PMWS-nonaffected farms on the island of Ireland over a 9-year period and the results of a study on PCV2 genogroup recovery from fecal samples taken from a farm in Northern Ireland from 2003 to 2005 that was first diagnosed as PMWS positive in August 2005. The results indicate that, although at least 2 distinct genogroups of PCV2 have been circulating on pig farms on the island of Ireland, there does not appear to be a direct relationship between infection with these different genogroups of PCV2 and the development of PMWS.     

The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: a case-control study

In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

A proposal on porcine circovirus type 2 (PCV2) genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). Despite first sequencing studies did not find any association between PCV2 sequences and PMWS occurrence, recent works have suggested the opposite. In the present study, 87 open reading frame 2 (ORF2) sequences obtained from pigs with different clinical conditions and coming from farms with different PMWS status were analyzed. Results further confirmed the existence of two genogroups and the definition of two PCV2 genotypes (1 and 2) is proposed. All sequences included in genotype 1 came from pigs from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Moreover, infection of single pigs from PMWS affected farms harbouring both genotypes is described. Present results suggest that PCV2 genotype 1 may potentially be more pathogenic than PCV2 genotype 2.     

猪圆环病毒研究进展

近年来猪圆环病毒感染给养猪业的健康发展带来巨大威胁。猪感染圆环病毒后可引起的疾病有猪断奶后多系统衰弱综合征(PMWS)、猪皮炎和肾病综合征(PDNS)、猪呼吸系统衰弱综合征(PRDC)等。近年来,基于PMWS而对PCV所做的研究工作不断深入,对病毒的相关特性有了较为清晰的认识。论文就近年来猪圆环病毒的致病性、流行病学、诊断及防控等方面的研究进展做一综述。     

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.     

Porcine circovirus 2-associated disease in Eurasian wild boar.

Porcine circovirus 2 (PCV2) was first identified in high-health herds of domestic swine and was associated with a debilitating disease called postweaning multisystemic wasting syndrome (PMWS). Most subsequent studies have indicated that PCV2 infects only swine but there is little information on porcids other than improved breeds of domestic swine. Multisystemic disease was reported in a group of Eurasian wild boars raised under free-range conditions. Affected young pigs had pneumonia and enteritis and were cachectic. Porcine circovirus 2 was identified in affected tissue by immunohistochemistry and in situ hybridization, and a PCV2-like virus was isolated from pooled organs. The open reading frame (ORF2) of the isolated PCV2 had a 98.7% homology with the ORF2 of a reference PCV2 isolate. These diagnostic data indicate that PCV2 can infect and cause disease in Sus scrofa subspecies other than domestic swine.     

Comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome

A comparative serologic and virologic study was performed in pigs from 5 herds with postweaning multisystemic wasting syndrome (PMWS) and 2 herds without PMWS in Quebec. In each herd, 60 blood samples were collected at 4-wk intervals from pigs from 3 to 23 wk of age. The serum was evaluated for the presence of antibodies to porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as for the presence of nucleic acid of PCV2, PRRSV, and porcine parvovirus (PPV), by means of the polymerase chain reaction (PCR). Serologic profiles for PCV2 were very similar in 6 of the 7 herds, including the 2 without PMWS, and were characterized by a gradual decrease in antibody titres from 3 until 11 wk of age, followed by seroconversion at 15 wk, and high PCV2 antibody titres thereafter in all pigs. Only starting at 11 to 15 wk of age could PCV2 viremia be detected, except in 1 herd, in which clinical signs were observed at 6 to 7 wk of age. A PCV2 viremia could be detected within the same pigs for a minimum of 8 wk, and the virus could still be detected in 41% of the serum samples obtained at 23 wk of age. The antibody level did not appear to influence the occurrence of disease, since titres were similar in pigs in the herds with or without PMWS. Infection with PRRSV, as demonstrated by PCR and seroconversion, preceded that of PCV2 by at least 1 mo in both types of herd. Both PRRSV and PCV2 were detected in some pigs in 5 of the 7 herds, including 1 herd without PMWS. Porcine parvovirus could be detected in serum by PCR in 2 herds with PMWS after the onset of clinical signs and also in 1 herd without PMWS. Genomic analysis of PCV2 strains identified in the herds without PMWS indicated complete or very high homology (99.4% to 100%) with the PCV2 strains identified in 4 herds with PMWS. In our field study, the triggering of PMWS in the herds could not be linked to coinfection with either PRRSV or PPV or to the use of a specific immunostimulant, such as vaccines, or to particular genomic differences between the PCV2 strains identified.     

猪圆环病毒感染及动物模型研究进展

1974年德国学者Tischer从多株连续传代的猪肾细胞(PK-15)中发现了猪圆环病毒(PCV),其致病性及其疾病直到1991年才由Clark报道,称为断奶仔猪多系统衰竭综合征(PMWS).随后,许多学者对PCV-2感染以及PMWS的动物模型建立进行了广泛研究,证实了免疫激发及PCV-2与其他病原混合感染对PMWS的...     

Study of the genetic variability of porcine circovirus type 2 detected in Serbia and Slovenia

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.