Detection and molecular characterization of porcine circovirus type 2 from piglets with Porcine Circovirus Associated Diseases in Colombia

Porcine circovirus type 2 (PCV2), a member of the Circoviridae family, is a small non-enveloped virus, containing a single-stranded circular deoxyribonucleic acid (DNA) genome [1], it is distributed worldwide and is considered to be an important emerging pathogen associated with several different syndromes and diseases in pigs, collectively grouped as porcine circovirus diseases (PCVD) [2]. PCV2 is the major infectious agent of PCVAD in pigs, a multifactorial disease, considered one of the most economically important swine diseases worldwide [3]. PCV2-associated systemic infection is clinically characterized by wasting, dyspnea, and lymphadenopathy and might be associated with diarrhea, pallor, and jaundice [4]. The most relevant histological lesions in this condition occur in lymphoid organs and consist of extensive lymphocytic depletion, macrophage infiltration, a few multinucleated giant cells, and botryoid basophilic cytoplasmic inclusion bodies [5].

PCV2 DNA genome is about 1767–1768 nucleotides long [6] and encodes for three open reading frames (ORFs) [7]. The ORF1 encodes the replication-related proteins: Rep (helicase) and Rep’ (nickase) that is the result of Rep-transcript alternative splicing process [8, 9]. The ORF2 encodes the capsid protein, the only structural viral protein that is also the most variable nucleotide sequence of the viral genome [10, 11]. Finally, the ORF3 embedded within ORF1, encodes a protein that is not essential for viral replication but is fundamental to the development of the viral pathogenesis [12, 13].

PCV2 is classified into two main genotypes, PCV2a and PCV2b which were further subdivided into different clusters, 1A–1C and 2A–2E for PCV2b and PCV2a, respectively [11]. A third genotype, PCV2c, has only been found in Denmark [14]. A new type of PCV referred to as PCV1/2a was reported in Canada in 2009, it was found to be a chimeric virus containing ORF1 of PCV1 and ORF2 of PCV2a [15]. Recently, two additional genotypes PCV2d and PCV2e, were described following sequence analysis of PCV2 isolates from China [16]. However, a subsequent analysis of data sequences failed to support the new genotypes reported [17].

In South America, PCV2 has been circulating in Brazil since 1988 [18], but it was also detected in Argentina [19, 20], and Chile [21]. PCV2-associated systemic infection was first reported in 2007 [22] in Colombia from pig farms located in different regions. In spite of that, genetic information about PCV2 strains in pig herds from the country had been unavailable until now. The main purpose of this work was to establish molecular detection and to achieve genetic and phylogenetic comparisons of full-length sequences of the ORF2 from PCV2 strains recovered from clinically healthy and PCV2-associated systemic infection affected pigs from different production systems in Colombia. Another purpose was to study the viral genotypes dynamics through time to trace temporal changes, in order to understand the molecular epidemiology of PCV2 in Colombia.

PVC2 was detected in Colombian pigs associated with a wide variety of clinical conditions as described previously [22, 23]. In this study, the presence of PCV2 was demonstrated by PCR in 22.4% of the serum and 83.6% of the tissue samples examined. The identification of PCV2 not only in cases associated with PMWS but also in healthy pigs, suggests that various risk factors may contribute to the exacerbation of PCV2 infection and the development of associated lesions. An epidemiological survey of PCVAD conducted between November 2008 and December 2009 in Colombia identified some factors associated with high mortality rates in the three major swine rearing areas in the country [22, 23]. Herds that did not have good management practice, presence of poliserositis in weaned pig and low feed intake resulted in a major risk of increased mortality. In that study it was found that vaccination against PCV2, authorized since 2008, represented a high effective intervention practice on controlling PCAVD outbreak even though PCV2 infection itself wasn’t considered a risk factor for PCVAD development. It remains unclear which factors contributed to maintain a subclinical PCV2 infection in Colombian swine herds and thus it needs to be further investigated.

Unfortunately, samples collected before 2002 were not available at the time of the study, but it is reasonable to assume that PCV2 was already present in the Colombian pig population prior to this time frame. The earliest confirmed detection of PCV2 worldwide was in 1962 in Germany [25], before that, the virus was probably causing subclinical infections or remained unknown for many decades before the description of PCVAD as a disease complex. An alternative to gain access to the knowledge of the PCV2 infection is the use of archived material, it is how in the UK investigations in archived formalin-fixed material established PCV2 detection using TaqMan1-PCR and immunohistochemistry in material originated from the 1970s [26]. The earliest PCV2 infection in Swiss archived material was found in 1986 by using immunohistochemistry, resulting in the recognition of the earliest histological lesions typical for PCV2-associated systemic infection [27]. Similarly, a study from Spain revealed the presence of PCV2 in archived material from 1985 onwards, and the occurrence of typical PCV2-associated systemic infection lesions as early as 1986 [28]. A retrospective study of PCV2 infection in Japan reported seven cases in 1989 [29]. Also, the earliest PCV2 infection in Thailand was reported in 1993 by using Nested PCR from formalin fixed tissues of PCV2-associated systemic infection affected pigs [30].

When considering Colombia, the data presented here showed an increase in the incidence of PCV2b infection between 2006-2007, in that period PCVAD was epizootic and caused problems in numerous farms in several provinces [22]. During that time, PCV2 positive samples by immunohistochemistry were collected from pigs showing characteristic lesions of PCV2-associated systemic infection and some of them were later studied by PCR. As mentioned earlier, 50 cases were collected from pigs with wasting problems during a time period between 2009 -2010. Forty of these samples were PCR positive and PCV2 DNA was identified in the 25.2% of the serum samples collected at that time from healthy pigs. It is well known that detection of PCV2 alone, without the three criteria for diagnostic of PCVAD, does not indicate PCVAD but merely PCV2 infection [3]. However, there was no evidence that could relate the PCV2 strain groups and pathogenic PCV2 isolates from PCV2-associated systemic infection cases and besides that it cannot be concluded that PCV2 isolates from healthy pigs are non-pathogenic.

This study characterized and reconstructed phylogenetic analysis of 23 ORF2 of PCV2 strains obtained from pigs with PCV2-associated systemic infection and healthy pigs during 2002 -2010. Molecular characterization of the isolates was based on the analysis of cap gene. This region is suitable for genotyping studies and is considered a reliable phylogenetic marker for PCV2 strains since it is possible to reconstruct the same tree as with the whole viral genome [11]. Porcine circovirus type 2 (PCV2) is divided into two major genotypes based on sequencing analysis. Recently, both genotypes were proposed and referred to as PCV2a and PCV2b [31].

The alignment of the amino acid sequences of the ORF2 PCV2 capsid protein performed in the present study has identified three major regions of amino acid heterogeneity located at amino acid positions 57 -89, 121 – 134 and 190- 210 within heterogenic regions (Figure 3) similar to previous reports [24, 32]. It is interesting to note that two of these regions (57-89 and 121-134) correspond with two dominant immunoreactive areas (65-87 and 113-139) as identified by Pepscan analysis [32]. These immunodominant regions of the capsid protein of PCV2 exposed to selective immune pressure could represent potential candidate regions involved in the emergence of PCV2 variants. However, no repeatable or characteristic amino acid motifs for these two regions of the capsid protein of PCV2 could be associated with strains identified from pigs with PCV2-associated systemic infection or healthy pigs. Whether the anti-PCV2 antiserum generated from Colombian PCV2 strains could recognize the same epitopes in strains from other countries is not yet known but the results presented here contribute to the knowledge of the variability of the immunoreactive regions among PCV2 strains.

In terms of PCV2 genotype and its dynamics over time, there was not a relationship between the genotype of PCV2 and year of detection. Among the 23 Colombian PCV2 strains in this study, the strain CO6602 collected in 2002 belonged to the genotype PCV2b, whereas in the period from 2002 to 2010, in spite of being genotype 2b more prevalent, the isolates were a mix of genotypes PCV2a and PCV2b. The results presented here suggest that PCV2b has become the main genotype acting in Colombia over time. Previous studies revealed that both genotypes were associated with PCVAD-affected and non-affected herds [24, 33‐35]. Nevertheless, PCV2b is currently prevailing in naturally occurring infections worldwide [36], a similar situation could be occurring in Colombian pig populations.

Furthermore, several recent publications have reported a shift from the genotype PCV2a to PCV2b which might be related to the occurrence of PCVAD outbreaks in Canada [37], Sweden [38], Switzerland [35] and Spain [39], indicating that PCV2b may be more virulent than PCV2a. However, in Colombia PCV2b has been present since 2002 in healthy animals and then it was associated to the PCVAD epizootic occurrence in farms of several regions of the country in the period 2006 – 2009. Nevertheless, in farm 2 a shift from PCV2a (2005) to PCV2b (2006) was found and, in farm 18 the variation was from PCV2b (2006) to PCV2a (2009). In addition, the PCV2b strains CO6602 and CO2906 collected in the same farm in the years 2002 and 2006 respectively showed less than 1.3% differences in the amino acid sequence of ORF2. It is well known that Colombia keeps a wide commercial exchange with North American countries, which includes the import of live animals and semen, so it is not surprising that the strains analyzed in this work were found to be closely related to Canadian and American strains isolated between 2004 - 2010, sharing 74,7% - 100% identity at the amino acid level. This coincides with findings in some countries, where the presence of PCV2 has been linked to imported pigs [40] and the movement of asymptomatic PCV2- infected pigs that occurs as a result of the swine trading which has been suggested to be responsible for the rapid spread of PCV2 around the globe. Unfortunately, there is a lack of information regarding the origin of breeding animals in the herds which limits the capacity to shed light over the potential source of infection and why it is not possible to determine the exact year of introduction of PCV2 in Colombian swine farms.

In Colombia, through the results presented here, it has been demonstrated that PCV-2 is associated with PCVAD. In this study, 23 ORF2 strains of PCV-2 were obtained from animals with confirmed diagnosis of systemic and subclinical infection originated from different farms in a time frame from 2002 to 2010. Although in South America data regarding molecular characterization of PCV2 strains is still scarce, based on genotyping studies it has been concluded that PCV2b is the predominant circulating genotype in the region. However in Colombia there was a mixture of both genotypes during the epizootic, but recently PCV2b became more common in cases of PCVAD. This finding contributes to the understanding of the molecular epidemiology of PCV2 in Latin-American countries and may also help to establish the bases necessary to study the emergence of new viral variants in this region.