PCV2 infection has a significant impact on the swine industry, leading to various losses for producers, with subclinical disease being of paramount importance [36]. There are nine recognized PCV2 genotypes: PCV2a, PCV2b, PCV2c, PCV2d, PCV2e, PCV2f, PCV2g, PCV2h and PCV2i [11], [44], [45]. Among these, PCV2a, PCV2b, and PCV2d are currently considered of greater importance and worldwide distribution, with PCV2d being the current predominant genotype [7], [46]. The other groups, including PCV2c, PCV2e, PCV2f, PCV2g, and PCV2h, largely represent small clusters (few isolates) of probably minor significance [47]. PCV2e, in particular, its viral structure contains 12 or 15 extra nucleotides at ORF2 sequences compared to those of PCV2a, PCV2b, and PCV2d. Due to the presence of these extra nucleotides at the 3' end of ORF2, PCV2e was considered a progenitor of PCV2a, 2b, and 2d. This distinct genetic characterization may affect the efficiency of PCV2e in vivo replication and deserves further investigation [47].
Herein, we analyzed clinical PCVD diagnostic samples that previously tested positive for PCV2 at the CEDISA laboratory. In our study, 79.88% (266/333) of the samples were genotyped for PCV2. The results demonstrated that PCV2b was the most prevalent, detected in 151 out of 266 analyzed samples, which is in agreement with several studies worldwide that demonstrate that PCV2b and PCV2d are currently the most prevalent genotypes of PCV2 infection in pigs [18], [37], [41]. Among them, a study conducted in Australia where this virus was detected in 7 out of 17 analyzed samples [41] and another study conducted in Austria in 2020 by [42] showed a higher prevalence of PCV2b in a retrospective report from 2002 to 2017, where PCV2b was found in all studied years, as well as in the retrospective study from 2015 to 2018 by [37] in China. Furthermore, our study showed that PCV2b and PCV2d genotypes were found in analyzed samples from all Brazilian states studied. This finding is similar to a study conducted by [43], where these two genotypes were found in all Chinese provinces analyzed. Previous studies have shown a shift in the most prevalent genotypes in pig production over the years [17] from the PCV2a genotype to PCV2b in 2002 [48], [49], and in 2012, a shift occurred from PCV2b to PCV2d [34], [50]. Since then, PCV2d has become the most prevalent in various regions of the world, such as China [37], South Korea [51], Austria [42], the United States [13], Colombia [52], and the Dominican Republic [53].
The presence of coinfections between genotypes (PCV2b and PCV2d) is becoming increasingly frequent, which was observed here in 27 samples. Previously published data [54] observed coinfections between the PCV2b and PCV2d genotypes in wild boars and domestic pigs in Ukraine. It was also possible to observe coinfection with PCV3 in our study in a total of 26 samples. Again, this result is related to other studies around the world, in which it was also possible to identify coinfection between PCV2 and PCV3 genotypes [33], [43], [55]. However, none of them relate to which PCV2 genotype was the most prevalent in coinfections with PCV3.
Lymph node samples provide a good indication of how PCV2 infection is in pig farms [22]. In our study, genotyping was possible for all lymph node samples received. In addition to careful diagnosis at the time of animal evaluation, the use of high-quality samples is of utmost importance. PCV2b was detected in all types of samples received, suggesting that this genotype causes systemic infection in animals. On the other hand, PCV2d was detected only in organ, lung, fetus, intestine, and blood serum samples. An important finding observed in this study was the identification of PCV2b in brain samples from finishing pigs. Brain lesions caused by PCV2 can be occasional [58]. In our study, it was not possible to correlate the clinical symptoms presented by the animals to the genotype of PCV2 detected.
PCV2d was the most common genotype in the finishing phase. This result is consistent with a study conducted in South Korea, in which [59] found a higher prevalence of PCV2d in pig lymph nodes at slaughter. On the other hand, in the nursery phase, PCV2b was the most common genotype. To our knowledge, this is the first study to find these data and seek a correlation between genotype and production phase. The finishing phase was the phase in which the most positive samples for PCV2 could be obtained and genotyped. This is probably due to the drop in vaccine immunity and maternal immunity, making pigs more susceptible to PCV2 infections [22].
Brazil is the fourth world pork producer, and PCV2 infection occurs broadly in Brazilian pig farms. Thus, PCVD is broadly controlled by commercial vaccines. Since the first detection of PCV2 infection associated with PCVD and reproduction of the disease, the evolution of the disease presentation and PCV2 genotype detection have been shown. A study of Brazilian swine herds analyzed clinical and PCV2-positive samples in 2019 from commercial pig farms from South, Southeast and Midwest Brazil [60]. The pig herds previously vaccinated against PCV2 presented clinical signs compatible with PCVD. A total of 75.47% was identified as PCV2d, 22.64% as PCV2b and 1.89% as coinfection of PCV2b and PCV2d [60]. There was also no detection of the PCV2a genotype. PCV2 clinical disease in vaccinated swine herds leads to the question of virulence variation. Specially after the new genotype identification and differences on disease presentation. However, only a single study comparing the three main PCV2 genotypes side by side found differences in virulence. Nonetheless, this was not repeatable by other groups. Recently, a comparison of the virulence of PCV2a, PCV2b and PCV2d was made in a model of PCV2 coinfection and porcine reproductive and respiratory syndrome virus (PRRSV). Interestingly, in pigs coinfected with PRRSV, PCV2d appeared to be more virulent than PCV2a and PCV2b, which was evident by an increased serum PCV2 load and a greater severity of lymph node depletion [61]. When the same study was repeated with Mycoplasma hyopneumoniae and concomitant infection of PCV2a, PCV2b or PCV2d, the results demonstrated once more that PCV2d was more virulent in the context of coinfection, as measured by serum PCV2 load and severity of lymphoid lesions [62]. In pigs inoculated at 42 days of age and then necropsied at 63 days of age, there were no significant differences between the groups of genotypes evaluated. Pigs inoculated with PCV2a, PCV2b or PCV2d had significant levels of PCV2 loads in the blood and lymph nodes compared to pigs inoculated with PCV2e. The results of this study indicated that PCV2a, PCV2b, and PCV2d are more virulent than PCV2e based on PCV2 blood and lymphoid viral load [63].
Other virulence studies compared PCV2 loads in the blood and lymph nodes and in the severity of lymphoid lesions. The isolated infected groups, PCV2a, PCV2b and PCV2d, resulted in similar virulence to each other, and all were more virulent than the PCV2e groups. Within the doubly infected groups, the combination of PCV2d and PRRSV was more virulent than the other PCV2 genotypes (2a, 2b, and 2e), each in combination with PRRSV [14]. Further studies have shown that the higher virulence of PCV2d compared to the other PCV2 genotypes (2a, 2b, and 2e) can be attributed to an extra amino acid (lysine residue) found in PCV2d ORF2.
PRDC is considered a multifactorial disease caused by the interaction of noninfectious components (environment, management, age, genetics, and nutrition) with viral and bacterial pathogens. Worldwide, the most relevant pathogens in PRDC are influenza A virus (IAV), PRRSV, Mycoplasma hyopneumoniae (Mhyo), PCV2 and pyogenic bacteria [29]. A study of respiratory agents in 74 lugs submitted for diagnosis at CEDISA showed that all lung samples were positive for IAV. Coinfection of IAV and Mhyo was seen in 31% of cases, and 14.9% of the lungs were positive for PCV [29]. In fact, Mhyo potentiated the severity of lymphoid lesions associated with PCV2 and increased the amount of PCV2 loads in the blood and lymph nodes, regardless of the PCV2 genotype [36].
Since there is no consensus on virulence variation among genotypes, PCV2 infection and disease in vaccinated herds remains unknown. There are several research studies on PCV2 vaccination and cross-protection. Most of the published knowledge of cross-protection between PCV2 genotypes results from the use of available vaccines (commonly commercial PCV2a vaccines) in commercial herds with continuous PCV2 field infections. Based on this, there is good cross-protection for many field isolates, such as PCV2b or PCV2d. Severe problems of PCVD in large percentages of pigs in a given herd are rare in vaccinated pig populations today. However, there is evidence that certain PCV2 genotypes, such as PCV2d, can replicate and increase prevalence in vaccinated herds and may be associated with clinical PCVD in swine herds [17], [64]. Many studies using PCV2a vaccines and cross-protection presented generally satisfactory results; that is, vaccinated pigs have a clear advantage over unvaccinated pigs. Such studies used a challenge with PCV2a [65], [66], PCV2b [65], [67] or PCV2d [56], [68]. However, a study that investigated chimeric live PCV1-2a and PCV1-2b found better protection against PCV2b challenge by the homologous vaccine [19], [68], possibly suggesting that homologous protection may be better than heterologous protection. Similarly, a field investigation was conducted in 2012 in the U.S. after PCV2a vaccination was used for approximately 5 years in almost all growing pigs [57]. Interestingly, PCV2b (9.9%) and non-PCV2a were found in the serum of vaccinated healthy pigs, while PCV2a and PCV2b were found in the serum of unvaccinated pigs [57]. In a recent field study, a PCV2d vaccine had some advantages over a PCV2a vaccine in protecting pigs from the simultaneous challenge of PCV2d/PRRSV 1-7-4 and endemic IAV and bacterial coinfections. In unvaccinated pigs, severe PCVD was reproduced [69].
Interestingly, by genotyping PCV2-positive samples from clinical cases, PCV2a was not detected. It is suggested that this is due to the impact of PCV2 vaccines and vaccination pressure [10], [70] favoring the emergence and selection of new genotypes, mainly PCV2b and PCV2d [42], [59]. Obviously, not all vaccines are equal, and there are differences among them, such as adjuvant and production technology, administration, doses, and protection coverage. However, for PCV2 infection, all commercial vaccines show protection, but their ability to control the disease and their use may vary.