By Piñeyro Pablo, Bailey L. Arruda, Eric R. Burrough, Phil Gauger and Rodger G. Main, Iowa State University
Circoviruses are small viruses with a circular, single-stranded DNA genome of approximately 2,000 bases, belonging to the family Circoviridae, genus Circovirus. Three types of porcine circovirus — PCV1, PCV2 and PCV3 — have been described thus far. PCV1 is a cell culture derived virus and is considered nonpathogenic for pigs1. In contrast, PCV2 is the primary etiological agent of porcine circovirus-associated disease which causes severe economic losses in the swine industry worldwide2.
Recently, collaborators from Iowa State University and Kansas State University reported the detection of a new species, PCV3, in typical lesions of porcine dermatitis and nephropathy syndrome, porcine circovirus associated disease and cases of reproductive failure3. Shortly after this description, PCV3 was also detected in a case of multisystemic disease without detection of any other significant pathogen4. PCV3 has subsequently been detected in pigs in most of the swine-producing countries worldwide. Despite the wide distribution of PCV3 and the apparent association with clinical disease similar to PCV2, the clinical implications of PCV3 are still being debated.
Porcine circovirus type 3: First description and current situation
During 2015, a sow farm in North Carolina described reproductive problems including increased numbers of abortions, mummies, stillbirths and low conception rates. The same farm also reported increased mortality in sows with clinical signs resembling those observed during PDNS outbreaks. All routine diagnostic testing for pathogens, including PCV2, was negative; however, metagenomic analysis demonstrated the presence of a new porcine circovirus genetically divergent from the previous PCVs described in pigs. This new PCV shares 37% genetic homology with PCV2. We performed a retrospective investigation that included 74 cases spanning clinical samples from 2010-16 with lesions representative of PDNS and PMWS (post weaning multisystemic wasting syndrome) without polymerase chain reaction or immunohistochemical detection of PCV2 and demonstrated the presence of PCV3 by PCR and immunofluorescence in approximately 65% of tissues evaluated. These findings were the first suggestion of a potential causative role for PCV3 in these two different clinical presentations. Following this first report, the University of Minnesota also presented more clinical evidence of PCV3 as a cause of lesions in pigs, detecting this new virus by in situ hybridization in piglets with myocarditis and multisystemic inflammation4.
After these preliminary reports, a large amount of information has been generated primarily trying to determine the presence and/or prevalence of PCV3 in most swine-producing countries. Numerous retrospective studies and clinical reports from Europe, including Denmark, England, Italy, Poland, Spain and Sweden have described the presence of PCV35-10. A retrospective study in Europe showed the presence of the virus in samples as far back as at least 19935. Virus circulation has also been reported in Brazil, China, Korea and Thailand11-13. The current information from some reports, if not all, lacks consistency of clinical signs associated with the presence of PCV3. For example, in studies conducted in China, Poland and Thailand, the virus has been detected by PCR in respiratory cases from weaning pigs, while other reports from China and Poland linked the presence of the virus with abortions14-16. There are additional reports from China that suggest an association of PCV3 with respiratory disease and cases of diarrhea in weaned pigs17. The information provided in some of these reports was obtained from asymptomatic animals without exhaustive diagnostic testing to rule out the presence of other enteric or respiratory pathogens.
Porcine circovirus type 3: Lesions and diagnosis
Lesions and clinical signs described in association with the presence of PCV3 are not specific. The diversity of clinical syndromes where PCV3 has been detected include respiratory disease, multisystemic disease, neurological clinical signs and reproductive failure. In addition, PCV3 has been described as a coinfection with PCV2, Torque teno sus virus 1 and 2, atypical porcine pestivirus, porcine reproductive and respiratory syndrome virus, porcine deltacoronavirus, porcine epidemic diarrhea virus, porcine kobuvirus, porcine pseudorabies virus, porcine sapelovirus, or as a single infection in some of these clinical presentations18,19.
We performed a retrospective study including 74 cases spanning samples from 2010-16 with lesions representative of PDNS and PMWS without immunohistochemical detection of PCV23. Microscopically, the lungs were characterized by variable degrees of lymphohistiocytic interstitial pneumonia with alveolar histiocytes and multinucleated giant cells and occasional secondary suppurative bronchopneumonia. In sections of skin, we observed severe necrotizing dermatitis with subcutaneous fibrinoid vasculitis (Figure 1). Lymph nodes often had diffuse granulomatous lymphadenitis characterized by cortical lymphoid depletion and follicular infiltration of histiocytes and multinucleated giant cells (Figure 2). Kidneys often had diffuse membranoproliferative glomerulonephritis and glomerulosclerosis. All samples were PCV3 positive by PCR and a large subset confirmed the presence of PCV3 by immunofluorescence. The fact that most of the lesions observed overlapped with different PCV2 clinical presentations make a clinical interpretation of PCV3 challenging.
Numerous laboratories have developed different diagnostic approaches for PCV3 detection. Different types of PCRs and in situ detection methods, such as in situ hybridization, are available for PCV3. At ISU, we offer a duplex PCV2/PCV3 PCR that allows our clients to detect both viruses in the same samples. In addition, PCV3 can be confirmed by a PCV3 in situ hybridization assay that utilizes a unique set of molecular probes targeting the PCV3 cap region and avoids cross-reaction with PCV2. Current data produced at ISU from 331 cases tested for PCV3 during 2017-18 suggest that the PCV3 is broadly endemic in U.S. swine (Figure 3). We have detected PCV3 in fetal lung and heart in a number of abortion cases by PCR in the absence of other known causes of porcine reproductive failures.
The virus has been confirmed by in situ hybridization in the lung, heart and kidney of aborted fetuses previously detected positive by PCR. However, despite these findings no consistent histological lesions in aborted fetuses have been observed. The virus has also been detected by PCR and confirmed by IHC and ISH in kidney and skin in cases with lesions consistent with PDNS (Figure 2). PCV3 is also commonly detected in processing fluids, oral fluids, lung, lymph nodes and serum (Table 1) further confirming its endemic nature. The clinical implications of the PCR findings in these samples remains unclear.
Based on currently available information, it can be concluded that PCV3 is distributed worldwide; however, its clinical implications are still under debate. Based on the diagnostic case submissions received by the ISU VDL, it seems that PCV3 is being more consistently detected in aborted fetuses and tissue with PDNS lesions. The current diagnostic tests are capable of detecting the virus in tissues but more information is necessary to understand the specific role of PCV3 in these clinical syndromes. More research is required to understand the clinical relevance of PCV3, the potential role of PCV3 in abortion and PDNS cases, the impact of PCV2 and PCV3 coinfection, the prevalence and seroprevalence of PCV3, and the role that previous exposure to PCV3, PCV2 or PCV2 vaccination may have on clinical observations.
Acknowledgment: All diagnosticians, faculty and histology and molecular departments staff at ISU-VDL for their collaboration generating most of the data presented in this review.
1. Allan, G., et al., Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2. Virus Research, 2012. 164(1-2): p. 4-9.