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Porcine Circovirus: A Historical Perspective J. Ellis Vet Pathol 2014 51: 315 DOI: 10.1177/0300985814521245 The online version of this article can be found at: http://vet.sagepub.com/content/51/2/315

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Review

Porcine Circovirus: A Historical Perspective

Veterinary Pathology 2014, Vol. 51(2) 315-327 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985814521245 vet.sagepub.com

J. Ellis1

Abstract Porcine circoviruses (PCVs) belong to the genus Circovirus and the family Circoviridae, and they are the smallest known viruses that replicate autonomously in mammalian cells. They are nonenveloped, and they have characteristic single-stranded, negative-sense, circular DNA. Two types of divergent PCVs are recognized: PCV1 and PCV2. About 20 years ago, PCV2 began to emerge as a major pathogen of swine around the world, leading to burgeoning knowledge about the virus and porcine circovirus–associated diseases. However, much of the history of its discovery, including the controversy related to its importance, is not recorded. This review examines current issues related to the biology of PCV2 in the context of the original studies related to determining its causal association with disease and to the evolving understanding of the complex pathogenesis of PCV2 infections. Keywords porcine circovirus, pigs, postweaning multisystemic wasting syndrome, virus

More is owing to what we call chance, that is philosophically speaking to the observation of events arising from unknown causes, than to any proper design, or preconceived theory in this business. Joseph Priestley, Experiments on Air (1790)

Porcine circovirus 2 (PCV2) is now recognized as an endemic infection of swine, worldwide, albeit still of questioned origin and longevity. In the space of less than 2 decades, this virus has gone from being a provincial oddity to one of the most economically important infectious agents in modern swine production and, fortunately, to date, one that can be effectively controlled with conventional vaccines. Because of its recognized importance, unique biology, and the burgeoning knowledge base, even given its relatively short history, there are many excellent reviews of various aspects of PCV2.2,21,61,66,70,77 The purpose of this article is not to be one more of those reviews but rather an attempt to put the current state of knowledge, as well as some of the many unanswered questions regarding the biology of this virus, in the context of the actual history—mostly untold—of its discovery and the initial studies to understand its biology. In many ways, this story is a celebration of the role of classical diagnostics, centered on the applications of the eyes and the microscope, in approaching a complex, economically important disease. A perusal of the now extensive literature on PCV2, especially one focusing on review articles, can leave the impression that the current state of knowledge was arrived at through a logical progression of seamless, rational scientific investigation. However, as in many discoveries,54 it was not that linear, and it was often impeded by groupthink and attendant entrenched dogmatic thinking.31

Chance Favors the Open Mind It has now been more than 20 years since Tracy Ward, a veterinarian in private practice, in Humboldt, Saskatchewan, and Chuck Rhodes, the swine medicine specialist at the herd investigation unit at the Western College of Veterinary Medicine (WCVM), investigated the first known outbreak of what came to be called postweaning multisystemic wasting syndrome (PMWS), the classic porcine circovirus–associated disease (PCVAD) entity. This incident occurred in a 40-sow farrow-to-feeder pig operation located in a remote part of northeastern Saskatchewan. In January 1990, the farm had been depopulated and restocked with breeding animals from a highhealth multiplier herd. One year later, in 1991, the farmer reported a dramatic increase in nursery mortality, up to 12% to 15%. Clinically, there was jaundice, diarrhea, and respiratory disease, as well as associated ill thrift, icterus, and sudden death. Chuck recalls submitting both live and dead weanlings, along with blood samples. He said that Ted Clark, a pathologist at the diagnostic laboratory at the WCVM, ‘‘did an extremely thorough workup on these pigs. We considered everything from aflatoxins, toxicities, and infectious agents, and the histopathology looked liked immunodeficiency.’’ This made sense since Ted reported that lymphoid depletion was consistently

1

Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada Corresponding Author: John Ellis, Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada. Email: [email protected]

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observed microscopically. No known pathogens were identified in multiple diagnostic submissions, and no final diagnosis was obtained. Thereafter, the problem appeared to resolve on that farm. A few years later in 1994, John Harding, at the time a swine consultant veterinarian in private practice, observed a similar disease syndrome in an intensive high-health swine operation, also in Saskatchewan. By definition, these ‘‘high-health’’ herds were free of common swine infections/diseases, including Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine dysentery, porcine reproductive and respiratory syndrome virus (PRRSV), and atrophic rhinitis. Although he and Tracy Ward worked in the same practice in 1991, he had no direct involvement in the initial outbreak, although he had a vague memory of her talking at the time about pigs with jaundice, a clinical sign that he specifically noted in the 1994 outbreak. The first submissions to the Diagnostic Laboratory at the WCVM relating to this new ‘‘outbreak’’ were made in October 1994. Chronic interstitial nephritis, severe focal hepatitis, and ileal muscular hypertrophy were found in 8- to 10-week-old pigs suffering loss of weight and body condition. The specific causes of these findings were unknown, but hepatitis associated with ascarid migration was proposed by Dr J. Orr, the attending diagnostic pathologist. Within months of these initial submissions, the farm experienced a fourfold increase in postweaning mortality, primarily in 2- to 4-month-old pigs associated with dyspnea, icterus, and weight loss. Numerous diagnostic submissions of carcasses, live animals, blood, serum, water, and feed were made over the course of the outbreak. All known swine pathogens were excluded, but with time, Ted Clark, the primary diagnostic pathologist who examined these further cases, began to recognize salient pathologic features common across the cases. He first documented many of what are now known as the classic, multisystemic lesions of PMWS and PCVAD,2,70 including, notably, the presence of characteristic botryoid basophilic intracytoplasmic inclusion bodies in cells of monocyte/macrophage lineage. On March 23, 1995, Lori Hassard, a technologist in the diagnostic virology laboratory at WCVM, which I supervised, reported that she had observed 17- to 19-nm dense particles that lacked the ‘‘hollow’’ appearance of parvoviruses, in electron micrographic preparations of material from John Harding’s cases. On the basis of her reading, she thought that they might be porcine circovirus (PCV). Ted and I discounted her seminal photographic evidence because we thought that the particles were probably parvoviruses; or, if they were circoviruses, ‘‘it was known’’—or at least we vaguely remembered from the literature—that PCV was not associated with disease.5 Nevertheless, Lori continued her efforts throughout 1995 and 1996 and demonstrated similar particles in more case material but not in normal pigs. In June 1995, Ted posted a brief description of the clinical disease and his morphologic findings on the American Association of Veterinary Laboratory Diagnosticians Listserv (@AAVLD.org) and, about the same time, sent pig-interested pathologists in North America and England representative slides. None of them at this stage reported seeing similar

lesions. In December 1995, Barbara Daft, a pathologist at a regional diagnostic laboratory in San Bernardino, California, responded to Ted’s posting, indicating that she had seen three 8- to 15-week-old pigs with clinical signs similar to the those that Ted described. Two had bacterial bronchopneumonia, but one had interstitial pneumonia and a generalized lymphadenopathy with intracytoplasmic inclusion bodies similar to what Ted described and what she had associated with circovirus infection. Dr Daft had dealt with many exotic avian cases and so had seen botryoid inclusions typical of circoviral infections in birds. She therefore also suggested that Ted’s cases may be associated with circovirus infection. This was further supported by evaluations of slides by Craig Riddell, an avian pathologist at WCVM, who mentioned that the inclusion bodies were similar to those he had seen in pigeons with circoviral disease. Concurrent with the initial pathologic and virologic investigations, representative samples of affected organs were submitted to the diagnostic bacteriology and immunohistochemistry laboratories at the WCVM for routine testing and screening for known swine pathogens. A number of bacteria and viruses were isolated or identified by immunohistochemistry, but none were consistently found in the case material. At the same time (May/June 1996) and following up on the lead provided by routine histopathologic examinations suggesting circoviral infection, Debbie Haines, who supervised the immunohistochemistry laboratory, identified 2 key investigators through cited publications on PCV: Irene Tischer, who had recently retired from the Robert Koch Institute in Berlin, and Gordon Allan, a virologist at Queen’s University and the diagnostic laboratory at Stormont, Belfast. These were the only 2 individuals who appeared repeatedly in the short list of references that she found. Both generously provided rabbit antisera and monoclonal antibodies raised against the virus that Dr Tischer had previously observed and isolated in porcine kidney cell (PK-15) cultures in the 1970s.89,91 By mid-October 1996, using the rabbit hyperimmune antisera, Debbie had visualized PCV antigen in typical lesions in a lymph node from a PMWS-affected pig. This rabbit hyperimmune antiserum from Dr Allan—labeled A/S—had been raised against PCV recovered from contaminated PK-15 cells. Other donated antisera and monoclonal antibodies to the contaminant PCV were unreactive. Thus, while Dr Allan was excited, he was skeptical of the preliminary findings, since he had already spent years of effort on circoviruses and had obtained consistently negative results concerning a possible association of PCV with disease in swine. Regardless, he was interested enough to continue working with us and provide more reagents and expertise. A few days later, armed with these preliminary immunohistochemical results, Drs Harding and Clark presented their preliminary clinical and pathologic findings at a regional meeting, the Western Canadian Association of Swine Practitioners in October 1996, and suggested the name of PMWS for the apparently new disease.18,42 It was immediately following these presentations that Dr John Strokappe, of Red Deer, Alberta, reported seeing similar clinical signs in a number of herds in Alberta and submitted cases to the WCVM. This was the first

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indication that PMWS was present in more than a single farm. It is also noteworthy that the original 40-sow farm from 1991 was retrospectively diagnosed with PMWS after 1997, indicating that the PCV was associated with disease before the outbreaks that we were investigating in the mid-1990s. Also in the fall of 1996, Barbara Daft, presented her findings on the pig with interstitial pneumonia and lymphadenopathy at the annual meeting of the American Association of Veterinary Laboratory Diagnosticians.20 With her collaborators, avian pathologists from Georgia, she reported the presence of paracrystalline arrays typical of circovirues by electron microscopy and demonstration of circoviral DNA by in situ hybridization (ISH) in lesions from that pig. The ISH was done with probes based on the genome of the PCV from PK-15 cells and a ‘‘generic’’ circoviral probe. The probes also detected circovirus-specific sequences in the affected pig by Southern blot hybridization but not from control pigs. On the basis of their observations, they reasonably concluded that this virus (PCV) could be associated with disease in pigs.20 This contrasted with Tischer’s earlier studies90 based on experimental infections in the 1980s using the PK-15-origin PCV and with the contemporary work from Gordon Allan’s group5 that included examinations of fetal material and experimental infections with the tissue culture–derived PCV. Since there was a lack of disease in pigs with documented PCV infections, both these investigators logically concluded that PCV was mildly or nonpathogenic in pigs. On January 27, 1997, Lori Hassard, after much trial and error over more than 2 years, finally isolated a circovirus from a PMWS-affected pig.30 She thought that it was ‘‘different’’ from the circovirus originally found by Dr Tischer. To increase the chances for isolation, she had worked with Debbie Haines to identify lymph nodes containing high amounts of PCV antigen on the basis of immunhistochemical staining. The conclusion that this PCV was different from the tissue culture–derived PCV isolated by Dr Tischer was based on differential staining of viruses from cases of disease compared to the staining of the PK-15 virus, with monoclonal antibodies and antisera obtained from Drs Tishcer and Allan. In addition, there was a strong reactivity of the PMWS-derived virus with convalescent serum from a Canadian pig (‘‘Blue-2’’). This pig had died of an inguinal hernia and was apparently free of PCV-associated disease. Furthermore, Lori demonstrated that this Blue-2 serum was free of antibodies to PRRSV and porcine parvovirus (PPV) and had questionable or no reactivity with the tissue culture derived PCV. These PCV isolates in cell culture also had in situ hybridization reactivity with a generic PCV probe prepared via the PCV cell culture contaminant. Further supporting the evidence that this was the first isolate of a new virus were the type of cells in which it was isolated. As luck would have it, we had previously (early 1990s) obtained PCV-negative pig kidney (‘‘Dulac’’)25 cells to grow bovine viral diarrhea virus for ELISA antigen, free of the confounding PCV contaminate that was in virtually all readily available isolates of porcine kidney cells. Use of these cells greatly reduced potential confusion and the likelihood that the PCV being identified was not the ‘‘old’’

(contaminant) PCV. In summary, we had a virus growing in cell cultures inoculated with material from PMWS-affected pigs that had DNA homology with the PCV contaminant by ISH but a different reactivity pattern when immunostained with PCV-reactive antibodies. This first isolation was the vitally important evidence that a different (new) PCV was causally associated with the apparently new disease in pigs. Lori then went on to isolate the new PCV from other field cases. In early 1997, shortly after a PCV was isolated from Canadian PCVAD cases, Gordon Allan, working closely with Francis McNeilly, isolated very similar viruses from Barbara Daft’s case material from the United States and from PMWS cases in Brittany, France,7 and then from lesional material from other sites in Europe.10 Antiserum raised against the first Canadian isolate, named ‘‘Stoon,’’ was used in addition to the monoclonal and polyclonal antibodies raised against the PK-15 PCV to clearly differentiate the phenotype of these new isolates from that of the tissue culture–derived PCV. As soon as isolates of the putatively new PCV were available in early 1997, Brian Meehan, a molecular biologist/virologist working with Gordon, provided the first, complete whole genomic sequences of PCVs from North America and Europe, indicating that the disease-associated PCVs shared >95% sequence identity but had

Porcine circovirus: a historical perspective.

Porcine circoviruses (PCVs) belong to the genus Circovirus and the family Circoviridae, and they are the smallest known viruses that replicate autonom...
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