Steven R. Bolin

Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein

Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.

Experimental Reproduction of Severe Disease in CD/CD Pigs Concurrently Infected with Type 2 Porcine Circovirus and Porcine Reproductive and Respiratory Syndrome Virus

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen–containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.

Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum- deprived piglets with type 2 porcine circovirus

Cesarean-derived, colostrum-deprived pigs (n = 23) were inoculated intranasally and subcutaneously with a low cell culture passage of type 2 porcine circovirus. In 11 pigs, a persistent fever that lasted 7–17 days began 12–15 days after inoculation with virus. Additional signs of disease in those 11 pigs included depression (11 of 11 pigs), palpable enlargement of inguinal, prefemoral, and popliteal lymph nodes (11 of 11), icterus (6 of 11), and hyperpnea (2 of 11). The remaining 12 pigs had fever that occurred intermittently for 2–4 days between days 12 and 20 postinoculation. Overt signs of disease in those pigs were limited to palpable enlargement of inguinal and popliteal lymph nodes (9 of 12 pigs). When compared with control pigs of similar age, the average daily rate of weight gain for all pigs inoculated with virus was less over a 2-week period that began 2 weeks post inoculation. At postmortem examination, lymph node enlargement was seen in 14 of 14 pigs euthanized between days 20 and 28 postinoculation. Lymph node enlargement was especially prominent in pigs that developed a persistent fever. Microscopic lesions noted in pigs that developed a persistent fever included cellular depletion in lymphoid tissues; hepatic cell necrosis; and lymphogranulomatous inflammation of lymph nodes, Peyers patches of the intestine, liver, kidney, and heart. Virus was isolated with varying frequency from nasal, rectal, or tonsil swab specimens, buffy coat, serum, urine, and lung lavage fluid obtained antemortem or postmortem. Virus was isolated from or viral DNA was detected in a variety of tissues obtained postmortem up to 125 days postinoculation. Antibody against type 2 porcine circovirus usually was detected in serum between 15 and 20 days postinoculation; however, antibody against virus was not detected in serum from 4 pigs euthanized 20–24 days postinoculation. Direct contact with pigs inoculated with virus 42 days previously resulted in transmission of virus to 3 of 3 control pigs.

Methods for Detection and Frequency of Contamination of Fetal Calf Serum with Bovine Viral Diarrhea Virus and Antibodies against Bovine Viral Diarrhea Virus

Methods used by the National Animal Disease Center to test fetal calf serum for contamination with bovine viral diarrhea virus (BVDV) and antibodies against BVDV are described. Using those methods, virus was isolated from 332 of 1,608 (20.6%) lots of raw fetal calf serum obtained specifically for the Center and 93 of 190 (49%) lots of commercially available fetal calf serum. Virus neutralization and immunoperoxidase staining tests were used to detect antibodies against BVDV in 224 of the 1,608 (13.9%) lots of raw fetal calf serum. Both BVDV and antibodies against BVDV were detected in 50 lots of raw serum. The molecular specificity of antibodies against BVDV was determined by radioimmunoprecipitation. Lots of fetal calf serum that contained BVDV-specific antibodies that did not neutralize virus were identified.

Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum

One thousand lots of pooled fetal bovine serum (FBS) were tested for contamination with bovine viral diarrhea virus (BVDV) and/or for contamination with neutralizing antibody against BVDV. Noncytopathic or cytopathic BVDV was isolated from 203 lots of FBS. Analysis of the viral isolates identified 115 type 1 and 65 type 2 BVDV isolates. An additional 23 virus isolates were mixtures of >2 BVDV isolates and were not classified to viral genotype. Further characterization of the type 1 viruses identified 51 subgenotype 1a and 64 subgenotype 1b BVDV isolates. Viral neutralizing antibody was detected in 113 lots of FBS. Differential viral neutralization indicated that type 1 BVDV induced the antibody detected in 48 lots of FBS and type 2 BVDV induced the antibody detected in 16 lots of FBS.

The Pathogenesis of Mucosal Disease

The pathogenesis of MD is complex and remains somewhat obscure. Clearly, the disease occurs in cattle persistently infected with noncytopathic BVDV. It also is clear that cytopathic BVDV is associated with MD, and is the likely trigger of the cellular destruction that leads to clinical disease. Whether the cellular destruction is attributable directly to the cytopathic virus, or occurs as the result of other mechanisms remains unclear. Although immunotolerance is involved in MD, it can be broken and its role in the disease process needs further research. It is logical, and well supported by research, that noncytopathic BVDV is the source of cytopathic BVDV. It also is likely that most outbreaks of MD are the result of a spontaneous mutation of noncytopathic to cytopathic virus within a PI animal. Antigenic homology between viruses would be expected in those outbreaks. MD also occurs when PI cattle are exposed with a cytopathic BVDV that is antigenically heterologous with the resident noncytopathic BVDV. In those situations, it may be a race between the cytopathic virus and the immune system.

Control of Bovine Viral Diarrhea Infection by use of Vaccination

Vaccination with either inactivated or modified live virus vaccines is beneficial for control of BVD in cattle. The advantages and/or disadvantages of each type of vaccine often influence vaccine selection. The frequency of vaccination depends on the herd management system, regional prevalence of BVDV, and required duration of protection. Vaccines for BVD likely will change in content as knowledge of BVDV increases and as new technologies are adapted for vaccine production.

Antigenic and genomic comparison between non-cytopathic and cytopathic bovine viral diarrhoea viruses isolated from cattle that had spontaneous mucosal disease

Antigenic and genomic relationships between five pairs of non-cytopathic and cytopathic bovine viral diarrhoea (BVD) viruses isolated from cattle with mucosal disease were examined. Antigenic similarity was evaluated by studying the binding characteristics of 10 monoclonal antibodies (MAbs) directed against the gp53 BVD virus glycoprotein. The MAb binding match between members of the same virus pair ranged from 10/10 to 7/10. The genomic relationships was evaluated by studying the hybridization characteristics of two cDNA probes and six complementary 20 base oligomer probes with virus DNA, selected on the basis of conservation between published BVD virus sequences; cDNA probes were hybridized at two stringencies (60 degrees C and 45 degrees C). The match of hybridization results between members of the same virus pair ranged from 4/4 to 1/4 with the cDNA probes and from 6/6 to 3/6 with the oligomer probes. The results indicate that members of the same virus pair can differ at both the antigenic and genomic levels.

A simple and rapid flow cytometric method for detection of porcine cell surface markers.

The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

Bovine Viral Diarrhea Serologic Diagnostic Reagents Prepared from Bacterially Expressed Recombinant Proteins

Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus and is classified in the Pestivirus genus of the family Flaviviridae. The BVDV genome has been cloned and sequenced, and a map of protein-encoding genes has also been established. According to this map, the BVDV genome encodes at least 4 primary gene products: p20, gp116, p125, and p133. There is evidence that the glycoprotein precursor gp116 gives rise to gp48, gp25, and gp53 through proteolytic processes, and p125 gives rise to p80 by proteolysis when cells are infected with cytopathic BVDV. The p125 is produced only in cells infected with noncytopathic BVDV. When an antibody against BVDV is analyzed by using radioimmunoprecipitation or western blots, gp48, gp25, gp53, and p80 (or p125) are detected. 1,2,8,9 Thus, these proteins are good candidates for specific enzyme-linked immunosorbent assays (ELISAs). Currently, virus neutralization (VN) is the standard serologic test for BVDV, and virus-neutralizing antibody binds primarily to gp53. 5,11 Other diagnostic tests, such as wholevirus ELISA, fluorescent antibody, and immunoperoxidase staining, also are used and likely detect additional viral proteins. All of these tests require specialized tissue culture equipment. Consequently, these procedures are often impractical for screening a large number of samples. Recombinant ELISA is now widely accepted for serodiagnosis of many important human and veterinary diseases. This test is a simple, rapid, highly sensitive, and inexpensive tool for screening for an antibody. In this report, we describe the production of large amounts of gp48, gp25, and p80 recombinant proteins, use of these recombinant proteins in the ELISA, and comparison of the ELISA with the standard VN test for the serodiagnosis of bovine viral diarrhea (BVD) in cattle. To express gp48, gp25, and p80, each gene fragment was cloned into the expression vector pGex2T or pGex3X. In each construct, the correct translational frame required for expression of glutathione-S-transferase (GST) fusion protein was created. The gp48 and p80 restriction fragments were isolated from pBV4-gp62 and pBV4-p80 clones. The construction and production of these recombinant proteins have been previously described. A 359-base pair (bp) fragment of the gp48 region (nucleotides 1295-1653) and a 918-bp