Dendritic cells harbor infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune responses, rendering them critical targets for virus infections. Porcine circovirus type 2 (PCV2) is associated with the development of postweaning multisystemic wasting syndrome (PMWS) in piglets. We demonstrate here that 80 to 90% of monocyte-derived and bone marrow-derived DCs interact with PCV2 similar to the early stages of an infection. There was no evidence for virus replication, but the virus did persist in DCs without loss of infectivity nor the induction of cell death. This could reflect an abortive infection, but there was no evidence of virus uncoating-the infectivity remained intact for at least 5 days. Alternatively, the results may reflect DC endocytosis of antigenic material. However, there was no modulation of DC surface major histocompatibility complex class I and class II, CD80/86, CD25, CD16, or CD14. Furthermore, infected DC did not transmit virus to syngeneic T lymphocytes, even when the latter were activated. Such coculture did not induce PCV2 replication or death of the lymphocytes or DCs. These results demonstrate that PCV2 can persist in DCs in the absence of virus replication or degradation. Such a silent virus infection presents a novel mechanism of not only immune evasion but also escaping the DC degradation pathway. Because of their migratory capacity, infection of DCs thus provides a potent vehicle for transport of the virus throughout the host without the need for replication. In addition, the lymphopenia seen in PMWS is not a direct effect of the virus on lymphocytes but would require additional events, as proposed by others.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system.

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.

Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2.

The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described.

Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome.

We report the isolation and characterisation of porcine circovirus 2 (PCV2) from cases of sow abortion and porcine dermatitis and nephropathy syndrome. The results suggest that the clinical scope of PCV2 infections requires continuous re-evaluation.

Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2.

We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation.

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.

RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: comparison with vaccine virus and field isolates from England, Scotland and the Republic of Ireland.

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.

A fluorimeter-based RT-PCR method for the detection and quantitation of porcine cytokines.

In this paper, we describe a fluorimeter-based, closed-tube, polymerase chain reaction (PCR) assay for the detection and quantification of the mRNA of porcine interleukin 1alpha (IL1alpha) and interleukin 2 (IL2) cytokines in peripheral blood leukocytes (PBLs) using melting curve analysis and compare it to a standard PCR performed in a block-based thermocycler.

A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS).

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified condition affecting pigs in North America and Europe. Porcine circovirus antigen and nucleic acid have been demonstrated associated with lesions, and a new porcine circovirus designated PCV2 has been recovered from tissues of these animals. In this study, in situ hybridisation and immunohistochemical protocols were developed, optimized and compared for their relative sensitivity in detecting PCV2 antigens and nucleic acid in tissues from cases of PMWS that had been fixed for up to 6 months in formalin. For both immunohistochemistry and in situ hybridization, an increase in specific signal was observed following increased exposure to both protease XIV and proteinase K. Maximum signal and minimal loss of tissue morphology was seen after 40 min treatment with protease XIV (0.5 mg/ml). After optimisation, a comparison of these techniques on sequential sections demonstrated that both techniques successfully detected antigen or nucleic acid in all of the tissues examined. More positive cells, with increased signal intensity, were detected following immunohistochemistry.

Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus.

Colostrum-deprived pigs were infected intranasally with a recent isolate of porcine circovirus (PCV2) and a porcine parvovirus (PPV), both from Canadian pigs with post-weaning multisystemic wasting syndrome (PMWS). Four pigs were inoculated with PCV2 alone, three with PPV alone, five with a combined PCV2/PPV inoculum, and two with a chloroform-treated combined PCV2/PPV inoculum. Pigs were killed 21-26 days after infection and tissue samples examined for gross and microscopical lesions and for the presence of viral antigens. No clinical signs, lesions or viral antigens were detected in two uninfected control pigs or in pigs inoculated with PPV alone. One pig inoculated with PCV2 alone became dull and thin. Mild to moderate histopathological lesions containing PCV2 antigen were detected in lymphoid tissues from the pigs inoculated with PCV2 alone. Pigs given the PCV2/PPV inoculum and the chloroform-treated PCV2/PPV inoculum became dull and two died. Jaundice and hepatomegaly were seen at post-mortem examination of most of the dually infected pigs. The latter showed large amounts of PCV2 antigen in numerous tissues; PPV antigen, which was less abundant, was detected in a few tissues, especially kidney. The lesions were similar to those seen in recently described field cases of porcine PMWS in North America and Europe.

Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland.

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).

Characterisation of strain-specific sequences from an abortifacient strain of ovine Chlamydia psittaci using subtraction hybridisation.

Enzootic abortion in ewes (EAE) is caused by strains of Chlamydia psittaci which have the ability to invade and colonise the placenta of sheep. In an attempt to improve diagnostic methods for the detection of EAE, subtraction hybridisation was used to isolate unique fragments of the genome of an abortifacient strain (S26/3) of C. psittaci. One S26/3 strain-specific sequence identified was shown to encode a putative helicase which is repeated throughout the EAE genome. The labelled strain-specific helicase gene fragment was used in the dot-blot hybridisation test for the detection of EAE DNA in ovine placental samples. We report the identification of C. psittaci S26/3 strain-specific sequence with potential as diagnostic probes for the detection of EAE.

Characterization of novel circovirus DNAs associated with wasting syndromes in pigs.

Porcine circovirus (PCV) was initially recognized as a contaminant of continuous pig kidney cell lines and was not thought to be pathogenic. Antibodies reactive to the cell culture isolate of PCV (PCV PK-15) are prevalent in the swine population worldwide. Recently, PCV PK-15-like antigen and nucleic acid were demonstrated in lesions associated with wasting syndromes in pigs in North America and Europe. Monoclonal antibodies raised to circoviruses isolated from pigs with wasting syndromes highlighted differences between these circoviruses and the PCV PK-15 cell culture isolate. This has led to speculation that a new pathogenic PCV may have emerged in the swine populations of several countries. We report the cloning and characterization of novel circovirus DNAs purified from virus isolates made from tissues of North American and European pigs with wasting syndromes. These North American and European circoviruses form a closely related group at the nucleotide sequence level (> 96% intra-group nucleotide sequence identity) but exhibit < 80% nucleotide sequence identity with the PCV PK-15 cell culture isolate. This report provides evidence for a new type of possibly pathogenic PCV. We propose that these new circoviruses should be referred to as PCV2 as opposed to the original PK-15 cell culture isolate, which should be referred to as PCV1.

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach.

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.

Close phylogenetic relationship between egg drop syndrome virus, bovine adenovirus serotype 7, and ovine adenovirus strain 287.

A cloned egg drop syndrome (EDS) virus genomic DNA fragment containing the protease gene has been identified and the complete nucleotide sequence of the protease and partial nucleotide sequence of the hexon genes has been determined. Phylogenetic analysis of the protease gene has revealed EDS virus to be genetically more closely related to bovine adenovirus type 7 (BAV-7) and ovine adenovirus isolate 287 (OAV287) than either of these two viruses are to other members of the genus Mastadenovirus or EDS virus is to an other member of the Aviadenovirus genus. The three viruses share further similarities in that they have a high percentage AT content in their genome and are characterized by having more compact genomes than other adenoviruses. The protease gene from all three viruses contained the active site residues (H55-D72-C122 triad) and C104 (providing a disulfide bond to cofactor pVIc). However, P137, found in all other members of the Mastadenovirus genus, and thought to be involved in trafficking, was missing from the protease of the EDS virus, BAV-7, and OAV287. These results suggest that EDS virus should be classified along with BAV-7 and OAV287 in a separate taxon.

Sequence of porcine circovirus DNA: affinities with plant circoviruses.

The complete nucleotide sequence (1759 nt) of the ssDNA genome of porcine circovirus (PCV) was determined from a cloned dsDNA replicative form isolated from PCV-infected cells. Sequence analysis detected no significant nucleic acid or protein similarity with another animal circovirus, chicken anaemia virus (CAV) but, surprisingly, the highest protein similarity was obtained between the product of the largest predicted PCV ORF (ORF1; encoding a potential protein of 35.7 kDa) and a putative protein encoded by the plant circovirus banana bunchy top virus (BBTV). High protein similarity was also detected with the other plant circoviruses subterranean clover stunt virus (SCSV) and coconut foliar decay virus (CFDV). This region of protein identity corresponds with the putative plant circovirus replication-associated protein (Rep). The presence of a nonanucleotide sequence at the apex of a potential-stem loop structure, identical to that found in the plant circoviruses CFDV and SCSV and similar (one mismatch) to that found in the plant circovirus BBTV and in the geminiviruses, suggests that rolling-circle replication may operate during PCV DNA replication. These findings show that PCV is unique in that it bridges the gap between animal and plant circoviruses. The taxonomic relationship of PCV with other members of the Circoviridae is discussed.

Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences.

The potential use of the ribosomal DNA internal transcribed spacer (ITS) sequences in understanding the phylogeny and systematics of Colletotrichum species has been evaluated. Sequence data from a limited number of isolates revealed that in Colletotrichum species the ITS 1 region (50.3% variable sites) shows a greater degree of intra- and inter-specific divergence than ITS 2 (12.4% variable sites). Nucleotide sequences of the ITS 1 region from 93 isolates representing 18 Colletotrichum species were determined. Data for 71 of these isolates where molecular and morphological identities concurred were used for phylogenetic analysis. The size of the ITS 1 region varied from 159 to 185 base pairs. Maximum intraspecific divergence was recorded with C. acutatum (5.8%), and C. capsici showed the greatest level of interspecific divergence (8.9-23.3%). Parsimony and distance analyses gave similar tree topologies. The bootstrapped consensus parsimony tree divided the 18 Colletotrichum species into six phylogenetic groups, designated 1-6. These groups, however, are not congruent with species clusterings based on spore shape. For example, the straight cylindrical spored species were represented both in groups 1 and 6; group 6 also included the falcate fusiform spored species C. capsici. The molecular evidence suggests refinement of the species concepts of some of the taxa examined. In group 6, divergence between C. gloeosporioides and C. fuscum (0.6-3.0%) or C. kahawae (0.6-3.0%) or C. fragariae (0.6-4.2%) overlap the divergence (3.6%) within C. gloeosporioides. It is suggested that C. fuscum as well as C. kahawae and C. fragariae fall within the group species C. gloeosporioides. ITS 1 data enabled clear distinction (7.1%) of Colletotrichum isolates from maize and sorghum into C. graminicola and C. sublineolum, respectively (group 2). Species such as C. acutatum, C. coccodes, C. dematium, and C. trichellum can be clearly distinguished based on ITS 1 sequence divergence, but C. destructivum cannot be confidently separated (98% homology) from C. linicola. Colletotrichum dematium f. truncatum is distinct (12.9%) from C. dematium and should probably be called C. truncatum.

Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments.

Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family, Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were "circularised" within the same 105 bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts.