Renée Larochelle

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions.

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990-2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs.

MOLECULAR EPIDEMIOLOGY OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) IN QUÉBEC

Porcine reproductive and respiratory syndrome virus (PRRSV) strains identified in samples from 226 field cases originating from Québec herds and submitted over a 4-year period (March 1998-July 2002) were studied. Sequencing of PRRSV strains was performed on the ORF5 gene amplified product and restriction fragment length polymorphism (RFLP) patterns for enzymes MluI, HincII and SacII were determined on these sequences. Twenty-four other PRRSV isolates including three vaccine strains were also included for comparison purposes and the total of 250 PRRSV strains were used in a phylogenetic analysis. Clinical and epidemiological data were collected through a questionnaire for each of the submitted field cases. About 75% of the cases were submitted during autumn and winter. Over 60% of the cases were submitted for reproductive problems, 33% for respiratory problems and 6% for increased PRRSV serological titers in the herd in absence of clinical signs. In 69% of the cases there was a PRRS vaccination program for the herd. However, only 26% of the animals from which samples were obtained had been vaccinated themselves. The genomic analysis of this large number of strains revealed a great variability of PRRSV ORF5 with 59% of amino acid positions being polymorphic. A total of 29 RFLP patterns were obtained. The main RFLP patterns obtained were 1-8-4 (28%), 1-4-4 (16%), 1-2-4 (9%) and 1-11-4 (9%). The global findings derived from the molecular analysis of 226 PRRSV strains suggest that PRRSV circulating in Québec represent a different sub-population of strains. Vaccine-like strains were identified in 10% of the cases. A phylogenetic tree enabled the identification of 44 groupings comprising two to 23 strains each. Of the 250 sequences analyzed, 183 (73%) could be included in one of these groupings. The data collected from the questionnaires were used to establish epidemiological links between strains within groupings. The main relationships between strains within a grouping were the introduction of infected animals (19%) and area spread (33%). In 40% of the cases from which an area spread was suspected, herds were located within 3 km from another. Aerosol transmission was suspected in several cases, more than half of which belonged to different owners. In 41 herds, more than one strain (2-8) were identified over a period varying from 3 months to 4 years. Data indicated that a PRRSV strain can persist in a herd up to 3.5 years displaying as little as 2% variation in ORF5 during this time. In 78% of the herds with multiple submissions genetically different strains were identified; often within 1 year of the original identification. These genetically distinct strains were often associated with a recrudescence of moderate to severe clinical signs. Coexistence on the same farm of different PRRSV strains was also observed.

Porcine reproductive and respiratory syndrome virus persistence in blood, spleen, lymph nodes, and tonsils of experimentally infected pigs depends on the level of CD8high T cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection suggesting an inefficient cellular immune response. The aim of the study was to evaluate the relationship between viral persistence and cytotoxic cells in blood, spleen, mediastinal lymph nodes (MLN) and tonsils of PRRSV experimentally infected pigs. Groups of four to six specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate, and blood and lymphoid organs were collected from 3 to 60 days post-infection (p.i.). Infectious particles and viral RNA were more or less rapidly eliminated in serum, spleen, lungs and MLN but persisted the longest in tonsils. Lymphocytes CD2+ CD4+, CD2+ CD8high, CD2+ CD8low and NK cells populations were phenotyped and their reactivity to PHA and ConA were tested. Analysis of T cell subsets in blood and lymphoid organs indicated that the percentages of CD2+ CD8+ T cells slightly increased in spleen at 17 days p.i, whereas no changes were observed in CD2+ CD4+ cells in blood or lymphoid organs. However, discrimination of CD8+ cells in CD8high and CD8low subsets revealed that the percentages of CD2+ CD8high cells increased in spleen and blood from 10 to 45 or 60 days p.i. while they transiently increased in MLN and decreased in tonsils. The CD8low/CD8high ratio increased in the blood of PRRSV-infected animals at three days p.i. due to a transient decrease of CD2+ CD8high cells. This same ratio decreased in the spleen of infected pigs from 10 to 45 days p.i. due to an increase of CD2+ CD8high cells. The CD2+ MIL-4+ cell subset (NK cells) was not significantly modified in blood or lymphoid organs. In addition, the ability of lymphoid T cells from blood and lymphoid organs to respond to ConA or PHA stimulation was transiently impaired in blood and spleen during the PRRSV persistent infection. Taken together, these results suggest that, in persistently infected pigs, an impaired CD2+ CD8high cell response in MLN and tonsils favors viral persistence in these organs, in contrast with the response seen in blood and spleen where viral elimination appears to occur sooner.

Evaluation of the presence of porcine reproductive and respiratory syndrome virus in packaged pig meat using virus isolation and polymerase chain reaction (PCR) method.

An investigation was carried out to assess the potential presence of porcine reproductive and respiratory syndrome virus (PRRSV) in packaged pig meat. Samples of meat were collected at the processing plants and were sent to the laboratory for testing by virus isolation and reverse transcription-polymerase chain reaction (RT-PCR). Samples collected at four plants were randomly selected from lots of packaged pig meat from different slaughtering days and were sent frozen to the laboratory. Homogenates of meat were prepared and were inoculated onto MARC-145 cells and after two passages the presence of PRRSV was monitored by indirect immunofluorescence staining using PRRSV specific monoclonal antibody. All pig meat samples (six pools of meat samples from 73 different lots = 438 total homogenates) tested were found negative by virus isolation. Primers from open reading frames 6 and 7 were designed and a RT-PCR assay was developed and was demonstrated to detect both North American and European PRRSV isolates. Using this assay virus was detected at a concentration as low as 0.355 infectious virions per ml in supernatant of PRRSV infected cells. This RT-PCR assay could detect PRRS viral nucleic acid from various tissue samples of experimentally infected pigs including muscle tissue, thus demonstrating its applicability on tissue samples. All meat sample homogenates tested by RT-PCR (one sample pool from the 73 lots) were also found negative for PRRS viral nucleic acid. The results suggest that pig meat does not retain detectable amounts of PRRSV and further support that the transmission of PRRSV through pig meat is unlikely.

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.

Detection of Porcine Reproductive and Respiratory Syndrome Virus in Cell Cultures and Formalin-Fixed Tissues by in Situ Hybridization using a Digoxigenin-Labeled Probe

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.

Comparison of Immunogold Silver Staining (IGSS) with Two Immunoperoxidase Staining Systems for the Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antigens in Formalin-Fixed Tissues

Laboratory diagnosis of porcine reproductive and respiratory syndrome (PRRS), a newly identified and economically important viral disease of swine, is based on the combination of serology, histopathology, and virus isolation. The isolation and identification of PRRS virus (PRRSV) is complex and labor intensive and may require many days or weeks to perform. The histological diagnosis of infection by PRRSV is limited since other viral pathogens may induce similar lesions. Furthermore, secondary bacterial infection commonly associated with PRRSV infection can complicate the interpretation of lesions. Detection of viral antigens in frozen lung sections using immunofluorescence (IF) has been reported, 1,11 but the IF procedure has shortcomings. The use of immunohistoch emical methods for the detection of viral antigens in formalin-fixe d paraffin-embed ded tissues offers several advantages over the IF technique. These procedures allow direct visualization of viral antigens along with simultaneous evaluation of histological lesions in the affected tissue using an ordinary light microscope. The ability to detect viral antigens in routinely fixed tissues facilitates convenient collection and transport of specimens, and potentially infectious specimens can be handled safely. Finally, since antigens are usually stable indefinitely in paraffin-embedded tissues, retrospective diagnoses can be made. The immunogold silver staining (IGSS) is an indirect 2-step nonenzyme-based method in which the secondary antibody labeled with colloidal gold particles is visualized following a silver precipitation reaction . 4

Immunohistochemical detection of porcine rotavirus using immunogold silver staining (IGSS).

Immunogold silver staining (IGSS) was applied for the detection of porcine group A rotavirus in formalin-fixed paraffin-embedded tissue sections of small intestine. Prior to the application of IGSS, the reactivity of protein A-gold as a marker was tested with group-specific antiserum in immunogold electron microscopy. Immune aggregates were intensely and specifically labeled with the gold complex. Application of IGSS to tissue sections resulted in specific dark staining of villous enterocytes infected by group A rotavirus. This method also proved effective for the detection of rotaviral antigen in infected cultured cells. The IGSS method may be suitable for routine diagnostic detection of rotaviral infections and may have application for detection of other viral pathogens of veterinary importance.