A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV.
Porcine circovirus type 2 (PCV2) is an etiological agent of porcine circovirus diseases (PCVDs). Post-weaning multisystemic wasting syndrome (PMWS) as the most important PCVD is considered a multifactorial disease. It was demonstrated that not only PCV2 but several viruses are associated with PMWS. Studies of viral co-infections in PMWS pigs led often to controversial results. The aim of this work was to determine the presence of emerging (PRRSV), re-emerging (PTV) and newly-emerging (TTSuV1, TTSuV2, PBoV1) viruses in samples of dead pigs suffering from PMWS. The impact of vaccination against PCV2 and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated.
Viruses were detected by PCR, RT-PCR and real-time PCR in the pooled tissue samples (lymph nodes, liver and spleen) of pigs with PMWS (n = 56) which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. In addition, lymph node samples were collected from apparently healthy fattening pigs (n = 59). The effect of vaccination against PCV2 with Ingelvac CircoFlex vaccine was also investigated. Between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with PMWS. Severe clinical disease was observed in swine co-infected with PCV2 and PRRSV. The prevalence of TTSuV1 and TTSuV2 was high in all groups of pigs and did not appear to have a significant effect on the syndrome. Simultaneous infection with TTSuV1 and PBoV1 was frequently confirmed in pigs with PMWS. No healthy pig was found to be infected with PRRSV, PTV or PBoV1. Vaccination against PCV2 did not influence the prevalence of TTSuVs, but significantly protected pigs against multiple viral infections.
Post-weaning PMWS pigs were more often co-infected with viral pathogens than suckling or fattening pigs. Co-infection with PRRSV enforces clinical signs of PMWS, the influence of other viral co-infections is not clear. Vaccination against PCV2 significantly reduced viral co-infections in pigs.
Post-weaning multisystemic wasting syndrome (PMWS); Multiple infections; Vaccination; Porcine circovirus type 2 (PCV2); Porcine reproductive and respiratory syndrome virus (PRRSV); Torque teno sus virus (TTSuV); Porcine teschovirus (PTV); Porcine bocavirus 1 (PBoV1)
Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.
artificial insemination; bioassay; boar; PCV2; semen
An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.
The objective of the present study was to evaluate polyclonal- and monoclonal-antibody-based immunohistochemical (IHC) tests for the detection of 2 genotypes of Porcine circovirus type 2 (PCV2), a and b, in formalin-fixed, paraffin-embedded lymph-node tissue from pigs with experimental or natural postweaning multisystemic wasting syndrome and to compare the IHC results with those of in-situ hybridization (ISH) assays. The ISH assays proved more sensitive than the IHC tests for the detection of PCV2a and PCV2b. According to these findings, polyclonal-antibody-based IHC testing is the most practical routine diagnostic method for the detection of PCV2 regardless of genotype because IHC testing is less technically complex than ISH testing. However, ISH assays are useful to differentiate between PCV2a and PCV2b in surveillance programs for the monitoring of PCV2 in swine herds.
Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics.
All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC).
Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC (κ = 0.38) and a moderate agreement in OSCC (κ = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach.
Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.
Head and neck squamous cell carcinoma; HN-SCC; OSCC; OPSCC; Human papillomavirus; HPV; DNA consensus PCR; Immunohistochemistry; IHC; p16-IHC; Epigenetic; Methylation-Specific PCR
Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszky's disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.
Individuals in natural populations are exposed to a diversity of pathogens which results in coinfections. The aim of this study was to investigate the relation between natural infection with tuberculosis (TB) due to infection by bacteria of the Mycobacterium tuberculosis complex and porcine circovirus type 2 (PCV2) in free-ranging Eurasian wild boar (Sus scrofa). Apparent prevalence for TB lesions and PCV2 infection was extremely high in all age classes, including piglets (51% for TB; 85.7% for PCV2). Modeling results revealed that the relative risk of young (less than 2 years old) wild boar to test positive to PCV2 PCR was negatively associated with TB lesion presence. Also, an interaction between TB, PCV2, and body condition was evidenced: in wild boar with TB lesions probability of being PCV2 PCR positive increased with body condition, whereas this relation was negative for wild boar without TB lesions. This study provides insight into the coinfections occurring in free-ranging host populations that are naturally exposed to several pathogens at an early age. Using TB and PCV2 as a case study, we showed that coinfection is a frequent event among natural populations that takes place early in life with complex effects on the infections and the hosts.
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 μg of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 μg of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 μg of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 μg of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.
We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.
Porcine circovirus type 2 (PCV2); Real-time PCR; Sensitivity
Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.
In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.
We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.
Virus-like particles; Porcine circovirus 2; Monoclonal antibodies
Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness.
Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b.
This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.
Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.
Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), and porcine dermatitis and nephropathy syndrome (PDNS). It has caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS.
A loop-mediated isothermal amplification (LAMP) assay was used to detect PCV2 in this study. Three pairs of primers were specially designed for recognizing eight distinct sequences of the ORF2 gene. This gene lies in the PCV2 virus genome sequence, and encodes the Rep protein that is involved in virus replication. Time and temperature conditions for amplification of PCV2 genes were optimized to be 55 min at 59°C. The analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection limit for PCV2 by the LAMP assay was 10 copies, whereas the limit by conventional PCR was 1000 copies. The assay did not cross-react with PCV1, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis of pigs virus or rotavirus. When 110 samples were tested using the established LAMP system, 95 were detected as positive.
The newly developed LAMP detection method for PCV2 was more specific, sensitive, rapid and simple than before. It complements and extends previous methods for PCV2 detection and provides an alternative approach for detection of PCV2.
Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4+, CD8+, CD4+ CD8+ (double-positive [DP]), and immunoglobulin M-positive (IgM+) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8+ and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM+ and CD8+ cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.
Intrinsic glioma subtypes (IGSs) are molecularly similar tumors that can be identified based on unsupervised gene expression analysis. Here, we have evaluated the clinical relevance of these subtypes within European Organisation for Research and Treatment of Cancer (EORTC) 26951, a randomized phase III clinical trial investigating adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in anaplastic oligodendroglial tumors. Our study includes gene expression profiles of formalin-fixed, paraffin-embedded (FFPE) clinical trial samples.
Patients and Methods
Gene expression profiling was performed in 140 samples, 47 fresh frozen samples and 93 FFPE samples, on HU133_Plus_2.0 and HuEx_1.0_st arrays, respectively.
All previously identified six IGSs are present in EORTC 26951. This confirms that different molecular subtypes are present within a well-defined histologic subtype. Intrinsic subtypes are highly prognostic for overall survival (OS) and progression-free survival (PFS). They are prognostic for PFS independent of clinical (age, performance status, and tumor location), molecular (1p/19q loss of heterozygosity [LOH], IDH1 mutation, and MGMT methylation), and histologic parameters. Combining known molecular (1p/19q LOH, IDH1) prognostic parameters with intrinsic subtypes improves outcome prediction (proportion of explained variation, 30% v 23% for each individual group of factors). Specific genetic changes (IDH1, 1p/19q LOH, and EGFR amplification) segregate into different subtypes. We identified one subtype, IGS-9 (characterized by a high percentage of 1p/19q LOH and IDH1 mutations), that especially benefits from PCV chemotherapy. Median OS in this subtype was 5.5 years after radiotherapy (RT) alone versus 12.8 years after RT/PCV (P = .0349; hazard ratio, 2.18; 95% CI, 1.06 to 4.50).
Intrinsic subtypes are highly prognostic in EORTC 26951 and improve outcome prediction when combined with other prognostic factors. Tumors assigned to IGS-9 benefit from adjuvant PCV.
Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.
The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.
The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.
Glioma; IDH1/2; Quantitative real-time PCR
Porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem for the swine industry in China. In this study, we investigated the genetic variation of PCV2 in China using strains isolated from 2004-2008. Viruses were isolated from samples collected from pigs with multi-systemic lesions and clinical signs of PMWS from different regions of China, and the genomes of these viruses were sequenced. The assembled sequences were used to define the genotypes of these strains; PCR-RFLP methodology was used to distinguish isolates and capture ELISA was used to demonstrate the antigenic changes resulted from ORF2 gene mutation of the isolates.
We identified 19 PCV2 isolates, including four newly emerging PCV2 mutant strains. The 19 isolates were designated into three genotypes (PCV2a, PCV2b and PCV2d). PCV2d represented a novel genotype and a shift from PCV2a to PCV2b as the predominant genotype in China was identified. This is the first report of 1766 nt PCV2 harboring a base deletion at other new different positions. Amino acid sequence analysis identified two novel ORF2 mutations (resulting in ORF2 sequences 705 and 708 nt in length) in three deletion strains (1766 nt) and one strain with a genome 1767 nt in length. Finding of two amino acids elongation of the ORF2-encoded Cap protein is firstly observed among PCV2 strains all over the world. The isolates were distinguished into different genotypes by PCR-RFLP methodology and antigenic changes were present in Cap protein of mutation isolates by capture ELISA.
The results of this study provide evidence that PCV2 is undergoing constant genetic variation and that the predominant strain in China as well as the antigenic situation has changed in recent years. Furthermore, the PCR-RFLP method presented here may be useful for the differential identification of PCV2 strains in future studies.
A competitive PCR (cPCR) assay was developed for monitoring porcine circovirus (PCV) DNA in serum samples from piglets. The cPCR was based on competitive coamplification of a 502- or 506-bp region of the PCV type 1 (PCV1) or PCV2 ORF2, respectively, with a known concentration of competitor DNA, which produced a 761- or 765-bp fragment, respectively. The cPCR was validated by quantification of a known amount of PCV wild-type plasmids. We also used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples. More than 50% of clinically healthy piglets could harbor both types of PCV. While PCV1 was detected in only 3 of 16 pigs with postweaning multisystemic wasting syndrome (PMWS), all the sick piglets contained PCV2. A comparison of the PCV2 DNA loads of healthy and sick animals revealed a significant difference, indicating that the development of PMWS may require a certain amount of PCV2.
Little information is known about viral distribution and transmission of porcine circovirus type 2 (PCV2) in species other than swine. It is still a debated topic whether the PCV2 could be infected and caused clinical lesions. Our study is aimed to estimate the susceptibility of Kunming mouse to PCV2. Forty-eight, 6-week-old Kunming mice were randomly divided into four groups. Group A (C1-C12) was inoculated with PK-15 cell culture as a control group. Group B (sPCV1-12) was inoculated orally and intramuscularly with PCV2 (106.2TCID50/ml). Group C (mPCV1-12) was inoculated orally and intramuscularly with PCV2 (106.2TCID50/ml) and a booster inoculation at days 14 and 28 after the first inoculation. Group D (MixPCV1-12) was unvaccinated but released into Group C. Each group was sacrificed at 7, 14, 28, and 42 days post-inoculation, respectively. Necropsy was checked on every mouse. Sera samples were collected for the test of PCV2 specific antibody. Tissues were collected for histopathology study and polymerase chain reaction (PCR).
The results showed that viral replication, seroconversion, and microscopic lesions were found in inoculated mice. Continuous existence of PCV2 viruses in lymph nodes have been confirmed by PCR, which took at least seven days for the virus to be transferred into other organs from the primary interface, and the diffusion to thymus had been retarded for seven days. Special PCV2 antibody could be found in PCV2 inoculation mice and was significantly higher than that in the control. Further more, microscopic lesions and the main target of PCV2 focused in the lymph nodes with a characteristic depletion and occasional necrosis of lymphocytes in the cortex and paracortex were found in inoculated mice.
The Kunming mouse could be infected by PCV2 virus and used as a PCV2 infected experimental model.
Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms.
Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc). Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples.
LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.
Presence of porcine circovirus type 1 (PCV1) and PCV2 was studied in sera and superficial inguinal lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs by using in situ hybridization and PCR. PCV1 and PCV2 were found in less than 3% and more than 50% of the samples, respectively. The most sensitive technique and site was PCR in superficial inguinal lymph nodes, but in situ hybridization correlated better with presence of characteristic lesions.
Accurate neuroendocrine neoplasia (NEN) staging is vital for determining prognosis and therapeutic strategy. The great majority of NENs express chromogranin A (CgA) which can be detected at a protein or transcript level. The current standards for lymph node metastasis detection are histological examination after Hematoxylin and Eosin (H&E) and CgA immunohistochemical (IHC) staining. We hypothesized that detection of CgA mRNA transcripts would be a more sensitive method of detecting these metastases.
We compared these traditional methods with PCR for CgA mRNA extracted from formalin fixed paraffin embedded slides of lymph nodes (n = 196) from small intestinal NENs, other gastrointestinal cancers and benign gastrointestinal disease. CgA PCR detected significantly more NEN lymph nodes (75%) than H&E (53%) or CgA IHC (57%) (p = 0.02). PCR detected CgA mRNA in 50% (14 of the 28) of SI-NEN lymph nodes previously considered negative. The false positive rate for detection of CgA mRNA was 19% in non-neuroendocrine cancers, and appeared to be due to occult neuroendocrine differentiation or contamination by normal epithelium during histological processing.
Molecular pathological analysis demonstrates the limitations of observer-dependent histopathology. CgA PCR analysis detected the presence of CgA transcripts in lymph nodes without histological evidence of tumor metastasis. Molecular node positivity (stage molN1) of SI-NEN lymph nodes could confer greater staging accuracy and facilitate early and accurate therapeutic intervention. This technique warrants investigation using clinically annotated tumor samples with follow-up data.
Chromogranin A; Immunohistochemistry; Histopathology; Lymph node; Metastasis; Micrometastasis; Neuroendocrine tumor; Neuroendocrine neoplasm; RNA; Staging
Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.
Infection of animals with a molecular viral clone is critical to study the genetic determinants of viral replication and virulence in the host. Type 2 porcine circovirus (PCV2) has been incriminated as the cause of postweaning multisystemic wasting syndrome (PMWS), an emerging disease in pigs. We report here for the first time the construction and use of an infectious molecular DNA clone of PCV2 to characterize the disease and pathologic lesions associated with PCV2 infection by direct in vivo transfection of pigs with the molecular clone. The PCV2 molecular clone was generated by ligating two copies of the complete PCV2 genome in tandem into the pBluescript SK (pSK) vector and was shown to be infectious in vitro when transfected into PK-15 cells. Forty specific-pathogen-free pigs at 4 weeks of age were randomly assigned to four groups of 10 each. Group 1 pigs served as uninoculated controls. Pigs in group 2 were each inoculated intranasally with about 1.9 × 105 50% tissue culture infective doses of a homogeneous PCV2 live virus stock derived from the molecular clone. Pigs in group 3 were each injected intrahepatically with 200 μg of the cloned PCV2 plasmid DNA, and pigs in group 4 were each injected into the superficial iliac lymph nodes with 200 μg of the cloned PCV2 plasmid DNA. Animals injected with the cloned PCV2 plasmid DNA developed infection resembling that induced by intranasal inoculation with PCV2 live virus stock. Seroconversion to PCV2-specific antibody was detected in the majority of pigs from the three inoculated groups at 35 days postinoculation (DPI). Viremia, beginning at 14 DPI and lasting 2 to 4 weeks, was detected in the majority of the pigs from all three inoculated groups. There were no remarkable clinical signs of PMWS in control or any of the inoculated pigs. Gross lesions in pigs of the three inoculated groups were similar and were characterized by systemically enlarged, tan lymph nodes and lungs that failed to collapse. Histopathological lesions and PCV2-specific antigen were detected in numerous tissues and organs, including brain, lung, heart, kidney, tonsil, lymph nodes, spleen, ileum, and liver of infected pigs. This study more definitively characterizes the clinical course and pathologic lesions exclusively attributable to PCV2 infection. The data from this study indicate that the cloned PCV2 genomic DNA may replace infectious virus for future PCV2 pathogenesis and immunization studies. The data also suggest that PCV2, although essential for development of PMWS, may require other factors or agents to induce the full spectrum of clinical signs and lesions associated with advanced cases of PMWS.