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Development of PCR for the Identification of Porcine Circovirus Type 2 (PCV-2) Genotype PCV-2a and PCV-2b
Dongsheng He,Yanzong Zhao,Danping Su,Xiaoyun Niu
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2011.2398.2401
Abstract: PCR was developed to evaluate for its ability to simultaneously detect viral infections of swine. Specific primers were designed for each sub-type of porcine circovirus type 2 (PCV-2); porcine circovirus type 2a (PCV-2a) and porcine circovirus type 2b (PCV-2b). Each target produced specific amplicon with a size of 229 bp (PCV-2a) and 785 bp (PCV-2b). The assay was sensitive and specific in detecting the target agent in clinical specimens. In conclusion, the PCR has the potential to be useful for routine molecular diagnosis and epidemiology.
Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of S?o Paulo, Brazil
Alessnadra M.M.G. Castro, Taís F Cruz, Vanessa R Salgado, Tatiana M Kanashiro, Karen L Ferrari, Jo?o P Araujo, Paulo E Brand?o, Leonardo J Richtzenhain
Acta Veterinaria Scandinavica , 2012, DOI: 10.1186/1751-0147-54-29
Abstract: Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of S?o Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n?=?1) or 2b (n?=?10).The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of S?o Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.
Loop-mediated isothermal amplification for detection of porcine circovirus type 2
Shun Zhou, Si Han, Jianli Shi, Jiaqiang Wu, Xiaoyuan Yuan, Xiaoyan Cong, Shaojian Xu, Xiaoyan Wu, Jun Li, Jinbao Wang
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-497
Abstract: The amplification of LAMP could be obtained at 63°C for 60 min. The detection limit was nearly 1 copy of DNA plasmid, more sensitive than PCR. There was no cross-reaction with porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) under the same conditions.LAMP is an useful rapid detection method with high sensitivity and specificity for PCV2.Porcine circovirus (PCV) is a non-enveloped, single-stranded circular DNA virus, which is divided into two distinct genotypes according to pathogenicity, antigenicity and DNA sequence [1]. PCV1 was considered non-pathogenic. But PCV2 is considered as the major pathogen of postweaning multisystemic wasting syndrome (PMWS) [2]. In addition, PCV2 was also associated with porcine respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS), proliferative and necrotizing pneumonia (PNP), etc [3]. Piglets infected with PCV2 were immunosuppressive beacause of virus invading immune system [4]. It makes other bacteria and viruses have chance to infect hosts. Epidemiological investigation showed PCV2 was fairly common in swine herds recent years. Pig industry in China suffered economic losses owing to diseases caused by PCV2 [5]. At present, the detection technique used widely in the laboratory is conventional PCR and quantitative real time polymerase chain reaction (Q-PCR). However, these methods have some shortcomings, such as costly PCR instruments and a long detection period. These limitations affect their applications to certain environments.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that was developed originally by Notomi et al in 2000 [6]. The LAMP assay has qualities of high sensitivity and specificity, moreover, it's a quite rapid detection technique. The method employs four primers specific to six regions and Bst DNA polymerase which has a function of strand displacement. The whole amplification could be finished w
Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method
Kai Zhao, Wei Shi, Fangting Han, Yan Xu, Lianlong Zhu, Yong Zou, Xiao Wu, Hong Zhu, Furong Tan, Shiru Tao, Xueming Tang
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-126
Abstract: 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.Porcine circovirus type 2 (PCV2) is a non-enveloped, circular, single-stranded DNA virus that belongs to the Circoviridiae family [1]. This virus is widespread in the commercial swine population, and is accepted as the causative agent of a number of diseases in these animals, such as postweaning multisystemic wasting syndrome (PMWS) [2], and porcine dermatitis and nephropathy syndrome (PDNS). These syndromes cause great losses to the pig industry. As a result, it is necessary to develop an effective method for detecting PCV2 to prevent these diseases. At present, many methods have been developed for the detection of this virus; among which, conventional PCR is commonly used [3]. However, the usefulness of PCR is limited by the presence of PCR inhibitors in the analysis of real biological samples. The wide range of inhibitors (including organic and inorganic
Global Gene Expression Profiling of Myeloid Immune Cell Subsets in Response to In Vitro Challenge with Porcine Circovirus 2b  [PDF]
Bettina Mavrommatis, Victoria Offord, Robert Patterson, Mick Watson, Theo Kanellos, Falko Steinbach, Sylvia Grierson, Dirk Werling
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091081
Abstract: Compelling evidence suggests that the early interaction between porcine circovirus 2 (PCV-2) and the innate immune system is the key event in the pathogenesis of Post-Weaning Multisystemic Wasting Syndrome (PMWS). Furthermore, PCV2 has been detected in bone-marrow samples, potentially enabling an easy spread and reservoir for the virus. To assess the gene-expression differences induced by an in-vitro PCV2b infection in different three different myeloid innate immune cell subsets generated from the same animal, we used the Agilent Porcine Gene Expression Microarray (V2). Alveolar macrophages (AM?s), monocyte-derived dendritic cells (MoDCs) and bone-marrow cells (BMCs) were generated from each animal, and challenged with a UK-isolate of a PCV2 genotype b-strain at a MOI of 0.5. Remarkably, analysis showed a highly distinct and cell-type dependent response to PCV2b challenge. Overall, MoDCs showed the most marked response to PCV2b challenge in vitro and revealed a key role for TNF in the interaction with PCV2b, whereas only few genes were affected in BMCs and AM?s. These observations were further supported by an enrichment of genes in the downstream NF-κB Signalling pathway as well as an up regulation of genes with pro-apoptotic functions post-challenge. PCV2b challenge increases the expression of a large number of immune-related and pro-apoptotic genes mainly in MoDC, which possibly explain the increased inflammation, granulomatous inflammation and lymphocyte depletion seen in PMWS-affected pigs.
Loop-mediated isothermal amplification for the detection of goose circovirus
Grzegorz J Wozniakowski, Wojciech Kozdrun, Elzbieta Samorek-Salamonowicz
Virology Journal , 2012, DOI: 10.1186/1743-422x-9-110
Abstract: The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.
Genetic Characterization of Porcine Circovirus Type 2 from Pigs with Porcine Circovirus Associated Diseases in Argentina  [PDF]
Ariel Pereda,Pablo Pi?eyro,Ana Bratanich,María Alejandra Quiroga,Danilo Bucafusco,María Isabel Craig,Javier Cappuccio,Mariana Machuca,Agustina Rimondi,Marina Dibárbora,Hector Ramón Sanguinetti,Carlos Juan Perfumo
ISRN Veterinary Science , 2011, DOI: 10.5402/2011/560905
Abstract: Porcine circovirus type 2 (PCV-2) has been associated with syndromes grouped by the term porcine circovirus associated diseases (PCVAD). The PCV-2 isolates have been grouped into two major groups or genotypes according to their nucleotide sequence of whole genomes and/or ORF-2: PCV-2b, which have, in turn, been subdivided into three clusters (1A–1C), and PCV-2a, which has been subdivided into five clusters (2A–2E). In the present study, we obtained 16 sequences of PCV-2 from different farms from 2003 to 2008, from animals with confirmatory diagnosis of PCVAD. Since results showed an identity of 99.8% among them, they were grouped within a common cluster 1A-B. This preliminary study suggests a stable circulation of PCV-2b among the Argentinean pig population. 1. Introduction Porcine circovirus (PCV) is a small nonenveloped virus that contains a single-stranded circular DNA of about 1.76?kb. PCV was originally isolated as a noncytopathic contaminant of the PK-15 cell line [1] and was classified as a member of Circoviridae family, genus Circovirus, based on its morphological and genomic characteristics [2, 3]. Two phenotypically different but genetically related strains of PCV have been identified in swine. PCV-1 was first detected as a contaminant of the porcine kidney PK-15 cell line [4] whereas PCV-2 has been associated with postweaning multisystemic wasting syndrome (PMWS) [5, 6], porcine dermatitis and nephropathy syndrome (PDNS) [7], proliferative necrotizing pneumonia [8], and reproductive disorders [9], all of them included by the term porcine circovirus associated diseases (PCVAD) [10]. PMWS is an emerging disease in pigs first described in a swine herd in Canada in 1991 [5] and also endemic in many swine-producing countries. In Argentina, PMWS was first reported in 2002 [11]. Diagnosis of PMWS is based on the presence of compatible clinical signs [12], characteristic histopathological lesions, and detection of PCV-2 antigen within typical lesions [10, 13]. The genomic structure of PCV-2 consists of two intergenic regions flanked by three open reading frames (ORFs): ORF-1, which encodes two replication proteins (Rep and Rep′), ORF2, which encodes the Cap protein containing the immunoreactive epitopes and is more variable at nucleotide sequence than ORF1, and ORF3, which encodes a proapoptotic protein [14]. Currently, PCV-2 genotype definition and nomenclature has been proposed according to the genome sequence of the whole genome and/or ORF2 [15, 16]. Five genotypes have been identified to date [17, 18]: PCV-2a and PCV-2b, which correspond to the
Filter Bank Common Spatial Pattern Algorithm on BCI Competition IV Datasets 2a and 2b  [PDF]
Kai Keng Ang,Zheng Yang Chin,Cuntai Guan,Haihong Zhang
Frontiers in Neuroscience , 2012, DOI: 10.3389/fnins.2012.00039
Abstract: The Common Spatial Pattern (CSP) algorithm is an effective and popular method for classifying 2-class motor imagery electroencephalogram (EEG) data, but its effectiveness depends on the subject-specific frequency band. This paper presents the Filter Bank Common Spatial Pattern (FBCSP) algorithm to optimize the subject-specific frequency band for CSP on Datasets 2a and 2b of the Brain-Computer Interface (BCI) Competition IV. Dataset 2a comprised 4 classes of 22 channels EEG data from 9 subjects, and Dataset 2b comprised 2 classes of 3 bipolar channels EEG data from 9 subjects. Multi-class extensions to FBCSP are also presented to handle the 4-class EEG data in Dataset 2a, namely, Divide-and-Conquer (DC), Pair-Wise (PW), and One-Versus-Rest (OVR) approaches. Two feature selection algorithms are also presented to select discriminative CSP features on Dataset 2b, namely, the Mutual Information-based Best Individual Feature (MIBIF) algorithm, and the Mutual Information-based Rough Set Reduction (MIRSR) algorithm. The single-trial classification accuracies were presented using 10 × 10-fold cross-validations on the training data and session-to-session transfer on the evaluation data from both datasets. Disclosure of the test data labels after the BCI Competition IV showed that the FBCSP algorithm performed relatively the best among the other submitted algorithms and yielded a mean kappa value of 0.569 and 0.600 across all subjects in Datasets 2a and 2b respectively.
A meta-analysis that compares the use of either peginterferon-α2a or peginterferon-α2b plus ribavirin for HCV infection
Nan Xiao, Shuang Shi, Hui Zhuang
Hepatic Medicine: Evidence and Research , 2010, DOI: http://dx.doi.org/10.2147/HMER.S11916
Abstract: meta-analysis that compares the use of either peginterferon-α2a or peginterferon-α2b plus ribavirin for HCV infection Original Research (3267) Total Article Views Authors: Nan Xiao, Shuang Shi, Hui Zhuang Published Date July 2010 Volume 2010:2 Pages 99 - 109 DOI: http://dx.doi.org/10.2147/HMER.S11916 Nan Xiao*, Shuang Shi*, Hui Zhuang Department of Microbiology, Peking University Health Science Center, Beijing, China, *These authors contributed equally to this work Background: Two kinds of peginterferons, peginterferon-α2a (PEG-IFN-α2a) and peginterferon-α2b (PEG-IFN-α2b), are used in the treatment of chronic hepatitis C virus (HCV) infection. However, it is unclear which is better in terms of virological responses and patient compliance. We conducted a meta-analysis to assess which peginterferon was better when used with ribavirin. Methods: Relevant clinical trials were identified through the PubMed and EMBASE databases. Primary outcomes included early virological response (EVR), end of treatment response (ETR) and sustained virological response (SVR). Secondary outcomes included biochemical and histological responses and the discontinuation of treatment after adverse events. Meta-analysis was performed using xed-effect or random-effect methods, depending on absence or presence of signi cant heterogeneity. Analyses were performed with Review Manager Version 4.2.2. Results: Seven clinical trials were included that involved 3,526 patients in total; six were randomized clinical trials (RCTs) and one was nonrandomized. PEG-IFN-α2a plus ribavirin was better than PEG-IFN-α2b plus ribavirin with regards to ETR (relative risk [RR] = 1.21, 95% confidence interval [CI]: 1.14–1.28). This advantage was less obvious for EVR (RR = 1.12, 95% CI: 1.06–1.19) and SVR (RR = 1.10, 95% CI: 1.02–1.18). Patients who received PEG-IFN-α2a were less likely to discontinue treatment for safety reasons (RR = 0.85, 95% CI: 0.52–1.38). Conclusion: We demonstrated that PEG-IFN-α2a was a better choice than PEG-IFN-α2b in terms of virological responses.
Efficacy of pegylated interferon α-2a and α-2b in patients with genotype 1 chronic hepatitis C: a meta-analysis  [cached]
Coppola Nicola,Pisaturo Mariantonietta,Tonziello Gilda,Sagnelli Caterina
BMC Infectious Diseases , 2012, DOI: 10.1186/1471-2334-12-357
Abstract: Background Two formulations of Pegylated interferon (Peg-IFN) are on the market for treatment of chronic hepatitis C virus (HCV) infection. The purpose of this meta-analysis was to assess the efficacy of Peg-IFN α-2a versus Peg-IFN α-2b in combination with ribavirin in anti-human immunodeficiency virus (HIV)-negative patients with genotype 1 chronic HCV infection. Methods The following criteria were to be met for inclusion in the meta-analysis: (a) original data from randomized and non-randomized clinical trials; (b) study on the efficacy of conventional doses of Peg-IFN α-2a (180 μg/week) versus Peg-IFN α-2b (1.5 μg/kg of body weight/week), both in combination with ribavirin, in antiviral therapy-na ve HCV-genotype 1 subjects; (c) at least one of these primary outcomes: Rapid Virological Response (RVR); Early Complete Virological Response (EVR); End of Treatment Response (ETR); Sustained Virological Response (SVR); (d) odds ratio estimates of relative risk (RR) and associated 95% confidence intervals (CIs) or at least data enabling them to be computed; (e) English language; and (f) published as a full paper up to December 2011. Results Seven published studies met the inclusion criteria, allowing a meta-analysis on 3,026 patients. Peg-IFN α-2a and Peg-IFN α-2b showed similar rate of RVR (RR = 1.05; 95% CI = 0.87-1.27, p = 0.62) and SVR (RR = 1.08; 95% CI = 0.99-1.18, p = 0.098). Peg-IFN α-2a more frequently than Peg-IFN α-2b achieved EVR (RR = 1.11; 95% CI = 1.02-1.21, p = 0.013) and ETR (RR = 1.22; 95% CI = 1.14-1.31, p < 0.0001). Conclusion The standard schedules of Peg-IFN α-2a and Peg-IFN α-2b, both in combination with ribavirin, can be used indifferently for patients with chronic HCV genotype 1 who are anti- to eliminate HIV-negative and antiviral treatment-na ve.
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