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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.
Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b  [cached]
Qiu Xiaohuo,Li Tian,Zhang Guorui,Cao Jingjing
Virology Journal , 2012, DOI: 10.1186/1743-422x-9-318
Abstract: Background 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. Results 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. Conclusions 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.
Porcine Circovirus type 2 (PCV2) causes apoptosis in experimentally inoculated BALB/c mice
Matti Kiupel, Gregory W Stevenson, Elizabeth J Galbreath, Adam North, Harm HogenEsch, Suresh K Mittal
BMC Veterinary Research , 2005, DOI: 10.1186/1746-6148-1-7
Abstract: PCV2 replicated and was associated with apoptosis in spleens, lymph nodes and Peyer's patches of infected BALB/c mice. Upregulation of caspase 1, 2, 3, 6, 7, 8, 11 and 12 and upregulation for the transcripts of apoptosis inhibitors bcl-2, bcl-w and bcl-X and apoptosis promoters' bax, bak and bad was detected in spleens of sPCV and mPCV mice, but not control mice. Apoptosis was further confirmed by light and electron microscopic morphology as well as by positive TUNEL assay and detection of activated caspase 3. PCV2 nucleic acid was detected by in-situ hybridization in the nuclei and cytoplasm of such apoptotic cells.The data presented here support the hypothesis that PCV2 induces apoptosis mediated through the activation of caspases 8 and 3 in the spleens of infected mice.Circoviruses, the smallest animal DNA viruses known so far, have a single copy of circular single-stranded ambisense DNA genome that varies in size between 1.7 and 2.3 kb. Animal circoviruses have been demonstrated in chickens (chicken anemia virus, ChAV, [49]), pigs (porcine circovirus, PCV, [45]), pigeons (pigeon circovirus, [47]) and psittacines (psittacine beak and feather disease virus, PBFDV, [36]). Porcine circovirus (PCV), an approximately 17 nm in diameter, non-enveloped virus with icosahedral symmetry, was originally identified as a noncytopathic contaminant of the PK-15 porcine kidney cell line [44]. The genome of PK-15 derived virus has been sequenced [28] and isolates of PCV that are genetically like PK-15 cell PCV are referred to as PCV1 [29]. Inoculation studies in pigs using PK-15 derived PCV1 did not result in clinical disease [1,46]. In 1990's, field strains of PCV have been found in lesions of pigs with postweaning multisystemic wasting syndrome (PMWS) [2,5,6,10,17,31,33,42]. Isolates of PMWS-associated PCV are genetically and antigenically different from the PK-15 cell PCV and are referred to as PCV2 [29].PMWS is clinically characterized by progressive weight loss, dyspnea, tach
Novel Genotypes of Type 2 Porcine Circovirus (PCV2) in PMWS Pigs in China Between 2008 and 2009
G.H. Zhao,W. Cheng,P.J. Zhang,Y.S. Han,D.K. Chen
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2010.3083.3091
Abstract: The present study analyzed genetic variation and genotypes of 17 strains of type 2 Porcine Circovirus (PCV2) from different epidemic regions in China in 2008 and 2009. All the genomic sequences were 1.767 bp in length. Sequence comparison of complete genomic sequences revealed 95.6-99.9% identity among 17 PCV2 strains and the most variable regions within 1.000-1.700 nt (located in the coding region of ORF2). Comparative analysis of amino acids of the two ORFs revealed that variation extend of ORF2 (93.1-100%) was greater than ORF1 (98.4-100%) and the third codon position showed much more variable than the first and second sites. Mutations in T and B lymphocyte epitopes were also detected by comparative analysis and it was found that T lymphocyte epitopes were more conserved than those of B lymphocytes. Phylogenetic analysis revealed 6 novel genotypes of PCV2 in addition to the 5 known geneotypes (PCV-2a, PCV-2b, PCV-2c, PCV-2d, PCV-2e) reported. Of these genotypes, the PCV-2b, PCV-2d and 3 unidentified genotypes were the most prevailing, within 13, 17 and 16 epidemic provinces, respectively. For the 17 Chinese PCV2 strains examined in this study, 5 strains represented PCV-2b genotype, 6 strains were PCV-2d, 1strain was PCV-2e and other 5 strains were novel genotypes while no strains were PCV-2a and PCV-2c genotype. These findings demonstrated the usefulness and attributes of complete genomic sequences for genetic variation and genotyping of PCV2 and have implications for the studies of population biology, molecular epidemiology and genetic structure of PCV2 and for the effective control of PMWS as well.
Reproduction in porcine circovirus type 2 (PCV2) seropositive gilts inseminated with PCV2b spiked semen
Sarli Giuseppe,Morandi Federico,Panarese Serena,Bacci Barbara
Acta Veterinaria Scandinavica , 2012, DOI: 10.1186/1751-0147-54-51
Abstract: Background Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen. Results Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen. Conclusions PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.
Porcine circovirus type 2 (PCV2): genetic variation and newly emerging genotypes in China
Long J Guo, Yue H Lu, Yan W Wei, Li P Huang, Chang M Liu
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-273
Abstract: 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.Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). This disease was first confirmed in Canada in 1997, and was then subsequently identified in pigs in the USA, France, Japan, Korea and other countries [1,2]. In recent years, PMWS has become a serious economic problem for the swine industry in China. PCV2 is a member of the genus Circovirus, of the family Circoviridae, the smallest non-enveloped, single-stranded, circular DNA viruses that replicate autonomously in mammalian cells. The viral DNA of PCV2, encapsulated by a single viral protein, is a single-stranded negative sense circularized molecule of 1767-1768 nucleotides (nt) [3-5]. Its genome contains two major open reading frames (ORFs), ORF1 which encodes two r
Rectal stenosis in pigs associated with Salmonella Typhimurium and porcine circovirus type 2 (PCV2) infection
Watanabe, Tatiane Terumi Negr?o;Zlotowski, Priscila;Oliveira, Luiz Gustavo Schneider de;Rolim, Ver?nica Machado;Gomes, Marcos José Pereira;Snel, Gustavo;Driemeier, David;
Pesquisa Veterinária Brasileira , 2011, DOI: 10.1590/S0100-736X2011000600009
Abstract: rectal stricture is an acquired annular fibrous constriction of the rectum that results from a variety of chronic necrotizing enteric diseases. in pigs, it is in most cases a sequel of salmonella infection. porcine circovirus type 2 (pcv2) is a known pathogen causing immunosuppression in pigs worldwide. pcv2 infected pigs may be predisposed to salmonellosis. in this report, rectal stenosis was observed in 160 pigs from a herd that experienced an outbreak of enteric salmonellosis over a 4-month period. distension of the abdominal wall and diarrhea were the main clinical signs observed. five animals were analyzed showing annular cicatrization of the rectal wall 5.0-7.0 cm anterior to the anorectal junction and salmonella-positive immunostaining in the large intestine. salmonella typhimurium was isolated from fragments of the large intestine. porcine circovirus type 2 antigen was observed in the mesenteric lymph-node in 4 pigs and in the large intestine in 3 pigs.
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
Transmission of porcine circovirus 2 (PCV2) by semen and viral distribution in different piglet tissues
Gava, Danielle;Zanella, Eraldo L.;Morés, Nelson;Ciacci-Zanella, Janice R.;
Pesquisa Veterinária Brasileira , 2008, DOI: 10.1590/S0100-736X2008000100011
Abstract: porcine circovirus infections are caused by the porcine circovirus 2 (pcv2). among six different clinical manifestations involving respiratory, enteric, nervous and reproductive signs, the postweaning multisystemic wasting syndrome (pmws) is the most important and studied disease. however, reproductive failures associated with pcv2 have been increasingly reported. some studies have shown the possible contamination of sows by semen of pcv2 positive boars. in order to investigate the transmission of pcv2 by contaminated semen and its ability to infect the sow and piglets, 20 pcv2 negative sows were inseminated, 10 with negative boar semen and 10 with previously nested-pcr tested positive boar semen. the sows were weekly monitored and blood samples were collected. based on the results, 4 out 20 sows were selected (1 sow was pcr negative and inseminated with a negative semen, 2 sows were pcr negative and inseminated with a positive semen and 1 sow was pcr negative and inseminated with a positive semen, but became pcr positive around the 30 days of pregnancy). after weaning, 12 male piglets, 3 of each sow, were selected and maintained under isolation. in order to investigate which organs harbored the virus, the young pigs were necropsied around 9 months of age. samples of serum collected monthly were tested by immunocitochemistry (icc), and all 12 pigs serum converted. samples of lymphoid, systemic and reproductive organs were analyzed by nested-pcr and immunohistochemistry (ihc). evaluation of the samples by nested-pcr, revealed that several tissues were positive in 10 of 12 pigs, mainly the lymph nodes, bone marrow and spleen. various samples were positive by ihc in 8 of 12 piglets, being the lymph nodes, tonsils and bulbourethral glands the most frequently positive. thus, the results of testing different samples, in the 3 tests (icc, nested-pcr and ihc) were complementary. these results show that pcv2 transmission through semen to the sows and piglets may occur and ma
First construction of infectious clone for newly emerging mutation porcine circovirus type 2 (PCV2) followed by comparison with PCV2a and PCV2b genotypes in biological characteristics in vitro
Long J Guo, Yue H Lu, Li P Huang, Yan W Wei, Hong L Wu, Chang M Liu
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-291
Abstract: Four PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 105.7 TCID50/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.Conclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.Porcine circovirus type 2 (PCV2) is a small non-enveloped virus with a single-stranded circular DNA genome of approximately 1.7 kb in size in the genus Circovirus, family Circoviridae [1-4]. Its genome contains at least two potentially functional open reading frames (ORFs): ORF1 (945 bp) encodes the Rep protein involved in viral replication and ORF2 (702 bp) encodes the immunogenic capsid protein [5-8]. PCV2 is generally considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for
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