Porcine circovirus type 3(PCV3)was initially identified in 2016 in pigs exhibiting unexplained cardiac and multi-organ inflammation in the USA(Palinski et al.2017).PCV3 has subsequently been identified in numerous cou...Porcine circovirus type 3(PCV3)was initially identified in 2016 in pigs exhibiting unexplained cardiac and multi-organ inflammation in the USA(Palinski et al.2017).PCV3 has subsequently been identified in numerous countries,including China,Brazil,Italy,and others,demonstrating widespread viral dissemination(Tan et al.2021).Notably,recent investigations have revealed PCV3 infection across multiple species,including pigs,cattle,dogs,wild boars,chamois,roe deer,and others(Tan et al.2021).This evidence suggests potential viral propagation beyond its primary host(pigs).展开更多
[Objectives]This study was conducted to find a convenient and reliable method for detecting the virus titer of porcine circovirus type 2(PCV2).[Methods]The reaction conditions of the immunoperoxidase monolayer assay(I...[Objectives]This study was conducted to find a convenient and reliable method for detecting the virus titer of porcine circovirus type 2(PCV2).[Methods]The reaction conditions of the immunoperoxidase monolayer assay(IPMA)method for detecting the viral titer of PCV2 were optimized,and the results were compared with those obtained by indirect immunofluorescence assay(IFA).[Results]PCV2-infected cells exhibited brownish-red staining in either the nucleus or cytoplasm when detected by IPMA,and the detection results were largely consistent with IFA detection.[Conclusions]Both IPMA and IFA methods can be effectively used for determination of PCV2 viral titer,providing reliable support for assessing viral content during PCV2 vaccine development and validating virus inactivation efficacy.展开更多
Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-i...Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.展开更多
Porcine circovirus type 3(PCV3),which was first detected in the United States of America in 2015,is a potential threat to the swine industry.However,the prevalence of PCV3 in Shanxi Province,China,is unclear.In this r...Porcine circovirus type 3(PCV3),which was first detected in the United States of America in 2015,is a potential threat to the swine industry.However,the prevalence of PCV3 in Shanxi Province,China,is unclear.In this research,the prevalence and genetic diversity of PCV3 were investigated in above area.Lung tissue samples(n=491)from 19 pig slaughterhouses across 11 cities throughout Shanxi Province were analyzed for PCV3 infection by PCR in 2019.The results showed that PCV3 positive rates in slaughterhouses and individuals were 100%(19/19)and 86.76%(426/491),respectively.PCV2 and PCV3 double-positive rates in slaughterhouses and individuals were 100%(19/19)and 59.27%(291/491),respectively.PCR positive samples were further sequenced and 8 PCV3 isolates were identified.The nucleotide homology of these isolates with other PCV3 isolates in NCBI database was 97.45-99.90%.A phylogenetic analysis,based on the complete genomic sequence and ORF2,divided these PCV3 strains into 2 major groups.Based on A24A/and R27/K amino acid mutations of capsid protein,the 8 identified PCV3 strains were separated to 2 clades.This was the first detailed investigation into the epidemiology of PCV3 in Shanxi Province.Our findings enabled us to assess the possibility of widespread transmission from this region.Thus,current findings establish a basis for further studies of genetic variations in PCV3 strains circulating in China.展开更多
Porcine circovirus type 2(PCV2),which serves as a major causative agent of PCV2-associated diseases and causes severe loss to the pig industry worldwide,can dysregulate the immune response and induce immunosuppression...Porcine circovirus type 2(PCV2),which serves as a major causative agent of PCV2-associated diseases and causes severe loss to the pig industry worldwide,can dysregulate the immune response and induce immunosuppression in PCV2-infected pigs.Similar to PCV2(porcine circovirus type 3(PCV3),a newly identified swine circovirus which might be closely associated with porcine dermatitis and nephropathy syndrome,reproductive disorder,and multisystemic inflammatoty responses,also interferes with host immune defense.Interaction between host immune system and PCVs is considered to be a crucial determinant of pathogenicity in pigs.Here,we sought to briefly discuss the current knowledge regarding the interaction of porcine circovirus type 2 and/or 3 with host immune cells and immune responses to better depict the viral immunomodulatory capacity,pathogenic mechanisms,and the future research direction in host immune responses to infection with PCV2 and PCV3.展开更多
Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zh...Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.展开更多
To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Seq...To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Sequence and phylogenetic analysis were carried out to compare these six strains with another 27 DuCV strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV, such as a stem-loop structure, three major open reading frames (Rep, Cap and ORF3), four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein. Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes. The DuCV strains with complete genomes containing 1988 and 1989 nucleotides clustered in genotype A, whereas the strains with complete genomes containing 1991, 1992, 1995 and 1996 nucleotides lay in genotype B. The six DuCV strains from Cherry Valley ducks were divided into the two groups. The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.展开更多
The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Bas...The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.展开更多
Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection ...Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.展开更多
Porcine circovirus type 2（PCV2）has recently been reported to elicit the unfolded protein response（UPR）via activation of the PERK/e IF2α（RNA-activated protein kinase-like endoplasmic reticulum（ER）kinase/eukaryo...Porcine circovirus type 2（PCV2）has recently been reported to elicit the unfolded protein response（UPR）via activation of the PERK/e IF2α（RNA-activated protein kinase-like endoplasmic reticulum（ER）kinase/eukaryotic initiation factor 2α）pathway.This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein.By transient expression,we found that both replicase（Rep）and capsid（Cap）proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eI F2α-ATF4（activating transcription factor 4）-CHOP（CCAAT/enhancer-binding protein homologous protein）axis.Cap expression,but not Rep,significantly reduced antiapoptotic B-cell lymphoma-2（Bcl-2）and increased caspase-3 cleavage,possibly due to increased expression of CHOP.Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression,caspase-3cleavage,and apoptotic cell death possibly by partially rescuing Bcl-2 expression,we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eI F2α/ATF4/CHOP/Bcl-2 pathway.This study,together with our earlier studies,provides insight into the mechanisms underlying PCV2 pathogenesis.展开更多
Four species of porcine circoviruses(PCV1–4)have been reported to circulate in Chinese domestic pigs,while the epizootiology of these viruses in free-ranging wild boars in China remains unknown.In this study,tissue a...Four species of porcine circoviruses(PCV1–4)have been reported to circulate in Chinese domestic pigs,while the epizootiology of these viruses in free-ranging wild boars in China remains unknown.In this study,tissue and serum samples collected from diseased or apparently healthy wild boars between 2018 and 2020 in 19 regions of China were tested for the prevalence of PCV1–4 infections.Positive rates of PCV1,PCV2,and PCV3 DNA in the tissue samples of Chinese wild boars were 1.6%(4/247),58.3%(144/247),and 10.9%(27/247)respectively,with none positive for PCV4.Sequence analysis of viral genome showed that the four PCV1 strains distributed in Hunan and Inner Mongolia shared 97.5%–99.6%sequence identity with global distributed reference strains.Comparison of the ORF2 gene sequences showed that 80 PCV2 strains widely distributed in 18 regions shared 79.5%–100%sequence identity with reference strains from domestic pigs and wild boars,and were grouped into PCV2a(7),PCV2b(31)and PCV2d(42).For PCV3,17 sequenced strains shared 97.2%–100%nucleotide identity at the genomic level and could be divided into PCV3a(3),PCV3b(2)and PCV3c(12)based on the phylogeny of ORF2 gene sequences.Serological data revealed antibody positive rates against PCV1 and PCV2 of 11.4%(19/167)and 53.9%(90/167)respectively.The data obtained in this study improved our understanding about the epidemiological situations of PCVs infection in free-ranging wild boars in China and will be valuable for the prevention and control of diseases caused by PCVs infection.展开更多
In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBan...In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.展开更多
Dear Editor,Mink circovirus (MiCV), which is clustered in the genus Circovirus of the family Circoviridae, was first described in minks from farms in Dalian, China in 2013 (Lian et al.2014). The complete single-strand...Dear Editor,Mink circovirus (MiCV), which is clustered in the genus Circovirus of the family Circoviridae, was first described in minks from farms in Dalian, China in 2013 (Lian et al.2014). The complete single-stranded circular genome of the virus is 1,753 nucleotides long and contains two major open reading frames (ORFs), designated ORF1 (Rep gene)and ORF2 (Cap gene)(Lian et al. 2014;Ge et al. 2018).展开更多
The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods....The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.展开更多
Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their ho...Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their hosts. The capsid-associated protein (Cap) of circoviruses is of interest because of its role in viral structure, immune evasion, host cell entry, and nuclear shuttling of viral components. The structure of the porcine circovirus 2 (PCV2) Cap has been solved and offered insight to these functions. Based on the crystallographic PCV2 Cap structure, models from circoviruses isolated from avian, fish, and mammalian hosts have been constructed and analyzed to better understand the roles of these proteins in the virus family. A high degree of conservation is observed in the models, however, the surface antigens differ among viruses. This is likely a reflection of the small genome harbored by circoviruses, and therefore the requirement of their few proteins to carry out specific vital functions, while maintaining enough variation to successfully infect their hosts. Here we describe the putative structures of a range of Cap proteins from circoviruses based on the crystallographic determination of porcine Cap, identifying key regions for function and inhibition of crystal formation.展开更多
PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2...PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.展开更多
We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 ...We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 in PK15 cells and obtained from sera of pigs with postweaning multisystemic wasting syndrome(PMWS). These subviral isolates ranged from 358 nt to 1 125 nt genomes. Investigating the complexity and diversity of PCV2 DI in vivo and in vitro can help elucidating the evolutionary history of PCV2.展开更多
Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult....Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult. With the rapid development of molecular biological and immunological technologies, the laboratory diagnostic methods for duck circovirus infection also advance greatly. The paper summarizes the research advances in various laboratory detection methods for duck cireovirus infection including PCR, nested PCR, multiple PCR, fluorescence quantitative PCR, LAMP, nucleic acid probe and ELISA, as well as their ad- vantages and shortages, aiming at providing reference for finding novel detection methods and for the diagnosis and comprehensive prevention and control.展开更多
Porcine cimovirus （PCV） is the smallest animal virus so far and has two serotypes. PCV1 is nonpathogenic, but PCV2 is pathogenic and causes post-weaning multisystemic wasting syndrome （ PMWS）. Factors to induce PM...Porcine cimovirus （PCV） is the smallest animal virus so far and has two serotypes. PCV1 is nonpathogenic, but PCV2 is pathogenic and causes post-weaning multisystemic wasting syndrome （ PMWS）. Factors to induce PMWS include immunity and infection status of sows, infec- tion time, mixed infection, PCV2 variants, physical status of gilts, and feeding management. For final diagnosis, histopathological changes and ex- istence of PCV2 in lymphoid tissues are professional standards, because fluorescence quantitative RT-PCR is not enough specific or sensitive. The commemial PCV2 vaccines can reduce occurrence of PMWS and PCV-related diseases. This paper reviews recent advances in epidemiology of PCV2 as well as diagnosis and control of PMWS.展开更多
[ Objective] This study aimed to investigate the molecular characteristics of porcine circovirus type 2 （PCV2） strains isolated from Hainan Province. [ Method] The complete genome of PCV2 was amplified from PMWS-sus...[ Objective] This study aimed to investigate the molecular characteristics of porcine circovirus type 2 （PCV2） strains isolated from Hainan Province. [ Method] The complete genome of PCV2 was amplified from PMWS-suspected samples by PCR for sequence analysis. [ Result] A total of eight PCV2 strains were isolated and identified. All the eight isolates belonged to genotype PCV2b, among which seven isolates belonged to subgenotype PCV2b-1 C, and one isolate be- longed to subgenotype PCV2b-IA/1B. ORF2 gene of PCV2 isolates from Hainan Province was 705 bp in length, encoding 234 amino acids. Antigenic epitopes of Cap protein exhibited certain changes. Nucleotide sequences and deduced amino acid sequences of ORF2 gene shared 95.3% -99.7% and 93.6% - 100% simi- larities among eight PCV2 isolates from Hainan Province, respectively. Moreover, nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 91.0% -99.9% and 91.0% -99.6% similarities with other PCV2 strain isolated from China （AY682994, AF381175, JX945577, JX682407, AY180397 ）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.0% - 97.0% and 88.0% -97.9% similarities with PCV2 isolates from other countries （ NC_005148, JQ994268, KJ187306, AF201307, AF454546, AY＂/Sd020）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 98.2% -100% and 94.9% -100% similarities with vaccine strain SH, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.6% -91.7% and 89.7% - 91.0% similarities with vaccine strain LG, respectively. [ Conclusion] This study provided theoretical hasis for the prevention and control of PCV2 and selection of vaccine strains in Hainan Province.展开更多
基金supported by the National Key Research and Development Program of China (2023YFD1800500)the Jiangsu Innovative and Entrepreneurial Talent Team Project,China (JSSCTD202224)+1 种基金the 111 Project D18007the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD),China
文摘Porcine circovirus type 3(PCV3)was initially identified in 2016 in pigs exhibiting unexplained cardiac and multi-organ inflammation in the USA(Palinski et al.2017).PCV3 has subsequently been identified in numerous countries,including China,Brazil,Italy,and others,demonstrating widespread viral dissemination(Tan et al.2021).Notably,recent investigations have revealed PCV3 infection across multiple species,including pigs,cattle,dogs,wild boars,chamois,roe deer,and others(Tan et al.2021).This evidence suggests potential viral propagation beyond its primary host(pigs).
基金Supported by General Project of Shandong Provincial Natural Science Foundation(ZR2022MC173)Binzhou Young Science and Technology Rising Star Program(QMX2023004)Binzhou Comprehensive Experimental Station of Shandong Swine Industry Technology System Project(SDAIT-08-13).
文摘[Objectives]This study was conducted to find a convenient and reliable method for detecting the virus titer of porcine circovirus type 2(PCV2).[Methods]The reaction conditions of the immunoperoxidase monolayer assay(IPMA)method for detecting the viral titer of PCV2 were optimized,and the results were compared with those obtained by indirect immunofluorescence assay(IFA).[Results]PCV2-infected cells exhibited brownish-red staining in either the nucleus or cytoplasm when detected by IPMA,and the detection results were largely consistent with IFA detection.[Conclusions]Both IPMA and IFA methods can be effectively used for determination of PCV2 viral titer,providing reliable support for assessing viral content during PCV2 vaccine development and validating virus inactivation efficacy.
基金supported by Grants from the National Key Research and Development Program (2016YFD0500703)Major Science and Technology Projects in Henan Province (171100110200)Luoyang HeLuo Talent Plan (Dr. Kegong Tian)
文摘Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.
基金This research was supported by funding from the Key Research and Development Projea Key Program of Shanxi Province,China(grant no.201703D211001).
文摘Porcine circovirus type 3(PCV3),which was first detected in the United States of America in 2015,is a potential threat to the swine industry.However,the prevalence of PCV3 in Shanxi Province,China,is unclear.In this research,the prevalence and genetic diversity of PCV3 were investigated in above area.Lung tissue samples(n=491)from 19 pig slaughterhouses across 11 cities throughout Shanxi Province were analyzed for PCV3 infection by PCR in 2019.The results showed that PCV3 positive rates in slaughterhouses and individuals were 100%(19/19)and 86.76%(426/491),respectively.PCV2 and PCV3 double-positive rates in slaughterhouses and individuals were 100%(19/19)and 59.27%(291/491),respectively.PCR positive samples were further sequenced and 8 PCV3 isolates were identified.The nucleotide homology of these isolates with other PCV3 isolates in NCBI database was 97.45-99.90%.A phylogenetic analysis,based on the complete genomic sequence and ORF2,divided these PCV3 strains into 2 major groups.Based on A24A/and R27/K amino acid mutations of capsid protein,the 8 identified PCV3 strains were separated to 2 clades.This was the first detailed investigation into the epidemiology of PCV3 in Shanxi Province.Our findings enabled us to assess the possibility of widespread transmission from this region.Thus,current findings establish a basis for further studies of genetic variations in PCV3 strains circulating in China.
基金This study was supported by a grant from the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD).
文摘Porcine circovirus type 2(PCV2),which serves as a major causative agent of PCV2-associated diseases and causes severe loss to the pig industry worldwide,can dysregulate the immune response and induce immunosuppression in PCV2-infected pigs.Similar to PCV2(porcine circovirus type 3(PCV3),a newly identified swine circovirus which might be closely associated with porcine dermatitis and nephropathy syndrome,reproductive disorder,and multisystemic inflammatoty responses,also interferes with host immune defense.Interaction between host immune system and PCVs is considered to be a crucial determinant of pathogenicity in pigs.Here,we sought to briefly discuss the current knowledge regarding the interaction of porcine circovirus type 2 and/or 3 with host immune cells and immune responses to better depict the viral immunomodulatory capacity,pathogenic mechanisms,and the future research direction in host immune responses to infection with PCV2 and PCV3.
基金Project supported by the Foundation of Agricultural Development of Hangzhou City, China
文摘Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.
基金the grant of Shandong Province Higher Educational Science and Technology Program (J08LF07)Shandong Provincial Natural Sciences Fund (Q2006D04)
文摘To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Sequence and phylogenetic analysis were carried out to compare these six strains with another 27 DuCV strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV, such as a stem-loop structure, three major open reading frames (Rep, Cap and ORF3), four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein. Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes. The DuCV strains with complete genomes containing 1988 and 1989 nucleotides clustered in genotype A, whereas the strains with complete genomes containing 1991, 1992, 1995 and 1996 nucleotides lay in genotype B. The six DuCV strains from Cherry Valley ducks were divided into the two groups. The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.
基金Shandong Provincial Excellent YoungScientist Fund (2004BS06002)Innovation Fund ofShandong Agricultural University
文摘The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.
基金Project supported by the Zhejiang Provincial Department of Science and Technology(No.2018C02028),China。
文摘Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.
基金supported by the National Natural Science Foundation of China(No.31272534)the Department of Education of Zhejiang Province(No.Y201635576),China
文摘Porcine circovirus type 2（PCV2）has recently been reported to elicit the unfolded protein response（UPR）via activation of the PERK/e IF2α（RNA-activated protein kinase-like endoplasmic reticulum（ER）kinase/eukaryotic initiation factor 2α）pathway.This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein.By transient expression,we found that both replicase（Rep）and capsid（Cap）proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eI F2α-ATF4（activating transcription factor 4）-CHOP（CCAAT/enhancer-binding protein homologous protein）axis.Cap expression,but not Rep,significantly reduced antiapoptotic B-cell lymphoma-2（Bcl-2）and increased caspase-3 cleavage,possibly due to increased expression of CHOP.Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression,caspase-3cleavage,and apoptotic cell death possibly by partially rescuing Bcl-2 expression,we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eI F2α/ATF4/CHOP/Bcl-2 pathway.This study,together with our earlier studies,provides insight into the mechanisms underlying PCV2 pathogenesis.
基金This work was supported by grants from the National Natural Science Foundation of China to Z Tu(31902307)National Key Research and Development Program of China to C Tu(2017YFD0500104)Wildlife Borne Infectious Diseases Monitoring Project of the State Forestry and Grassland Administration of China.
文摘Four species of porcine circoviruses(PCV1–4)have been reported to circulate in Chinese domestic pigs,while the epizootiology of these viruses in free-ranging wild boars in China remains unknown.In this study,tissue and serum samples collected from diseased or apparently healthy wild boars between 2018 and 2020 in 19 regions of China were tested for the prevalence of PCV1–4 infections.Positive rates of PCV1,PCV2,and PCV3 DNA in the tissue samples of Chinese wild boars were 1.6%(4/247),58.3%(144/247),and 10.9%(27/247)respectively,with none positive for PCV4.Sequence analysis of viral genome showed that the four PCV1 strains distributed in Hunan and Inner Mongolia shared 97.5%–99.6%sequence identity with global distributed reference strains.Comparison of the ORF2 gene sequences showed that 80 PCV2 strains widely distributed in 18 regions shared 79.5%–100%sequence identity with reference strains from domestic pigs and wild boars,and were grouped into PCV2a(7),PCV2b(31)and PCV2d(42).For PCV3,17 sequenced strains shared 97.2%–100%nucleotide identity at the genomic level and could be divided into PCV3a(3),PCV3b(2)and PCV3c(12)based on the phylogeny of ORF2 gene sequences.Serological data revealed antibody positive rates against PCV1 and PCV2 of 11.4%(19/167)and 53.9%(90/167)respectively.The data obtained in this study improved our understanding about the epidemiological situations of PCVs infection in free-ranging wild boars in China and will be valuable for the prevention and control of diseases caused by PCVs infection.
基金Fifteenth National Science and Technology Support Program of China(2007BAD86B04-4)
文摘In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.
基金financially supported by the National Key Research and Development Program of China (No. 2017YFD0500103)the National Natural Science Foundation of China (No. 31772747)+2 种基金the Science and Technology Development Project of Jilin Province (No. 20170623043TC)the Program for JLU Science and Technology Innovative Research Team (JLUSTIRT, No. 2017TD-28)the Fundamental Research Funds for the Central Universities
文摘Dear Editor,Mink circovirus (MiCV), which is clustered in the genus Circovirus of the family Circoviridae, was first described in minks from farms in Dalian, China in 2013 (Lian et al.2014). The complete single-stranded circular genome of the virus is 1,753 nucleotides long and contains two major open reading frames (ORFs), designated ORF1 (Rep gene)and ORF2 (Cap gene)(Lian et al. 2014;Ge et al. 2018).
基金supported by the National Science and Technology Support Program(2015BAD12B05)the China Agricultural Research System(CARS-43-8)+2 种基金the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province,China(2014NZ0030)the Ministry of Education Program of China(20125103110013)the Sichuan Province Research Programs,China(2013HH0042/2013 TD0015/2014-002)
文摘The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.
文摘Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their hosts. The capsid-associated protein (Cap) of circoviruses is of interest because of its role in viral structure, immune evasion, host cell entry, and nuclear shuttling of viral components. The structure of the porcine circovirus 2 (PCV2) Cap has been solved and offered insight to these functions. Based on the crystallographic PCV2 Cap structure, models from circoviruses isolated from avian, fish, and mammalian hosts have been constructed and analyzed to better understand the roles of these proteins in the virus family. A high degree of conservation is observed in the models, however, the surface antigens differ among viruses. This is likely a reflection of the small genome harbored by circoviruses, and therefore the requirement of their few proteins to carry out specific vital functions, while maintaining enough variation to successfully infect their hosts. Here we describe the putative structures of a range of Cap proteins from circoviruses based on the crystallographic determination of porcine Cap, identifying key regions for function and inhibition of crystal formation.
基金National Key Research and Development Program of China,Grant/Award Number:2017YFD0500103National Natural Science Foundation of China,Grant/Award Number:31772747,31272385+3 种基金Jilin Province Science and Technology Development Projects,Grant/Award Number:20150204077NYGraduate Innovation Fund of Jilin Universitythe Program for Changjiang Scholarsthe University Innovative Research Team,Grant/Award Number:IRT1248
文摘PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.
基金Supported by National Natural Science Foundation of China(31272574,30972184)Fund for Independent Innovation of Agricultural Sciences in Jiangsu Province[CX(11)2060]
文摘We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 in PK15 cells and obtained from sera of pigs with postweaning multisystemic wasting syndrome(PMWS). These subviral isolates ranged from 358 nt to 1 125 nt genomes. Investigating the complexity and diversity of PCV2 DI in vivo and in vitro can help elucidating the evolutionary history of PCV2.
文摘Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult. With the rapid development of molecular biological and immunological technologies, the laboratory diagnostic methods for duck circovirus infection also advance greatly. The paper summarizes the research advances in various laboratory detection methods for duck cireovirus infection including PCR, nested PCR, multiple PCR, fluorescence quantitative PCR, LAMP, nucleic acid probe and ELISA, as well as their ad- vantages and shortages, aiming at providing reference for finding novel detection methods and for the diagnosis and comprehensive prevention and control.
基金funded by the Chinese Academy of Science Knowledge Innovation Project ( KZCX2-EW-QN411,Kscx2-Yw-N-051)the National Basic Research Program of China( 2009CB118800)
文摘Porcine cimovirus （PCV） is the smallest animal virus so far and has two serotypes. PCV1 is nonpathogenic, but PCV2 is pathogenic and causes post-weaning multisystemic wasting syndrome （ PMWS）. Factors to induce PMWS include immunity and infection status of sows, infec- tion time, mixed infection, PCV2 variants, physical status of gilts, and feeding management. For final diagnosis, histopathological changes and ex- istence of PCV2 in lymphoid tissues are professional standards, because fluorescence quantitative RT-PCR is not enough specific or sensitive. The commemial PCV2 vaccines can reduce occurrence of PMWS and PCV-related diseases. This paper reviews recent advances in epidemiology of PCV2 as well as diagnosis and control of PMWS.
基金Supported by Natural Science Foundation of Hainan Province(Grant No.313100)
文摘[ Objective] This study aimed to investigate the molecular characteristics of porcine circovirus type 2 （PCV2） strains isolated from Hainan Province. [ Method] The complete genome of PCV2 was amplified from PMWS-suspected samples by PCR for sequence analysis. [ Result] A total of eight PCV2 strains were isolated and identified. All the eight isolates belonged to genotype PCV2b, among which seven isolates belonged to subgenotype PCV2b-1 C, and one isolate be- longed to subgenotype PCV2b-IA/1B. ORF2 gene of PCV2 isolates from Hainan Province was 705 bp in length, encoding 234 amino acids. Antigenic epitopes of Cap protein exhibited certain changes. Nucleotide sequences and deduced amino acid sequences of ORF2 gene shared 95.3% -99.7% and 93.6% - 100% simi- larities among eight PCV2 isolates from Hainan Province, respectively. Moreover, nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 91.0% -99.9% and 91.0% -99.6% similarities with other PCV2 strain isolated from China （AY682994, AF381175, JX945577, JX682407, AY180397 ）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.0% - 97.0% and 88.0% -97.9% similarities with PCV2 isolates from other countries （ NC_005148, JQ994268, KJ187306, AF201307, AF454546, AY＂/Sd020）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 98.2% -100% and 94.9% -100% similarities with vaccine strain SH, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.6% -91.7% and 89.7% - 91.0% similarities with vaccine strain LG, respectively. [ Conclusion] This study provided theoretical hasis for the prevention and control of PCV2 and selection of vaccine strains in Hainan Province.
