TypeⅡdiabetes mellitus(T2DM)has experienced a dramatic increase globally across countries of various income levels over the past three decades.The persistent prevalence of T2DM is attributed to a complex interplay of...TypeⅡdiabetes mellitus(T2DM)has experienced a dramatic increase globally across countries of various income levels over the past three decades.The persistent prevalence of T2DM is attributed to a complex interplay of genetic and environmental factors.While numerous pharmaceutical therapies have been developed,there remains an urgent need for innovative treatment approaches that offer effectiveness without significant adverse effects.In this context,the exploration of the gut microbiome presents a promising avenue.Research has increasingly shown that the gut microbiome of individuals with T2DM exhibits distinct differences compared to healthy individuals,suggesting its potential role in the disease’s pathogenesis and progression.This emerging field offers diverse applications,particularly in modifying the gut environment through the administration of prebiotics,probiotics,and fecal microbiome transfer.These interventions aim to restore a healthy microbiome balance,which could potentially alleviate or even reverse the metabolic dysfunctions associated with T2DM.Although current results from clinical trials have not yet shown dramatic effects on diabetes management,the groundwork has been laid for deeper investigation.Ongoing and future clinical trials are critical to advancing our understanding of the microbiome’s impact on diabetes.By further elucidating the mechanisms through which microbiome alterations influence insulin resistance and glucose metabolism,researchers can develop more targeted interventions.The potential to harness the gut microbiome in developing new therapeutic strategies offers a compelling prospect to transform the treatment landscape of T2DM,potentially reducing the disease’s burden significantly with approaches that are less reliant on traditional pharmaceuticals and more focused on holistic,systemic health improvements.展开更多
Diabetes mellitus is associated with foot ulcers,which frequently pave the way to lower-extremity amputation.Neuropathy,trauma,deformity,high plantar pressures,and peripheral vascular disease are the most common under...Diabetes mellitus is associated with foot ulcers,which frequently pave the way to lower-extremity amputation.Neuropathy,trauma,deformity,high plantar pressures,and peripheral vascular disease are the most common underlying causes.Around 15%of diabetic patients are affected by diabetic foot ulcer in their lifetime.64 million people are affected by diabetics in India and 40000 amputations are done every year.Foot ulcers are evaluated and classified in a systematic and thorough manner to assist in determining the best course of therapy.This paper proposes a novel model which predicts the threat of diabetic foot ulcer using independent agents for various input values and a combination of fuzzy expert systems.The proposed model uses a classification system to distinguish between each fuzzy framework and its parameters.Based on the severity levels necessary prevention,treatment,and medication are recommended.Combining the results of all the fuzzy frameworks derived from its constituent parameters,a risk-specific medication is recommended.The work also has higher accuracy when compared to other related models.展开更多
Based on the observation data of meteorological stations,Doppler radar observation data of Ulanqab City,and ERA-5 reanalysis data,a snowstorm process in Ulanqab City from March 17 to 18,2022 was analyzed.The results s...Based on the observation data of meteorological stations,Doppler radar observation data of Ulanqab City,and ERA-5 reanalysis data,a snowstorm process in Ulanqab City from March 17 to 18,2022 was analyzed.The results show that this was a type Ⅱ snowstorm process generated under the joint influence of upper trough and ground low inverted trough and frontal cyclone.The main period of snowfall can be divided into two time stages,and the total snowfall was more in the south and less in the north,which was consistent with that of average specific humidity field.Water vapor conditions provided by strong water vapor transport and convergence,strong upward movement shown by large vertical velocity field,and the suction action of high-and low-layer divergence and convergence were the reasons for the hourly heavy snowfall on the 18^(th).During the process,radar echoes were mainly sheet-shaped,and composite reflectivity was 15-25 dBZ in most areas.The zero speed line in the first period was positively"S"-shaped,and there was warm advection and southwest wind.On the morning of the 18^(th),after the cold front transited the city,Ulanqab City was gradually controlled by northwest wind,and the snow tended to end.展开更多
BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 (PCV2) as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secretin...BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 (PCV2) as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay (IFA) indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.展开更多
[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 (PCV2) in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by P...[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 (PCV2) in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low (102.67); the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases (PCVAD) in East China.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
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
BACKGROUND Osteopetrosis is a rare genetic disorder characterized by increased bone density due to defective bone resorption of osteoclasts.Approximately,80%of autosomal dominant osteopetrosis type II(ADO-II)patients ...BACKGROUND Osteopetrosis is a rare genetic disorder characterized by increased bone density due to defective bone resorption of osteoclasts.Approximately,80%of autosomal dominant osteopetrosis type II(ADO-II)patients were usually affected by heterozygous dominant mutations in the chloride voltage-gated channel 7(ClCN7)gene and present early-onset osteoarthritis or recurrent fractures.In this study,we report a case of persistent joint pain without bone injury or underlying history.CASE SUMMARY We report a 53-year-old female with joint pain who was accidentally diagnosed with ADO-II.The clinical diagnosis was based on increased bone density and typical radiographic features.Two heterozygous mutations in the ClCN7 and Tcell immune regulator 1(TCIRG1)genes by whole exome sequencing were identified in the patient and her daughter.The missense mutation(c.857G>A)occurred in the CLCN7 gene p.R286Q,which is highly conserved across species.The TCIRG1 gene point mutation(c.714-20G>A)in intron 7(near the splicing site of exon 7)had no effect on subsequent transcription.CONCLUSION This ADO-II case had a pathogenic CLCN7 mutation and late onset without the usual clinical symptoms.For the diagnosis and assessment of the prognosis for osteopetrosis,genetic analysis is advised.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
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
Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detectio...Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.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
[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were desig...[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.展开更多
In September 2011, an infectious disease suspected to be postweaning multisystemic wasting syndrome (PMWS) broke out in some pig farm in Taizhou. The inguinal lymph node, liver and lung tissues were collected and gr...In September 2011, an infectious disease suspected to be postweaning multisystemic wasting syndrome (PMWS) broke out in some pig farm in Taizhou. The inguinal lymph node, liver and lung tissues were collected and grinded into tissue suspension. The suspension was subjected to PCR detection, and the positive product was sequenced. The suspension of positive samples was filtered with 0.22 μm filter membrane, and the filtrate was inoculated onto PK15 cells. After five generations of blind passages, the cell viral liquid was collected and extracted for DNA, which was subjected to PCR detection and indirect immunofluorescence. The results showed that the isolate was porcine circovirus type 2 (PCV2) and designated as TAIZ110926. The target sequence was committed to NCBI with a serial number: KF039888.展开更多
[Objective]The aim of this study is to explore the dynamic changes of Porcine circovirus type 2 (PCV2) and the propagation differences between different PCV2 strains.[Method]A rapid, sensitive and SYBR Green I-based r...[Objective]The aim of this study is to explore the dynamic changes of Porcine circovirus type 2 (PCV2) and the propagation differences between different PCV2 strains.[Method]A rapid, sensitive and SYBR Green I-based real-time quantitative PCR assay was developed to detect PCV2 in mice. The Balb/c mice were inoculated with 200 μL of 1×10^6 TCID 50 /mL different strains of PCV2;the serum and tissues of mice were collected at different time. SYBR Green I fluorescent quantitative PCR was used to determine the viral titer in the serum and tissues of mice.[Result]The results indicated that the SYBR Green I PCR could speci fically detect PCV2 with high specificity. All strains were detected in the serum 14 d after infection, and 2007HA strain had the highest level of 1.21×10^8 copies/mL. The titers of all strains decreased 21 d after infection and then increased 28 d after infection. In addition, 2010NJ strain had the highest titer in serum 28 d after infection. The two PCV2b isolates, 2010NJ and 2009ZJ, had the highest titer in lungs and spleens. [Conclusion]All results showed that different PCV2 isolates have different proliferation ef ficiencies in Balb/c mice, even if they belong to the same subtype. In addition, the proliferation rate of 2009ZJ in visceral organs was significantly higher than that in serum. However, this phenomenon is not obvious for other strains. These results laid a foundation for the analysis of proliferation characteristics and pathogenicity of different PCV2 strains in vivo.展开更多
PCV2 (Porcine circovirus type 2) is considered as the essential infectious agent of PMWS (post weaning multisystemic wasting syndrome) in pigs. Serological studies have shown that the virus is ubiquitous. Currentl...PCV2 (Porcine circovirus type 2) is considered as the essential infectious agent of PMWS (post weaning multisystemic wasting syndrome) in pigs. Serological studies have shown that the virus is ubiquitous. Currently, there are many reports about the epidemiology and genetic characteristics of this virus around the world, but in Mexico it has not been studied. More than 3,500 samples of serum, rectal swabs, tissues and semen of 34 Mexican porcine farms from 10 important producer regions were analyzed by PCR. Results show that 97% of the farms were positive. Both genotypes, PCV2a and PCV2b were detected. It also found that the most prevalent genotype in Mexico is PCV2b. Regarding to amino acid sequence; three major heterogenic regions were present in the positions 59-91,123-136 and 185-210.展开更多
文摘TypeⅡdiabetes mellitus(T2DM)has experienced a dramatic increase globally across countries of various income levels over the past three decades.The persistent prevalence of T2DM is attributed to a complex interplay of genetic and environmental factors.While numerous pharmaceutical therapies have been developed,there remains an urgent need for innovative treatment approaches that offer effectiveness without significant adverse effects.In this context,the exploration of the gut microbiome presents a promising avenue.Research has increasingly shown that the gut microbiome of individuals with T2DM exhibits distinct differences compared to healthy individuals,suggesting its potential role in the disease’s pathogenesis and progression.This emerging field offers diverse applications,particularly in modifying the gut environment through the administration of prebiotics,probiotics,and fecal microbiome transfer.These interventions aim to restore a healthy microbiome balance,which could potentially alleviate or even reverse the metabolic dysfunctions associated with T2DM.Although current results from clinical trials have not yet shown dramatic effects on diabetes management,the groundwork has been laid for deeper investigation.Ongoing and future clinical trials are critical to advancing our understanding of the microbiome’s impact on diabetes.By further elucidating the mechanisms through which microbiome alterations influence insulin resistance and glucose metabolism,researchers can develop more targeted interventions.The potential to harness the gut microbiome in developing new therapeutic strategies offers a compelling prospect to transform the treatment landscape of T2DM,potentially reducing the disease’s burden significantly with approaches that are less reliant on traditional pharmaceuticals and more focused on holistic,systemic health improvements.
文摘Diabetes mellitus is associated with foot ulcers,which frequently pave the way to lower-extremity amputation.Neuropathy,trauma,deformity,high plantar pressures,and peripheral vascular disease are the most common underlying causes.Around 15%of diabetic patients are affected by diabetic foot ulcer in their lifetime.64 million people are affected by diabetics in India and 40000 amputations are done every year.Foot ulcers are evaluated and classified in a systematic and thorough manner to assist in determining the best course of therapy.This paper proposes a novel model which predicts the threat of diabetic foot ulcer using independent agents for various input values and a combination of fuzzy expert systems.The proposed model uses a classification system to distinguish between each fuzzy framework and its parameters.Based on the severity levels necessary prevention,treatment,and medication are recommended.Combining the results of all the fuzzy frameworks derived from its constituent parameters,a risk-specific medication is recommended.The work also has higher accuracy when compared to other related models.
文摘Based on the observation data of meteorological stations,Doppler radar observation data of Ulanqab City,and ERA-5 reanalysis data,a snowstorm process in Ulanqab City from March 17 to 18,2022 was analyzed.The results show that this was a type Ⅱ snowstorm process generated under the joint influence of upper trough and ground low inverted trough and frontal cyclone.The main period of snowfall can be divided into two time stages,and the total snowfall was more in the south and less in the north,which was consistent with that of average specific humidity field.Water vapor conditions provided by strong water vapor transport and convergence,strong upward movement shown by large vertical velocity field,and the suction action of high-and low-layer divergence and convergence were the reasons for the hourly heavy snowfall on the 18^(th).During the process,radar echoes were mainly sheet-shaped,and composite reflectivity was 15-25 dBZ in most areas.The zero speed line in the first period was positively"S"-shaped,and there was warm advection and southwest wind.On the morning of the 18^(th),after the cold front transited the city,Ulanqab City was gradually controlled by northwest wind,and the snow tended to end.
基金Supported by National Natural Science Foundation of China(31302071)AgriculturalScience and Technology Independent Innovation Fund of Jiangsu Province(TechnicalInnovation)[CX(13)3065]~~
文摘BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 (PCV2) as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay (IFA) indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.
基金Supported by National Natural Science Foundation of China(31302071)Special Fund for Agro-scientific Research in the Public Interest(201303046)+1 种基金Independent Innovation Project of Jiangsu Province[CX(13)3065]Project of the Fourth Period of "333" High-level Personnel Training Program of Jiangsu Province(BRA2012194)~~
文摘[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 (PCV2) in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low (102.67); the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases (PCVAD) in East China.
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Supported by the Science and Technology Plan Program of Sichuan of China,No.2018JY0608。
文摘BACKGROUND Osteopetrosis is a rare genetic disorder characterized by increased bone density due to defective bone resorption of osteoclasts.Approximately,80%of autosomal dominant osteopetrosis type II(ADO-II)patients were usually affected by heterozygous dominant mutations in the chloride voltage-gated channel 7(ClCN7)gene and present early-onset osteoarthritis or recurrent fractures.In this study,we report a case of persistent joint pain without bone injury or underlying history.CASE SUMMARY We report a 53-year-old female with joint pain who was accidentally diagnosed with ADO-II.The clinical diagnosis was based on increased bone density and typical radiographic features.Two heterozygous mutations in the ClCN7 and Tcell immune regulator 1(TCIRG1)genes by whole exome sequencing were identified in the patient and her daughter.The missense mutation(c.857G>A)occurred in the CLCN7 gene p.R286Q,which is highly conserved across species.The TCIRG1 gene point mutation(c.714-20G>A)in intron 7(near the splicing site of exon 7)had no effect on subsequent transcription.CONCLUSION This ADO-II case had a pathogenic CLCN7 mutation and late onset without the usual clinical symptoms.For the diagnosis and assessment of the prognosis for osteopetrosis,genetic analysis is advised.
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.
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.
文摘Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.
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.
基金Supported by Shandong Province Natural Science Fund Project
文摘[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.
基金Supported by Key Project of Jiangsu Agri-animal Husbandry Vocational College(ZD201104)College Students'Innovation and Enterpreneurship Training Program of Jiangsu Province(201312806014Y)Special Fund for Fenghuang talents in Jiangsu Agri-animal Husbandry Vocational College
文摘In September 2011, an infectious disease suspected to be postweaning multisystemic wasting syndrome (PMWS) broke out in some pig farm in Taizhou. The inguinal lymph node, liver and lung tissues were collected and grinded into tissue suspension. The suspension was subjected to PCR detection, and the positive product was sequenced. The suspension of positive samples was filtered with 0.22 μm filter membrane, and the filtrate was inoculated onto PK15 cells. After five generations of blind passages, the cell viral liquid was collected and extracted for DNA, which was subjected to PCR detection and indirect immunofluorescence. The results showed that the isolate was porcine circovirus type 2 (PCV2) and designated as TAIZ110926. The target sequence was committed to NCBI with a serial number: KF039888.
文摘[Objective]The aim of this study is to explore the dynamic changes of Porcine circovirus type 2 (PCV2) and the propagation differences between different PCV2 strains.[Method]A rapid, sensitive and SYBR Green I-based real-time quantitative PCR assay was developed to detect PCV2 in mice. The Balb/c mice were inoculated with 200 μL of 1×10^6 TCID 50 /mL different strains of PCV2;the serum and tissues of mice were collected at different time. SYBR Green I fluorescent quantitative PCR was used to determine the viral titer in the serum and tissues of mice.[Result]The results indicated that the SYBR Green I PCR could speci fically detect PCV2 with high specificity. All strains were detected in the serum 14 d after infection, and 2007HA strain had the highest level of 1.21×10^8 copies/mL. The titers of all strains decreased 21 d after infection and then increased 28 d after infection. In addition, 2010NJ strain had the highest titer in serum 28 d after infection. The two PCV2b isolates, 2010NJ and 2009ZJ, had the highest titer in lungs and spleens. [Conclusion]All results showed that different PCV2 isolates have different proliferation ef ficiencies in Balb/c mice, even if they belong to the same subtype. In addition, the proliferation rate of 2009ZJ in visceral organs was significantly higher than that in serum. However, this phenomenon is not obvious for other strains. These results laid a foundation for the analysis of proliferation characteristics and pathogenicity of different PCV2 strains in vivo.
文摘PCV2 (Porcine circovirus type 2) is considered as the essential infectious agent of PMWS (post weaning multisystemic wasting syndrome) in pigs. Serological studies have shown that the virus is ubiquitous. Currently, there are many reports about the epidemiology and genetic characteristics of this virus around the world, but in Mexico it has not been studied. More than 3,500 samples of serum, rectal swabs, tissues and semen of 34 Mexican porcine farms from 10 important producer regions were analyzed by PCR. Results show that 97% of the farms were positive. Both genotypes, PCV2a and PCV2b were detected. It also found that the most prevalent genotype in Mexico is PCV2b. Regarding to amino acid sequence; three major heterogenic regions were present in the positions 59-91,123-136 and 185-210.