Canine parvovirus 2(CPV-2)is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies.The genetic variability and antigenic diversity of CPV-2 hin...Canine parvovirus 2(CPV-2)is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies.The genetic variability and antigenic diversity of CPV-2 hinder its efective prevention of infection by vaccination.To investigate the epidemiology and genetic characteristics of CPV-2 in China,rectal swabs from afected dogs were collected from diferent animal clinics in Kunshan from 2022 to 2023.Preliminary detection and capsid gene sequencing of CPV-2 were performed using previously described primers and protocols.The overall detection rate for CPV-2 was 16.5%(33/200).A signifcant association was found between the CPV-2-positivity and clinical signs,age,breed and vaccination status.Sequence analysis revealed the presence of CPV-2c genotypes in all positive samples,which were genetically similar to other Asian CPV-2c strains.Notably,four key mutations(A5G,F267Y,Y324I and Q370R)were detected in all isolates,and one novel mutation(I447M)was detected in three CPV-2 isolates.These mutations in the CPV-2 strains could impact vaccine efcacy and the efectiveness of the virus immune evasion.Surprisingly,no recombination events were observed between the identifed CPV-2c strains and reference strains from China.Our data revealed that amino acid residues 324,426 and 440 of VP2 may under strong selection pressure.This pattern of genetic variation in the CPV-2 lineage warrants continuous laboratory-based surveillance programs in other parts of China to better understand the pattern of seasonal distribution and association between emerging genotypes and the intensity of disease severity.展开更多
A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as th...A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.展开更多
Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bac...Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.展开更多
A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination...A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.展开更多
The canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). It is clinically characterized by severe vomiting,hemorrhagic enteritis,significant reduction in white blood cells and myo...The canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). It is clinically characterized by severe vomiting,hemorrhagic enteritis,significant reduction in white blood cells and myocarditis. The disease with high incidence,highly infectious and high mortality has become one of the serious infectious diseases threatening dog raising industry in China. In this research,260 cases of canine parvovirus case from an Aite Pet Clinic in Taizhou City during January 2010 and March 2011 were analyzed. This study discloses the epidemiology of CPV in Taizhou region of Jiangsu Province,i. e.,the incidence of CPV and canine motility are closely correlated with age,breed,immune inoculation and season. This study provides useful guide for the clinical treatment of CPV in the future.展开更多
[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification （LAMP）, improve the detection rate of canine parvovirus （CPV） and reduce the testing cost. [ Method] Acco...[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification （LAMP）, improve the detection rate of canine parvovirus （CPV） and reduce the testing cost. [ Method] According to the conserved regions of the VP2 gene of CPV, six primers were designed to amplify the special DNA sequences by LAMP. In addition, the reaction conditions of LAMP were optimized, and the sensitivity, specificity, repeatability and stability were verified. [ Result] The optimal reaction time of the LAMP method for CPVwas 60 min. The products obtained by LAMP had high specificity without cross-reaction with other generic viruses. The sensitivity of the LAMP was 100 times higher than that of PCR. [ Conclusion] The LAMP method for detecting CPV has high practical value. It has many advantages such as high specificity, high sensitivity, simple operation, low cost and rapid analysis, and it does not require special equipment. Therefore, this method is more suitable for the detection of CPV.展开更多
In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) ...In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.展开更多
Ability of a synthetic Aluminium-Magnesium Silicate [AMS] to inhibit activities of canine parvovirus [CPV] was investigated in vitro and in vivo. Five samples of CPV isolated in Nigeria, were each incubated with equal...Ability of a synthetic Aluminium-Magnesium Silicate [AMS] to inhibit activities of canine parvovirus [CPV] was investigated in vitro and in vivo. Five samples of CPV isolated in Nigeria, were each incubated with equal amount of a synthetic AMS on a volume to weight [v/w] basis, for one hour and then centrifuged. Viral titres of the supernatants were tested by the haemagglutination [HA] test and their mean titre compared with mean titre of portions of same viral samples, not incubated with the AMS. Also, five puppies and five adult dogs infected with the parvovirus isolates were treated by dosing each with 400 mg/kg of a drug formulation that has 12% AMS per os for seven days. As control, five puppies and five adult dogs from same class as the experimental dogs were similarly infected but were not treated. Incubating parvovirus with AMS reduced its load from mean HA titre 825.6 ± 261.1 to mean HA, 270.8 ± 132.1 [p < 0.05]. Also treating parvovirus infected dogs with a 12% AMS drug formulation reduced mortality due to the virus from 100% to zero [p < 0.01].展开更多
A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tiv...A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.展开更多
基金Kunshan Municipal Government research funding(Grant Number:22KKSGR075).
文摘Canine parvovirus 2(CPV-2)is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies.The genetic variability and antigenic diversity of CPV-2 hinder its efective prevention of infection by vaccination.To investigate the epidemiology and genetic characteristics of CPV-2 in China,rectal swabs from afected dogs were collected from diferent animal clinics in Kunshan from 2022 to 2023.Preliminary detection and capsid gene sequencing of CPV-2 were performed using previously described primers and protocols.The overall detection rate for CPV-2 was 16.5%(33/200).A signifcant association was found between the CPV-2-positivity and clinical signs,age,breed and vaccination status.Sequence analysis revealed the presence of CPV-2c genotypes in all positive samples,which were genetically similar to other Asian CPV-2c strains.Notably,four key mutations(A5G,F267Y,Y324I and Q370R)were detected in all isolates,and one novel mutation(I447M)was detected in three CPV-2 isolates.These mutations in the CPV-2 strains could impact vaccine efcacy and the efectiveness of the virus immune evasion.Surprisingly,no recombination events were observed between the identifed CPV-2c strains and reference strains from China.Our data revealed that amino acid residues 324,426 and 440 of VP2 may under strong selection pressure.This pattern of genetic variation in the CPV-2 lineage warrants continuous laboratory-based surveillance programs in other parts of China to better understand the pattern of seasonal distribution and association between emerging genotypes and the intensity of disease severity.
文摘A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.
基金Supported by the National Key R&D Program of China(2017YFD0501102,2016YFD0501003)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department of Heilongjiang Province(TSTAU-R2018017)。
文摘Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.
文摘A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.
基金Supported by the Special Fund for Special Fund for Veterinary Public Health Security and Management Innovation Team of Chinese Academy of Agricultural Science and Technology Innovation Project(ASTIP-IAS11)the National High Technology Research and Development Program of China(2012AA101302)the Special Fund for Agro-scientific Research in the Public Interest(201303042)
文摘The canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). It is clinically characterized by severe vomiting,hemorrhagic enteritis,significant reduction in white blood cells and myocarditis. The disease with high incidence,highly infectious and high mortality has become one of the serious infectious diseases threatening dog raising industry in China. In this research,260 cases of canine parvovirus case from an Aite Pet Clinic in Taizhou City during January 2010 and March 2011 were analyzed. This study discloses the epidemiology of CPV in Taizhou region of Jiangsu Province,i. e.,the incidence of CPV and canine motility are closely correlated with age,breed,immune inoculation and season. This study provides useful guide for the clinical treatment of CPV in the future.
基金supported by Independent Innovation Specific Projects of Shandong Province ( 2008ZHZX1A1103)
文摘[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification （LAMP）, improve the detection rate of canine parvovirus （CPV） and reduce the testing cost. [ Method] According to the conserved regions of the VP2 gene of CPV, six primers were designed to amplify the special DNA sequences by LAMP. In addition, the reaction conditions of LAMP were optimized, and the sensitivity, specificity, repeatability and stability were verified. [ Result] The optimal reaction time of the LAMP method for CPVwas 60 min. The products obtained by LAMP had high specificity without cross-reaction with other generic viruses. The sensitivity of the LAMP was 100 times higher than that of PCR. [ Conclusion] The LAMP method for detecting CPV has high practical value. It has many advantages such as high specificity, high sensitivity, simple operation, low cost and rapid analysis, and it does not require special equipment. Therefore, this method is more suitable for the detection of CPV.
文摘In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.
文摘Ability of a synthetic Aluminium-Magnesium Silicate [AMS] to inhibit activities of canine parvovirus [CPV] was investigated in vitro and in vivo. Five samples of CPV isolated in Nigeria, were each incubated with equal amount of a synthetic AMS on a volume to weight [v/w] basis, for one hour and then centrifuged. Viral titres of the supernatants were tested by the haemagglutination [HA] test and their mean titre compared with mean titre of portions of same viral samples, not incubated with the AMS. Also, five puppies and five adult dogs infected with the parvovirus isolates were treated by dosing each with 400 mg/kg of a drug formulation that has 12% AMS per os for seven days. As control, five puppies and five adult dogs from same class as the experimental dogs were similarly infected but were not treated. Incubating parvovirus with AMS reduced its load from mean HA titre 825.6 ± 261.1 to mean HA, 270.8 ± 132.1 [p < 0.05]. Also treating parvovirus infected dogs with a 12% AMS drug formulation reduced mortality due to the virus from 100% to zero [p < 0.01].
文摘A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.
