应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动...应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。展开更多
Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mu...Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence,immunogenicity,host selection,and other abilities.In this study,the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifcally,diarrhea or vomiting during the period spanning from 2018 to 2022.The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%.Capsid(virion)protein 2(VP2)gene of each FPV-positive sample was sequenced and amplifed,yielding 65 VP2 sequences.Among them,six VP2 gene sequences were detected in the majority of the samples test positive for FPV,and these positive samples originated from a diverse range of geographical locations.These isolates were named FPV-6,FPV-10,FPV-15,FPV-251,FPV-271 and FPV-S2.Additionally,the substitution of Ala300Pro(A300P)in VP2 was detected for the frst time in feline-derived FPV(FPV-251).FPV-251 isolate,with this substitution in VP2 protein,exhibited stable proliferative capacity in Madin-Darby canine kidney(MDCK)cells and A72 cells.FPV-271 was selected as the FPV control isolate due to its single amino acid diference from VP2 protein of FPV-251 at position 300(FPV-271 has alanine,while FPV-251 has proline).After oral infection,both FPV-251 and FPV-271 isolates caused feline panleukopenia,which is characterized by clinical signs of enterocolitis.However,FPV-251 can infect dogs through the oral route and cause gastrointestinal(GI)symptoms with lesions in the intestine and mesenteric lymph nodes(MLNs)of infected dogs.This is the frst report on the presence of an A300P substitution in VP2 protein of feline-derived FPV.Additionally,FPV isolate with a substitution of A300P at VP2 protein demonstrated efcient replication capabilities in canine cell lines and the ability to infect dogs.展开更多
In order to understand the variation of FPV strains in the Jinan area, Shandong Province, China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional...In order to understand the variation of FPV strains in the Jinan area, Shandong Province, China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional site variation was carried out. It was found that FPV-XY2, FPV-XY3 and FPV-XY6 were the same strain with 100% homology, and also close to FPV-XY1, and the homology between FPV-XY4 and FPV-XY5 was close. The homology between the reference strain and the test strain was over 99.3%. According to the evolutionary analysis, the genetic relationship among FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 was close, and the genetic relationship between FPV-XY4 and FPV-XY5 was close, and the result was similar to the homologous result. Compared with the VP2 amino acid sequence of the standard strain FPV-CU4, the VP2 protein of all the test strains changed from I to t on the 101st Amino acid, this may be the cause of immune failure in these 6 cases;the change of a to s in the 91st amino acid position of FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 may be the cause of enhanced virulence of FPV. This study provides a reference for exploring the epidemic law of FP in the Jinan area, the standard of FP treatment plan and the research and development of FPV subunit vaccine.展开更多
Objective:To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361,0613158,061060 and 0715880) and cell culture adapted(06361-CA,0613158-CA.061060- CA and 0715880-CA) G1P[8]rotavirus stra...Objective:To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361,0613158,061060 and 0715880) and cell culture adapted(06361-CA,0613158-CA.061060- CA and 0715880-CA) G1P[8]rotavirus strains.Methods:Full-length VP4 genes of each of the four wild type G1P[8]rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing. Results:All four cell culture adapted G1P[8]rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains.The number of substitutions however,varied from 1-64 and 1-13 respectively.The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilizalion and hemagglutinaling activity. The presence of unique amino acid substitutions was identified in two of the four wild type(V377G. S387N in 061060 and 1644L in 0715880) and all four cell culture adapted(A46V in 0613158-CA. T60R in 06361-CA,L237V.G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8]specificity.Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.Conclusions:Amino acid substitutions detected in the VP4 genes of G1P[8]rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation,immunogenicity and conformation is needed for the development of newer rolavirus vaccines.展开更多
[ Abstracts ] In order to investigate the effect of chicken Akirin2 gene on the immune response induced by VP2 DNA vaccine of infectious bursal disease virus （IBDV）. [ Methods] The 14-day-old SPF chickens were immun...[ Abstracts ] In order to investigate the effect of chicken Akirin2 gene on the immune response induced by VP2 DNA vaccine of infectious bursal disease virus （IBDV）. [ Methods] The 14-day-old SPF chickens were immunized with recombinant plasmids expressing VP2 protein and Akirin2 protein, and strength- ened immunization was conducted at the 14＇~ day after the first immunization. Finally, test chickens were challenged with IBDVBC6-85 virulent strain. [ Resultss ] Test results showed that Akirin2 gene could enhance the specific immune response induced by VP2 DNA vaccine, improve the proliferation of peripheral blood lym- phocytes and ＇affect the expressing of cytokines TNF-a, IFN-Y, IL-1β, IL-2, IL-4, IL 6, IL-9, IL-10, IL-17 and IL-18. Effects of recombinant plasmids co-ex- pressing Akirin2 protein and VP2 protein on cytokine expression showed some differences with the recombinant plasmids expressing Akirir/2 protein or VP2 protein along. [ Conclusions] Chicken Akirin2 gene could significantly enhance the humoral immune response and cellular immune response induced by VP2 DNA vaccine of IBDV.展开更多
According to the complete nucleotide sequence of Muscovy duck parvovirus registered in the gene bank, two modified primers (LHMP7/LHMP8) were designed and, for each of them, a restriction endonuclease recognition site...According to the complete nucleotide sequence of Muscovy duck parvovirus registered in the gene bank, two modified primers (LHMP7/LHMP8) were designed and, for each of them, a restriction endonuclease recognition site, SacⅡ or KpnⅠ, was included respectively. The DNA encoding VP2 structural protein of the wild strain MDPV Q and the attenuated strain MDPV 26 which had been derived by continued passage of virulent wild type (MDPV Q) in Muscovy duck embryo were amplified by PCR and recombined into T vector and sequenced respectively. It shows that both DNA encoding VP 2 structural protein of MDPV Q and MDPV which has been registered in the gene bank had the high homology (97.9%). MDPV 26 and MDPV Q displayed 99.7% identity, and shared 98.3% homology with that of reported MDPV.展开更多
文摘应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。
基金the Experimental Animal Research Project of Hubei Province(Grant No.2023CFA005).
文摘Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence,immunogenicity,host selection,and other abilities.In this study,the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifcally,diarrhea or vomiting during the period spanning from 2018 to 2022.The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%.Capsid(virion)protein 2(VP2)gene of each FPV-positive sample was sequenced and amplifed,yielding 65 VP2 sequences.Among them,six VP2 gene sequences were detected in the majority of the samples test positive for FPV,and these positive samples originated from a diverse range of geographical locations.These isolates were named FPV-6,FPV-10,FPV-15,FPV-251,FPV-271 and FPV-S2.Additionally,the substitution of Ala300Pro(A300P)in VP2 was detected for the frst time in feline-derived FPV(FPV-251).FPV-251 isolate,with this substitution in VP2 protein,exhibited stable proliferative capacity in Madin-Darby canine kidney(MDCK)cells and A72 cells.FPV-271 was selected as the FPV control isolate due to its single amino acid diference from VP2 protein of FPV-251 at position 300(FPV-271 has alanine,while FPV-251 has proline).After oral infection,both FPV-251 and FPV-271 isolates caused feline panleukopenia,which is characterized by clinical signs of enterocolitis.However,FPV-251 can infect dogs through the oral route and cause gastrointestinal(GI)symptoms with lesions in the intestine and mesenteric lymph nodes(MLNs)of infected dogs.This is the frst report on the presence of an A300P substitution in VP2 protein of feline-derived FPV.Additionally,FPV isolate with a substitution of A300P at VP2 protein demonstrated efcient replication capabilities in canine cell lines and the ability to infect dogs.
文摘In order to understand the variation of FPV strains in the Jinan area, Shandong Province, China, the VP2 gene of 6 FPV strains was sequenced, and the analysis of the genetic relationship, evolution and main functional site variation was carried out. It was found that FPV-XY2, FPV-XY3 and FPV-XY6 were the same strain with 100% homology, and also close to FPV-XY1, and the homology between FPV-XY4 and FPV-XY5 was close. The homology between the reference strain and the test strain was over 99.3%. According to the evolutionary analysis, the genetic relationship among FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 was close, and the genetic relationship between FPV-XY4 and FPV-XY5 was close, and the result was similar to the homologous result. Compared with the VP2 amino acid sequence of the standard strain FPV-CU4, the VP2 protein of all the test strains changed from I to t on the 101st Amino acid, this may be the cause of immune failure in these 6 cases;the change of a to s in the 91st amino acid position of FPV-XY1, FPV-XY2, FPV-XY3, FPV-XY6 may be the cause of enhanced virulence of FPV. This study provides a reference for exploring the epidemic law of FP in the Jinan area, the standard of FP treatment plan and the research and development of FPV subunit vaccine.
基金supported by National Institute of Virology(NIV,Indian Council of Medical Research, Govt.of India),Pune
文摘Objective:To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361,0613158,061060 and 0715880) and cell culture adapted(06361-CA,0613158-CA.061060- CA and 0715880-CA) G1P[8]rotavirus strains.Methods:Full-length VP4 genes of each of the four wild type G1P[8]rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing. Results:All four cell culture adapted G1P[8]rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains.The number of substitutions however,varied from 1-64 and 1-13 respectively.The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilizalion and hemagglutinaling activity. The presence of unique amino acid substitutions was identified in two of the four wild type(V377G. S387N in 061060 and 1644L in 0715880) and all four cell culture adapted(A46V in 0613158-CA. T60R in 06361-CA,L237V.G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8]specificity.Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.Conclusions:Amino acid substitutions detected in the VP4 genes of G1P[8]rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation,immunogenicity and conformation is needed for the development of newer rolavirus vaccines.
基金Supported by Guangdong Province Application of Science and Technology Research and Development of Special Funds(2015B020230011)
文摘[ Abstracts ] In order to investigate the effect of chicken Akirin2 gene on the immune response induced by VP2 DNA vaccine of infectious bursal disease virus （IBDV）. [ Methods] The 14-day-old SPF chickens were immunized with recombinant plasmids expressing VP2 protein and Akirin2 protein, and strength- ened immunization was conducted at the 14＇~ day after the first immunization. Finally, test chickens were challenged with IBDVBC6-85 virulent strain. [ Resultss ] Test results showed that Akirin2 gene could enhance the specific immune response induced by VP2 DNA vaccine, improve the proliferation of peripheral blood lym- phocytes and ＇affect the expressing of cytokines TNF-a, IFN-Y, IL-1β, IL-2, IL-4, IL 6, IL-9, IL-10, IL-17 and IL-18. Effects of recombinant plasmids co-ex- pressing Akirin2 protein and VP2 protein on cytokine expression showed some differences with the recombinant plasmids expressing Akirir/2 protein or VP2 protein along. [ Conclusions] Chicken Akirin2 gene could significantly enhance the humoral immune response and cellular immune response induced by VP2 DNA vaccine of IBDV.
文摘According to the complete nucleotide sequence of Muscovy duck parvovirus registered in the gene bank, two modified primers (LHMP7/LHMP8) were designed and, for each of them, a restriction endonuclease recognition site, SacⅡ or KpnⅠ, was included respectively. The DNA encoding VP2 structural protein of the wild strain MDPV Q and the attenuated strain MDPV 26 which had been derived by continued passage of virulent wild type (MDPV Q) in Muscovy duck embryo were amplified by PCR and recombined into T vector and sequenced respectively. It shows that both DNA encoding VP 2 structural protein of MDPV Q and MDPV which has been registered in the gene bank had the high homology (97.9%). MDPV 26 and MDPV Q displayed 99.7% identity, and shared 98.3% homology with that of reported MDPV.
