犬细小病毒JL09-11株的分离鉴定及免疫研究
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摘要
犬细小病毒病是一种感染犬和野生犬科动物的急性接触性传染病,对我国乃至世界的养犬业造成了较大的危害,因此,在初生幼犬的免疫程序中,犬细小病毒病的免疫是必不可少的。同时,犬细小病毒是一种变异性极强的病毒,自从1978年CPV-2被成功分离以来,人们已经发现了数种变异变种,包括CPV-2a,CPV-2b,和CPV-2c等。这些变种主要是由于犬细小病毒粒子表面主要抗原位点上的氨基酸序列发生变异造成的。这些位点上的氨基酸变异使得病毒具有了不同的抗原性,这种抗原性上的差异可以用单克隆抗体进行鉴别。虽然人们已经证实在大多数情况下,犬细小病毒的不同变种毒株之间具有完全的免疫保护力,但是由于疫苗株病毒与流行毒株抗原性的差异而造成免疫失败的案例也时有报道。因此,对本病进行防控的主要任务就是跟踪病毒的流行变异趋势,研制针对正在流行的病毒株的疫苗,以免由于新型病毒的出现或异种抗原类型毒株的侵入而有可能造成的大规模流行,对家犬或野生犬科动物造成较严重的危害。
本研究取吉林省境内一只疑似患有犬细小病毒病的病犬肠内容物作为病料,对其进行PCR检测,结果为阳性。之后将病料研磨,过滤后接种F81细胞系进行犬细小病毒的分离。在接种4天后,细胞出现了犬细小病毒特异性的病变,同时正常细胞对照未产生类似的细胞病变。将病变的细胞上清冻融三次收毒后在F81细胞系上继续传代,连续传代三次后病变依然稳定。我们分别用PCR方法,血凝和血凝抑制试验对细胞培养物中的犬细小病毒进行检测,结果均为阳性。其中在应用PCR的方法进行检测的过程中,我们采用的是肉食类动物犬细小病毒的通用引物,结果出现了目的条带;血凝试验的结果显示细胞培养物中犬细小病毒的血凝效价为212
为了对所分离的犬细小病毒毒株进行进化分析和氨基酸序列分析,我们首先用有限稀释法对病毒进行了纯化,将纯化后的病毒作为模板,对其主要抗原基因VP2进行扩增,并测序。结果发现我们分离到的犬细小病毒是一株具有新型抗原特性的犬细小病毒,这表现在其VP2基因的主要抗原位点上的氨基酸序列较以前分离得到的任何一个变种都不完全一致。我们收集在NCBI数据库中存在的国内犬细小病毒分离株的VP2基因序列,与JL09-11株一并进行了进化分析,结果发现JL09-11株在进化树中与国内的CPV-2a变种具有较近的进化关系。但是在对本株CPV进行氨基酸序列分析的过程中,我们发现它并不能被归为CPV-2a这一变种,;血凝抑制试验的结果表明,细胞培养物的血凝作用可以被本实验室保存的抗犬细小病毒的阳性血清所抑制。至此我们确定已经分离得到犬细小病毒,并将其命名为JL09-11株。原因是其297位氨基酸与CPV-2b变种相同,是A而不是S,对国内分离到的各个CPV毒株进行进一步的分析发现,国内所分离到的大多数被定义为CPV-2a变种的毒株其297位氨基酸是A而非S。同S297A变种比较又有555位氨基酸的差异,我们认为我国流行的大多数CPV毒株具有这样的特点,它们是介于CPV-2和CPV-2c之间的进化变种。因此以具有本国CPV抗原特性的CPV毒株为毒种,研制灭活疫苗,对我国犬细小病毒病的流行具有很好的针对性。我们将JL09-11株犬细小病毒灭活后辅以本实验室配制的纳米佐剂对其免疫效果进行了评价,为JL09-11株毒种灭活疫苗的研制奠定基础。
在进行JL09-11株犬细小病毒进行免疫效果评价之前,我们首先在F81细胞系上建立了中和试验,用以测定血清中犬细小病毒的中和抗体。为以后灭活疫苗的免疫效果评价做准备。
将JL09-11株犬细小病毒的细胞培养悬液于37℃使用1%的甲醛灭活24小时后,对其进行TCID50
我们采用未接种任何疫苗,且外周血中犬细小病毒中和抗体滴度较低的健康家犬来对我们灭活犬细小病毒JL09-11株的免疫效果进行评价。在免疫试验过程中,每条试验犬注射灭活疫苗的剂量为1.5ml,其中含有大约5.0×10的测定,结果显示病毒已经完全被灭活。然后以4:1的比例与本实验室制备的纳米佐剂进行混合后制成犬细小病毒的灭活疫苗。5个TCID50
通过本研究我们分离到了一株新的犬细小病毒,进化分析显示,本株犬细小病毒与国内的CPV-2a变种具有较近的进化关系。并且我们发现,国内的大多数被归为CPV-2a变种的毒株与JL09-11一样,主要抗原蛋白VP2的第297位氨基酸均为A,同时其555位氨基酸发生了由I到V的回复突变。这与CPV-2b和CPV-2c相同。我们发现国内流行的主要就是带有这两个突变的CPV-2a变种。我们利用本研究所分离到的JL09-11株犬细小病毒进行了灭活和免疫试验,结果证明,我们分离到的JL09-11株犬细小病毒具有较好的免疫原性,具有用于研制灭活疫苗的应用潜力。经过灭活的犬细小病毒。在免疫前以及免疫后14天,分别对试验犬外周血中的犬细小病毒中和抗体滴度进行测定。结果显示,在免疫后14天试验犬外周血中的犬细小病毒中和抗体较免疫前得到了显著提高。证明了本研究所分离得到的犬细小病毒具有研制灭活疫苗的潜力。
Canine parvovirus(CPV)infection is a kind of acute, infectious disease of domestic dogs and wild canidae, which causes large loss to the worldwide dog industry. As a result, the immunfaction for this disease is a compulsory immune progress for puppies. CPV is a kind of virus with high mutation rate; several antigenic variants have been identified after the isolation of the first canine parvovirus isolate CPV-2, including CPV-2a, CPV-2b, CPV-2c and so on. Those variants were labeled by the variety of the amino acid sequences in the key antigenic sites of the VP2, which is the main protein constructing the nucleocapsid of this virus. The mutations of the amino acid in those antigenic sites brought various antigenic properties to different variants, which can be identified by Mabs. Although the cross immune protection among different variants has been confirmed, several cases of immune failure due to the difference of the vaccine strains and the prevalent strains were reported. As a result, the prime task for the prevention and control of this disease is to identify the antigenic property of the prevalent virus, as well as exploit vaccines.
In this research, the bowel content of a dog in suspension of canine parvovirus infection was collected and detected by PCR to confirm the CPV infection. After grinding and filing, the bowel content was inoculate to F81 cell line for virus isolation. The specific pathological change of the CPV on F81 cell line was appeared 4 days after inoculation. After three cycles of alternative freezing and thawing, the cell culture supernatant was inoculate on F81 cell for another two generations. At each generation, the typical pathological change of the CPV infecting F81 cell, such as rounding, connecting among each other and shedding were appeared. PCR, HA and HI test were performed to identify the isolated CPV in the cell culture supernatant. The PCR identification employed the universal primers of carnivorous animal CPV got positive result; the results of the HA test indicated the HA titer of the cell culture supernatant was 212
To get a better understand to the evolutional relationship of the JL09-11 with that exist in China mainland, as well as the amino acid mutations at the key antigenic sites ; the results of the HI test showed the hemagglutination of the cell culture supernatant could be inhibited by the CPV antibody positive serum stored at our lab, which confirmed that the pathogen we isolated from the bowel content was CPV. The isolated CPV strain was named JL09-11. of the main nucleocapsid protein of this strain, limiting-dilution assay was performed for virus purification. The gene of the main nucleocapsid protein VP2 was amplified through PCR and sequenced. The results showed the JL09-11 is a strain of CPV with new antigenic property, as the amino acid sequences in the key epitopes were not identical with any variant as former published. The VP2 sequences of CPV isolated in China mainland were collected from the NCBI database and involved in the phylogenetic tree reconstruction. In the phylogenetic tree, the JL09-11 strain clustered together with strains which were defined as CPV-2a, although the amino acid analysis indicated the amino acids at site 297 and 555 were A and V orderly. Further analysis indicated those mutations were character of the CPV isolated in China mainland. Then the strain JL09-11 was involved in the development of an inactivated vaccine against canine parvovirus.
Before exploiting the inactivated canine parvovirus vaccine, the neutralizing test was established on F81 cell line, which was used in the test of the neutralizing antibody against the CPV in the canine serum samples.
The cell culture supernatant of the CPV JL09-11 was inactivated with 1% formalin at 37℃for 24 hours. Then the TCID50
The immune effect assessment test was performed on health domestic dogs with no or low titters of neutralizing antibody against CPV in the serum. Each experimental dog was vaccinated with 1.5ml inactivated vaccine, containing about 5.0×10test was performed on the inactivated samples to detect if they were inactivated completely. The results showed the CPV in the cell culture supernatant was inactivated completely. At last the inactivated CPV was mixed with nanometer adjuvant with the proportion of 4:1 to form the inactivated canine parvovirus vaccine. 5 TCID50
This study isolated a new CPV strain JL09-11. Phylogenic analysis indicated the JL09-11 shared high phylogenic homology with strains of CPV-2a variant isolated in China mainland. Amino acid analysis was performed and focused on the VP2 of the JL09-11. The result showed the 297 site of this protein was A(alanine), which is a inactivated CPV. We collected the serum samples before and 14 days after vaccination. The neutralizing antibody against the CPV in the serum samples were tested through neutralizing test as former established. The results indicated that the neutralizing antibody against CPV in the serum of experimental dogs was elevated significantly 14 days after vaccination. specific amino acid of S297A variant, at the same time the amino acid at site 555 was also vary from variant S297A variant. There was a retrieve mutation at site 555 from I (isoleucine) to V (valine). Then we recognized that most CPV strains isolated in China mainland and labeled as CPV-2a have this character. Although cross immune protection among different CPV variants has been reported, exception cases happened from time to time. As a result, it is necessary to develop a vaccine based on the endemic CPV variant of China mainland to prevent and control the canine parvovirus infection. So a kind of inactivated vaccine based on the CPV JL09-11 strain with high immune effect was developed in this study. Assessment test showed this vaccine has the potential to be employed in clinical CPV prevention.
本研究取吉林省境内一只疑似患有犬细小病毒病的病犬肠内容物作为病料,对其进行PCR检测,结果为阳性。之后将病料研磨,过滤后接种F81细胞系进行犬细小病毒的分离。在接种4天后,细胞出现了犬细小病毒特异性的病变,同时正常细胞对照未产生类似的细胞病变。将病变的细胞上清冻融三次收毒后在F81细胞系上继续传代,连续传代三次后病变依然稳定。我们分别用PCR方法,血凝和血凝抑制试验对细胞培养物中的犬细小病毒进行检测,结果均为阳性。其中在应用PCR的方法进行检测的过程中,我们采用的是肉食类动物犬细小病毒的通用引物,结果出现了目的条带;血凝试验的结果显示细胞培养物中犬细小病毒的血凝效价为212
为了对所分离的犬细小病毒毒株进行进化分析和氨基酸序列分析,我们首先用有限稀释法对病毒进行了纯化,将纯化后的病毒作为模板,对其主要抗原基因VP2进行扩增,并测序。结果发现我们分离到的犬细小病毒是一株具有新型抗原特性的犬细小病毒,这表现在其VP2基因的主要抗原位点上的氨基酸序列较以前分离得到的任何一个变种都不完全一致。我们收集在NCBI数据库中存在的国内犬细小病毒分离株的VP2基因序列,与JL09-11株一并进行了进化分析,结果发现JL09-11株在进化树中与国内的CPV-2a变种具有较近的进化关系。但是在对本株CPV进行氨基酸序列分析的过程中,我们发现它并不能被归为CPV-2a这一变种,;血凝抑制试验的结果表明,细胞培养物的血凝作用可以被本实验室保存的抗犬细小病毒的阳性血清所抑制。至此我们确定已经分离得到犬细小病毒,并将其命名为JL09-11株。原因是其297位氨基酸与CPV-2b变种相同,是A而不是S,对国内分离到的各个CPV毒株进行进一步的分析发现,国内所分离到的大多数被定义为CPV-2a变种的毒株其297位氨基酸是A而非S。同S297A变种比较又有555位氨基酸的差异,我们认为我国流行的大多数CPV毒株具有这样的特点,它们是介于CPV-2和CPV-2c之间的进化变种。因此以具有本国CPV抗原特性的CPV毒株为毒种,研制灭活疫苗,对我国犬细小病毒病的流行具有很好的针对性。我们将JL09-11株犬细小病毒灭活后辅以本实验室配制的纳米佐剂对其免疫效果进行了评价,为JL09-11株毒种灭活疫苗的研制奠定基础。
在进行JL09-11株犬细小病毒进行免疫效果评价之前,我们首先在F81细胞系上建立了中和试验,用以测定血清中犬细小病毒的中和抗体。为以后灭活疫苗的免疫效果评价做准备。
将JL09-11株犬细小病毒的细胞培养悬液于37℃使用1%的甲醛灭活24小时后,对其进行TCID50
我们采用未接种任何疫苗,且外周血中犬细小病毒中和抗体滴度较低的健康家犬来对我们灭活犬细小病毒JL09-11株的免疫效果进行评价。在免疫试验过程中,每条试验犬注射灭活疫苗的剂量为1.5ml,其中含有大约5.0×10的测定,结果显示病毒已经完全被灭活。然后以4:1的比例与本实验室制备的纳米佐剂进行混合后制成犬细小病毒的灭活疫苗。5个TCID50
通过本研究我们分离到了一株新的犬细小病毒,进化分析显示,本株犬细小病毒与国内的CPV-2a变种具有较近的进化关系。并且我们发现,国内的大多数被归为CPV-2a变种的毒株与JL09-11一样,主要抗原蛋白VP2的第297位氨基酸均为A,同时其555位氨基酸发生了由I到V的回复突变。这与CPV-2b和CPV-2c相同。我们发现国内流行的主要就是带有这两个突变的CPV-2a变种。我们利用本研究所分离到的JL09-11株犬细小病毒进行了灭活和免疫试验,结果证明,我们分离到的JL09-11株犬细小病毒具有较好的免疫原性,具有用于研制灭活疫苗的应用潜力。经过灭活的犬细小病毒。在免疫前以及免疫后14天,分别对试验犬外周血中的犬细小病毒中和抗体滴度进行测定。结果显示,在免疫后14天试验犬外周血中的犬细小病毒中和抗体较免疫前得到了显著提高。证明了本研究所分离得到的犬细小病毒具有研制灭活疫苗的潜力。
Canine parvovirus(CPV)infection is a kind of acute, infectious disease of domestic dogs and wild canidae, which causes large loss to the worldwide dog industry. As a result, the immunfaction for this disease is a compulsory immune progress for puppies. CPV is a kind of virus with high mutation rate; several antigenic variants have been identified after the isolation of the first canine parvovirus isolate CPV-2, including CPV-2a, CPV-2b, CPV-2c and so on. Those variants were labeled by the variety of the amino acid sequences in the key antigenic sites of the VP2, which is the main protein constructing the nucleocapsid of this virus. The mutations of the amino acid in those antigenic sites brought various antigenic properties to different variants, which can be identified by Mabs. Although the cross immune protection among different variants has been confirmed, several cases of immune failure due to the difference of the vaccine strains and the prevalent strains were reported. As a result, the prime task for the prevention and control of this disease is to identify the antigenic property of the prevalent virus, as well as exploit vaccines.
In this research, the bowel content of a dog in suspension of canine parvovirus infection was collected and detected by PCR to confirm the CPV infection. After grinding and filing, the bowel content was inoculate to F81 cell line for virus isolation. The specific pathological change of the CPV on F81 cell line was appeared 4 days after inoculation. After three cycles of alternative freezing and thawing, the cell culture supernatant was inoculate on F81 cell for another two generations. At each generation, the typical pathological change of the CPV infecting F81 cell, such as rounding, connecting among each other and shedding were appeared. PCR, HA and HI test were performed to identify the isolated CPV in the cell culture supernatant. The PCR identification employed the universal primers of carnivorous animal CPV got positive result; the results of the HA test indicated the HA titer of the cell culture supernatant was 212
To get a better understand to the evolutional relationship of the JL09-11 with that exist in China mainland, as well as the amino acid mutations at the key antigenic sites ; the results of the HI test showed the hemagglutination of the cell culture supernatant could be inhibited by the CPV antibody positive serum stored at our lab, which confirmed that the pathogen we isolated from the bowel content was CPV. The isolated CPV strain was named JL09-11. of the main nucleocapsid protein of this strain, limiting-dilution assay was performed for virus purification. The gene of the main nucleocapsid protein VP2 was amplified through PCR and sequenced. The results showed the JL09-11 is a strain of CPV with new antigenic property, as the amino acid sequences in the key epitopes were not identical with any variant as former published. The VP2 sequences of CPV isolated in China mainland were collected from the NCBI database and involved in the phylogenetic tree reconstruction. In the phylogenetic tree, the JL09-11 strain clustered together with strains which were defined as CPV-2a, although the amino acid analysis indicated the amino acids at site 297 and 555 were A and V orderly. Further analysis indicated those mutations were character of the CPV isolated in China mainland. Then the strain JL09-11 was involved in the development of an inactivated vaccine against canine parvovirus.
Before exploiting the inactivated canine parvovirus vaccine, the neutralizing test was established on F81 cell line, which was used in the test of the neutralizing antibody against the CPV in the canine serum samples.
The cell culture supernatant of the CPV JL09-11 was inactivated with 1% formalin at 37℃for 24 hours. Then the TCID50
The immune effect assessment test was performed on health domestic dogs with no or low titters of neutralizing antibody against CPV in the serum. Each experimental dog was vaccinated with 1.5ml inactivated vaccine, containing about 5.0×10test was performed on the inactivated samples to detect if they were inactivated completely. The results showed the CPV in the cell culture supernatant was inactivated completely. At last the inactivated CPV was mixed with nanometer adjuvant with the proportion of 4:1 to form the inactivated canine parvovirus vaccine. 5 TCID50
This study isolated a new CPV strain JL09-11. Phylogenic analysis indicated the JL09-11 shared high phylogenic homology with strains of CPV-2a variant isolated in China mainland. Amino acid analysis was performed and focused on the VP2 of the JL09-11. The result showed the 297 site of this protein was A(alanine), which is a inactivated CPV. We collected the serum samples before and 14 days after vaccination. The neutralizing antibody against the CPV in the serum samples were tested through neutralizing test as former established. The results indicated that the neutralizing antibody against CPV in the serum of experimental dogs was elevated significantly 14 days after vaccination. specific amino acid of S297A variant, at the same time the amino acid at site 555 was also vary from variant S297A variant. There was a retrieve mutation at site 555 from I (isoleucine) to V (valine). Then we recognized that most CPV strains isolated in China mainland and labeled as CPV-2a have this character. Although cross immune protection among different CPV variants has been reported, exception cases happened from time to time. As a result, it is necessary to develop a vaccine based on the endemic CPV variant of China mainland to prevent and control the canine parvovirus infection. So a kind of inactivated vaccine based on the CPV JL09-11 strain with high immune effect was developed in this study. Assessment test showed this vaccine has the potential to be employed in clinical CPV prevention.
引文
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[15] Ikeda Y, Mochizuki M, Naito R, Nakamura K, Miyazawa T, Mikami T, Takahashi E, Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats,Virology,2000,278:13-19.
[16] Mochizuki M, Harasawa R, Nakatani H, Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan, Vet.Microbiol,1993,38:1-10.
[17] Gamoh K, Shimazaki Y, Makie H, Senda M, Itoh O, Inoue Y, The pathogenicity of canine parvovirus type-2b, FP84 strain isolated from a domestic cat, in domestic cats, J. Vet. Med. Sci,2003,65:1027–1029.
[18] Ikeda Y, Nakamura K, Miyazawa T, Takahashi E, Mochizuki M., Feline host range of canine parvovirus: recent emergence of new antigenic types in cats, Emerg. Infect. Dis,2002,8:341–346.
[19] Colin R. Parrish, Host range relationships and the evolution of canine parvovirus, Vet.Microbiol,1999,69:29-40.
[20] Chinchkar S.R, Mohana Subramanian B, Hanumantha Rao N, Rangarajan P.N, Thiagarajan D, Srinivasan V.A, Analysis of VP2 gene sequences of canine parvovirus isolates in India, Arch. Virol,2006,151:1881–1887.
[21] Doki M, Fujita K, Miura R, Yoneda M, Ishikawa Y, Taneno A, Kai C, Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Japan in 1999 and 2000,Infect.Dis,2006,29:199-206.
[22] Hirayama K, Kano R, Hosokawa-Kanai T, Tuchiya K, Tsuyama S, Nakamura Y,Sasaki Y, Hasegawa A,VP2 gene of a canine parvovirus isolate from stool of a puppy, VP2 gene of a canine parvovirus isolate from stool of a puppy,J. Vet. Med. Sci,2005,67:139–143.
[23] Parrish C.R, Aquadro C, Strassheim M.L, Evermann J.F, Sgro J.-Y., Mohammed H., Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus, J. Virol,1991,65:6544-6552.
[24] Decaro N, Martella V, Desario C, Bellacicco AL, Camero M, Manna L, d'Aloja D, Buonavoglia C, First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain, Vet Med B Infect Dis Vet Public Health, 2006, 53:468-472.
[24] Hong C, Decaro N, Desario C, Tanner P, Pardo MC, Sanchez S, Buonavoglia C, Saliki JT, Occurrence of canine parvovirus type 2c in the United States, J Vet Diagn Invest,2007,19:535-539.
[25] Costa A.P, Leite J.P, Labarthe N.V, Garcia R.C., Genomic typing of canine parvovirus circulating in the state of riode janeiro, Brazil from 1995 to 2001 using polymerase chain reaction assay, Vet. Res. Commun, 2005,29:735-743.
[26] Pereira C.A, Leal E.S, Durigon E.L, Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus, Infect Genet Evol,2007,7:399-409.
[27] Martella V, Decaro N, Elia G, Buonavoglia C., Surveillance activity for canine parvovirus in Italy, J.Vet. Med. B Infect. Dis. Vet. Public Health,2005, 52:3 12–315.
[28] Decaro N, Desario C, Parisi A, Martella V, Lorusso A, Miccolupo A, Genetic analysis of canine parvovirus type 2c, Virology,2009,385:5-10.
[29] Larson L, Schultz R.D, Do two current canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant, Vet. Ther,2008,9:94-101.
[30] Hoelzer K, Shackelton L.A, Parrish C.R, Holmes E.C, Phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses, J. Gen. Virol,2008, 89:2280–2289.
[31] Motohiro Horiuchi, Yumi Yamaguchi, Takashi Gojobori, Masami Mochizuki,Hideyuki Nagasawa, Yutaka Toyoda, Naotaka Ishiguro, and Morikazu Shinagawa, Differences in the Evolutionary Pattern of Feline Panleukopenia Virus and Canine Parvovirus,Viorology,1998,249:440-452.
[32] Barker I.K, Parrish C.R, Infectious diseases of wild mammals, Blackwell Publishing,2001,1:668-705.
[33] Driciru M, Siefert L, Prager K.C, Dubovi E, Sande R, Princee F, A serosurvey of viral infections in lions (Panthera leo), from Queen Elizabeth National Park, Uganda, J. Wildl. Dis, 2006, 42:667–671.
[34] Biek R, Zarnke R.L, Gillin C, Wild M, Squires J.R, Poss ., Serologic survey for viral and bacterial infections in western populations of Canada lynx (Lynx canadensis), J. Wildl. Dis, 2002 38:840–845.
[35] Biek R, Ruth T.K, Murphy K.M, Anderson C.R. Jr, Johnson M, DeSimone R., et al., Factors associated with pathogen seroprevalence and infection in Rocky Mountain cougars, J. Wildl. Dis, 2006, 42:606–615.
[36] Burger D, Gorham J.R, Ott R.L, Protection of cats against feline panleukopenia following mink virus enteritis vaccination, Small Anim. Clin, 1963, 3:611–614.
[37] Truyen U, Parrish C.R, Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo, J. Virol,1992,66:53991–5408.
[38] Hoelzer K, Parrish CR, The emergence of parvoviruses of carnivores, Vet Res,2010,41:39.
[39] Veijalainen P, Characterization of biological and antigenic properties of raccoon dog and blue fox parvoviruses: a monoclonal antibody study, Vet. Microbiol,1988,16:219–230.
[40] Nandi S,Anbazhagan R, Molecular characterisation and nucleotide sequence analysis of canine parvovirus strains in vaccines in India. J Vet. Italiana,2010,46(1):69-81.
[41] Kumar M, Nandi S, Molecular Typing of Canine Parvovirus Variants by Polymerase Chain Reaction and Restriction Enzyme Analysis, Transbound Emerg Dis,2010,57(6):458-463.
[42] Turiso. J.A.L, Cones, E, Martinez, C, Recombinant vaccine for canine parvovirus in dogs,J.Viral,1992,66:2748-2753.
[43] Langeveld, J.P, Casal, J.I, Osterhaus,A.D,First peptide vaccine providing, protection against viral infection in the target animal: studies of canine parvovirus in dogs. J. Viral. 1994, 68:4506-4513.
[44] Patial S, Chaturvedi VK, Rai A, Saini M, Chandra R, Saini Y, Gupta PK, Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2, Vaccine, 2007, 25:4020-4028.
[45]刘忠华,钟翎,黄韧,半嵌套式PCR技术区别检测犬细小病毒疫苗株和强毒株的研究,中国病毒学,2003,18,4:401～403.
[46] Buonavoglia C,Martella V,et al., Evidence for evolution of canine parvovirus type-2 in Italy. J Gen. Virol,2001,82:3021-3025.
[47] Decaro N,Desario C,et al., Evidence for immunization failure in vaccinated adult dogs infected with canine parvovirus type 2c,New Microbiol,2008,31,1:125-130.
[48]刘维全,范泉水,江禹,夏咸柱,黄耕,王吉贵,房金波,王蕾,肉食兽细小病毒通用PCR诊断技术的建立,中国兽医学报,2001,3:249～250.
[49]邱薇,夏咸柱,范泉水,王春华,黄耕,何洪彬,范秀军,乔军,余春,武银莲,犬细小病毒血凝抑制抗体和中和抗体的关系,中国兽医科技,2000,30,10:3～4 .
[2] Tullis G E,Burger L R,Pintel D J,et al., The minor capsid portein VP1 of Autonomous parvovirus minute virus of mice is dispensable for encapsidation of progeny sinle-stranded DNA but is required for infectivity, J Virol,1993,67:131-141.
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[5] Chinchkar S.R, Mohana Subramanian B, Hanumantha Rao N, Rangarajan P.N., Thiagarajan D., Srinivasan V.A., Analysis of VP2 gene sequences of canine parvovirus isolates in India, Arch Virol, 2006, 151:1881–1887.
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[8] Parrish C.R, Host range relationships and the evolution of canine parvovirus,Vet. Microbiol,1999,69:29–40.
[9] Truyen U, Gruenberg A, Chang S.F, Obermaier B, Veijalainen P, Parrish C.R, Gruenberg A, Chang S.F, Obermaier B, Veijalainen P., Parrish C.R., Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo, J. Virol,1995, 69:4702–4710.
[10] Steinel A, Parrish C.R, Bloom M.E, Truyen U, Parvovirus infections in wild carnivores, J. Wildl. Dis, 2001,37:594–607.
[11] Colin R, Parrish James A, Host range relationships and the evolution of canineparvovirus, Vet Microbiol,1999,69:29-40.
[12] Truyen U,Emergence and recent evolution of canine parvovirus,Vet Microbiol,1999,69,1-2:47-50.
[13] Truyen U, Parrish C.R, Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo,J.Virol,1992,66:5399–5408.
[14] Ikeda Y, Miyazawa T, Nakamura K, Naito R,Inoshima Y, Tung K.C, et al., Serosurvey for selected virus infections of wild carnivores in Taiwan and Vietnam, J. Wildl. Dis,1999,35:578–581.
[15] Ikeda Y, Mochizuki M, Naito R, Nakamura K, Miyazawa T, Mikami T, Takahashi E, Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats,Virology,2000,278:13-19.
[16] Mochizuki M, Harasawa R, Nakatani H, Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan, Vet.Microbiol,1993,38:1-10.
[17] Gamoh K, Shimazaki Y, Makie H, Senda M, Itoh O, Inoue Y, The pathogenicity of canine parvovirus type-2b, FP84 strain isolated from a domestic cat, in domestic cats, J. Vet. Med. Sci,2003,65:1027–1029.
[18] Ikeda Y, Nakamura K, Miyazawa T, Takahashi E, Mochizuki M., Feline host range of canine parvovirus: recent emergence of new antigenic types in cats, Emerg. Infect. Dis,2002,8:341–346.
[19] Colin R. Parrish, Host range relationships and the evolution of canine parvovirus, Vet.Microbiol,1999,69:29-40.
[20] Chinchkar S.R, Mohana Subramanian B, Hanumantha Rao N, Rangarajan P.N, Thiagarajan D, Srinivasan V.A, Analysis of VP2 gene sequences of canine parvovirus isolates in India, Arch. Virol,2006,151:1881–1887.
[21] Doki M, Fujita K, Miura R, Yoneda M, Ishikawa Y, Taneno A, Kai C, Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Japan in 1999 and 2000,Infect.Dis,2006,29:199-206.
[22] Hirayama K, Kano R, Hosokawa-Kanai T, Tuchiya K, Tsuyama S, Nakamura Y,Sasaki Y, Hasegawa A,VP2 gene of a canine parvovirus isolate from stool of a puppy, VP2 gene of a canine parvovirus isolate from stool of a puppy,J. Vet. Med. Sci,2005,67:139–143.
[23] Parrish C.R, Aquadro C, Strassheim M.L, Evermann J.F, Sgro J.-Y., Mohammed H., Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus, J. Virol,1991,65:6544-6552.
[24] Decaro N, Martella V, Desario C, Bellacicco AL, Camero M, Manna L, d'Aloja D, Buonavoglia C, First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain, Vet Med B Infect Dis Vet Public Health, 2006, 53:468-472.
[24] Hong C, Decaro N, Desario C, Tanner P, Pardo MC, Sanchez S, Buonavoglia C, Saliki JT, Occurrence of canine parvovirus type 2c in the United States, J Vet Diagn Invest,2007,19:535-539.
[25] Costa A.P, Leite J.P, Labarthe N.V, Garcia R.C., Genomic typing of canine parvovirus circulating in the state of riode janeiro, Brazil from 1995 to 2001 using polymerase chain reaction assay, Vet. Res. Commun, 2005,29:735-743.
[26] Pereira C.A, Leal E.S, Durigon E.L, Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus, Infect Genet Evol,2007,7:399-409.
[27] Martella V, Decaro N, Elia G, Buonavoglia C., Surveillance activity for canine parvovirus in Italy, J.Vet. Med. B Infect. Dis. Vet. Public Health,2005, 52:3 12–315.
[28] Decaro N, Desario C, Parisi A, Martella V, Lorusso A, Miccolupo A, Genetic analysis of canine parvovirus type 2c, Virology,2009,385:5-10.
[29] Larson L, Schultz R.D, Do two current canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant, Vet. Ther,2008,9:94-101.
[30] Hoelzer K, Shackelton L.A, Parrish C.R, Holmes E.C, Phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses, J. Gen. Virol,2008, 89:2280–2289.
[31] Motohiro Horiuchi, Yumi Yamaguchi, Takashi Gojobori, Masami Mochizuki,Hideyuki Nagasawa, Yutaka Toyoda, Naotaka Ishiguro, and Morikazu Shinagawa, Differences in the Evolutionary Pattern of Feline Panleukopenia Virus and Canine Parvovirus,Viorology,1998,249:440-452.
[32] Barker I.K, Parrish C.R, Infectious diseases of wild mammals, Blackwell Publishing,2001,1:668-705.
[33] Driciru M, Siefert L, Prager K.C, Dubovi E, Sande R, Princee F, A serosurvey of viral infections in lions (Panthera leo), from Queen Elizabeth National Park, Uganda, J. Wildl. Dis, 2006, 42:667–671.
[34] Biek R, Zarnke R.L, Gillin C, Wild M, Squires J.R, Poss ., Serologic survey for viral and bacterial infections in western populations of Canada lynx (Lynx canadensis), J. Wildl. Dis, 2002 38:840–845.
[35] Biek R, Ruth T.K, Murphy K.M, Anderson C.R. Jr, Johnson M, DeSimone R., et al., Factors associated with pathogen seroprevalence and infection in Rocky Mountain cougars, J. Wildl. Dis, 2006, 42:606–615.
[36] Burger D, Gorham J.R, Ott R.L, Protection of cats against feline panleukopenia following mink virus enteritis vaccination, Small Anim. Clin, 1963, 3:611–614.
[37] Truyen U, Parrish C.R, Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo, J. Virol,1992,66:53991–5408.
[38] Hoelzer K, Parrish CR, The emergence of parvoviruses of carnivores, Vet Res,2010,41:39.
[39] Veijalainen P, Characterization of biological and antigenic properties of raccoon dog and blue fox parvoviruses: a monoclonal antibody study, Vet. Microbiol,1988,16:219–230.
[40] Nandi S,Anbazhagan R, Molecular characterisation and nucleotide sequence analysis of canine parvovirus strains in vaccines in India. J Vet. Italiana,2010,46(1):69-81.
[41] Kumar M, Nandi S, Molecular Typing of Canine Parvovirus Variants by Polymerase Chain Reaction and Restriction Enzyme Analysis, Transbound Emerg Dis,2010,57(6):458-463.
[42] Turiso. J.A.L, Cones, E, Martinez, C, Recombinant vaccine for canine parvovirus in dogs,J.Viral,1992,66:2748-2753.
[43] Langeveld, J.P, Casal, J.I, Osterhaus,A.D,First peptide vaccine providing, protection against viral infection in the target animal: studies of canine parvovirus in dogs. J. Viral. 1994, 68:4506-4513.
[44] Patial S, Chaturvedi VK, Rai A, Saini M, Chandra R, Saini Y, Gupta PK, Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2, Vaccine, 2007, 25:4020-4028.
[45]刘忠华,钟翎,黄韧,半嵌套式PCR技术区别检测犬细小病毒疫苗株和强毒株的研究,中国病毒学,2003,18,4:401～403.
[46] Buonavoglia C,Martella V,et al., Evidence for evolution of canine parvovirus type-2 in Italy. J Gen. Virol,2001,82:3021-3025.
[47] Decaro N,Desario C,et al., Evidence for immunization failure in vaccinated adult dogs infected with canine parvovirus type 2c,New Microbiol,2008,31,1:125-130.
[48]刘维全,范泉水,江禹,夏咸柱,黄耕,王吉贵,房金波,王蕾,肉食兽细小病毒通用PCR诊断技术的建立,中国兽医学报,2001,3:249～250.
[49]邱薇,夏咸柱,范泉水,王春华,黄耕,何洪彬,范秀军,乔军,余春,武银莲,犬细小病毒血凝抑制抗体和中和抗体的关系,中国兽医科技,2000,30,10:3～4 .