湖北省鸭肝炎病毒的分离鉴定及生物学特性研究
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摘要
鸭病毒性肝炎(Duck Viral Hepatitis,DVH)是雏鸭的一种高度致死性的、传播迅速的病毒性疾病,以肝炎为其主要特征。DHV有1型、2型和3型3个血清型。在我国流行的主要是1型鸭肝炎病毒。该病是目前危害养鸭业的主要疾病之一,给养鸭业造成了巨大的经济损失。本研究旨在从湖北地区分离鸭肝炎病毒,并通过鸭胚中和试验、保守区基因序列的测定、动物回归试验、动物保护试验对病毒进行鉴定,通过对鸭胚、雏鸭的致死性试验,对试验雏鸭组织病理变化的观察及组织病变的电镜观察,分析研究鸭肝炎病毒的致病性,同时对分离株进行系统的生物学特性研究,为鸭肝炎的流行病学调查和分类学研究提供素材,并为有效地预防和控制鸭病毒性肝炎提供理论依据。研究的结果如下:
1.通过雏鸭尿囊腔接种,对湖北采集的20份疑似鸭病毒性肝炎的病鸭肝组织进行病原分离,获得2株病毒。将2株分离株经鸭胚传至第5代,收集鸭胚尿囊液病毒测定这些毒株的ELD_(50)分别为10~(-3.88)/0.2ml,10~(-4.59)/0.2ml。经鸭胚中和试验鉴定,2株均为1型鸭肝炎病毒,分别命名为鸭肝炎病毒株DHV-1 JX和DHV-1 DY。
2.根据GenBank中的DHV序列,设计引物,经RT-PCR扩增病毒保守区基因组,对其进行序列测定。与Genebank已发表的1型鸭肝炎病毒的核苷酸序列同源性介于92%～97%之间,进化树分析JX株和DY株的亲源关系较远,不在同一分支上。
3.JX和DY株病毒对11日龄鸭胚的致死性为100%,人工感染雏鸭的致死率分别为50%和70%。死亡鸭呈角弓反张姿态,剖检主要可见肝脏肿胀,有大量的出血点。镜下观察2株病毒主要侵害雏鸭肝、胰、脾、肾、脑等组织,造成实质器官的病变,肝脏均呈出血性坏死性炎症,DY株胰脏出现明显的坏死灶。肝组织超微结构观察到细胞胞浆的膜状结构中有双层单位膜包绕的圆形似病毒样颗粒,直径约为30～40nm。
4.对JX株和DY株病毒的理化特性表现为:病毒在56℃、30min部分灭活,62℃、30min能完全灭活,对乙醚、氯仿、胰酶有抵抗力。同时病毒不凝集鸡、兔、鸭、小鼠的红细胞。
Duck viral hepatitis(DVH) is a fatal and rapidly spreading disease of young ducklings. The major pathologic change in infected ducklings is hepatitis.Three different viruses, duck hepatitis virus types 1,2 and 3,have been found to be associated with these symptoms.Duck virus hepatitis type 1was popular with in China.DHV was the biggest trouble maker of the development of the poultry industry.The present study describes isolating and identifying duck hepatitis of Hubei strain,the specific gene cloned and sequenced,neutralization test,and recurrence test,and protection test.A virulent field strains of DHV-1 were identified and their pathogenicity to both duck embryo and duckling were tested.The pathogeny of DHV was analysed through observation electron microscope and pathological change of duckling's internal organ,its bionomics.The study provided information for the epidemiology and taxology of DVH,and was useful for providing technological means and academic basis to effectively prevent.Gained some results as follow:
Isolation two virus were obtained through uscovy duck embryo allantoic sacinoculation from duckling liver samples collected from Hubei provinces in China.The ELD_(50) of the two virus strains detected with duck embryos were 10~(-388)/0.2mL and 10~(-459)/0.2mL respectively.Two virus were identified DVH type 1 and named JX strain, DYstrain.
DHV-1 of conservation region fragments obtained in RT-PCR were sequenced and aligned 92-97%similarity at the nucleotide level with that of other DHV-1 strains listed in GenBank.Phylogenetic tree revealed that JX strains and DY strain was grouped into different lineage.
The lethal rate of these isolates was 100 percent when inoculating 11-day-old duck embryos,and the mortality rate was 50 and 70 percent respectively When inoculating 4-day-old ducklings intramuscularly with these isolates.Dead ducklings showed the sign of opisthotonos,with swollen and punctuate hemorrhage in their livers.The histopathological changes of inculated duckling were observed.The result showed that the histopathological changes were orrhagic necrotic hepatitis,pancreas limitation coagulation necrosis.Particle resemble virus in membrane structure was shown about 30～40nm in diameter.
The studies of physical and chemical properties about two strains showed: aether-resistant,chloroform-resistant,enzyme-resistant,heat,pH3.0-resistant and absent-aggiutination.
1.通过雏鸭尿囊腔接种,对湖北采集的20份疑似鸭病毒性肝炎的病鸭肝组织进行病原分离,获得2株病毒。将2株分离株经鸭胚传至第5代,收集鸭胚尿囊液病毒测定这些毒株的ELD_(50)分别为10~(-3.88)/0.2ml,10~(-4.59)/0.2ml。经鸭胚中和试验鉴定,2株均为1型鸭肝炎病毒,分别命名为鸭肝炎病毒株DHV-1 JX和DHV-1 DY。
2.根据GenBank中的DHV序列,设计引物,经RT-PCR扩增病毒保守区基因组,对其进行序列测定。与Genebank已发表的1型鸭肝炎病毒的核苷酸序列同源性介于92%～97%之间,进化树分析JX株和DY株的亲源关系较远,不在同一分支上。
3.JX和DY株病毒对11日龄鸭胚的致死性为100%,人工感染雏鸭的致死率分别为50%和70%。死亡鸭呈角弓反张姿态,剖检主要可见肝脏肿胀,有大量的出血点。镜下观察2株病毒主要侵害雏鸭肝、胰、脾、肾、脑等组织,造成实质器官的病变,肝脏均呈出血性坏死性炎症,DY株胰脏出现明显的坏死灶。肝组织超微结构观察到细胞胞浆的膜状结构中有双层单位膜包绕的圆形似病毒样颗粒,直径约为30～40nm。
4.对JX株和DY株病毒的理化特性表现为:病毒在56℃、30min部分灭活,62℃、30min能完全灭活,对乙醚、氯仿、胰酶有抵抗力。同时病毒不凝集鸡、兔、鸭、小鼠的红细胞。
Duck viral hepatitis(DVH) is a fatal and rapidly spreading disease of young ducklings. The major pathologic change in infected ducklings is hepatitis.Three different viruses, duck hepatitis virus types 1,2 and 3,have been found to be associated with these symptoms.Duck virus hepatitis type 1was popular with in China.DHV was the biggest trouble maker of the development of the poultry industry.The present study describes isolating and identifying duck hepatitis of Hubei strain,the specific gene cloned and sequenced,neutralization test,and recurrence test,and protection test.A virulent field strains of DHV-1 were identified and their pathogenicity to both duck embryo and duckling were tested.The pathogeny of DHV was analysed through observation electron microscope and pathological change of duckling's internal organ,its bionomics.The study provided information for the epidemiology and taxology of DVH,and was useful for providing technological means and academic basis to effectively prevent.Gained some results as follow:
Isolation two virus were obtained through uscovy duck embryo allantoic sacinoculation from duckling liver samples collected from Hubei provinces in China.The ELD_(50) of the two virus strains detected with duck embryos were 10~(-388)/0.2mL and 10~(-459)/0.2mL respectively.Two virus were identified DVH type 1 and named JX strain, DYstrain.
DHV-1 of conservation region fragments obtained in RT-PCR were sequenced and aligned 92-97%similarity at the nucleotide level with that of other DHV-1 strains listed in GenBank.Phylogenetic tree revealed that JX strains and DY strain was grouped into different lineage.
The lethal rate of these isolates was 100 percent when inoculating 11-day-old duck embryos,and the mortality rate was 50 and 70 percent respectively When inoculating 4-day-old ducklings intramuscularly with these isolates.Dead ducklings showed the sign of opisthotonos,with swollen and punctuate hemorrhage in their livers.The histopathological changes of inculated duckling were observed.The result showed that the histopathological changes were orrhagic necrotic hepatitis,pancreas limitation coagulation necrosis.Particle resemble virus in membrane structure was shown about 30～40nm in diameter.
The studies of physical and chemical properties about two strains showed: aether-resistant,chloroform-resistant,enzyme-resistant,heat,pH3.0-resistant and absent-aggiutination.
引文
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76. Kaleta E F. Duck viral hepatitis type 1 vaccination: Monitoring of immune response with a microneutralization test in pekin duck embryo kidney cell culture.Avian Pathol, 1988, 17: 325-332
77. Kim M C, Kwon Y K, Joh S J, Lindberg A M, Kwon J H, Kim J H, Kim S J. Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae. J Gen Virol, 2006, 87(11): 3307-3316
78. Korolkov V I, Tishchenko G P. Laboratory trials of a duck hepatitis vaccine. Tr Beloruss NI Vet Inst, 1975, 13: 79-83
79. Levine P P, Fabricant J. A hitherto-undescribed virus disease of ducks in North America. Cornell Vet, 1950,40(1): 71-86
80. Levine P P, Hofstad M S. Duck disease investigation. Annual Report of the New York State Veterinary College, Ithaca, 1945, 55-56
81. Lu Y S, Lin D F, Le Y L, Liao Y K, Tsai H J. Infectious bill atrophy syndrome caused by parvovirus in a co-outbreak with duck viral hepatitis in ducklings in Taiwan. Avian Dis, 1993, 37: 591-596
82. Maiborda A D. Cultivation of duck hepatitis virus in cell culture.Russian Veterinarian, 1972, (8): 50-52
83. Maiboroda A D, Kontrimavichus L M. Propagation of duck hepatitis virus in goose-embryo cells. Byull Vses Inst Eksp Vet, 1968, 4: 5-7
84. Maiboroda A D. Formation of duck hepatitis virus in culture cells. Veterinariya Mosolow, 1972, 8: 50-52
85. Murty D K, Hanson I E. A modified microgel diffusion method and its application in the study of the virus of duck hepatitis. Am J Vet Res, 1961, 22: 274-278
86. Pollard M, Starr T J. Propagation of duck hepatitis virus in tissue culture. Proc Soc Exp Biol Med, 1959, 101: 521-524
87. Polyakov A A, Volkovskii G D.Survival of duckling hepatitis virus outside the host and methods of disinfection. Vses Inst Vet Sanit, 1969, 34: 278-290
88. Priz N N. Comparative study of virus hepatitis in animal(dogs and ducks)using different routes of influence. Vopr Virusol, 1973, 6: 696-700
89. Reuss U. Versuche zur aktiven und passiven Immunisierung bei der Virushepatitis der Entenkuken. Zentralbl Veterinaermed, 1959, 6: 808-815
90. Reuss U. Virusbiologische Untersuchungen bei der Entenhepatitis.Zentralbl Veterinaermed, 1959, 6: 209-248
91. Sandhu T S, Calnek BW, Zeman L. Pathologic and Serologic characterization of a variant of duck hepatitis type 1 virus. Avian Disease, 1992, 36(4): 932-936
92. Schoop G, Staub H, Erguney K. Virus hepatitis of ducks V Attempted adaptation of the virus to chicken embryos. Monatsh Tierheilked, 1959, 11: 99-106
93. Taurso N M, Coghill G E, Klutch M J.Properties of the attenuated vaccine strain of duck hepatitis virus. Avian Dis, 1969, 13: 321-329
94. Toth T E, Norcross N L. Humoral immune response of the duck hepatitis virus:Virus-neutralizing vs virus-precipitating antibodies. Avian Dis, 1981, 25: 17-38
95. Toth T E. Studies of an agent casuing mortality among ducklings immune to duck virus hepatitis. Avian Dis, 1969, 13: 834-846
96. Toth T E. Chicken-embryo-adapted duck hepatitis virus growth curve in embryonated chicken eggs. Avian Dis, 1969, 13: 535-539
97. Vashhenko V. The use of indirect hemagglutination reaction for the diagnosis of virus hepatitis of ducklings. Eksp Inf Inst Ptits, 1982, 109: 32-34
98. Woolcock P R, Fabricant J. Duck virus hepatitis. Diseases of Poultry, 9th ed. Iowa State University Press, Ames, 1991, IA 597-600
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93. Taurso N M, Coghill G E, Klutch M J.Properties of the attenuated vaccine strain of duck hepatitis virus. Avian Dis, 1969, 13: 321-329
94. Toth T E, Norcross N L. Humoral immune response of the duck hepatitis virus:Virus-neutralizing vs virus-precipitating antibodies. Avian Dis, 1981, 25: 17-38
95. Toth T E. Studies of an agent casuing mortality among ducklings immune to duck virus hepatitis. Avian Dis, 1969, 13: 834-846
96. Toth T E. Chicken-embryo-adapted duck hepatitis virus growth curve in embryonated chicken eggs. Avian Dis, 1969, 13: 535-539
97. Vashhenko V. The use of indirect hemagglutination reaction for the diagnosis of virus hepatitis of ducklings. Eksp Inf Inst Ptits, 1982, 109: 32-34
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