Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (＜2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

The Effect of Gankang Suppository on Duck Hepatitis B Virus, Serum Biochemistry and Liver Histology in Ducklings

To examine the effect of Gankang Suppository on duck hepatitis B virus （DHBV）, the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups： a Gankang Suppository treatment group, an acyclovir （ACV） group and a DHBV model group （control）, with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medica- tion and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values （log） of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment （P=0.0092, P=0.0070, P=0.0080, respectively）. Compared with DHBV model control group, the ALT level was significantly decreased （P=0.0020, P=0.0019, respectively） on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment （P=0.0298）. Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal （P=0.0016）. Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group （P=0.0282, P=0.0084, P=0.0195, respectively）. It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

Isolation,Identification and Pathogenic Characteristics of Duck Hepatitis Virus Isolates

[ Objective] The aim of this study was to determine and analyze the pathogen and the reasons of duck viral hepatitis which is prevalent recently and difficult to control. [Method] Viruses were isolated from livers and spleens of ducks with typical clinical symptoms in Linyi, Weifang, Binzhou and other regions of Shandong Province. The pathogenic characteristics were observed by inoculation in chicken or duck embryo, RT- PCR, serological test, and duck regression. [ Result] Four duck hepatitis virus （DHV） strains were isolated, and the 5th passage allantoic fluid contained 10^3.41 -10^5.20 ELD50/mI. The serum cross protection rate was 20% -80% between the DHV stains and DHV type I. The mortalities of 4- day-old healthy ducks challenged by these four stains were 50% -100%. All challenged ducks had typical lesions of duck viral hepatitis, and the death peak appeared after 24-48 h. [Conclusion] The virulence of different DHV isolates has regional difference.

Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1

To determine the genomic sequence of a duck hepatitis virus type 1 （DHV-1） strain, real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5＇ untranslated region （UTR）-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3＇ UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.

Complete Genomic Sequence of a Chinese Isolate of Duck Hepatitis Virus

The complete genomic sequence of Duck hepatitis virus 1(DHV-1) ZJ-V isolate was sequenced and determined to be 7 691 nucleotides(nt) in length with a 5'-terminal un-translated region(UTR) of 626 nt and a 3'-terminal UTR of 315 nt(not including the poly(A) tail).One large open reading frame(ORF) was found within the genome(nt 627 to 7 373) coding for a polypeptide of 2 249 amino acids.Our data also showed that the poly(A) tail of DHV-1 has at least 22 A's.Sequence comparison revealed significant homology(from 91.9% to 95.7%) between the protein sequences of the ZJ-V isolate and those of 21 reference isolates.Although DHV-1 has been classified as an unassigned virus in the Picornaviridae family,its genome showed some unique characteristics.DHV-1 contains 3 copies of the 2A gene and only 1 copy of the 3B gene,and its 3'-NCR is longer than those of other picornaviruses.Phylogenetic analysis to do sequence homology based on the VP1 protein sequences showed that the ZJ-V isolate shares high sequence homology with the reported DHV-1 isolates(from 92.9% to 99.2%),indicating that DHV-1 is genetically stable.

The Potential of Duck Hepatitis Virus(DHV-1) Stimulating the Body Weight Gain and the Effects of Silymarin on It in Duckling

To evaluate the effects of duck hepatitis virus-1 （DHV-1） on the body weight gain in duck and the effects of silymarin on it in vivo, 100 10-d-old ducks, both male and female, were collected to be subjected to the test. The experiments were conducted in 8 groups： in group 1-3, the animals were inoculated with 1：105 diluted duck hepatitis virus （DHV-1） infected allantoic fluid and given 0, 30, and 50 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 4-6, the animals were inoculated with 1：5 × 105 diluted DHV-1 infected allantoic fluid and given 0, 10, and 30 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 7, the animals were given 10 mg kg^-1 BW d^-1 silymafin only. Group 8 was the control one treated by injecting sterillized saline into the leg muscles. All the silymarin was given from 0 to 4 d after inoculation of the virus. By the 5th d after inoculation, the vein blood was drawn from the dorsal foot vein and the plasma samples were collected and stored at -20℃. The body weight gain （BWG） was measured from 0 to 10 d after inoculation. The plasma IGF-I, T3, and T4 concentrations were measured by radioimmunoassay （RIA）. At the virus dose of 1：5 ×105 diluted virus infected allantoic fluid, the inoculation of the virus enhanced the BWG significantly compared with that of the control （P〈 0.01）, while 10-50 mg kg^-1 BW d^-1 silymarin could counteract the effects of the virus on the BWG dose-dependently. The plasma IGF-I levels showed no correlation with the BWG, but the T3 levels showed a same tropism with the body weight gain. The present results indicated that sublethal DHV-1 enhanced the body weight gain of ducklings significantly, and the silymarin could counteract this effect in vivo.

Molecular Characterization of Duck Hepatitis B Virus Isolated from Hubei Brown Ducks

The objective of this study was to characterize the genome structure of duck hepatitis B virus （DHBV） isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction （PCR） and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 % This strain （GenBank accession number DQ276978） had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the ＂Chinese＂ DHBV strains than those of the ＂Western＂ country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.

Development of RT PCR Assay for Detection of Duck Hepatitis Virus Type 1

In this study,a RT-PCR assay for rapid detection of duck hepatitis virus type 1( DHV-1) was established in the presence of a pair of primers designed based on vp1 gene sequence,using the genomic RNA of DHV-1 or other common viruses as the template. The template was serially diluted 10-fold to determine the reaction sensitivity. Finally,seven liver samples of ducks with suspected DHV-1 infection,which were collected from different regions of Jiangsu Province,were detected with this method. The results showed that a DNA fragment of about 360 bp was specifically amplified with the RT-PCR system,and the detection limit was1 fg/μL. The detection rate of clinical samples with this method was 100%. The results revealed that the RT-PCR system which had high specificity and high sensitivity in the detection of DHV-1 was successfully established.

Therapeutic effect of Styela plicata on duck hepatitis B virus in vivo

Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to receive the alcohol extract of Styela plicata or lamivudine for 30 consecutive days. The DHBV DNA of sera was detected by RT-PCR. and the histological analysis of duckling liver was evaluated. Results:Thirty days after therapy,histological analysis of duckling liver showed that the ducklings receiving the alcohol extract of Styela plicata or lamivudine exhibited catabatic status in the degree of liver cell degeneration and inflammation compared with the ducklings receiving normal diet. DHBV DNA of sera from alcohol extract of Styela plicata-treated ducklings and lamivudine-treated ducklings all produced significantly lower levels compared with ducklings receiving normal diet (P<0. 01 ). Although these treatment groups all exhibited a rebound phenomenon 10 d after withdrawal of medication, they still exhibited a significant lower level of serum DHBV DNA compared with the control group responded to normal diet (P<0. 05, P<0. 01). Conclusion:Styela plicata may be an effective antiviral medicine in treating chronic hepatitis B. The data of this experiment will be valuable in studying the therapeutic role and the potential therapeutic mechanism of Styela plicata.

DUCK HEPATITIS B VIRUS DNA WITHIN HEPATIC MULTICENTRIC CANCER AND/OR METASTATIC CANCER

Duck hepatitis B vims (DHBV) DNA was detected in different tumorous nodules of ducks with hepatic multicentric cancer or intrahepatic metastasis by Southern blot technique. Among 7 ducks with hepatocellular carcinoma of multiple tumor nodules, the hybridization pattern of Integrated DHBV DNA In different tumorous nodules was identical in 3 cases and different in 2 cases. One case showed a similar hybridization pattern in two tumorous nodules and other one was negative tor DHBV DNA. Integrated DHBV DNA was also identified in a metastatic lung cancer of ducks with hepatocellular carcinoma. The hybridization pattern of metastasis of lungs was as the some as that in primary hepatocellular carcinoma. The same discrete hybridization bands In the different tumorous nodules indicate that these nodules might arise from one transformed cell. The different hybridization patterns In various tumorous nodules show that these tumorous nodules might arise from various transformed cells. The results suggest that the hybridization pattern of different nodules of hepatocellular carcinoma with viral DNA probe could make a cell clone origin marker of tumor nodule to differentiate hepatic multlcentric cancer from Intrahepatic metastatic cancer.

Detection of the covalently closed circular DNA of duck hepatitis B virus by Taq-Man fluorescent quantitative PCR assay

To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.

芒果苷抗DHAV-1致鸭胚肝细胞炎性损伤的研究

【目的】探究芒果苷(mangiferin,Man)对鸭甲型肝炎病毒1型(Duck hepatitis A virus-1,DHAV-1)致鸭胚肝细胞(DEHCs)炎性损伤的影响。【方法】从14日龄SPF级麻鸭鸭胚肝脏组织分离原代DEHCs,用含不同浓度芒果苷(0、5、10、20、40、80和160μmol/L)的培养液培养48 h后,通过CCK-8法检测其安全浓度范围;用安全浓度的芒果苷培养DEHCs 12、24和48 h后,检测培养液上清中乳酸脱氢酶(LDH)的活力,判定芒果苷对DEHCs的毒性作用。将DEHCs分为对照组(Mock)、模型组(Model)、芒果苷组(Man10、Man20和Man40)和利巴韦林组(Rib),每组3个重复,所有组细胞血清饥饿培养12 h后,Mock组加入含10%胎牛血清(FBS)的DMEM培养基,Model、Man10、Man20、Man40和Rib组用DHAV-1(MOI=1.0)攻毒2 h后,Model组更换为含10%FBS的DMEM培养基,Man10、Man20、Man40培养基中分别添加10、20和40μmol/L芒果苷,Rib组添加1μmol/L利巴韦林。48 h后收集各组细胞,用比色法检测丙二醛(MDA)含量及过氧化氢酶(CAT)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、总一氧化氮合酶(T-NOS)和谷胱甘肽过氧化物酶(GSH-Px)的活性;免疫荧光(IF)法检测DHAV-1在各组DEHCs中的分布情况;Western blotting法检测NLRP3/Pro-Caspase1/IL-1β通路蛋白的表达情况。提取2日龄SPF麻鸭的新鲜全血,分离鸭外周血单个核细胞(duPBMCs),duPBMCs分组及处理同DEHCs,ELISA法检测各组duPBMCs上清液中白介素8(IL-8)和IL-1β的含量。【结果】与0μmol/L芒果苷组相比,80和160μmol/L芒果苷组细胞的增殖能力显著降低(P<0.05),因此5~40μmol/L芒果苷为处理DEHCs的安全给药浓度。20和40μmol/L芒果苷处理48 h细胞上清液中LDH活力显著低于处理12和24 h(P<0.05),20和40μmol/L芒果苷处理12和24 h细胞上清液中LDH活力显著高于5和10μmol/L芒果苷处理(P<0.05),各浓度芒果苷处理48 h细胞上清液中LDH活力均差异不显著(P>0.05),故48 h为最适处理时间。与Mock组相比,Model组DEHCs中CAT、T-AOC、SOD和GSH-Px活性均显著降低(P<0.05),DEHCs中MDA含量、T-NOS活性、DHAV-1拷贝数、DHAV-1阳性率、NLRP3表达量及duPBMCs中IL-8、IL-1β分泌量均显著升高(P<0.05)。与Model组相比,Man10、Man20和Man40组DEHCs中CAT、T-AOC、SOD和GSH-Px活性均显著提高(P<0.05),DEHCs中MDA含量、T-NOS活性、DHAV-1拷贝数、DHAV-1阳性率、NLRP3表达量及duPBMCs中IL-8、IL-1β分泌量均显著降低(P<0.05)。【结论】芒果苷可通过增加抗氧化能力、降低DHAV-1的复制水平、抑制NLRP3通路的激活、降低促炎细胞因子的释放抗DHAV-1感染DEHCs造成的炎性损伤。

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Is hepatitis B-virucidal validation of biocides possible with the use of surrogates?

The hepatitis B virus(HBV)is considered to be a major public health problem worldwide,and a significant number of reports on nosocomial outbreaks of HBV infections have been reported.Prevention of indirect HBV transmission by contaminated objects is only possible through the use of infection-control principles,including the use of chemical biocides,which are proven to render the virus non-infectious.The virucidal activity of biocides against HBV cannot be predicted;therefore,validation of the virucidal action of disinfectants against HBV is essential.However,feasible HBV infectivity assays have not yet been established.Thus,surrogate models have been proposed for testing the efficacy of biocides against HBV.Most of these assays do not correlate with HBV infectivity.Currently,the most promising and feasible assay is the use of the taxonomically related duck hepatitis B virus(DHBV),which belongs to the same Hepadnaviridae virus family.This paper reviews the application of DHBV,which can be propagated in vitro in primary duck embryonic hepatocytes,for the testing of biocides and describes why this model can be used as reliable method to evaluate disinfectants for efficacy against HBV.The susceptibility levels of important biocides,which are often used as ingredients for commercially available disinfectants,are also described.

鸭病毒性肝炎病毒VP1基因表达及其抗体检测ELISA方法的建立

【目的】研究DHV VP1基因在大肠杆菌中的表达,并以纯化的重组蛋白为抗原建立鸭病毒性肝炎抗体检测ELISA方法。【方法】采用RT-PCR技术,扩增VP1基因,与pMD18-T载体进行连接,构建DHV VP1基因克隆重组质粒。然后定向插入到pET-32a(+)表达载体,筛选原核表达载体pET-32a-VP1,进行ITPG诱导表达分析。以纯化的重组蛋白为抗原建立间接ELISA方法并初步应用于临床。【结果】DHV VP1基因可在大肠杆菌中稳定、高效地表达。Western blot检测表明,表达的重组蛋白能与鸭肝炎阳性血清发生特异性反应。确定间接ELISA方法的抗原最佳包被浓度为5μg/孔,血清最佳稀释度为1∶100,临界值为OD450值≥0.302,建立的ELISA方法具有较好的敏感性、特异性和重复性。通过对80份血清样品的检测表明,该方法与中和试验的符合率为97.5%,初步临床应用结果表明该方法可用于雏鸭母源抗体和免疫后抗体的消长变化的检测。【结论】以大肠杆菌表达的DHV VP1重组蛋白为抗原建立的间接ELISA方法可用于鸭病毒性肝炎抗体的检测。

基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立

以纯化的基因C型鸭甲肝病毒(DHAV-C)VP1重组蛋白作为包被抗原,建立检测DHAV-C抗体的间接ELISA方法。结果表明,试验的最佳条件:用pH9.6、0.05 mol·L-1的碳酸盐缓冲液将包被抗原稀释成5μg·mL-1,4℃包被过夜,抗体稀释度为1∶100,HRP酶标羊抗鸭IgG稀释度为1∶5 000,封闭液为含10%脱脂乳的0.01mol·L-1 PBS,37℃封闭2h,血清稀释液为含1%BSA的PBS,抗体反应时间为60min,酶标羊抗鸭IgG反应时间为30min,底物作用时间为15min,临界值为OD450nm≥0.474。该方法重复性好,批内变异系数为2.03%~2.95%,批间变异系数为3.28%~7.38%,与鸭圆环病毒(DUCV)、鸭乙型肝炎病毒(DHBV)、鸭瘟病毒(DPV)、新城疫病毒(NDV)、禽流感病毒(AIV)、鸭抗大肠杆菌、鸭抗金黄色葡萄球菌、鸭抗沙门菌、鸭抗禽多杀性巴氏杆菌以及鸭疫里默杆菌阳性血清无交叉反应,而与基因A型鸭甲肝病毒(DHAV-A)阳性血清有交叉反应,表明该方法可以作为检测DHAV-C和DHAV-A抗体的通用ELISA方法。与中和试验相比,阳性血清检测符合率为80%,阴性血清检测符合率为100%。初步临床应用表明:该方法可用于雏鸭母源抗体和免疫后抗体消长的检测。本研究基于DHAV-C VP1重组蛋白建立的间接ELISA方法可用于基因A型和C型鸭甲型肝炎的血清流行病学调查和抗体检测。

复方六月雪对鸭乙型肝炎病毒DNA的抑制作用

目的:观察复方六月雪(CLYX)对鸭乙型肝炎病毒(DHBV)DNA的抑制作用,为研究和开发新的抗HBV药物提供实验依据。方法:采用1日龄雏鸭接种广西麻鸭DHBV强阳性血清,13天后用PCR法筛选出DHBV强阳性鸭。随机分成5组:CLYX高、中、低剂量组、模型组和阳性对照组,每组10只,各组雏鸭均灌胃给药14天。于用药前(T0)、用药7天(T7)、14天(T14)及停药后3天(P3)分别采血,采用荧光定量PCR(FQ-PCR)法检测血清DHBVDNA的含量。结果:CLYX各剂量组用药后血清DHBV DNA含量显著降低(P<0.01),停药3天后,CLYX低剂量组和阳性对照组血清DHBV DNA有反跳现象,而CLYX中、高剂量组血清DHBV DNA没有反跳现象。CLYX抑制DHBV DNA的作用有明显的量效和时效反应关系。结论:CLYX可有效地抑制DHBV DNA的复制。

间接ELISA检测鸭肝炎病毒抗体的研究

以蔗糖密度梯度离心法纯化的病毒作为包被抗原，建立了检测鸭肝炎病毒（ＤＨＶ）抗体的间接ＥＬＩＳＡ方法。经特异性及重复性试验，效果良好。ＥＬＩＳＡ效价与琼扩、中和效价存在平行关系。经ＥＬＩＳＡ检测，１日龄雏鸭免疫后，４日龄可检出ＥＬＩＳＡ抗体，１０日龄达到峰值。ＤＨＶ高免血清在雏鸭体内作用维持时间为１０ｄ左右。攻毒保护试验表明，攻毒前雏鸭的血清抗体水平与攻毒后雏鸭存活率具直接相关性。

鸭Ⅰ型肝炎病毒RT-LAMP可视化检测方法的建立

为建立一种快速检测鸭Ⅰ型肝炎病毒(DHV I)的方法,本研究根据基因库中DHV Ⅰ基因的保守序列,设计特异性环介导等温扩增(LAMP)引物,建立了DHV I的RT-LAMP可视化检测方法。该方法的敏感性可达10fg,高于常规PCR方法 100倍;全部反应可在1 h内完成;可以通过肉眼观察颜色直接判定结果;对其它鸭常见病原体的检测结果均为阴性。实验结果表明建立的RT-LAMP方法简便、快速、灵敏、特异,可用于DHV Ⅰ感染的快速检测。