Serological survey on canine coronavirus antibodies in giant pandas by virus neutralization test

In order to survey the infectious situation of canine coronavirus (CCV) in giant panda population, a virus neutralization test detecting specific antibodies against CCV in giant panda抯 sera was established by using two-fold dilutions of serum and 100 TCID50 of the virus. The 62 sera samples of giant pandas, which were gathered from zoos and reserve region of Sichuan Province, China were detected. The neutralization antibody titer of 1:4 was recognized as the positive criterion, 8 sera samples were detected to be positive, and the positive rate was 12.9%. The titers of neutralizing antibody ranged from 1:8 to 1:32. It was the first comprehensive investigation on neutralization antibodies against CCV in giant panda population in China. The results of study showed that the infection of CCV in giant panda population was universal, which has posed a threat to the health of giant panda. Therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against CCV infection.

Comparative Studies on Detection of Antibodies against Infectious Bursal Disease Virus with Test Strips and Agar Gel Immunodiffusion Method

[Objective] This study aimed to compare the detection results of antibodies against infectious bursal disease virus with test strips and agar gel immunodiffusion method. [Method] Antibodies against infectious bursal disease virus in chicken serum were detected using test strips developed in our laboratory, and the results were comparad^with that using traditional agar diffusion method. [Result] The comparative study of the two methods showed that the sensitivity of test strips was eight times over agar gel immunodiffusion; test strips showed higher detection rate in the deter- mination test of 216 clinical samples, with high specificity, easy preservation, and simple and rapid operation, thereby being more suitable for the monitoring of clinical antibodies. [Conclusion] Test strips could replace the existing serological methods, having great promotion and application value in antibody monitoring.

养殖场生物制品的保存及使用(上)

在畜禽养殖生产过程中,使用各种兽用生物制品对动物进行有计划的免疫接种,是动物疫病综合性防控中的重要环节,也是构建动物安全体系的重要措施。生物制品的保存及使用应注意以下方面:一、生物制品的种类和特点1.弱毒苗弱毒苗是利用毒力减弱的细菌或病毒等微生物经大量繁殖后制成的疫苗,也称活苗。

High Serum Resistin Level may be an Indicator of the Severity of Coronary Disease in Acute Coronary Syndrome

Objective To investigate the correlation between serum resistin level, cardiovascular risk factors and severity of coronary disease in acute coronary syndrome （ACS）. Methods Alter evaluated by clinical history, electrocardiography, exercise tolerance tests, laboratory tests, and coronary angiography, 220 consecutive patients with suspected chest pain were divided into normal control group, stable angina pectoris （SAP） group, and ACS group, respectively. Baseline clinical characteristics, including height, weight, waist circumference, hip circumference, white blood cell count, high-sensitive C-reactive protein （hsCRP）, total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, were compared among three groups. ELISA was used to detect serum resistin levels. Pearson＇s correlation coefficient analysis was used to assess association between resistin and other traditional cardiovascular risk factors. Multinomial logistic regression analyses were used to define the relationship between serum resistin level and SAP or ACS. Results Serum resistin level in ACS group （1.18±0.48 μg/L） was significantly higher than that in normal control and SAP groups （0.49±0.40 and 0.66±0.40 μg/L; P〈0.01）. Only in ACS group, increased serum resistin level was significantly correlated with hsCRP （r=0.262, P=0.004） and white blood cell count （r=0.347, P=0.001）. Furthermore, serum resistin levels showed a stepwise increase with the number increase of 〉 50% stenosed coronary vessels. Multinomial logistic regression test demonstrated that serum resistin was a strong risk factor for ACS （OR=29.132, 95 % CI： 10.939-77.581, P〈0.001）. Conclusion These findings suggested the potential role of resistin in atherosclerosis and especially its involvement in ACS.

High serum leptin is an independent risk factor for nonresponse patients with low viremia to antiviral treatment in chronic hepatitis C

AIM： To determine whether body weight and/or serum leptin were independent predictors of response to antiviral treatment in patients with chronic hepatitis C. METHODS： A retrospective evaluation was performed in 139 patients with chronic hepatitis C treated with interferon （IFN） from 1996 to 2000. Sustained response was defined as negative by hepatitis C virus （HCV） RNA analysis using PCR and normal transaminase at 24 wk after cessation of IFN therapy. Patients who remained positive for HCV RNA at the end of IFN treatment were defined as resistant to IFN therapy. Sex, age, body mass index （BMI） （≥ 25 vs 〈 25）, complication of diabetes mellitus, serum leptin level （≥ 8.0μg/L vs 〈8.0μg/L）, and the stage of liver fibrosis by needle biopsy （F1/F2 vs F3/F4） were examined. RESULTS： Sustained response was achieved in 33 patients （23.7%）, while others failed to show a response to IFN therapy. Overall, the factors associated with sustained antiviral effects were HCV-RNA load, HCV genotype, serum leptin level, and stage of liver fibrosis evaluated by univariate analysis. BMI was not associated with any therapeutic effect of IFN. Multivariate analysis indicated that HCV-RNA load was a significant risk factor, but among the patients with low viremia （HCV-RNA 〈 100 MU/L）, leptin level was an independent risk factor for IFN resistance. Namely, a high level of serum leptin attenuated the effect of IFN on both male and female patients with low viremia. CONCLUSION： High serum leptin level is a negative predictor of response to antiviral treatment in chronic hepatitis C with low viremia.

Utility of serological markers in inflammatory bowel diseases: Gadget or magic?

The panel of serologic markers for inflammatory bowel diseases （IBD） is rapidly expanding. Although antiSaccharornyces cerev/siae antibodies （ASCA） and atypical perinuclear antineutrophil cytoplasmic antibodies （P-ANCA） remain the most widely investigated, an increasing amount of experimental data is available on newly discovered antibodies directed against various microbial antigens. The role of the assessment of various antibodies in the current IBD diagnostic algorithm is often questionable due to their limited sensitivity. In contrast, the association of serologic markers with disease behavior and phenotype is becoming increasingly well-established. An increasing number of observations confirms that patients with Crohn＇s disease expressing multiple serologic markers at high titers are more likely to have complicated small bowel disease （e.g. stricture and/or perforation） and are at higher risk for surgery than those without, or with low titers of antibodies. Creating homogenous disease sub-groups based on serologic response may help develop more standardized therapeutic approaches and may help in a better understanding of the pathomechanism of IBD. Further prospective clinical studies are needed to establish the clinical role of serologic tests in IBD.

Prokaryotic Expression of P1 Gene of Type Asia1 Foot and Mouth Disease Virus(FMDV)and the Preparation of Its Antiserum

[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.

Long-term treatment outcomes of clevudine in antiviral-naive patients with chronic hepatitis B

AIM:To evaluate the treatment outcomes of clevudine compared with entecavir in antiviral-naive patients with chronic hepatitis B(CHB).METHODS:We retrospectively analyzed the clinical data of CHB patients treated with clevudine 30 mg/d and compared their clinical outcomes with patients treated with entecavir 0.5 mg/d.The biochemical response,as assessed by serum alanine aminotransferase(ALT) activity,virologic response,as assessed by serum hepatitis B virus DNA(HBV DNA) titer,serologic response,as assessed by hepatitis B e antigen(HBeAg) status,and virologic breakthrough with genotypic mutations were assessed.RESULTS:Two-hundred and fifty-four patients [clevudine(n = 118) vs entecavir(n = 136)] were enrolled.In clevudine-treated patients,the cumulative rates of serum ALT normalization were 83.9% at week 48 and 91.5% at week 96(80.9% and 91.2% in the entecavir group,respectively),the mean titer changes in serum HBV DNA were-6.03 and-6.55 log 10 copies/mL(-6.35 and-6.86 log 10 copies/mL,respectively,in the entecavir group),and the cumulative non-detection rates of serum HBV DNA were 72.6% and 83.1%(74.4% and 83.8%,respectively,in the entecavir group).These results were similar to those of entecavir-treated patients.The cumulative rates of HBeAg seroconversion were 21.8% at week 48 and 25.0% at week 96 in patients treated with clevudine,which was similar to patients treated with entecavir(22.8% and 27.7%,respectively).The virologic breakthrough in the clevudine group occurred in 9(7.6%) patients at weeks 48 and 15(12.7%) patients at week 96,which primarily corresponded to genotypic mutations of rtM204I and/or rtL180M.There was no virologic breakthrough in the entecavir group.CONCLUSION:In antiviral-naive CHB patients,longterm treatment outcomes of clevudine were not inferior to those of entecavir,except for virologic breakthrough.

KINETIC CHANGES OF ANTIBODY TO M.LEPRAE AND ITS RELATION WITH CLINICAL DISEASE

A sero epidemiological survey on 1 833 healthy residents was carried out in 6 villages of a leprosy high endemic area in Wenshan and Guangnan counties, Yunnan Province. The part of the residents with initially antibody positive as well as the part of residents with initially antibody negative have been followed up for 3 consecutive years by serology and clinical examination for studying kinetic changes of antibody to M.leprae and its relation with clinical disease. The results showed that the rates of subclinical infection of leprosy in a high endemic area are different from village to village, and the risk of developing clinical disease does not associate with subclinical infection rate. It correlates with the number of cured accumulative leprosy cases and active cases within the village. The authors consider that in leprosy high endemic villages, especially those cropped up new multi bacillary leprosy cases frequently in recent years, it may be helpful to use serology to detect early leprosy cases.

Human chromosome pellicle antibody recognizing centromere protein-C(CENP-C),the main component of the kinetochore

Recently the antichromosome antisera from several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes. In order to identify the pellicle components, we used these antichromosome antisera to screen a human embryonic cDNA library. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C), a human centromere autoantigen. This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

Herpes Simplex Virus Type 1 ICP27 Protein:Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni 2+ -NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.

Detection of anti-Helicobacter pyloriantibodies in serum and duodenal fluid in peptic gastroduodenal disease

AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid. METHODS:Data were collected from 93 patients submitted to upper digestive endoscopy due to dyspeptic symptoms. The patients were either negative(group A)or positive (group B)to H pylori by means of both histological detection and urease tests.Before endoscopy,peripheral blood was collected for the investigation of anti-H pylori IgG and IgA antibodies.To perform the urease test,biopsies were obtained from the gastric antrum.For the histological evaluation,biopsies were collected from the gastric antrum (greater and lesser curvatures)and the gastric body. Following this,duodenal fluid was collected from the first and second portions of the duodenum.For the serological assaying of anti-Hpylori IgG and IgA,and anti-Hpylori IgA in duodenal fluids,the ELISA method was utilized. RESULTS:The concentration of serum IgG showed sensitivity of 64.0%,specificity of 83.7%,positive predictive value of 82.0%,negative predictive value of 66.6% and accuracy of 73.1% for the diagnosis of H pylori infection.For the same purpose,serum IgA showed sensitivity of 72.0%, specificity of 65.9%,positive predictive value of 72.0%, negative predictive value of 67.4% and accuracy of 69.8%. If the serological tests were considered together,i.e.when both were positive or negative,the accuracy was 80.0%, sensitivity was 86.6%,specificity was 74.2%,positive predictive value was 74.2% and negative predictive value was 86.6%.When values obtained in the test for detecting IgA in the duodenal fluid were analyzed,no significant difference(P=0.43)was observed between the values obtained from patients with or without H pylori infection. CONCLUSION:The results of serum IgG and IgA tests for H pylori detection when used simultaneously,are more efficient in accuracy,sensitivity and negative predictive value, than those when used alone.The concentration of IgA antibodies in duodenal fluid is not useful in identifying patients with or without H pylori.

Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.

Effects of Chinese Herbmedicines as Feed Additives on Resistance of Trionyx sinensis to Diseases

In the-comparison between two experimental turtle (Trionyx sinensis) groups fed on the feed containing 1 % or 2% Chinese medicinal herb powder and the contrast group to which no Chinese herb was fed,no obvious difference of γ-globulin level in the turtles' serum was found,but the survival rate of the experimental groups was obviously higher than that of the contrast group,and cell immunity of the experimental groups was significantly higher when tested by subcutaneous injection of PHA.Meanwhile,the Chinese herb promoted the growth of the turtles.

Sustained virologic response following HCV eradication in two brothers with X-linked agammaglobulinaemia

X-linked agammaglobulinaemia (XLA) is a humoral immunodeficiency syndrome characterized from childhood by the absence of circulating B lymphocytes, absent or reduced levels of serum immunoglobulin and recurrent bacterial infections. For many affected patients, regular treatment with immunoglobulin is life saving. Hepatitis C viral (HCV) infection acquired through contaminated blood products is widely described in this patient cohort. The natural history of HCV infection in patients with XLA tends to follow a more rapid and aggressive course compared to immunocompetent individuals. Furthermore, standard anti-viral therapy appears to be less efficacious in this patient cohort. Here we report the cases of two brothers with XLA who contracted HCV through contaminated blood products. They were treated with a six month course of Interferon alpha-2b and Ribavirin. We report a sustained virologic response five years after completing treatment.

Lyme Disease in Iraq： First Detection of IgM Antibodies to Borrelia burgdorferi in Human Sera

The sera of 180 human samples were tested for the presence of antibodies against Borrelia burgdorferi using the ELISA （enzyme-linked immunosorbent assay） technique. Out of 180 sero-samples, 46 （25.55%） were positive. Females of the age range 18-35 years had the highest rate of sero-positive samples 14 （38.88%）, while the highest percentage of sero-negative samples was found in males of the age range 50-80 years. The other sero-positive samples were： 6 （26.08%）, 6 （25%） and 3 （11.53%） in males of ages between 18-35, 35-50 and 50-80 years, respectively, and 11 （29.72%） and 6 （17.64%） in females in the age ranges 35-50 and 50-80 years, respectively. The mean concentration of Anti- B. burgdorferi antibody was higher （16.7 U/mL） when compared with mean concentration of normal value （5.5 U/mL）, P 〈 0.001.

Highly sensitive serological approaches for Pepino mosaic virus detection

Pepino mosaic virus(PepMV)causes severe disease in tomato and other Solanaceous crops around globe.To effectively study and manage this viral disease,researchers need new,sensitive,and high-throughput approaches for viral detection.In this study,we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies(mAbs).A panel of highly specific and sensitive murine mAbs(15B2,8H6,23D11,20D9,3A6,and 8E3)could be produced through cell fusion,antibody selection,and cell cloning.Using the mAbs as the detection antibodies,we established double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),Dot-ELISA,and Tissue print-ELISA for detecting PepMV infection in tomato plants.Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1310720 and 1:20480(weight/volume ratio(w/v),g/mL),respectively.Among the three methods developed,the Tissue print-ELISA was found to be the most practical detection technique.Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction(RT-PCR)and DNA sequencing,dem on strati ng all three serological methods are reliable and effective for monitoring PepMV.An ti-PepMV mAbs and the newly developed DAS-ELISA,Dot-ELISA,and Tissue print-ELISA can benefit PepMV detection and field epidemiological study,and management of this viral disease,which is already widespread in tomato plants in Yunnan Province of China.