An unusual COVID-19 case with over four months of viral shedding in the presence of low neutralizing antibodies: a case report

The ongoing coronavirus disease 2019(COVID-19)pandemic is a global public health crisis,causing social and economic disasters in many countries.In China,two-consecutive negative results of nucleic acid tests for SARS-CoV-2 from the respiratory samples are required to end the quarantine of COVID-19 patients.However,clinicians face a dilemma in case of patients with long-term viral shedding.This report described an unusual COVID-19 case who had persistent viral RNA positivity for more than 4 months after initial illness in the presence of low neutralizing antibodies,but without prolonged clinical symptoms.Multiple anti-viral drug treatments had no impact and there was no evidence of re-infection.When the patient was self-quarantined at home,no infection occurred to the three family members living with her for 15 to 19 days.Sputum viral culture in BSL-3 laboratory on the 102nd day after symptom onset was negative.From the 129th day on,8 continuous nucleic acid tests of sputum samples showed negative results.The patient was discharged on 137th days since symptom onset.In conclusion,viral RNA shedding in the sputum of the COVID-19 patient may last over 4 months.As no evidence shows the existence of infectious virus,two-consecutive negative nucleic acid tests may not be the prerequisite for ending quarantine of COVID-19 patients with prolonged viral shedding.

Characteristics of Viral Shedding in Respiratory Samples and Specific Antibodies Production in 564 COVID-19 Patients

Positive nucleic acid(NA)results have been found in recovered and discharged COVID-19 patients,but the proportion is unclear.This study was designed to analyze the recurrent positive rate of NA results after consecutively negative results,and the relationship between the specific antibody production and positive NA rate.According to Strengthening the Reporting of Observational Studies in Epidemiology guidelines,data of inpatients in Sino-French New City Branch of Tongji Hospital between Jan.28 and Mar.6,2020 were collected.A total of 564 COVID-19 patients over 14 years old who received the examinations of NA and antibodies against SARS-CoV-2 were included.Days of viral shedding and specific antibodies were recorded and assessed.Among NA tests in respiratory samples(throat swabs,nasopharyngeal swabs,sputum and flushing fluid in alveoli),the patients with all-negative NA results accounted for 17.20%,those with single-positive results for 46.63%,and those with multiple-positive results for 36.17%respectively.Besides,the recurrent positive NA results after consecutively negative results appeared in 66 patients(11.70%).For multiple-positive patients,median viral shedding duration was 20 days(range:1 to 57 days).Of the 205 patients who received 2 or more antibody tests,141(68.78%)had decreased IgG and IgM concentrations.IgM decreased to normal range in 24 patients,with a median of 44 days from symptom onset.Viral shedding duration was not significantly correlated with gender,age,disease severity,changes in pulmonary imaging,and antibody concentration.It is concluded that antibody level and antibody change had no significant correlation with the positive rate of NA tests and the conversion rate after continuous negative NA tests.In order to reduce the recurrent positive proportion after discharge,3 or more consecutive negative NA test results with test interval more than 24 h every time are suggested for the discharge or release from quarantine.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

AIM To prepare hybridoma cell lines which secrete anti HCV recombinant NS3 and NS5 proteins′ monoclonal antibodies, and to evaluate their usage in the study of the distribution of HCV NS3 and NS5 antigen in liver tissues. METHODS The hybridoma cell lines were raised using the spleen cells of BALB/C mouse immunized with recombinant NS3 and NS5 proteins according to the conventional protocols. The antibody secreting cells were screened using solid phase ELISA and cloned by limited dilution method. In order to determine the specificity of these hybridoma cell lines, the culture supernatant of these cells was western blot assayed with expression and nonexpresion E. coli and ELISA with other antigens, including HCV core and NS3 and HBsAg. Immunohistochemistry of 51 cases paraffin embedded liver tissues was performed to determine the distribution of HCV NS5 antigen as well as NS3 antigen in liver tissues. RESULTS Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were raised. They are named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Among them only 2B6 against NS3 protein can react with the polypipetides of C7 that is another recombinant polypipetides of NS3 gene. Others have no reaction with HCV core and HBsAg of HBV, and there is no cross reaction between NS3Ag and anti NS5Ag McAb and between NS5Ag and anti NS3 McAb. The immunohistochemistry results indicate that no HCV antigen was detected in the specimens of HBV infection in 20 cases. In 31 HCV infected specimens the positive rate of NS3Ag and NS5Ag are 51 6% (16/31) and 54 9% (17/31), respectively. There were six pure HCV infected specimens in these 31 specimens and half of them were HCV NS3Ag and NS5Ag positive. In the co infection of HBV and HCV group the positive rate of NS3Ag and NS5Ag were 52% (13/25) and 56% (14/25), respectively, almost the same with that of pure HCV infected group. The positive rates of HCV antigens were 70 6% (12/17) and 76 5% (13/17) in CAC patients. CONCLUSION The monoclonal antibodies we prepared are specific to the recombinant HCV NS3 and NS5 proteins and can be used in the clinical immunohistochemistry diagnosis.

Neutralizing antibodies in hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.

Research review Study and application of monoclonal antibodies against herpes simplex virus

The results of research work completed in recent years in the authors’ laboratory withmonoclonal antibodies (McAbs) against herpes simplex virus (HSV) are reported in this paper as fol-lows.(1) Eighteen hybridoma cell lines steadily producing McAbs against HSV were established byhybridoma technic.(2) The mechanisms of neutralization in vitro and animal protection in vivo medi-ated by different McAbs were investigated.(3) A method of detecting virus antigua was developedwith the McAbs.(4) Tne isolates of virus were typed and antigenically analysed by type-specificand type-common McAbs.(5) HSV antigen in clinical spectimens was detected and directly typed byan ELISA method coating with type-specific and type-common McAb.(6) The target antigens ofMcAbs were identified,purified,and their genes were localized.(7) Experimental rabbitHSV keratitis was treated with McAbs.

Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

Quantitative hepatitis B core antibody and quantitative hepatitis B surface antigen:Novel viral biomarkers for chronic hepatitis B management

The management of hepatitis B virus(HBV)infection now involves regular and appropriate monitoring of viral activity,disease progression,and treatment response.Traditional HBV infection biomarkers are limited in their ability to predict clinical outcomes or therapeutic effectiveness.Quantitation of HBV core antibodies(qAnti-HBc)is a novel non-invasive biomarker that may help with a variety of diagnostic issues.It was shown to correlate strongly with infection stages,hepatic inflammation and fibrosis,chronic infection exacerbations,and the presence of occult infection.Furthermore,qAnti-HBc levels were shown to be predictive of spontaneous or treatment-induced HBeAg and HBsAg seroclearance,relapse after medication termination,re-infection following liver transplantation,and viral reactivation in the presence of immunosuppression.qAnti-HBc,on the other hand,cannot be relied on as a single diagnostic test to address all problems,and its diagnostic and prognostic potential may be greatly increased when paired with qHBsAg.Commercial qAnti-HBc diagnostic kits are currently not widely available.Because many methodologies are only semi-quantitative,comparing data from various studies and defining universal cut-off values remains difficult.This review focuses on the clinical utility of qAnti-HBc and qHBsAg in chronic hepatitis B management.

Anal human papilloma viral infection and squamous cell carcinoma:Need objective biomarkers for risk assessment and surveillance guidelines

High grade anal intraepithelial neoplasia due to human papilloma viral(HPV)infections is a precursor lesion for squamous cell carcinoma especially in high risk populations.Frequent examination and anal biopsies remain unpopular with patients;moreover they are also risk factors for chronic pain,scarring and sphincter injury.There is lack of uniform,surveillance methods and guidelines for anal HPV specifically the intervals between exam and biopsies.The aim of this editorial is to discuss the intervals for surveillance exam and biopsy,based on specific HPV related biomarkers?Currently there are no published randomized controlled trials documenting the effectiveness of anal screening and surveillance programs to reduce the incidence,morbidity and mortality of anal cancers.In contrast,the currently approved screening and surveillance methods available for HPV related cervical cancer includes cytology,HPV DNA test,P16 or combined P16/Ki-67 index and HPV E/6 and E/7 mRNA test.There are very few studies performed to determine the efficacy of these tests in HPV related anal precancerous lesions.The relevance of these biomarkers is discussed in this editorial.Longitudinal prospective research is needed to confirm the effectiveness of these molecular biomarkers that include high risk HPV serotyping,P16 immuno-histiochemistry and E6/E7 mRNA profiling on biopsies to elucidate and establish surveillance guidelines.

牛病毒性腹泻病毒E2蛋白的真核表达及间接ELISA抗体检测方法的建立

为建立检测牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白,将纯化后的E2蛋白作为包被抗原,用方阵滴定方法对影响ELISA的各个因素进行优化,并进行特异性、敏感性和重复性试验。结果表明,在昆虫细胞中表达了BVDV E2蛋白,Western blot证实目的蛋白可与BVDV阳性血清发生特异性反应。ELISA优化结果显示,E2蛋白最佳包被浓度为0.5μg/mL,最佳封闭液为1%明胶,最佳血清稀释度为1∶400,最佳血清作用方式为37℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37℃作用30 min,最佳底物作用时间为室温20 min,阳性临界值为OD 450≥0.423。与血清中和试验法进行比较,总符合率为97.8%,板内和板间重复性试验的变异系数均小于10%。该方法与牛常见病毒阳性血清均无交叉反应。说明建立的间接ELISA抗体检测方法特异性、敏感性和重复性良好,可用于大批量样本的临床检测和流行病学研究。

牛病毒性腹泻病毒E^(rns)-ELISA抗体检测试剂盒的制备

为建立快速检测牛病毒性腹泻病毒(BVDV)的血清学方法,本试验以BVDV NADL毒株结构蛋白E^(rns)作为包被抗原,优化反应条件并组装BVDV酶联免疫吸附试验(ELISA)抗体检测试剂盒,通过重复性试验、灵敏性试验、符合率试验和保存期试验验证该试剂盒的准确性、灵敏性和稳定性。结果显示,抗原包被浓度为4μg/mL,1%酪蛋白溶液37℃封闭45 min,被检血清以1∶400稀释孵育30 min,兔抗牛HRP标记二抗以1∶20000稀释孵育30 min,TMB底物反应5 min时,ELISA的反应背景下降,区分度最好。优化后方法的批内重复性试验和批间重复性试验的变异系数均在10%以内,表明该试剂盒具有较好的稳定性;灵敏性试验结果显示,当阳性血清稀释度达到1∶6400时仍可以检测为阳性,说明该试剂盒灵敏度较高;该试剂盒与美国爱德士生物科技公司(IDEXX)BVDV总抗体检测试剂盒共同检测466份临床血清样品,两者总符合率为89.1%;保存期试验结果显示,第4个月时该试剂盒仍能检测到阳性血清,说明稳定性较好。综上表明,本试验利用E^(rns)蛋白建立的BVDV间接ELISA抗体检测试剂盒灵敏度高且重复性好,可为BVDV抗体检测和流行性调查提供新的方法。

自身免疫性肝炎临床误诊分析

目的探讨自身免疫性肝炎(AIH)的诊治措施及误诊原因、防范措施。方法回顾性分析2020年1月—2022年5月收治的AIH误诊为病毒性肝炎21例的临床资料。结果21例主要症状为食欲缺乏、乏力、黄疸、发热,伴腹胀12例,恶心5例,胸闷和胸痛4例,呕吐及关节痛各3例。体形消瘦,巩膜及皮肤黏膜黄染明显。查血丙氨酸转氨酶和天冬氨酸转氨酶升高;7例γ-谷氨酰转肽酶升高,碱性磷酸酶和总胆红素升高各6例。腹部B超检查示肝大18例,肝内回声不均。初期外院诊断为病毒性肝炎,予相应治疗15 d无好转,遂转我院。查血抗平滑肌抗体(SMA)、抗核抗体(ANA)阳性,血γ-球蛋白、IgG升高,结合肝炎病毒血清学检测阴性及相关病史,遂明确诊断为AIH。误诊时间18~21 d。确诊后,18例予泼尼松单独治疗,3例予泼尼松联合硫唑嘌呤治疗。治疗1年后随访,患者病情稳定,无复发。结论AIH发病较隐匿,以年轻女性高发,临床表现多样且无特异性,易误诊为病毒性肝炎,行肝炎病毒血清学检查及ANA、SMA、抗肝肾微粒体、免疫球蛋白或肝组织病理检查可区分二者,确诊后应及时予有效治疗,以改善患者预后。

2.1亚型猪瘟病毒流行株E2蛋白单克隆抗体的制备及其抗原表位的鉴定

为获得仅与我国基因2.1亚型猪瘟病毒(CSFV)流行株反应的单克隆抗体(MAb),并揭示疫苗株与流行株E2蛋白抗原氨基酸的差异,本研究利用基因2.1亚型重组质粒p FastBac1-JL23 E2通过杆状病毒表达重组E2蛋白(rE2),并采用Ni柱纯化后经SDS-PAGE检测r E2的表达及纯化效果;采用western blot鉴定r E2的反应原性。SDS-PAGE及western blot结果表明经杆状病毒表达了基因2.1亚型CSFV E2蛋白,且其纯化效果和反应原性均较好。采用杂交瘤技术制备2.1基因亚型CSFV E2蛋白的单克隆抗体(MAb),采用亲和层析法纯化该MAb,采用BCA法测定其浓度及亚类。将79株1.1、2.1、2.2和2.3基因亚型的CSFV及1.1基因亚型的HCLV株和SM株感染PK-15细胞,72 h后采用IFA检测MAb与不同基因型CSFV的反应性;采用western blot鉴定MAb与10个基因亚型共18种CSFV代表株E2蛋白的反应性。采用IFA鉴定MAb与不同基因型CSFV的中和活性。结果显示,经IFA筛选后共获得16株分泌E2蛋白MAb的杂交瘤细胞株。其中仅一株MAb TCH061与所有2.1基因亚型(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j、2.1k、2.1l、2.1m、2.1n)CSFV感染的细胞反应后出现绿色荧光,与基因1.1亚型HCLV疫苗株和SM株以及其他基因型CSFV感染的细胞反应后均无绿色荧光;western blot结果显示,该MAb能够识别2.1基因亚型CSFV E2蛋白(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j),在90 ku处出现特异性条带,而与其他基因型CSFV E2蛋白均不反应。表明TCH061为2.1基因亚型CSFV E2蛋白特异性的MAb。MAb的纯化结果显示,在50 ku和25 ku处有明显条带,其浓度为1.70μg/μL且该MAb TCH061重链为Ig G2a,轻链为κ链。中和活性结果显示,TCH061对JL^(23)株(2.1b)、GDLF1株(2.1c)有一定的中和活性,但不能中和C株。分别以GD53株E^(24)个不同区域的截短蛋白为抗原通过western blot初步确定MAb识别抗原表位的区域。利用CLC Sequence Viewer比对与MAb反应的CSFV E2蛋白氨基酸序列的差异,通过SWISS-MODEL预测MAb抗原表位的关键氨基酸位点。利用杆状病毒表达CSFV JL^(23)株各位点突变后的E2蛋白,采用western blot鉴定各突变的E2蛋白与MAb TCH061的反应性。Western blot结果显示,TCH061的抗原表位位于E2蛋白的aa1~aa90,且其识别CSFV E2蛋白P^(20)不识别L^(20)的CSFV。通过SWISS-MODEL预测E2蛋白氨基酸序列的I18、G^(19)、P^(20)、L^(21)、G^(22)、A^(23)和E^(24)在空间上比较接近;MAb TCH061与JL^(23)株I18M突变的E2蛋白反应,与G^(19)K、P^(20)L和L^(21)P突变的E2蛋白均不反应,与G^(22)K突变的E2蛋白反应性较弱,与HCLV E2蛋白不反应,但与HCLV L^(20)P突变的E2蛋白反应。证实TCH061的抗原表位为^(19)GPLG^(22),经Py MOL结构模拟确定其为空间构象表位;且除了aa^(20),其他位点在各基因型CSFV中均非常保守,表明P^(20)是2.1基因亚型CSFV流行株E2蛋白抗原表位的关键氨基酸。综上所述,本研究首次获得一株区别于CSFV疫苗株和2.1基因亚型CSFV流行株的MAb TCH061,揭示了疫苗株与流行株E2蛋白氨基酸位点的差异,为研发猪瘟疫苗和CSFV流行株感染的血清学鉴别检测方法的建立提供了重要的MAb资源。

Early hepatitis B viral DNA clearance predicts treatment response at week 96

AIM To investigate whether hepatitis viral DNA load at 24 wk of treatment predicts response at 96 wk in patients with chronic hepatitis B.METHODS A total of 172 hepatitis B envelope antigen(HBe Ag)-positive chronic hepatitis B patients who received initial treatment at 16 tertiary hospitals in Hunan Province, China were enrolled in this study. All patients received conventional doses of lamivudine and adefovir dipivoxil, telbivudine, entecavir dispersible tablets, or entecavir tablets for 96 wk. Patients who used other antiviral drugs or antitumor and immune regulation therapy were excluded. Patients were stratified into three groups according to their viral DNA load at 24 wk: < 10 IU/m L(group 1), 10-103 IU/m L(group 2), and > 103 IU/m L(group 3). Correlations of 24-wk DNA load with HBe Ag negative status and HBe Ag seroconversion at 96 wk were analyzed. Receiver operating characteristic curve analysis was used to test the predictive value of the HBV DNA load at 24 wk for long-term response.RESULTS The rates of conversion to HBe Ag negative status and HBe Ag seroconversion rates were 53.7% and 51.9%, respectively, in group 1; 35.21% and 32.39% in group 2; and 6.38% and 6.38% in group 3. The receiver operating characteristic curves for the three subgroups revealed that the lowest DNA load(< 10 IU/m L) was better correlated with response at 96 wk than a higher DNA load(10-103 IU/m L). Nested PCR was used for amplifying and sequencing viral DNA in patients with a viral DNA load > 200 IU/m L at 96 wk; resistance mutations involving different loci were present in 26 patients, and three of these patients had a viral DNA load 10-103 IU/m L at 96 wk. CONCLUSION Hepatitis B viral DNA load at 24 wk of antiviral treatment in patients with chronic hepatitis B is a predictor of the viral load and response rate at 96 wk.

牛病毒性腹泻净化效果研究

为了解吉林省某规模牛场BVDV的流行情况,并制定净化方案,应用BVDV ELISA试剂盒对2022年3月—2023年10月采集吉林省某规模牛场的血液和粪便样本进行病原学调查。结果表明:该牛场全群BVDV抗体、抗原阳性率分别为2.72%、0.85%,抗原阳性5头(其中成年牛1头,后备牛1头,犊牛3头),抗体阳性16头(其中成年牛5头,后备牛5头,犊牛6头)。对检测为BVDV阳性的牛直接淘汰,并通过3次复检,筛选出BVDV持续性感染牛,对其进行淘汰,并对检测阴性牛免疫。经过长达1年的监测及淘汰,该规模牛场BVDV病原学检出率为0,取得了良好净化效果。

牛病毒性腹泻病毒NS5B蛋白的截短表达及多克隆抗体制备

本研究旨在克隆牛病毒性腹泻病毒(BVDV)的非结构蛋白NS5B截短基因,采用原核表达系统表达获得截短蛋白,并进一步制备其多克隆抗体。构建的原核表达质粒pET-28a-NS5B转化至BL21感受态细胞,经IPTG诱导后,发现NS5B蛋白以包涵体为主。纯化蛋白免疫小鼠制备的多克隆抗体具有良好的反应性,能够特异性识别真核表达的NS5B蛋白。本研究中NS5B蛋白的截短表达及其多克隆抗体的制备为进一步研究NS5B蛋白的生物学功能,以及建立BVDV鉴别诊断的血清学检测方法奠定了基础。

雏鸭母源抗体对新型鸭呼肠孤病毒保护效果研究

研究旨在采用新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)主动感染不同母源抗体组的商品雏鸭,对雏鸭泄殖腔排毒、体重、肝脏、脾脏组织病变和病理指标进行检测、观察,以期探究母源抗体对雏鸭早期感染新型鸭呼肠孤病毒的保护效果从而为该病的防控奠定基础。试验用58只商品雏鸭在相同环境条件下饲养,检测1日龄雏鸭NDRV母源抗体,根据母源抗体进行分组并于3日龄攻毒,攻毒后3、5、7 d采集泄殖腔棉拭子检测排毒情况,并在雏鸭2、8、10日龄进行称重。在攻毒后7 d处死鸭只并剖检,观察肝脏和脾脏病变,计算脾脏指数并制备部分肝脏和脾脏病理切片。结果表明,攻毒鸭泄殖腔均检测到排毒,高母源抗体组无法阻止排毒;攻毒鸭体重增重极显著低于对照组(P<0.01);攻毒鸭脾脏坏死,肝脏均有散在出血点伴有坏死灶;攻毒鸭脾脏指数极显著高于对照组(P<0.01);攻毒后不同母源抗体组雏鸭脾脏指数、体重差异不显著(P>0.05);肝脏、脾脏细胞均有不同程度病理受损。该试验说明高母源抗体无法给雏鸭提供对该NDRV分离株的有效保护,通过免疫种鸭来保护雏鸭免受感染的方法有待商榷。

血清富亮氨酸α_(2)糖蛋白1、新蝶呤与病毒抗体检测对病毒性脑膜炎患儿病情评估及预后预测的价值

目的探讨血清富亮氨酸α_(2)糖蛋白1(LRG1)、新蝶呤(NPT)与病毒抗体交互作用在病毒性脑膜炎(VM)患儿病情进展中的作用及其对VM患儿病情评估及预后预测的价值。方法选择2020年8月至2023年2月开封市儿童医院收治的130例VM患儿为观察组,另选择同期收治的130例有VM症状但经腰椎穿刺脑脊液检查确诊为非中枢系统感染性疾病患儿为对照组。入院后采集2组患儿2 mL外周静脉血,采用酶联免疫吸附法检测血清LRG1、NPT水平;治疗前行腰椎穿刺,采集1 mL脑脊液标本,采用酶联免疫吸附法检测脑脊液中单纯疱疹病毒Ⅰ(HSV-Ⅰ)、巨细胞病毒(CMV)、EB病毒(EBV)、副流感病毒(PIV)的特异性IgG抗体。比较2组患儿的血清LRG1、NPT水平及病毒抗体IgG阳性率。依据病情严重程度将VM患儿分为轻症组(n=76)和重症组(n=54),依据治疗2周后的预后将VM患儿分为预后良好组(n=37)和预后不良组(n=93);比较不同病情严重程度组、不同预后组患儿血清LRG1、NPT水平及病毒抗体IgG阳性率,分析血清LRG1、NPT和病毒抗体与VM患儿病情严重程度、病情进展的相关性,并分析其对VM患儿病情进展的交互作用,采用受试者操作特征(ROC)曲线分析血清LRG1、NPT与病毒抗体对VM患儿预后的预测价值。结果观察组患儿血清LRG1、NPT水平及病毒抗体IgG阳性率均显著高于对照组(P<0.05);重症组患儿血清LRG1、NPT水平及病毒抗体IgG阳性率均显著高于轻症组(P<0.05);预后不良组患儿血清LRG1、NPT水平及病毒抗体IgG阳性率均显著高于预后良好组(P<0.05)。高血清LRG1水平与高病毒抗体IgG阳性率、高血清NPT水平与高病毒抗体IgG阳性率导致VM患儿预后不良中呈正向交互作用(比值比:15.238、9.684);血清LRG1高表达与高病毒抗体IgG阳性率(比值比:15.238)、血清NPT高表达与高病毒抗体IgG阳性率(比值比:9.684)为次相乘模型。血清LRG1、NPT水平与病毒抗体IgG阳性率预测治疗2周VM患儿预后不良的曲线下面积(AUC)分别为0.786、0.794、0.919,三者联合预测的AUC为0.933;LRG1、NPT与病毒抗体IgG阳性率联合预测治疗2周VM患儿预后不良的AUC大于三者单独预测(P<0.05)。结论NPT、LRG1可能参与了VM的发生发展,血清NPT、LRG1水平与VM患儿病情严重程度、病情进展均呈正相关,血清NPT、LRG1水平及病毒抗体IgG阳性率对VM的病情评估及预后预测有一定价值,且三者联合对VM预后的预测价值更高;VM患儿高LRG1与高病毒抗体IgG阳性率、高NPT与高病毒抗体IgG阳性率同时存在时具有正向交互作用。

牛病毒性腹泻抗体制剂的制备及其应用研究

抗体制剂是牛病毒性腹泻最为有效的临床治疗药剂,但现有制剂易在瘤胃中降解而药效不佳。本研究利用抗体包被技术开发了一种能有效避免瘤胃降解的牛病毒性腹泻抗体制剂的制备方法,对抗体制剂筛选的6个组方体系进行稳定性、安全性和药效综合考察,表明体系1(抗体5.0g、卵磷脂20.0g、聚乙二醇80015g、β-谷甾醇1.0g、鞣云实素3.0g、吐温-2015.0g、去离子水41.0g)为最优组方,临床疗效显著高于普通抗体制剂。该制剂具有稳定性强和安全性高的特点,能有效解除瘤胃液对口服抗体的影响,促进其在肠道中的高效吸收和利用,对临床防控牛病毒性腹泻有着重要意义。

抗体类产品病毒安全控制探讨

病毒安全控制对于抗体类产品的生产质量至关重要。本文汇总了在药学审评及现场检查中抗体类产品在病毒安全控制方面的相关问题,结合最新修订的ICH Q5A(R2)指导原则、连续制造技术的发展情况以及平台化验证的技术要求,主要从厂房与仪器设备、细胞库、原材料与辅料及耗材、上游生产过程、下游纯化过程、变更控制等方面对抗体类产品病毒安全控制进行分析探讨,以期为相关生产企业及监管部门提供参考借鉴。