843例儿童急性呼吸道感染血清IgM测定与分析

目的对儿童常见呼吸道感染病原体作统计分析并了解其流行趋势。方法采用ELISA法对843例急性呼吸道感染(ARI)患儿血清中的IgM抗体进行测定后,按性别、年龄、发病季节分组后进行统计分析。结果286人次IgM测定阳性,总阳性率33.9%;以呼吸道合胞病毒(RSV)检出率最高(38.1%),其在性别组、季节组间差异无统计学意义;在年龄组(尤其是小于3岁以下组)间差异有统计学意义。肺炎支原体(34.3%)、肺炎衣原体(11.5%)在年龄组和季节组中差异有统计学意义。结论RSV、肺炎支原体为衡阳地区儿童ARI常见病原体,RSV为新生儿常见病原体,肺炎支原体、肺炎衣原体感染多见于3岁以上儿童,流行于冬春季。

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.

Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP1 Protein of Asia 1 Type Foot-and-Mouth Disease Virus

Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.

Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

Comparison of Multiplex Fluorescent PCR with Serum Type-specific Antibody Detection in Diagnosis of Genital Herpes

Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.

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.

Application of high-titred IgY antibodies in orthopox virus diagnostics

Layer chickens were immunized with three species of inactivated orthopox virus （vaccinia virus, calpox virus and cowpox virus）. Antibodies （IgY） were purified from egg yolks by improved polyethylene glycol precipitation. The development of IgY directed against orthopox virus antigens was followed by immunofluorescence assay, plaque reduction neutraliztion test and immunoelectron microscopy. Cross-reactivity of two IgY antibodies with cells infected by the different strains of the pox viruses was also investigated by different methods （immunofluorescence assay, plaque reduction neutraliztion test and Western blot）. Even in very high dilutions in immunofluorescence assay （titres up to 1：10^6 and 1：10^5, respectively） and persisted on a plateau over 10 months after four booster injections, it was showed that anti-vaccinia virus IgY and anti-calpox virus IgY were positive. Neutralizing activity and ultra-structural detection of antigen with gold-labelled antibodies were respectively observed in plaque reduction neutralization test and immunoelectron microscopy. Western blot analysis revealed specific binding of IgY to virus proteins. Thus, there was cross-reactivity between different orthopox viruses. Finally, orthopox virns-specific IgY antibodies bounded magnetic beads （Dynabead） were used to concentration of orthopox viruses. This study suggests that anti-pox virus IgY could serve as a useful tool for orthopox viruses 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.

Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells

AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2 000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

Antiviral Activity of the Effective Monomers from Folium Isatidis Against Influenza Virus in Vivo

In order to evaluate the anti-influenza virus activity of the effective monomer from Folium Isatidis (FI) in vivo,we established mice model with viral pneumonia and divided them into 3 different dose groups,then observed their lung indexes,pulmonary pathological changes,pulmonary virus hemagglitination titers,living time and death rates.The results showed that the monomer could reduce the pulmonary index from 2.64 to 1.93,1.63 and 1.40 (P<0.01) and decrease the hemagglitination titer from 1.15 to 0.84,0.70 and 0.59 (P<0.01).In addition,different groups of FI could significantly lessen the mortality rate from 100% to 30%,25% and 15%,and prolong the living time from 5.1d to 6.5d,8.4d and 8.9d respectively(P<0.01).The high dose (75 mg/kg/d) has the similar effect with 100 mg/kg/d dose of virazole(P>0.05),and more effective than 200 mg/kg/d dose of antiviral liquor (P<0.05).

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

Antiviral Activity of GuiQi Polysaccharides against Enterovirus 71 in vitro

In this study,we have investigated the antiviral activity of GuiQi polysaccharides (GQP) upon enterovirus 71 (EV71) in vitro.An assay using methyl thiazolyl tetrazolium (MTT),and analyses of cytopathic effects (CPE)were used to examine the antiviral activity of GQP upon Vero cells infected with EV71.The results revealed that GQP at concentrations below 31.2μg/mL exhibited significant antiviral effects upon EV71 when applied under three different experimental protocols.GQP was most strongly active in preventing the adsorption of EV71 to target cells and in this respect it was significantly more effective than ribavirin.In addition,it was clear that GQP could inhibit viral replication when added to cells 2 h after infection,but if added at the point of infection its effect was weak.GQP is considered to be less toxic than ribavirin,and may warrant further evaluation as a possible agent in the treatment of hand,foot and mouth disease (HFMD).

Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection

AIM： To evaluate the prevalence of isolated anti-HBc in patients with chronic hepatitis C virus （HCV） infection, and its relation to disease severity. METHODS： We screened all patients with chronic HCV infection referred to King Faisal Specialist Hospital and Research Center for hepatitis B surface antigen （HBsAg）, antibody to hepatitis B surface antigen （anti- HBs）, and anti-HBc. One hundred and sixty nine patients who tested negative for both HBsAg and anti-HBs were included in this study. RESULTS： Pathologically, 59 had biopsy-proven cirrhosis and 110 had chronic active hepatitis （CAH）. Of these 169 patients, 85 （50.3%） tested positive for anti-HBc. Patients with CAH had significantly higher prevalence of isolated anti-HBc than patients with cirrhosis, 71 （6d.5%） and 14 （23.7%） respectively （P 〈 0.001）. Twenty-five patients were tested for HBV DNA by qualitative PCR. The test was positive in 3 of them （12%; occult HBV infection）. CONCLUSION： Isolated anti-HBc alone is common in Saudi patients with chronic HCV infection, and is significantly more common in those with CAH than those with cirrhosis. Therefore, a screening strategy that only tests for HBsAg and anti-HBs in these patients will miss a large number of individuals with isolated anti-HBc, who may be potentially infectious.

Spontaneous elimination of hepatitis C virus infection: A retrospective study on demographic, clinical, and serological correlates

To find correlates to spontaneous clearance of hepatitis C virus （HCV） infection, this study compared individuals with self-limited and chronic infection with regard to clinical, demographic, and serological pa- rameters. METHODS： Sixty-seven anti-HCV positive and repeatedly HCV RNA negative individuals were considered to have resolved HCV infection spontaneously. To determine the viral genotype these patients had been infected with HCV serotyping was performed. For comparison reasons, 62 consecutive patients with chronic hepatitis C were enrolled. Cases and controls were compared stratified for age and sex. RESULTS： Retrospective analysis showed （1） a lower humoral reactivity to HCV in patients with self-limited compared to chronic HCV-infection and （2） that younger age, history of iv drug use, and acute/post-acute hepatitis A or B co-infections, but not viral genotypes, are independent correlates for spontaneous HCV clearance. CONCLUSION： The stronger humoral reactivity to HCV in patients with persistent infections and in those with a history of iv drug use is supposed to be due to continuous or repeated contact（s） to the antigen. Metachronous hepatitis A or hepatitis B infections might favor HCV clearance.

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay(ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells(HPDC).

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiforrnis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides （PGL）, Gracilaria lemaneiformis polysaccharide fraction-1 （GL-1）, Gracilaria lemaneiformis polysaccharide fraction-2 （GL-2） and Gracilaria lemaneiformis polysaccharide fraction-3 （GL-3） were studied by the Methyl thiazolyl tetrazolium （MTT） method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect （CPE） on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit （PFU）, MTT and CPE methods. The results showed： i） Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii） Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides （PGL and GL-2） both of which exhibited higher antiviral activity; iii） A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions

Human Monoclonal Antibodies as Candidate Therapeutics Against Emerging Viruses and HIV-1

More than 40 monoclonal antibodies (mAbs) have been approved for a number of disease indications with only one of these (Synagis) - for a viral disease, and not for therapy but for prevention. However, in the last decade novel potent mAbs have been discovered and characterized with potential as therapeutics against viruses of major importance for public health and biosecurity including Hendra virus (HeV), Nipah virus (NiV), severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus (EBOV), West Nile virus (WNV), influenza virus (IFV) and human immunodeficiency virus type 1 (HIV-1). Here, we review such mAbs with an emphasis on antibodies of human origin, and highlight recent results as well as technologies and mechanisms related to their potential as therapeutics.