我国1998～1999年流行的婴幼儿腹泻轮状病毒的分型研究

轮状病毒是世界范围内引起婴幼儿腹泻的主要病原。根据病毒外壳蛋白VP4和VP7抗原性的不同可区分为不同型：P（VP4,protease sensitive）型和G（VP7,glycoprotein）型。1998－1999年在中国8个城市（长春、秦皇岛、北京、杭州、福州、广州、成都、昆明）采集了急性腹泻患儿的1093份非细菌性腹泻粪便标本,先进行A组轮状病毒（HRV）的筛选,其中阳性标本433份（39.6％）,电泳型长型占优势（96％）。对HRV标本,再利用血清型特异的MAbELISA和／或RT－PCR进行G分型。结果表明,在1998－1999年,在上述8城市非细菌性腹泻行季节,以HRV G1型为主要流行株,占阳性的83.4％,其次为G3（12.0%）、G4（3.5%)和G2(3.2%）。此外,有3份（0.7％）HRV标本未能分型,12（2.8％）份标本为混合感染,还结合1982－1996年全国12个地区1382份HRV标本的分型资料,分析了我国HVR G血清型的流行规律。实验中又抽样选取了124份GHRV标本,用RT－PCR进行P分型,其中P[8]型76份（61.3％）,P[4]型14份（11.3%）,P[6]型12份(9.7%),P[9]型8份(6.4%)。另外15份（12.1%)未能分出P型,有待进一步检定,实验中HRV分离株除了觉见的P[8]G1(51.4%)、P[4]G2(4.6%)毒株外,还检测到P[8]G3(11.0%)、P[8]G4(6.4%) 和其它较少见的病毒型。以上结果为我国轮状病毒疫苗的应用和开发提供了较系统、清晰的流行病学背景资料。     

Validity of an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies for serotyping human rotavirus in stool specimens

We recently developed a method for serotyping human rotavirus (HRV) by an enzyme-linked immunosorbent assay with HRV serotype-specific neutralizing monoclonal antibodies (ELISA serotyping). In the present study this method was compared with the fluorescent focus neutralization test with serotype-specific rabbit antisera (NT serotyping) in the sensitivity and specificity of the test. Direct serotyping of HRVs which were contained in stool specimens indicated that while only 37% of the samples were successfully serotyped in NT, 78% of the samples could be serotyped in ELISA. Regarding the samples whose serotype could be determined in the two tests, the assigned serotypes were identical in both tests. The results obtained indicated the utility of ELISA serotyping in clinical and epidemiologic studies of HRV infection.     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.     

Quantification of capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples

Streptococcus pneumoniae is a major cause of mortality in underdeveloped countries, where more than one million people die from pneumococcal disease every year. Vaccines are the most efficient method for preventing the infection and are based on the capsular polysaccharide (PS) protection. The serotype 14 is the most frequent in pediatric infections worldwide. This study aimed to establish a quantification protocol for PS present in culture broth samples of S. pneumoniae serotype 14 (PS14) and use this protocol for selection of the best PS14 producer strain. Phenol-sulfuric, HPSEC, competitive ELISA, and sandwich ELISA methods were tested for PS14 quantification. Sandwich ELISA was the method with the best reproducibility and sensitivity and the least susceptible to interferences. The quantification limit and detection limit of this method were 0.99 and 0.57 ng/mL, respectively. Statistical analysis was performed to calculate the coefficient of variation (CV) intraassay (1-3% intraplate and 2-6% interplate) and interassay (11-15%) and the reproducibility in different days (CV<20%). The sandwich ELISA allows us to select, among six strains evaluated, the strain 5287 as the best PS14 producer (11.68 mg PS14/biomass) and it was shown to be the best choice for measurement of pneumococcal polysaccharides in culture broth samples.     

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.     

Efficacy,safety and immunogenicity of hexavalent rotavirus vaccine in Chinese infants

A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy and safety of a hexavalent live human-bovine reassortant rotavirus vaccine (HRV) against rotavirus gastroenteritis (RVGE). A total of 6400 participants aged 6–12 weeks were enrolled and randomly assigned to either HRV (n = 3200) or placebo (n = 3200) group. All the subjects received three oral doses of vaccine four weeks apart. The vaccine efficacy (VE) against RVGE caused by rotavirus serotypes contained in HRV was evaluated from 14 days after three doses of administration up until the end of the second rotavirus season. VE against severe RVGE, VE against RVGE hospitalization caused by serotypes contained in HRV, and VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were also investigated. All adverse events (AEs) were collected for 30 days after each dose. Serious AEs (SAEs) and intussusception cases were collected during the entire study. Our data showed that VE against RVGE caused by serotypes contained in HRV was 69.21% (95%CI: 53.31–79.69). VE against severe RVGE and RVGE hospitalization caused by serotypes contained in HRV were 91.36% (95%CI: 78.45–96.53) and 89.21% (95%CI: 64.51–96.72) respectively. VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were 62.88% (95%CI: 49.11–72.92), 85.51% (95%CI: 72.74–92.30) and 83.68% (95%CI: 61.34–93.11). Incidences of AEs from the first dose to one month post the third dose in HRV and placebo groups were comparable. There was no significant difference in incidences of SAEs in HRV and placebo groups. This study shows that this hexavalent reassortant rotavirus vaccine is an effective, well-tolerated, and safe vaccine for Chinese infants.     

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.     

昆明婴幼儿腹泻的轮状病毒分子基因特征研究

目的了解昆明婴幼儿腹泻中轮状病毒感染情况。方法收集昆明医学院第一附属医院儿科2004年10-12月住院的腹泻的患儿粪便标本60份,采用聚丙烯酰胺凝胶电泳（PAGE）法和RT—PCR法进行轮状病毒分型。结果60份粪便标本中,29份检测到A组轮状病毒RNA基因,阳性检出率为48.3％,未发现B组及C组轮状病毒。其中RNA长型有29份（100％）,未发现短型和混合型。对29份有明确编号的轮状病毒阳性标本来源的患儿分析显示,平均发病年龄10.5个月。29份标本中,有20份可用RT—PCR分型,均为G3型,未发现其他型。结论A组轮状病毒是昆明地区5岁以下婴幼儿腹泻病的主要病原,3月龄-2岁婴幼儿是轮状病毒的易感人群,以冬季10月份至12月份为流行高峰,基因组以长型为主,血清型为G3型。     

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL-receptor family including the very low density lipoprotein (VLDL)-receptor (VLDL-R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL-R to 15 A resolution by cryo-electron microscopy. The receptor fragments, which include the first three ligand-binding repeats of the VLDL-R (V1-3), bind to the small star-shaped dome on the icosahedral 5-fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM-1 is located at the base of a depression around each 5-fold axis. Homology models of the three domains of V1-3 were used to explore the virus-receptor interaction. The footprint of VLDL-R on the viral surface covers the BC- and HI-loops on VP1.     

Human rotavirus K8 strain represents a new VP4 serotype.

The complete VP4 gene of the human rotavirus (HRV) K8 strain (G1 serotype) was cloned and inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. A K8VP4 recombinant baculovirus was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with transfer vector DNA containing the K8VP4 gene and wild-type baculovirus DNA. Infection of Sf9 cells with this VP4 recombinant baculovirus resulted in the production of a protein that is similar in size and antigenic activity to the authentic VP4 of the K8 strain. Guinea pigs immunized with the expressed VP4 developed antibodies that neutralized the infectivity of the K8 strain. This antiserum neutralized HRV strains belonging to VP4 serotypes 1A, 1B, and 2 with efficiency eightfold or lower than that of the homologous virus, indicating that the human rotavirus K8 strain represents a distinct VP4 serotype (P3). In addition, low levels of cross-immunoprecipitation of the K8VP4 and its VP5 and VP8 subunits with hyperimmune antisera to HRV strains representing different VP4 serotype specificities also suggested that the K8 strain possesses a unique VP4 with few epitopes in common with other P-serotype strains.     

2A proteinase of human rhinovirus cleaves cytokeratin 8 in infected HeLa cells

Rhino- and enteroviruses encode two proteinases, 2A and 3C, which are responsible for the processing of the viral polyprotein and for cleavage of several cellular proteins. To identify further targets of the 2A proteinase of human rhinovirus serotype 2 (HRV2), an in vitro cleavage assay followed by two-dimensional electrophoresis was employed. Cytokeratin 8, a member of the intermediate filament group of proteins, was found to be proteolytically cleaved in vitro by the 2A proteinase of HRV2 and of coxsackievirus B4 and in vivo during HRV2 infection of HeLa cells. The cleavage results in removal of 14 amino acids from the N-terminal head domain of cytokeratin 8. However, other intermediate filament proteins (cytokeratins 7 and 18 and vimentin) were not cleaved in the course of the HRV2 infection. Compared with the processing of the eucaryotic translation initiation factors 4GI and 4GII, cleavage of cytokeratin 8 occurs late in the infection cycle at the time of the onset of the cytopathic effect.     

Synthesis and antirhinovirus activity of new 3-benzyl chromene and chroman derivatives

A series of 3-benzyl chromenes and chromans were synthesized and tested in vitro against human rhinovirus (HRV) 1B and 14, two representative serotypes for rhinovirus group B and A, respectively. All the new compounds, with the exception of 3-benzyl-2H-chromene (3a), showed a potent activity against HRV serotype 1B within micro or submicromolar range (IC₅₀s from 0.11 to 6.62 μM). The low cytotoxicity of all the derivatives resulted in compounds with high therapeutic index (TI). On the contrary, HRV 14 infection was only weakly inhibited by the majority of these compounds. The 3-benzylidenechromans 2b and 2c showed the highest anti-HRV 1B activity (IC₅₀ 0.12 and 0.11 μM, respectively) coupled with remarkable TI (625.00 and 340.91, respectively). Mechanism of action studies on (Z)-3-(4-chlorobenzylidene)chroman (2b) suggest that the new compounds behave as capsid binders and interfere with very early stages of HRV 1B replication, similarly to related flavanoids.     

Clinical significance of 4 patients with chronic hepatitis B achieving HBsAg clearance by treated with pegylated interferon alpha-2a for less than 1 year: a short report

This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007–June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients ≤ 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Cross-reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants.

We analyzed cross-reactive neutralization epitopes on protein VP3 of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs), which showed a variety of interserotypic reactivity patterns when examined in a neutralization test and an enzyme-linked immunosorbent assay against 15 HRV and 2 animal RV strains. Serological study with the six cross-reactive N-MAbs revealed antigenic variations in some HRV strains within the same serotype as well as a marked antigenic difference between serotype 2 strains and serotype 1, 3, and 4 strains. Epitope analysis of the antigenic variants resistant to the six individual cross-reactive N-MAbs suggested the existence of at least three distinct cross-reactive neutralization epitopes on VP3 of HRV.     

Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes

A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.     

Group A human rotavirus genomics: evidence that gene constellations are influenced by viral protein interactions

Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication.