Effects of human parvovirus B19 VP1 unique region protein on macrophage responses

Background  

Activity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage.     

人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。     

Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE.     

人博卡病毒基因克隆及衣壳编码基因序列变异分析

为了解人博卡病毒(Human Bocavirus,HBoV)VP1基因进化关系;阐明HBoV目前具体的变化规律,用PCR的方法扩增了1株HBoV的全基因和9株HBoV的VP1基因,克隆并测序,在此基础上,将HBoV的全基因序列和衣壳序列分别与细小病毒亚科其他14个有代表性的病毒进行遗传分析,构建进化树,对目前所有可得到的HBoV的17个衣壳蛋白进行二级结构分析和抗原性分析。结果显示:HBoV全基因序列与B19关系较远,但衣壳序列遗传关系较近。以有典型性的猫瘟细小病毒(Feline parvovirus,FPV)衣壳蛋白为参照,分析多个HBoV衣壳序列之间的变异情况,显示HBoV衣壳的二级结构基础表现出较高的保守性,序列之间的变化主要发生在高抗原区域和感染活性区域。衣壳病毒变异情况显示HBoV在稳定自身的情况下表现出一定的活跃性以逃避免疫反应,也表现出一定的感染适应力。     

B19病毒XA株VP1独特区真核表达系统的建立及鉴定

目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体.方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体plRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体plRES2-EGFP-VP1u.将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达.结果:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达.结论:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础.     

Exacerbating Effects of Human Parvovirus B19 NS1 on Liver Fibrosis in NZB/W F1 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling.     

TNF-α Mediated Increase of HIF-1α Inhibits VASP Expression,Which Reduces Alveolar-Capillary Barrier Function during Acute Lung Injury (ALI)

Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar-capillary barrier function and increased pulmonary vascular permeability. Vasodilator-stimulated phosphoprotein (VASP) is widely associated with all types of modulations of cytoskeleton rearrangement-dependent cellular morphology and function, such as adhesion, shrinkage, and permeability. The present studies were conducted to investigate the effects and mechanisms by which tumor necrosis factor-alpha (TNF-α) increases the tight junction permeability in lung tissue associated with acute lung inflammation. After incubating A549 cells for 24 hours with different concentrations (0–100 ng/mL) of TNF-α, 0.1 to 8 ng/mL TNF-α exhibited no significant effect on cell viability compared with the 0 ng/mL TNF-α group (control group). However, 10 ng/mL and 100 ng/mL TNF-α dramatically inhibited the viability of A549 cells compared with the control group (*p<0.05). Monolayer cell permeability assay results indicated that A549 cells incubated with 10 ng/mL TNF-α for 24 hours displayed significantly increased cell permeability (*p<0.05). Moreover, the inhibition of VASP expression increased the cell permeability (*p<0.05). Pretreating A549 cells with cobalt chloride (to mimic a hypoxia environment) increased protein expression level of hypoxia inducible factor-1α (HIF-1α) (*p<0.05), whereas protein expression level of VASP decreased significantly (*p<0.05). In LPS-induced ALI mice, the concentrations of TNF-α in lung tissues and serum significantly increased at one hour, and the value reached a peak at four hours. Moreover, the Evans Blue absorption value of the mouse lung tissues reached a peak at four hours. The HIF-1α protein expression level in mouse lung tissues increased significantly at four hours and eight hours (**p<0.001), whereas the VASP protein expression level decreased significantly (**p<0.01). Taken together, our data demonstrate that HIF-1α acts downstream of TNF-α to inhibit VASP expression and to modulate the acute pulmonary inflammation process, and these molecules play an important role in the impairment of the alveolar-capillary barrier.     

Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity

The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.     

Th17-Related Cytokines in Systemic Lupus Erythematosus Patients with Dilated Cardiomyopathies: A Possible Linkage to Parvovirus B19 Infection

Dilated cardiomyopathies (DCM) are a major cause of mortality in patients with systemic lupus erythematosus (SLE). Immune responses induced by human parvovirus B19 (B19) are considered an important pathogenic mechanism in myocarditis or DCM. However, little is known about Th17-related cytokines in SLE patients with DCM about the linkage with B19 infection. IgM and IgG against B19 viral protein, and serum levels of Th17-related cytokines were determined using ELISA in eight SLE patients with DCM and six patients with valvular heart disease (VHD). Humoral responses of anti-B19-VP1u and anti-B19-NS1 antibody were assessed using Western blot and B19 DNA was detected by nested Polymerase Chain Reaction (PCR). Levels of interleukin (IL)-17, IL-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly higher in SLE patients with DCM (mean ± SEM, 390.99±125.48 pg/ml, 370.24±114.09 pg/ml, 36.01±16.90 pg/ml, and 183.84±82.94 pg/ml, respectively) compared to healthy controls (51.32±3.04 pg/ml, p<0.001; 36.88±6.64 pg/ml, p<0.001; 5.39±0.62 pg/ml, p<0.005; and 82.13±2.42 pg/ml, p<0.005, respectively). Levels of IL-17 and IL-6 were higher in SLE patients with DCM versus those with VHD (both p<0.01). Five (62.5%) of DCM patients had detectable anti-B19-NS1 IgG and four (50.0%) of them had anti-B19-VP1u IgG, whereas only one (16.7%) of VHD patients had detectable anti-B19-NS1 IgG and anti-B19-VP1u IgG. Serum levels of IL-17, IL-6 and IL-1β were markedly higher in SLE patients with anti-B19-VP1u IgG and anti-B19-NS1 IgG compared to those without anti-B19-VP1u IgG or anti-B19-NS1 IgG, respectively. These suggest a potential association of B19 with DCM and Th17-related cytokines implicated in the pathogenesis of DCM in SLE patients.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

细小病毒B19壳抗原VP2在大肠杆菌中的表达及血清学检测

为了进行B19感染临床的血清学诊断,利用原核表达载体PQE31克隆和表达B19壳蛋白VP2,酶切鉴定PCR产物及PQE31-VP2克隆的正确性,Western-blot证明表达蛋白的特异性,并对其表达条件和纯化条件进行了优选。在D600为0.7,诱导时间为5h时表达量最高。Ni2+亲和色谱,用0.5mol/L咪唑洗脱液洗脱,获得纯化蛋白。利用纯化蛋白检测100份人群血清,免疫斑点法结果为阳性94例,阴性6例;ELISA结果为阳性84例,阴性16例,两种方法结果一致(0.25>P>0.1)。     

Conserved B-Cell Epitopes among Human Bocavirus Species Indicate Potential Diagnostic Targets

Background

Human bocavirus species 1–4 (HBoV1–4) have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs.

Methodology/Principal Findings

We generated HBoV1–3 VP2 gene fragment phage display libraries (GFPDLs) and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides–P1 (¹MSDTDIQDQQPDTVDAPQNT²⁰), and P2 (¹⁶²EHAYPNASHPWDEDVMPDL¹⁸⁰)–that are conserved among HBoV1–4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1–4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4). Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide.

Conclusions/Significance

The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools.     

Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity

The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A₂ (PLA₂) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA₂ motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA₂ motif as well as a proportional increase of the PLA₂ activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA₂ motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA₂ activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA₂ constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.     

Boswellia serrata Preserves Intestinal Epithelial Barrier from Oxidative and Inflammatory Damage

Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD), however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM) use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H₂O₂ or INF-γ+TNF-α, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER) and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin) immunofluorescence. The expression of phosphorylated NF-κB and reactive oxygen species (ROS) generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE) and its pure derivative acetyl-11-keto-β-boswellic acid (AKBA), were tested at 0.1-10 μg/ml and 0.027μg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H₂O₂ or INF-γ+TNF-α treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-κB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H₂O₂ exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study elucidates the pharmacological mechanisms mediated by BSE, in protecting intestinal epithelial barrier from inflammatory damage and supports its use as safe adjuvant in patients affected by IBD.     

Identification and nearly full-length genome characterization of novel porcine bocaviruses

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2) (sPLA(2)) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+)-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.     

Human Bocavirus Capsid Structure: Insights into the Structural Repertoire of the Parvoviridae

Human bocavirus (HBoV) was recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) on the basis of genomic similarity to bovine parvovirus and canine minute virus. HBoV has been implicated in respiratory tract infections and gastroenteric disease in children worldwide, yet despite numerous epidemiological reports, there has been limited biochemical and molecular characterization of the virus. Reported here is the three-dimensional structure of recombinant HBoV capsids, assembled from viral protein 2 (VP2), at 7.9-Å resolution as determined by cryo-electron microscopy and image reconstruction. A pseudo-atomic model of HBoV VP2 was derived from sequence alignment analysis and knowledge of the crystal structure of human parvovirus B19 (genus Erythrovirus). Comparison of the HBoV capsid structure to that of parvoviruses from five separate genera demonstrates strong conservation of a β-barrel core domain and an α-helix, from which emanate several loops of various lengths and conformations, yielding a unique surface topology that differs from the three already described for this family. The highly conserved core is consistent with observations for other single-stranded DNA viruses, and variable surface loops have been shown to confer the host-specific tropism and the diverse antigenic properties of this family.Human bocavirus (HBoV), a newly discovered member of the family Parvoviridae, was originally isolated in randomly selected nasopharyngeal aspirates (5). Since this initial discovery, HBoV has also been detected worldwide, predominantly in children under the age of 2 years with respiratory infections, in serum, urine, and fecal samples (40). Symptomatic children commonly exhibit acute diseases of the upper and lower respiratory tracts (7, 36, 44, 56) and, possibly, gastroenteritis (31, 56) though the link to gastroenteritis outbreaks has been questioned (12). It is still unclear if HBoV is the sole etiologic agent of respiratory disease as higher rates of coinfections with other respiratory pathogens such as human rhinovirus and Streptococcus spp. are often observed (4). However, Allander et al. recently reported (4) that HBoV was found in 19% of children with acute wheezing, thereby making it the fourth most common virus, after rhinoviruses, enteroviruses, and respiratory syncytial virus, detected in children exhibiting this symptom. These findings suggest that, at high viral load, HBoV could be an etiologic agent of respiratory tract disease (4). HBoV infection is common in the first few years of life, and clinical research suggests it may follow the primary period for acquisition of human parvovirus B19 (B19) though there is no antigenic cross-reactivity between B19 and HBoV (28, 30). By age 5, most people have circulating antibodies against HBoV, as is also true for other respiratory viruses such as respiratory syncytial virus, rhinoviruses, and human metapneumovirus (17). HBoV has also been identified in adults, with ∼63% of samples tested being seropositive, showing a positive correlation with age and a slight positive bias toward women (14).The Parvoviridae is a family of small, nonenveloped viruses that package a single-stranded DNA (ssDNA) genome of ∼5,000 bases. These viruses are subdivided into two subfamilies: Parvovirinae and Densovirinae (Table ​(Table1).1). The Parvovirinae are further subdivided into five genera, all of whose members infect vertebrates. The Densovirinae (four genera) infect only invertebrates. Phylogenetic analysis places HBoV in the recently classified Bocavirus genus (Table ​(Table1).1). In addition to HBoV, numerous parvoviruses circulate among the human population. Among these are the following: several dependoviruses; adeno-associated virus (AAV) serotypes AAV1 to AAV3, AAV5, and AAV9; the Erythrovirus B19; and the newly discovered human parvovirus genotypes 4 (Parv4) and 5 (Parv5) (23, 27, 50). Of these, only B19 had been implicated in disease until the discovery of HBoV and Parv4, which has been isolated from patients who present symptoms of acute HIV infection (50).

TABLE 1.

Selected properties of representative members of the Parvoviridae

抗细小病毒B19-VP2单克隆抗体的建立

制备抗细小病毒B19－VP2单克隆抗体,用于检测人血清中的B19抗原,辅助诊断相关疾病;也可用于制备人类细小病毒基因工程疫苗。用纯化的基因工程表达的B19－VP2蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0细胞融合,有限稀释法克隆细胞。ELISA及IF证明抗体特异性。克隆筛选出4株细胞,并初步建立了检测B19－VP2抗原的双抗体夹心酶联免疫吸附试验,为双抗体夹心法检测B19抗原为临床相关疾病诊断提供了检测手段。