Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and Chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.     

原位杂交检测微小病毒B19方法的建立及临床应用

目的 建立原位杂交（ＩＳＨ）方法检测人微小病毒Ｂ１９,并确定其在先天性心脏病９ＣＨＤ）心脏组织细胞的定位分布。方法 以Ｂ１９特异的衣壳蛋白ＶＰＩ ＤＮＡ内１１１２ｂｐ片段为模板,采用随机引物标记探针法,建立了ＩＳＨ检测Ｂ１９ ＤＮＡ浓度于０．１ｂｇ／μ１以上呈阳性。在６６例先天性心脏病心脏组织中,检测至Ｂ１９ ＤＮＡ阳性７例,并发现Ｂ１９ ＤＮＡ阳性信号主要定位于心肌细胞核内,３８例对照组健康心肌     

先天性心脏病心肌活检组织中微小病毒感染的定位

目的 了解人微小病毒Ｂ１９（Ｐａｖｏｖｉｎｕｓ Ｂ１９）感染在先天性心脏病（Ｃｏｎｇｅｎｔｉａｌ ｈｅａｒｔ ｄｉｓｅａｓｅ，ＣＨＤ）心脏组织中的定位。方法 采用巢式聚合酶链反应（ＰＣＲ）和组织原位杂交（ＩＳＨ）技术，对３７例ＣＨＤ患者外周血及手术活检的心肌组织和２８例非先天畸形心肌组织进行Ｂ１９ ＤＮＡ的检测，结果 ３７例ＣＨＤ组中Ｂ１９ ＤＮＡ在血清及心肌组织中阳性率分别为１６．２２％（６／３     

Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V‐IgM and ‐IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V‐specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non‐infected, and 29 from other viral recent infections (Epstein‐Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well‐established Biotrin EIAs.     

结直肠癌组织中人细小病毒B19的检测

目的探讨人细小病毒B19感染与结直肠癌发生的关系。方法运用原位杂交对50例石蜡包埋结直肠癌患者的肿瘤组织,癌周结直肠组织以及10例正常成人结肠组织中B19病毒进行检测。激光捕获显微切割肿瘤细胞及癌周正常肠上皮细胞,巢式PCR扩增B19DNA。结果50例结直肠癌标本中,原位杂交示B19阳性信号在肿瘤组织为78％（39／50）,癌周结直肠组织为40％（20／50）,正常结肠组织中5例（n=10）,经统计分析肿瘤与癌周组织B19感染差异有显著性（P〈0．01）,正常结肠组织与癌周组织间未见统计学差异（P=1．000）。显微切割进一步证实B19病毒DNA存在于肿瘤细胞内。结论结直肠组织中人细小病毒B19感染较常见,主要存在于结直肠癌上皮细胞内,该病毒可能在结直肠癌的发生过程中起一定作用。     

In situ hybridisation and in situ polymerase chain reaction detection of parvovirus B19 DNA within cells

Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.     

病毒性心肌炎患儿微小病毒B19感染的近期研究

目的进一步探讨病毒性心肌炎患儿微小病毒B19(HPVB19)感染的状况及其相关性。方法应用巢式聚合酶链反应的方法以及ELISA法,对60例病毒性心肌炎患儿(观察组)及30例随机挑选本院门诊健康体检儿童(对照组)血浆中微小病毒B19-DNA检测,同时进行B19-VP2-IgM检测。对观察组中HPCB19-DNA检测阳性的与阴性的两组中血CK、CK-MB及心功能指标进行比较。统计方法采用χ2和t检验。结果60例观察组,16例B19-DNA检测阳性,30例健康儿童B19-DNA检测均为阴性,阳性检出率为26.7%(16/60),两组比较有显著差别(P<0.01);60例观察组,B19-VP2-IgM阳性25%(15/60),对照组30例均为阴性极显著(P<0.01)。60例观察组中,B19 DNA及B19VP2 M均阳性15例;1例仅B19 DNA阳性;B19 DNA和B19 VP2 IgM同时阴性43例,B19 DNA和19-VP2-IgM一致率为93%,有一致性(P<0.01)。观察组中HPCB19-DNA检测阳性的与阴性的两组中血CK、CK-MB值变化无显著性差异,P>0.05。但心功能指标LVSF比较有明显差异(P<0.01),SV比较亦有明显差异(P<0.005)。结论小儿病毒性心肌炎与HPVB19感染有关,HPVB19是小儿病毒性心肌炎主要病原之一,而且本研究发现HPVB19感染所致小儿病毒性心肌炎的心功能改变中左室功能受累程度较重。     

Seroepidemiology of human parvovirus B19 in Taiwan

In order to determine the prevalence and risk factors of human parvovirus B19 (B19) infection in Taiwan, a seroepidemiological study was carried out in 19 townships. Serum samples were collected from 862 healthy residents, who were selected by stratified random sampling from various study areas. They were chosen from four different ethnic groups including aborigines, Fukien Taiwanese, Hakka Taiwanese, and mainland Chinese. Serum samples were screened for B19 IgG antibody by indirect antibody capture enzyme-linked immunosorbent assay (ELISA) and B19 IgM by IgM antibody capture (MAC)-ELISA, respectively. The overall prevalence of anti-B19 IgG and anti-B19 IgM was 32.8% and 0.35%, respectively. The anti-B19 seropositive rate in females was significantly higher than that of males (36.4% vs. 29.4%, P < .001). The age-sex-adjusted seropositive rate in urban townships (39.9%) was higher than that in aboriginal townships (30.5%, P < .001). The seropositive rate increased significantly with age showing a dose–response relationship (P = 0.0001 based on a trend test). Blood transfusion was found to be associated with an increased seropositive rate showing a multivariate-adjusted odds ratios of 1.6. J. Med. Virol. 57:169–173, 1999. © 1999 Wiley-Liss, Inc.     

Tissue persistence of parvovirus B19 genotypes in asymptomatic persons

Parvovirus B19 (B19V) can persist in immunocompetent symptomatic and non-symptomatic individuals, as demonstrated by the finding of viral DNA in different tissues, in absence of viremia and of anti-B19V IgM. The spread and the nature of this phenomenon have not been clearly determined. In order to investigate the frequency of persistence and the tissue distribution of the three genotypes of B19V, the viral load of the persistent virus and its expression in the affected tissues, 139 tissue samples and 102 sera from 139 asymptomatic individuals have been analyzed by consensus PCRs and genotype specific PCRs for B19V detection and genotyping. Viral load was measured by real time PCR and viral mRNAs were detected by RT-PCR. Altogether, 51% individuals carried B19V DNA, more frequently in solid tissues (65%) than in bone marrow (20%). Genotype 1 was found in 28% tissue samples, genotype 2 in 68% and genotype 3 in 3% only. Viral load ranged from less then 10 copies to 7 x 10(4) copies per 10(6) cells, with the exception of two samples of myocardium with about 10(6) copies per 10(6) cells. mRNA of capsid proteins was present in two bone marrow samples only. In conclusion, in asymptomatic individuals B19V persistence is more common in solid tissues than in bone marrow, and genotype 2 persists more frequently than genotype 1. The results suggest that the virus persists without replicating, at sub-immunogenic levels.     

Detection of IgE anti-parvovirus B19 and increased CD23+ B cells in parvovirus B19 infection: relation to Th2 cytokines

The immune profile of a parvovirus B19-infected patient (male, 8 years old) was studied on day 0 (initial presentation) and on days 14 and 210 post symptom presentation (psp). Before infection, the patient was skin test positive to various allergens, including ragweed and tree and grass pollens, and had a serum IgE level of 150 IU/mL. On day 0, the patient was diagnosed as parvovirus B19 infected, as judged by the presence of IgG anti-parvovirus Abs in serum (EIA) and presentation of "slap cheek" rash. The patient's serum IgE level increased from 150 IU/mL before infection to 256 IU/mL on day 0, was 233 IU/mL on day 14, and returned to preinfection levels on day 210. In contrast, there was little change in the levels of serum IgM, IgG, or IgA (nephelometry). IgE anti-parvovirus B19 protein (VP-N) was detected in serum (Western blot) on days 0, 14, and 210, despite the decrease in total IgE on day 210. Although there was no increase in total numbers of blood CD23+ B cells on day 0, by day 14 the numbers of these cells increased dramatically (93%), remaining high on day 210. In contrast, there were virtually no changes in total numbers of CD4+ and CD8+ T cells or CD16/56+ NK precursor cells on days 0-210. On day 0, when IgG and IgE anti-parvovirus were detected in serum, patient's peripheral blood mononuclear cells (PBMC) expressed mRNA for the Th2 cytokines IL-4 and IL-10, but not for the Th1 cytokines IFN-gamma or IL-2. However, by day 14 psp, PBMC expressed mRNA for the Th1 cytokines IFN-gamma and IL-2, as well as for IL-4 and IL-10. This is the first demonstration of the existence of IgE anti-parvovirus B19 Ab. The presence of IgE anti-parvovirus B19 Ab in serum on day 0 and its persistence in serum 7 months psp suggests that IgE anti-parvovirus may be useful in prognosis of parvovirus B19 infection. Our results reinforce the idea that IgE, in general, may play a major role in anti-viral immunity, perhaps in conjunction with CD23+ cells. The results further suggest that clearance of this infection is accompanied by a switch to Th1 cytokines.     

Possible transmission of parvovirus B19 from intravenous immune globulin

To look for genetic changes in human parvovirus B19 that might be associated with chronic infection, we sequenced B19 DNA obtained from serum specimens collected over an approximately 1-year period from a patient with systemic vasculitis. A comparison of the nucleotide sequences of the VP1/VP2 gene from four specimens revealed an abrupt change in the B19 genotype that coincided with initiation of intravenous immune globulin (IVIG) therapy. We suspect that one or more of the lots of IVIG administered to the patient were contaminated with B19. If true, this finding suggests that investigators must be careful in linking B19 infection to disease based on detection of B19 DNA in persons who have received multiple unit blood products. J. Med. Virol. 53:233–236, 1997. Published 1997 Wiley-Liss, Inc.

1 This article was prepared by a group consisting of both United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Persistence of parvovirus B19 DNA in synovium of patients with haemophilic arthritis

A progressive arthropathy develops commonly in haemophiliacs and its pathogenesis is not fully understood. Human parvovirus B19 has been associated with several diseases including acute and chronic arthropathy and some studies suggest its implication in chronic inflammatory diseases of the joints such as rheumatoid arthritis. In haemophiliacs parvovirus B19 infection occurs very frequently because of its transmission with plasma derivatives. In order to assess a role of B19 virus in haemophilic arthritis, synovial tissue samples from patients with haemophilia with arthritis and from patients, nonhaemophiliacs, with arthrosis or with joint trauma were examined for B19 DNA by nested PCR. In addition, the prevalence of antibody to parvovirus B19 NS1 protein as a possible serological marker of persistent B19 infection was tested and the association of the outcome of parvovirus infection with genetic diversity of B19 P6 promoter sequences was investigated. B19 DNA was detected in the synovial tissue of 31% of haemophiliacs with progressive arthropathy and of 5% of control patients. Fourteen out of 17 patients (82%) with haemophilic arthritis and with B19 DNA in their synovial membranes had IgG antibodies against the nonstructural protein NS1 of parvovirus B19. On the other hand, 19% of patients with haemophilia with B19 PCR negative synovial tissue and 21% of controls showed anti-NS1 antibodies. The P6 promoter presented specific sites of point mutations shared frequently by isolates from patients with haemophilia and arthritis. These results indicate that B19 DNA can persist in the synovial membranes of patients with haemophilic arthritis significantly more frequently in comparison to control individuals with arthrosis or joint trauma and show a correlation between anti- NS1 antibody presence and B19 DNA persistence in the synovial tissue.     

Survival of parvovirus B19-infected cells by cellular autophagy

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.     

人细小病毒B19在结直肠癌中表达增强

目的探讨人类细小病毒B19感染与结直肠癌的关系。方法运用巢式PCR检测50例结直肠癌患者的肿瘤组织和对应的癌周结直肠组织以及10例正常成人结肠组织中的B19DNA,免疫组化检测B19结构蛋白VP1/VP2。结果肿瘤组织B19DNA阳性率为96%,显著高于癌周(60%)及正常对照(60%)。免疫组化显示,腺癌组织82%VP1/VP2蛋白表达,显著高于癌周组织(30%)及正常对照(20%)。结论人细小病毒B19可能在结直肠癌的发生中起一定作用,为探讨结直肠癌的发病机制提供新思路。     

Detection of parvovirus B19 in the lower respiratory tract

BackgroundHuman parvovirus B19 infection generally displays a self-limiting course followed by viral clearance; although, in some cases, persistent infection may occur. Few cases of severe pulmonary disease following primary infection in both immunocompetent and immunocompromised patients were reported.ObjectivesTo investigate the prevalence and clinical impact of parvovirus B19 in the lower respiratory tract.Study designThe prevalence of parvovirus B19-DNA was evaluated by Real-Time PCR in 264 bronchoalveolar lavages (BAL) from 189 adult patients over a full-year period and related to demographic characteristics, underlying pathologies, immune status, admission to intensive care unit, mortality within 28 days, and discharge diagnosis.ResultsParvovirus B19-DNA was detected in 7/189 (3.7%) patients, without significant association to demographic characteristics, immune status, transplant versus non-transplant status, admission to intensive care unit, presence of haematological conditions. In two lung transplant recipients surveillance specimens were positive to B19. Four of the remaining five patients presented respiratory insufficiency. A significant association to mortality was found, as 3/7 (42.9%) positive patients died within 28 days. No patient presented serological evidence of recent or acute infection and viremia.ConclusionsParvovirus B19 may be detected at low frequency in BAL specimens from patients with different pathological backgrounds. This finding could be due to chronic infection with virus persistence in the lower respiratory tract, also in the absence of symptoms unequivocally attributable to B19. The high rate of mortality warrants the need for further studies to evaluate the opportunity to consider parvovirus B19 in the diagnostic work-up of lower respiratory tract infections.     

A clinical and epidemiological study of human parvovirus B19 infection in fetal hydrops using PCR southern blot hybridization and chemiluminescence detection

Ninety-eight samples from 80 cases of spontaneous abortions after fetal death or hydrops fetalis from 12,000 pregnant women were examined using PCR. DNA was extracted from amniotic fluid, fetal blood, ascitic fluid and fetal biopsies or placenta specimens using QIA amp kits (QIAGEN). A 270-bp length fragment located within the B19 gene NS1 was amplified using PCR followed by electrophoresis and southern-blot hybridization assay using a horseradish peroxidase-labelled probe and chemiluminescence detection. This assay was able to detect 1 to 10 DNA copies in a 10 |gml sample. Parvovirus B19 was identified in 11 cases (14% of fetal hydrops; 1 case for 1,100 pregnancies). Amniotic fluid was the most common and reliable sample to assess the diagnosis. Gestational age ranged from 17 to 28 weeks (mean 23 weeks). IgM antibodies were detected in 3 maternal sera, 2 patients of which reported an exposure to B19 infection during pregnancy. In 2 cases, intrauterine blood transfusions led to the cessation of symptoms and to birth of normal babies. J. Med. Virol. 54:140–144, 1998. © Wiley-Liss, Inc.     

Genetic drift of parvovirus B19 is found in AIDS patients with persistent B19 infection

It is generally thought that parvovirus B19 is stable genetically. Consistently, genetic drift has not been found in patients with persistent B19 infection. In this report, longitudinal genetic changes in NS1 and VP1 gene of B19 isolates from three AIDS patients with persistent B19 infection were studied. One of the three patients was not treated with highly active anti-retroviral therapy (HAART). B19 viral DNA from these patients was amplified by polymerase chain reaction (PCR) and then sequenced directly. A single genetic change was found in the B19 isolate obtained from the patient not treated with HAART on Day 10 after intravenous immunoglobulin (IVIG) treatment. The nucleotide sequences of B19 isolated from this patient, then remained unchanged over a period of 11 months. Analysis of NS1 clones derived from his longitudinal viral isolates showed the existence of quasi-species but genetic drift was not found. One of the other two patients treated with HAART experienced treatment failure; he was later treated with mega-HAART. In contrast to the genetic stability of B19 isolates from the patient not treated with HAART, multiple genetic changes were discovered in the viral isolates from the two other patients after HAART and mega-HAART, respectively. Through analysis of B19 clones, the frequency of clones containing these mutations confirmed the genetic drift. Nucleotide substitutions seen in VP2 gene of isolates with genetic drift from both patients were all non-conserved, suggesting that they are positively selected.     

Large-scale screening for human parvovirus B19 DNA in clinical specimens by dot blot hybridization and polymerase chain reaction

Large-scale screening for human parvovirus B19 (619) DNA in serum samples was carried out by both dot blot hybridization and the polymerase chain reaction (PCR). Dot blot hybridization was undertaken with a digoxigenin-labeled DNA probe. Serum samples from four patients were pooled and tested by a dot blot hybridization assay. When a dot was positive, each of the four samples was tested separately to identify the positive sample. The PCR template was the DNA extracted from mixed serum samples from 10 patients. When B19 DNA was positive by PCR, each of the ten samples was tested separately. A total of 7, 969 serum samples were tested by dot blot hybridization and 15 samples (11 patients) were positive for B19 DNA; 7, 038 serum samples were tested by PCR and 71 samples (50 patients) were positive. Large-scale screening for B19 DNA by PCR suggested a broader spectrum of clinical manifestations associated with B19 infection. © Wiley-Liss, Inc.