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Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma.

Human parvovirus B19 is a frequent contaminant of blood and plasma-derived medicinal products and transmission of this virus has been shown to occur through the administration of contaminated products. Inactivation of the virus has proved difficult and as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus B19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (NAT). We present data comparing the performance of two commercially available kits for the detection and quantitation of parvovirus B19 DNA using the LightCycler, and determine their applicability for the detection of recently discovered variants of the virus. Parvovirus B19 DNA was readily quantified using both commercial assays. However, one kit failed to detect any of the variant viruses. The second kit detected the variant viruses although there was a marked difference in the sensitivity of detection of the different virus genotypes. To improve the detection of these variant viruses a novel assay has been developed and data are presented to show its use for screening pooled plasma for the presence of parvovirus B19 DNA and the variants identified recently.

Base Sequence↗

Real-time PCR protocol for the detection of porcine parvovirus in field samples.

This report describes a real-time polymerase chain reaction assay with SYBR Green for detection of a broad range of porcine parvoviruses (PPV) and accurate virus quantification in porcine tissues. The assay targets the VP2 gene of PPV and the porcine genomic c-myc gene for normalization. The detection limit of the SYBR Green reaction was shown to be equivalent to 6 x 10(0) to 6 x 10(1) PPV copies/reaction and the overall detection limit equivalent to 0.1 TCID(50)/100 microl. The assay was linear over a 10(7) dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders such as porcine circovirus 2 (PCV-2), porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (PRV) and other parvoviruses such as feline parvovirus (FPV), canine parvovirus (CPV), minute virus of canines (MVC) and a human parvovirus (B19) were not detected by this assay.

Animals↗

Parvovirus B19 in pregnancy.

Parvovirus B19 is a widespread infection that may affects 1-5% of pregnant women, mainly with normal pregnancy outcome. The prevalence of infection is higher during epidemics - between 3 and 20% with sero-conversion rate of 3-34%. Infection during pregnancy can cause a variety of other signs of fetal damage. The risk of adverse fetal outcome is increased if maternal infection occurs during the first two trimesters of pregnancy but may also happen during the third trimester. It is a significant cause of fetal loss throughout pregnancy, but has a higher impact in the second half of pregnancy when spontaneous fetal loss from other causes is relatively rare. Parvovirus infection can cause severe fetal anemia as a result of fetal erythroid progenitor cells infection with shortened half life of erythrocytes, causing high output cardiac failure and therefore nonimmune hydrops fetalis (NIHF). The P antigen expressed on fetal cardiac myocytes enables the Parvovirus B19 to infect myocardial cells and produce myocarditis that aggravates the cardiac failure. Although there are several reports of major congenital anomalies among offspring of mothers infected by Parvovirus, the virus does not seem to be a significant teratogen. Since Parvovirus B19 infection can cause severe morbidity and mortality, it should be part of the routine work up of complicated pregnancies. Risk assessment for maternal infection during pregnancy is especially important during epidemics when sero-conversion rates are high.

Female↗

Do canine parvoviruses affect canine neurons? An immunohistochemical study.

In cats (most of which died from panleukopenia), cerebral neurons have recently been shown to be susceptible to canine parvovirus infection. In addition to positive immunostaining and distinct in situ hybridization signals, signs of neurodegeneration were identified by histopathology, mainly in the diencephalic area. Similar histological lesions of the diencephalic regions in dogs have also attracted attention; therefore, an immunohistochemical study was initiated to determine the possible infection of canine neurons with canine parvoviruses. The study was carried out on formalin-fixed and paraffin-embedded brain tissue, with and without signs of neurodegeneration, from 40 dogs, most of them dying from parvovirus enteritis. Immunohistochemistry, using polyclonal antiserum against canine parvoviruses, was negative in all 40 cases, suggesting that, unlike cats, canine parvoviruses do not seem capable of infecting canine neurons.

Animals↗

A study by means of a new assay of the relationship between an outbreak of erythema infectiosum and non-immune hydrops fetalis caused by human parvovirus B19.

An enzyme-linked immunosorbent assay (ELISA), used to detect IgG and IgM antibody specific for human parvovirus B19, was established by use of human parvovirus B19 capsid protein VP-1 expressed in Escherichia coli. Paired samples of serum derived from 44 mothers and single samples derived from 24 babies having unexplained non-immune hydrops fetalis (NIHF), were tested by means of the assay. Five cases (11%) of NIHF were suspected of having been induced by intrauterine human parvovirus B19 infection because the samples of maternal serum were positive for parvovirus B19 IgM antibody. Four of the five cases arose during an outbreak of erythema infectiosum. According to our study and previous reports over 90% of NIHF caused by parvovirus B19 intrauterine infection have become clinically overt in the second trimester of pregnancy during a period ranging from 15 to 27 weeks of gestation.

Blotting, Western↗

Detection of parvovirus B19 IgM by antibody capture enzyme immunoassay: receiver operating characteristic analysis.

Parvovirus B19 infection can cause severe effects in high-risk groups including pregnant women and immunocompromised individuals. Although serological detection of B19 infection is commonplace, minimal information is available on the absolute performance characteristics of various tests for the detection of B19 IgM. The performance of the first parvovirus B19 IgM enzyme immunoassay to be cleared by the US Food and Drug Administration (FDA) is described. The immunoassay cut-off has been established using receiver operating characteristic (ROC) analysis giving a sensitivity and specificity of detection of 89.1 and 99.4%, respectively. No cross-reactivity is observed with rubella or other viral disease IgM which cause similar symptomologies to parvovirus B19. Multi-site reproducibility studies have shown high immunoassay reproducibility with detection rates (observed/expected result) of 100% for nonreactive specimens (N=324) and strongly reactive (N=403), respectively. Immunoassay reproducibility ranged from 11.76 to 17. 46% coefficient of variation for all reactive specimens tested (N=12) whereby each specimen was assayed a total of 81 times. Parvovirus B19 IgM seroprevalence of 1% was observed in a US blood donor population (N=399). In the absence of international performance criteria, this study will be of major benefit to the clinical virologist in assessing immunoassay reliability for the detection of recent infection with parvovirus B19.

Antibodies, Viral↗

Comparison of penaeid shrimp and insect parvoviruses suggests that viral transfers may occur between two distantly related arthropod groups.

The DNA and putative amino acid sequences of representative insect and shrimp parvoviruses (subfamily Densovirinae) were analyzed using computer programs. Shrimp viruses included hepatopancreatic parvovirus (HPV) of Penaeus monodon (HPVmon) and P. chinensis (HPVchin), spawner-isolated mortality virus from P. monodon (SMVmon) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from P. vannamei. Insect viruses included Aedes aegypti densovirus (AaeDNV), Aedes albopictus densovirus (AalDNV), Junonia coenia densovirus (JcDNV), Galleria mellonella densovirus (GmDNV), Bombyx mori densovirus 5 (BmDNV), Diatraea saccharalis densovirus (DsDNV) and Periplaneta fuliginosa densovirus (PfDNV). Virion size for all these viruses ranged between 18 and 30 nm diameter and ssDNA genome length was between 4 and 6 kb. Using BLAST or Clustal W with the sequence fragments available, no significant DNA homology was found except for 77% DNA identity between HPVmon and HPVchin. However, phylogenetic trees constructed by comparing DNA genome sequences for putative viral polypeptides, capsid proteins and nonstructural proteins placed the parvoviruses into two Clades: Clade 1 with SMVmon, PfDNV, DsDNV, GmDNV, JcDNV, and BmDNV; and Clade 2 with HPVmon, HPVchin, IHHNV, AalDNV and AaeDNV. The four shrimp parvoviruses fell into two different clades that grouped with different insect parvoviruses.

Aedes↗

Expression of parvovirus LuIII NS1 from a Sindbis replicon for production of LuIII-luciferase transducing virus.

In order to develop an alternative packaging system for recombinant parvoviruses, the gene for the major nonstructural protein (NS1) of parvovirus LuIII was inserted into a Sindbis replicon vector. Cells infected with recombinant SinNS1 virus produced NS1 RNA from the Sindbis 26S promoter and expressed NS1 protein which was able to transactivate a parvovirus P38 promoter. Co-transfections of Sindbis-NS1 RNA together with a packageable LuIII transducing genome and a coat protein expression plasmid generated detectable levels of LuIII-luciferase transducing virus. These levels could be increased by a capsid expression plasmid that was also capable of expressing NS2. These results show that a multi-functional parvovirus protein expressed from a Sindbis RNA molecule can be used to produce recombinant parvoviruses.

Genetic Vectors↗

[Primary parvovirus B19 infection in immunocompetent adults: clinical and biological manifestations. Retrospective study of 16 patients].

PURPOSE: The spectrum of clinical and biological expressions of parvovirus B19 infection is incompletely described. METHODS: We report 16 retrospective cases of primary parvovirus B19 infection that occurred in immunocompetent adults and were diagnosed by serology. RESULTS AND CONCLUSION: Most cases conformed to the classical descriptions of parvovirus-induced arthropathy (transient polyarthritis often accompanied by erythema, dysesthesia and pruritus). However, the diagnosis of primary parvovirus B19 infection should also be considered in other clinical and biological situations such as edema, acute anemia or cytopenia, vascular purpura, neuro-ophthalmologic manifestations, and hepatitis. Parvovirus infection may mimic diseases such as lupus erythematosus or systemic necrotizing vasculitis. The risk for confusion with both diseases is increased, as immunological abnormalities such as hypocomplementemia, when they are systematically searched for, prove to be common.

Adult↗

[Acute transitory intrafamilial erythroblastopenia and hereditary spherocytosis: role of parvovirus B19].

INTRODUCTION: Acute parvovirus B19 infection induces a transient inhibition of erythroid cell formation, which may induce an erythroblastopenia crisis in patients suffering from chronic hemolytic anemia. EXEGESIS: We report here an exceptional observation of acute erythroblastopenia crisis with good outcome, occurring at the same time in a mother and her son, both suffering from hereditary spherocytosis. Diagnosis of parvovirus infection is based on detection of serum parvovirus B19-specific immunoglobulin M antibodies in the mother and her son and by the positivity of parvovirus B19 DNA detected by PCR in serum in the mother. Outcome was good, with the end of the erythroblastopenia crisis obtained 7 to 10 days later, but requiring blood transfusion in the mother. CONCLUSION: Our observation is a reminder that the contagiosity of parvovirus B19 is high in household contacts and that protection of family members should rapidly be considered in hereditary spherocytosis.

Acute Disease↗

Recombinant human parvovirus B19 vectors.

In an attempt to exploit the remarkable tissue-tropism of the human parvovirus B19 to target human hematopoietic cells of the erythroid lineage, recombinant human adeno-associated virus 2 genomes were encapsidated in parvovirus B19 capsids. Although efficient transduction of primary human hematopoietic cells in the erythroid lineage occurred, a low-level of transgene expression in non-erythroid cells was also detected. These studies suggest that cell surface expression of P antigen, the primary receptor for parvovirus B19, is necessary but not sufficient for parvovirus B19 vector-mediated transduction of human hematopoietic cells. These studies also suggest the existence of a putative cell surface co-receptor, which is required for successful infection of human hematopoietic cells by parvovirus B19.

DNA, Recombinant↗

Parvovirus B19 infection in thoracic organ transplant recipients.

BACKGROUND: Clinical manifestations of parvovirus B19 infection in immunocompromised patients are mostly reported as acute or chronic hematologic disorders. More recently, respiratory or renal involvement has been described. OBJECTIVE: We started in 1994 a prospective study of parvovirus B19 infection in a group of lung (LTP) and heart-lung (HLTP) transplanted patients, including occasionally heart transplanted (HTP) patients. STUDY DESIGN: 62 patients (49 LTP, 11 HLTP, 2 HTP) were included in a serological survey and DNA detection by PCR was performed on each serum sample of the first 29 patients; later we performed it only when serology could suggest an acute episode, or when parvovirus infection could be suspected on clinical or biological observations. A total of 1655 sera were examined by serological tests and DNA detection was done in 500 samples. Specific IgM, seroconversion, significant increase of specific IgG levels, and/or parvovirus B19 DNA detection, were considered as markers of viral infection. RESULTS: We observed the presence of both markers of infection in 24 patients (39%), with an individual combination of positive antibody and PCR results. Acute or chronic anaemia, neutropenia were associated to these laboratory findings in 19 patients, but in five cases, an asymptomatic clinical infection suggested viral persistence. CONCLUSIONS: We report parvovirus associated acute or chronic anaemia and pancytopenia in a group of LTP, HLTP and HTP patients, as well as asymptomatic cases of infection. In the hypothesis of a parvoviral persistent or latent infection, current diagnosis methods may be unreliable to identify any other clinical manifestations.

Adolescent↗

Acute parvovirus B19 infection mimicking juvenile myelomonocytic leukemia.

An 11-month-old patient with parvovirus infection mimicking juvenile myelomonocytic leukemia (JMML) is presented. The patient's history, presenting physical and laboratory features, was suggestive of JMML and consisted of fever, hepatosplenomegaly, lymphadenopathy, desquamation of the skin, anemia, leukocytosis with monocytosis and trilineage dysplastic findings of the peripheral blood and bone marrow. However, positive IgM titers for parvovirus B19 followed by seroconversion, negative cytogenetics and the benign follow-up of the patient suggested acute parvovirus infection as an etiologic factor for development of dysplastic features in the patient, and thus is recommended for consideration in the differential diagnosis of MDS. Although parvovirus B19 infection mimicking MDS has previously been shown in two patients with spherocytosis and one with subclinical immune deficiency; to our knowledge, the present report is the first describing the association of acute parvovirus B19 infection with dysplastic features mimicking myelodysplasia (MDS) in a child without a demonstrable underlying hematolymphoid disorder.

Acute Disease↗

Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors.

Autonomous parvoviruses preferentially replicate in and kill in vitro-transformed cells and reduce the incidence of spontaneous and implanted tumors in animals. Because of these natural oncotropic and oncolytic properties, parvoviruses deserve to be considered as potential antitumor vectors. Here, we assessed whether parvovirus H1 is able to kill human hepatoma cells by induction of apoptosis but spares primary human liver cells, and whether the former cells can efficiently be transduced by H1 virus-based vectors. Cell death, infectivity, and transgene transduction were investigated in Hep3B, HepG2, and Huh7 cells and in primary human hepatocytes with natural and recombinant H1 virus. All hepatoma cells were susceptible to H1 virus-induced cytolyis. Cell death correlated with H1 virus DNA replication, nonstructural protein expression, and with morphological features of apoptosis. H1 virus-induced apoptosis was more pronounced in p53-deleted Hep3B and p53-mutated Huh7 cells than in HepG2 cells which express wild-type p53. In Hep3B cells, apoptosis was partially inhibited by DEVD-CHO, a caspase-3 inhibitor. In contrast, H1 virus-infected primary hepatocytes were neither positive for nonstructural protein expression nor susceptible to H1 virus-induced killing. Infection with a recombinant parvovirus vector carrying the luciferase gene under control of parvovirus promoter P38 led to higher transgene activities in hepatoma cells than in the hepatocytes. Taken together, H1 virus kills human hepatoma cells at low virus multiplicity but not primary hepatocytes. Thus, recombinant H1 viruses carrying antitumor transgenes may be considered as potential therapeutic options for the treatment of hepatocellular carcinomas.

Apoptosis↗

Neurodevelopmental outcome after intrauterine red cell transfusion for parvovirus B19-induced fetal hydrops.

OBJECTIVE: To assess long term neurodevelopmental outcome of children after intrauterine intravascular red cell transfusion (JUT) for Parvovirus B19-induced fetal hydrops. DESIGN: Data of study children were investigated retrospectively. Neurodevelopmental evaluation was performed by appropriate standard tests (Griffiths, Snijders-Oomen, Kaufmann Assessment Battery for Children tests). SETTING: Tertiary care university teaching hospital. SAMPLE: Twenty children who had Parvovirus-induced fetal hydrops and intrauterine transfusion of packed red blood cells (IUT). METHODS: Retrospective chart analysis and standard neurodevelopmental testing. MAIN OUTCOME MEASURES: Developmental quotient (DQ) and intelligence quotient (IQ) according to the age at testing. RESULTS: Twenty survivors of Parvovirus B19-induced fetal hydrops successfully treated by IUT were followed until 13 months to nine years of age. On clinical follow up, no neurologic sequelae were evident. Neurodevelopmental scores of all children ranged within two standard deviations of a normal population (median 101, range 86-116) and exceeded one standard deviation in three children. There was no significant neurodevelopmental delay. CONCLUSION: Children having survived successful IUT for Parvovirus B19-induced fetal anaemia and hydrops have a good neurodevelopmental prognosis. Our results support the use of IUT for correction of Parvovirus B19-induced fetal anaemia and subsequent hydrops.

Blood Transfusion, Intrauterine↗

Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen).

BACKGROUND: The presence of a specific cellular receptor is thought to be necessary for susceptibility to viral infection. The erythrocyte P antigen is the cellular receptor for parvovirus B19. We hypothesized that the rare persons with the p phenotype, whose erythrocytes do not have this receptor, would be naturally resistant to B19 infection, which causes erythema infectiosum. METHODS: Blood samples were collected from two populations in cross-sectional studies. We determined the P antigen phenotype of the red cells and tested plasma for anti-B19-specific antibodies. Bone marrow from donors of known P antigen phenotype was inoculated with parvovirus B19. Infectivity was measured by assays of erythroid progenitor cells, dot blot analysis, and in situ hybridization for B19 DNA, and an immunofluorescence assay for viral-capsid proteins. RESULTS: Of the 17 subjects with the p red-cell phenotype, who did not have P antigen on their erythrocytes, none (0 of 11 and 0 of 6) had serologic evidence of previous parvovirus B19 infection. In contrast, the seropositivity rates in the two control groups were 71 percent (53 of 75, P < 0.001) and 47 percent (32 of 68, P = 0.03). In vitro, bone marrow from donors with the p phenotype maintained normal erythropoiesis despite very high concentrations of virus, with no evidence of infection of erythroid progenitor cells by parvovirus B19. CONCLUSIONS: People who do not have P antigen, which is the cellular receptor for parvovirus B19, are naturally resistant to infection with this pathogen.

Adolescent↗

Association of symptomatic acute human parvovirus B19 infection with human leukocyte antigen class I and II alleles.

To determine the effect of the major histocompatibility complex on the development of symptoms during acute human parvovirus B19 infection, we compared human leukocyte antigen (HLA) class I and II alleles in 36 patients with symptomatic acute B19 infection with those in >900 control subjects from northwestern England. The frequency of each of HLA-DRB1*01 (P=.016), DRB1*04 (P=.007), and DRB1*07 (P<.0001) alleles was significantly higher in parvovirus B19 patients than in control subjects. In the parvovirus group, 63.9% carried the rheumatoid arthritis-associated shared epitope sequence, compared with 45% of control subjects (odds ratio [OR], 2.2; 95% confidence interval [CI], 0.97-4.8; P=.04), and carriage was associated with fatigue during the acute phase (OR, 4.2; 95% CI, 0.8-23.9; P=.047). All symptomatic parvovirus-associated HLA-DRB1 molecules carry a neutrally charged glutamine at position 10 and a positively charged lysine at position 12 of the first hypervariable region. HLA-B49 was associated with parvovirus infection independently of HLA-DRB1*01, DRB1*04, and DRB1*07.

Acute Disease↗

Active, fulminant, lethal myocarditis associated with parvovirus B19 infection in an infant.

We report a case of fulminant myocarditis in an 11-month-old female infant who had no other clinical signs of parvovirus infection. The patient presented with severe respiratory distress and died in sudden cardiac arrest 3 h after admission. The clinical presentation was similar to that of an asthmatic attack. Autopsy revealed signs of acute lymphocytic myocarditis. Parvovirus DNA was demonstrated by polymerase chain reaction (PCR) analysis of tissue sections obtained from the heart, lungs, liver, kidneys, and spleen. Transmission electron microscopy of myocardial tissue showed crystalline arrays with the appearance of parvovirus. The results of immunohistochemical analysis for the detection of parvovirus antigens were negative, and no viral inclusions were demonstrable. We suggest that the current diagnostic procedure underestimates the prevalence of parvovirus-associated myocarditis. PCR analysis should be used as a complement in suspected cases, to enhance the rate of detection of the infection and to reach a correct diagnosis.

DNA, Viral↗