Biosecurity in Scandinavia.

This article investigates the extent to which biosecurity measures are recognized and have been implemented in the Nordic countries, in the absence of formalized security standards and legislation. Two trials were undertaken: first, a broad combined biosafety and biosecurity questionnaire survey of the Nordic countries, and, second, a focused on-site audit of 22 facilities, with 94 laboratories, in Denmark. Both trials indicated that external security had been partially implemented but that little attention had been paid to internal security and the establishment of biosecurity. It was demonstrated that the backgrounds and identities of insiders were rarely checked and that they could have gained access to both pathogen inventory lists and freezers in many facilities. In 81% of pathogen-containing facilities, pathogens were not routinely and centrally accounted for. The authors recommend the establishment of a legal framework congruent with international standards and obligations; novel governmental national biosecurity authorities, requiring a fusion of both microbiological and technical expertise and legislative powers; and the formulation of a new code of conduct termed "Good Biosecurity Practice."

Identification and characterization of a second novel human erythrovirus variant, A6.

Parvovirus B19 (B19), currently the only accepted member of the Erythrovirus genus, is the only parvovirus known to be pathogenic in humans. Recently a viral sequence, tentatively termed V9 which showed 11% variability from the published B19 sequences, was described from a patient with aplastic crisis. To search for additional parvovirus variants, we used the new NS1/7.5EC PCR assay whose primers were designed from a conserved region of the B19/V9 sequence and encompasses an MfeI restriction enzyme site that would allow differentiation between B19- and V9-like sequences. Screening of 225 serum and bone marrow samples and 62 plasma pools identified one new atypical parvovirus sequence, A6, from an anemic HIV-positive patient. A6 exhibited 88% similarity to B19 and 92% to V9, compared to >98% correspondence between reported B19 isolates. Based on the genome similarity to B19, an RT-PCR for A6 capsid transcripts was developed and used to test for A6 infectivity of UT7/Epo/S1 cells. Despite high viral titers, A6 viral transcripts were not detected. Thus, although the prevalence of B19 variants probably is low, the true clinical significance remains unknown. Current PCR analyses are unlikely to detect novel variants without the design of specific primers to the A6/V9/B19 common sequences.

Serologic study on parvovirus b19 infection in childhood acute lymphoblastic leukemia during chemotherapy: clinical and hematologic implications.

PURPOSE: To present a first descriptive serologic study on the clinical and hematologic implications of parvovirus B19 (B19) infection in children with acute lymphoblastic leukemia from the time of initial admission until discontinuation of chemotherapy. PATIENTS AND METHODS: Seventy-five patients were studied by polymerase chain reaction, enzyme-linked immunosorbent assay, sequencing, and immunodiffusion. RESULTS: During the period of observation, 8% (4/48) of B19-seronegative patients seroconverted and infection triggered profound anemia and thrombocytopenia. B19-specific IgG disappeared in 26% (8/31) of B19-seropositive patients, and these patients were significantly younger and the B19 IgG titers were lower on admission compared with patients who continuously displayed B19 IgG. B19 DNA was detected in the seroconverting patients, and this helped in determining the time of infection, which coincided with a B19 epidemic in 75% (3/4) of patients. Patients typically presented with fever and myalgia; a rash, indicative of B19 infection, was observed in only one patient. CONCLUSIONS: B19 infection was able to mimic a leukemic relapse or therapy-induced cytopenia and led to hospital admission, frequent blood sampling, renewed bone marrow aspirates, multiple transfusions of red blood cells or platelets, and cessation of maintenance chemotherapy for up to 3 weeks. The peculiar disappearance of B19-specific IgG, which could not be ascribed to a generalized low level of serum immunoglobulins, has not been previously reported. The results indicate that B19 should be assayed at diagnosis of leukemia to avoid subsequent diagnostic uncertainty, and during treatment in B19-seronegative patients exhibiting unexplained cytopenia.

Detection of parvovirus B19 NS1-specific antibodies by ELISA and western blotting employing recombinant NS1 protein as antigen.

Human parvovirus B19 (B19) encodes a number of nonstructural proteins, including the major protein, NS1, and two structural proteins, VP1 and VP2. The use of denatured NS1 in enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assay has provided an opportunity to study some of the immunologic properties of NS1, but the results have been equivocal and the diagnostic sensitivity poor, probably because of the absence of conformational epitopes. Various viral isolates and baculovirus vectors were employed to produce recombinant B19 NS1 under nondenaturing conditions for the first time. To assess the antigenicity of purified B19 NS1, the reaction patterns of 252 samples were compared by B19 NS1 and VP2 ELISA. In sera from individuals with past infection (VP2 IgG-positive), the use of this new antigen increased significantly the sensitivity of ELISA compared with WB (78% vs. 33%, P = 0.001), contradicting perpetuated claims that B19 NS1 IgG is detected primarily in patients with arthralgia or chronic infection. Previous reports of the absence of NS1 IgG during the initial phase of infection (< 6 weeks) were proved incorrect by the detection of NS1 IgG in 60% of samples from patients recently infected by B19. Including conformational epitopes in the ELISA increases the diagnostic sensitivity, although immunologically, a temporal (years) attenuation of NS1 antibodies appears to take place. This novel diagnostic tool may be useful as a supplement in case of borderline results by VP2 ELISA and for monitoring the efficacy of future capsid-based B19 vaccines.

Human parvovirus B19.

Parvovirus B19 (B19) was discovered in 1974 and is the only member of the family Parvoviridae known to be pathogenic in humans. Despite the inability to propagate the virus in cell cultures, much has been learned about the pathophysiology of this virus, including the identification of the cellular receptor (P antigen), and the control of the virus by the immune system. B19 is widespread, and manifestations of infection vary with the immunologic and hematologic status of the host. In healthy immunocompetent individuals B19 is the cause of erythema infectiosum and, particularly in adults, acute symmetric polyarthropathy. Due to the tropism of B19 to erythroid progenitor cells, infection in individuals with an underlying hemolytic disorder causes transient aplastic crisis. In the immunocompromised host persistent B19 infection is manifested as pure red cell aplasia and chronic anemia. Likewise, the immature immune response of the fetus may render it susceptible to infection, leading to fetal death in utero, hydrops fetalis, or development of congenital anemia. B19 has also been suggested as the causative agent in a variety of clinical syndromes, but given the common nature, causality is often difficult to infer. Diagnosis is primarily based on detection of specific antibodies by enzyme-linked immunosorbent assay or detection of viral DNA by dot blot hybridization or PCR. Treatment of persistent infection with immunoglobulin reduces the viral load and results in a marked resolution of anemia. Vaccine phase I trials show promising results.

Prevalence of parvovirus B19 and parvovirus V9 DNA and antibodies in paired bone marrow and serum samples from healthy individuals.

Parvovirus B19 (hereafter referred to as B19) exhibits a marked tropism to human bone marrow (BM), and infection may lead to erythema infectiosum, arthropathy, hydrops fetalis, and various hematologic disorders. Recently, a distinct parvovirus isolate termed V9 with an unknown clinical spectrum was discovered. In contrast to the many studies of B19 serology and viremia, valid information on the frequency of B19 or V9 DNA in the BM of healthy individuals is limited. To develop a reference value, paired BM and serum samples from healthy subjects were tested for the presence of B19 and V9 DNA and specific antibodies. Immunoglobulin M (IgM) was not found in any of the serum samples. The prevalence of IgG showed a gradual and steady increase from 37% in children aged 1 to 5 years to 87% in people aged >50 years. When 190 well-characterized subjects were examined, B19 DNA was detected in the BM of 4 individuals (2.1%; 95% confidence interval, 0.58 to 5.3%) while none of the paired serum samples showed evidence of circulating viral DNA. V9 DNA was not found in any of the BM or serum samples. The finding of B19 DNA probably indicated a primary infection in one 7-year-old individual and reinfection or reactivation of persistent infection in the remaining three persons, aged 47 to 58 years. Serving as a benchmark for future studies, these findings are useful when interpreting epidemiologic data, performing BM transplantation, or considering clinical implications of parvovirus infection.

Baculovirus expression of erythrovirus V9 capsids and screening by ELISA: serologic cross-reactivity with erythrovirus B19.

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).

Parvovirus B19 and parvovirus V9 are not associated with Henoch-Schönlein purpura in children.

BACKGROUND: Based on single case reports, parvovirus B19 (B19) has repeatedly been proposed as an etiologic agent in patients with Henoch-Schönlein purpura (HSP), perhaps causing vasculitis by direct invasion of vascular endothelial cells because of the tissue distribution of the cellular B19 receptor. A cohort of children with HSP and other vasculitic diseases was investigated and compared with healthy control children to assess the role of B19 as well as parvovirus V9 (a putative emerging B19-like virus). PATIENTS AND METHODS: Serum samples from 36 children with HSP (n = 29) or other vasculitic diseases (n = 7) were examined, and 38 healthy bone marrow donors were used as controls. The presence of specific B19 and V9 IgM and IgG antibodies was determined with a recently developed enzyme-linked immunosorbent assay, and viral DNA was detected by a novel nested PCR. RESULTS: Specific IgM was not present in any of the patient or control serum samples. B19 DNA was detected in one patient, a previously healthy 8-year-old boy diagnosed with HSP, whereas none of the controls was B19-positive. V9 was not detected in any of the clinical or control samples. It seems likely that B19 infection might have triggered the development of HSP in the B19-positive patient, because B19 viremia is otherwise uncommon. CONCLUSIONS: Although causality is difficult to construe in single cases, the data indicate that B19 is not a common contributing factor in the pathogenesis of vasculitis and that this pathogen is only rarely associated temporally with HSP or vasculitic diseases in children.