The detection of parvoviruses.

Parvovirus B19 is a single-stranded DNA virus which causes severe disease in immunocompromised patients and foetal loss in pregnant women. It is classified as an Erythrovirus and this genus also comprises two related viral genotypes (so-called LaLi/A6 (genotype 2) and V9 (genotype 3)) which appear to be immunologically indistinguishable from Parvovirus B19. Serological and nucleic acid test (NAT) systems to detect Parvovirus B19-mediated infection are commercially available; however, some NAT systems are genotype-specific. International standard preparations of Parvovirus B19 IgG and DNA have been produced for assay standardisation purposes, and to ensure consistency of assay manufacture and performance. Immunological assays, such as B-cell ELISpot, T-cell stimulation, and cytokine detection can also be used to confirm exposure to Parvovirus B19. Immunohistochemical techniques, employing commercially available monoclonal antibodies, are used to localise the virus in infected tissue and Parvovirus B19 viral antigen can also be detected in serum and plasma using antigen-specific ELISA. NAT systems have also been described to detect newly identified parvoviruses such as human bocavirus (HBoV), PARV4, and PARV5, although absolute confirmation of clinical diseases associated with these agents is required. This chapter describes the current status of detection systems for all the aforementioned parvoviruses, with particular emphasis on Erythrovirus detection by serological, NAT, and immunological approaches.

Seroprevalence of erythrovirus B19 IgG antibody among paediatric patients in Makkah and Jeddah, Kingdom of Saudi Arabia.

OBJECTIVE: To estimate the prevalence of IgG antibodies against B19 virus (B19V) in Makkah and Jeddah, Saudi Arabia. METHODS: B19V-specific IgG antibodies were detected by a commercial indirect enzyme-linked immunosorbent assay in sera of 400 paediatric patients (185 males and 215 females) aged 1-17 years. RESULTS: Of the 400 patients, 80 (20%) had sera positive for B19V-specific IgG. The difference in the prevalence of the antibodies between genders was not statistically significant (p = 0.9). The prevalence of anti-B19V antibodies increased significantly in the age group of 12-17 years as compared to younger patients (37.5 vs. 18% in those aged 1-11 years; p = 0.006). CONCLUSION: This study indicated a high prevalence of IgG antibodies against B19V in paediatric patients with an increase in age.

Identification and characterization of persistent human erythrovirus infection in blood donor samples.

The presence of human erythrovirus DNA in 2,440 blood donations from the United Kingdom and sub-Saharan Africa (Ghana, Malawi, and South Africa) was screened. Sensitive qualitative and real-time quantitative PCR assays revealed a higher prevalence of persistent infection with the simultaneous presence of immunoglobulin G (IgG) and viral DNA (0.55 to 1.3%) than previously reported. This condition was characterized by a low viral load (median, 558 IU/ml; range, 42 to 135,000 IU/ml), antibody-complexed virus, free specific IgG, and potentially infectious free virus. Human erythrovirus genotype 1 (formerly parvovirus B19) was prevalent in the United Kingdom, Malawi, and South Africa. In contrast, only human erythrovirus genotype 3 (erythrovirus variant V9) was prevalent in Ghana. Genotype 3 had considerable genetic diversity, clustering in two probable subtypes. Genotype 1-based antibody assays failed to detect 38.5% of Ghanaian samples containing antibodies to genotype 3 virus but did not fail to detect cases of persistent infection. This study indicates a potential African origin of genotype 3 human erythrovirus and considerable shortcomings in the tools currently used to diagnose erythrovirus infection.

Lack of association between B19 or V9 erythrovirus infection and ANCA-positive vasculitides: a case-control study.

OBJECTIVES: To examine the potential association of human B19 or V9 erythrovirus infection and onset of ANCA-positive vasculitides. METHODS: We tested the sera of 13 adults with newly diagnosed ANCA-positive vasculitides. Each was age- and sex-matched to three sera obtained from healthy controls. All samples were tested for B19- and V9-specific immunoglobulin (Ig) G and IgM antibodies (Ab) (third-generation ELISA), and B19 or V9 DNA was sought with the polymerase chain reaction. Statistical analysis was performed by conditional logistic regression. RESULTS: Patient diagnoses comprised six cases of Wegener's granulomatosis, six of microscopic polyangiitis and one of Churg-Strauss syndrome. IgG Ab to B19 were detected equally in patient and control sera (77 and 79% respectively) (odds ratio=0.84, P=0.84). All 13 cases and 39 controls were negative for IgM Ab and viral DNA. CONCLUSION: These results suggest that neither acute nor chronic B19 or V9 infection is an aetiological factor in ANCA-associated vasculitides.

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).