Combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus B19 infection

Abstract

Parvovirus B19 is a single-stranded DNA virus with a specific tropism for human erythroid precursor cells. The virus codes for two overlapping structural (capsid) proteins and one non-structural protein which is thought to perform essential functions in viral replication, transcription and packaging. The ultrastructural localization of these proteins was achieved in cultured haemopoietic cells derived from fetal liver which had been infected in vitro and subsequently embedded in LR White acrylic resin. Postembedding immunogold detection of B19 structural and non-structural proteins was combined with localization of viral nucleic acid by in situ hybridization using a digoxigenin-labelled probe and different sized gold labels. The majority of the B19 capsid protein and DNA present in cells harvested 48 hours post-infection co-localized within the centri-nuclear region of erythroid cells demonstrating characteristic chromatin margination. Relatively little DNA hybridization signal was present over paracrystalline inclusions strongly labelled with anti-capsid protein monoclonal antibody R92F6. Viral DNA and capsid protein were co-localized in apparent egress from the nucleus through nuclear pores. B19 non-structural protein was detected in association with both nuclear and cytoplasmic arrays of capsids, supporting the view that this protein plays an important role in viral packaging and remains associated with the complete viral particle until its release from the cell. Co-localization of viral nucleic acid and proteins at the ultrastructural level is a flexible, rapid and highly specific tool for examination of viral life-cycles within cells.

Human parvovirus B19: A review

“…Patients with transient aplastic crisis may need supportive therapy with blood transfusions until neutralizing antibody response can clear the virus and hematopoiesis is restored (Bonvicini et al, 2007). In cases of fetal infections and hydrops, intrauterine transfusions are indicated when the hemoglobin concentration in the fetal circulation falls below a threshold level, and case series report improved survival rates of hydropic fetuses (Morey et al, 1995;Bonvicini et al, 2006). There are several options for the treatment of pure red cell aplasia and persistent infection in immunocompromised patients, in whom B19V-specific antibody response is absent or minimal (Lunardi et al, 1998).…”

“…There are several options for the treatment of pure red cell aplasia and persistent infection in immunocompromised patients, in whom B19V-specific antibody response is absent or minimal (Lunardi et al, 1998). Commercial Ig (IVIG), a significant source of anti-B19V antibodies, has proved to be efficacious, although no controlled studies have been carried out (Morey et al, 1995;Moudgil et al, 1997;Murer et al, 2000;Egbuna et al, 2006). Various regimens have been reported with favorable outcomes, but on the basis of the pooled data, 400 mg/kg per day for 5 to 10 consecutive days seems to be clinically useful in most cases.…”

Advances in Human B19 Erythrovirus Biology

“…The link between toxicity and apoptosis was first suggested by ultrastructural examination of B19V-infected fetal erythroid progenitors (63,64) and then in an erythroid cell line transfected with the NS1 gene, where activation of caspase 3 was demonstrated (61). The nucleoside triphosphate-binding domain of the NS1 protein of B19V was considered to be involved in B19V-mediated apoptosis in erythroid and nonerythroid cells (61,62,80).…”

Resin Tissue Microarrays: a Universal Format for Immunohistochemistry

“…Although resin TMAs have not been processed using this method, there is evidence that fixation by formalin or glutaraldehyde and embedding in acrylate resin allows the demonstration of DNA and mRNA transcripts (Le Guellec et al 1992;Morey et al 1995).…”