Kati Hokynar

Persistence of human parvovirus B19 in human tissues.

Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation.

Integrity and full coding sequence of B19 virus DNA persisting in human synovial tissue.

Primary infection by human parvovirus B19 is often accompanied by arthropathy of varying duration, of which the most severe cases can be indistinguishable from rheumatoid arthritis (RA). While this might seem to imply a role in RA pathogenesis, recent studies have verified long-term persistence of B19 DNA in synovial tissue not only in patients with rheumatoid or juvenile arthritis, but also in immunocompetent, non-arthritic individuals with a history of prior B19 infection. However, the latter data are based on PCR amplification of short segments of DNA, with little sequence information. We determined the nucleotide sequence and examined the integrity of the protein-coding regions of B19 genomes persisting in synovial tissue and compared the results with data from synovial tissues of recently infected patients. In synovium of both previously and recently infected subjects, the viral coding regions were found to be present in an apparently continuous, intact DNA molecule. Comparison with sequences reported from blood or bone marrow showed that the synoviotropism or persistence of the B19 virus DNA was not due to exceptional mutations or particular genotype variants. The synovial retention of full-length viral genomes may represent a physiological process functioning in long-term storage of foreign macromolecules in this tissue.

Detection and Differentiation of Human Parvovirus Variants by Commercial Quantitative Real-Time PCR Tests

ABSTRACT Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies

Summary APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Biological and Immunological Relations among Human Parvovirus B19 Genotypes 1 to 3

ABSTRACT The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ∼10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.

T Helper Cell–Mediated In Vitro Responses of Recently and Remotely Infected Subjects to a Candidate Recombinant Vaccine for Human Parvovirus B19

T cell proliferation to human parvovirus B19 antigen was measured in 6 patients with recent B19 infection (1 with pneumonia and pleuritis), 1 patient with symptoms persisting >180 days after onset, 18 nonsymptomatic subjects with remote B19 immunity, and 12 B19-seronegative control subjects. Recombinantly expressed virus-like particles (VP1/2 capsids), a candidate B19 vaccine, were used as antigen. Virus-specific T helper cell proliferation was detectable in all the recently infected patients and in most (17/18) of the remotely infected subjects but not in the seronegative control subjects. The B19-specific T cell responses, in general, were most vigorous among the recently infected patients. However, such strong B19-specific proliferation was not confined within the acute phase, as 28% (5/18) of the remotely infected healthy individuals had B19-specific reactivity persisting at acute-phase levels, apparently for years or decades. These data indicate that B cells recognizing the VP1/2 capsids receive class II-restricted help from CD4(+) T lymphocytes.

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2=several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER-mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.

Tissue persistence and prevalence of B19 virus types 1–3

Human parvovirus B19 is a minute ssDNA virus that causes a wide variety of diseases, including erythema infectiosum, arthropathy, anemias and fetal death. In addition to the B19 prototype, two new variants (B19 types 2 and 3) have been identified. After primary infection, B19 genomic DNA has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. The viral DNA persists as an intact molecule without persistence-specific mutations, and via a storage mechanism with life-long capacity. Thus, the mere presence of B19 DNA in tissue cannot be used as a diagnostic criterion, although a possible role in the pathology of diseases, for example through mRNA or protein production, cannot be excluded. The molecular mechanism, host-cell type and possible clinical significance of tissue persistence are yet to be elucidated.

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis

Background: Ca2+-independent phospholipase A-β (iPLAβ) has been repeatedly implicated as a homeostatic enzyme. Results: Activity of iPLAβ correlates inversely with the hydrophobicity of the phospholipid substrate. Conclusion: Efflux of the phospholipid substrate from a membrane mainly determines the activity of iPLAβ. Significance: Results support the role of iPLAβ as a homeostatic enzyme that responds to increased phospholipid efflux proposed to occur at particular “critical” compositions. The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.

Genoprevalence in human tissues of TT-virus genotype 6

Summary.TT virus (TTV) is a newly discovered human virus of high genotypic diversity. TTV is widely distributed among humans, but the possible genotype-related differences in TTV biology are not well known. The prevalence and amount of TTV-DNA, especially of genotype 6, was determined by nested-PCR in various human tissues, and human parvovirus B19, another ssDNA virus, was used as a reference. TTV DNA was detected simultaneously in bile, peripheral blood mononuclear cells (PBMC) and plasma of 77% subjects, in 38% skin samples, in 38% synovial samples and in all (100%) adenoids, tonsils and liver samples. The relative concentrations of TTV-DNA did not vary significantly among the different samples. Genotype 6 TTV-DNA was detected in bile and plasma of one subject (3%), in skin and serum of one subject (8%) and in one liver (5%). The overall prevalence of TTV genotype 6 was 4% in subjects and 4% in sera. TTV genotype 6 was shown to occur in human tissues with no obvious tissue-type or symptom specificity. Parvovirus B19 DNA was detected overall in 38% subjects, and bile was the only sample type tested that did not persistently harbour B19 DNA.