Clinical aspects of parvovirus B19 infection

Authors

  • K. BROLIDEN,

    1. From the Department of Medicine, Solna, Unit of Infectious Diseases, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
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  • T. TOLFVENSTAM,

    1. From the Department of Medicine, Solna, Unit of Infectious Diseases, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
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  • O. NORBECK

    1. From the Department of Medicine, Solna, Unit of Infectious Diseases, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
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Prof. Kristina Broliden, Department of Medicine, Solna, Unit of Infectious Diseases, B2:00, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Solna, 171 76 Stockholm, Sweden.
(fax: +46 8 7178501; e-mail: kristina.broliden@karolinska.se).

Abstract.

Parvovirus B19 is a significant human pathogen that causes a wide spectrum of clinical complications ranging from mild, self-limiting erythema infectiosum in immunocompetent children to lethal cytopenias in immunocompromised patients and intrauterine foetal death in primary infected pregnant women. The infection may also be persistent and can mimic or trigger autoimmune inflammatory disorders. Another important clinical aspect to consider is the risk of infection through B19-contaminated blood products. Recent advances in diagnosis and pathogenesis, new insights in the cellular immune response and newly discovered genotypes of human parvoviruses form a platform for the development of modern therapeutic and prophylactic alternatives.

Viral characteristics

Parvovirus B19 (‘B19’) is a member of the erythroviruses, named so because of a pronounced tropism for erythroid precursor cells. It is a single-stranded nonenveloped DNA virus and one of the smallest viruses known to infect mammalian cells [1]. B19 is genetically stable, and sequenced isolates have shown low variability in the range of a few percentage for the two capsid proteins VP1 and VP2, and even lower for the nonstructural protein NS1 [2–4] (Fig. 1). Differences in clinical manifestations of B19 infection have not been explained by B19 sequence variability [5–8]. Novel viral isolates have now also been sequenced that are similar to B19 only to about 88–90%, and are proposed to compose two novel genotypes within the erythrovirus genus. These variants are not readily detected by available B19 serology and polymerase chain reaction (PCR) assays [9–13]. Recently, another human parvovirus provisionally named human bocavirus was cloned by molecular screening of respiratory tract samples from children with lower respiratory tract infections [14]. Although these novel genotypes are capable of human infection and were initially detected in individuals with B19-related disease, the potential pathology has yet to be established [8, 12, 15].

Figure 1.

 Parvoviruses are symmetrical icosahedral particles. They form small capsids and contain a DNA genome. The viral genome encodes only three proteins with known function, the nonstructural protein NS-1 and two capsid proteins VP1 and VP2.

Infection and tropism

B19 is thought to exclusively infect humans, and shows a pronounced tropism for erythroid precursors [16–18]. The virus is very stable and often survives in blood products despite standard procedures for viral elimination [19, 20]. B19 uses at least three cellular receptors for cell attachment and entry. The first to be identified was the glycolipid globoside, also known as the blood group P antigen (P-ag) [21, 22]. P-ag is present on the haematopoietic precursors, erythroblasts and megakaryocytes that B19 shows tropism for and also on a variety of other cells including endothelial cells, foetal myocytes, and placental trophoblasts [23–27]. These latter cells have been thought to represent nonpermissive cells into which B19 can enter but not produce complete virions. For a productive infection to occur, a co-receptor has been defined, the α5β1-integrin. This integrin is involved in cell adhesion and is expressed on erythroid progenitors, which might explain the tropism of B19 and the active replication seen in these cells [28, 29]. Another molecule, the Ku80 autoantigen, has also been suggested as a co-receptor for B19 allowing entry into cells [30].

Epidemiology

B19 is a common virus that is spread worldwide, and the seroprevalence increases with age, so that 15% of preschool children, 50% of younger adults and about 85% of the elderly show serologic evidence of past infection [31–35]. In developing countries the seroprevalence has been shown to be a little higher, probably because of poor and crowded living standards, whereas in isolated tribal communities seroprevalence figures are below 10% [36–38]. Infection appears to confer lifelong immunity to immunocompetent hosts. Although the seroprevalence is high, viraemia or presence of viral DNA is rare in healthy individuals. The frequency of B19 viraemia in voluntary blood donors has been estimated at rates of 1 : 167 to 1 : 35 000 [35, 39–43]. The frequency varies greatly depending on epidemic periods and sensitivity of the methodology used. Although B19 viraemia in blood is rare, presence of B19 DNA in bone marrow (BM) samples can be found by PCR in 2% of healthy individuals and in up to 10% of children with haematologic malignancies without concomitant viraemia [31, 44, 45]. The persistency of B19 DNA may represent both infectious virus and residual DNA from remote infection.

The incidence of infection shows a seasonal variation in temperate climates, being more common during winter and early spring [46]. Epidemics are noted at intervals of about 3–4 years, with outbreaks of erythema infectiosum (EI) and B19-related disease. B19 is normally transmitted through the respiratory route, but can also be transmitted vertically from the mother to the foetus, through BM and organ transplantations, and via transfused blood products [42, 47–51]. As most infections occur in children aged 5–15 years, adults at risk are parents of children in that age group, or those working at day care centres or schools [52–54]. The secondary attack rate during epidemics of EI is about 50% in susceptible children and 25% in susceptible teachers [53, 55]. Nosocomial transmission may occur and although rare, is a potential risk in paediatric wards for immunocompromised children [56–58].

Blood product safety

The genomic stability and the absence of a lipid envelope make B19 resistant to heat inactivation and solvent detergents normally used to inactivate viral concomitants in blood products. The risk of transmission through blood transfusions and plasma-derived products has been known for many years [59–63] and many manufacturers of plasma derivatives screen their products by quantitative PCR [64]. Quantitative measurements of B19 DNA should also be considered in blood transfusions intended for immunosuppressed individuals and other risk groups, which has been reviewed by Corcoran and Doyle [65]. Indeed, recommendations have been made in the Netherlands that blood donors with high anti-B19 immunoglobulin G (IgG) titres should be used for donations to high-risk recipients [66].

We thus still need to learn more about risk judgement in recipients of blood products. The susceptibility depends not only on host factors but also on the relation of viral load and anti-B19 IgG titres in the blood product [67, 68]. Life-threatening B19 infection has even been transmitted by intravenous immunoglobulin (IVIG) [69].

Clinical manifestations

After the discovery in 1974, it was not until 1981 a distinct disease was associated with infection of B19 when it was detected in a sickle-cell anaemic patient with transient aplastic crisis (TAC) [70]. It was subsequently shown to be the cause of EI in 1983, a disease first described in 1799 [71].

The features of B19 infection have been studied in experimental infections of healthy individuals [72, 73]. After intranasal inoculation of B19, there was a biphasic clinical course. The viraemia peaked after 8–9 days at 1011 virions per mL, with viral excretion through the respiratory tract, but not via urine or faeces, accompanied with mild symptoms of fever, malaise, myalgia and pruritus. About three weeks after infection, a second phase of symptoms developed, with typical maculopapular rash for the B19 infection and in some cases arthralgia. The BM was normal after 6 days, but on day 10 there was an almost complete loss of erythroid precursors, and a subsequent marked drop in the reticulocyte counts was noted in peripheral blood. The effects on the haemoglobin level and other blood cell counts were less pronounced.

Since the time of these experiments, the wide clinical spectrum of B19-associated complications has been dissected and the different outcomes depend heavily on the immune status of the host.

Erythema infectiosum

Erythema infectiosum is the most common clinical manifestation of B19 infection in immunocompetent hosts although asymptomatic infection is seen in 25–50% of infected individuals [55, 74]. Another name for EI is the ‘Fifth disease’, referring to a fifth place in a listing of the common infections during childhood. Classically, EI affects school-aged children with low-grade fever, malaise and a characteristic facial rash that has given rise to the name ‘Slapped cheek syndrome’. Arthralgia may occur in some children with EI but is not as common as in adults. The slapped-cheek appearance is followed by the spread of a maculopapular rash on the trunk, back and extremities (Fig. 2). The infection is normally self-limiting within a week or two but the rash can be recurrent for some months following exposure to sunlight, heat, emotion, or exercise [75]. The rash and the joint symptoms are most likely caused by immune complex deposition.

Figure 2.

 The pathogenesis of erythema infectiosum is probably a result of antibody-antigen immune complex depositions in skin, blood vessels and synovia. The rash typically appears on the cheeks followed by a lace-like maculopapular rash on the upper part of the body. Joint symptoms are more common in adults than in children. In addition to deposition of immune complexes, the inflammatory response in synovial tissue may be a result of the secreted phospholipase A2 motif in the unique region of the B19 minor capsid protein [89].

Arthropathy

On average, 50% of adult cases of EI have associated joint manifestations that may persist for weeks to months, and in a few cases for years. The typical facial exanthema and fever of EI is only seen in a minority of adult cases. The arthropathy is particularly common in middle-aged women, and is characterized by a polyarthritis typically involving the metacarpophalangeal joints, knees, wrists, or ankles [76–79]. Both arthralgia and inflammatory arthritis may occur and the arthropathy sometimes even mimics classical rheumatoid arthritis (RA). The joint involvement is however not erosive and is probably immune-mediated as it appears simultaneously with circulating antibodies. B19 DNA has indeed been detected in synovial fluid and biopsies in both acute and remotely infected individuals [80–83]. Although B19 infection may mimic RA, and even trigger a positive test for rheumatoid factor, its role in the aetiology of this disease has not been proved [84, 85].

Kerr et al. [86] have described an association between development of symptoms during B19 infection and carriage of the HLA-DRB1*01, *04, and *07 alleles. B19-associated arthritis may thus at least partly be genetically associated and has, in earlier studies, been shown to be more common in individuals with HLA DR4 or B27 [87, 88]. The mechanism of the arthropathy is partly unknown. In addition to immune-mediated inflammation, one hypothesis include the activation of synoviocytes by the secreted phospholipase A2 motif in the B19 VP1 unique region. This activity would thus accelerate the inflammatory response in synovial tissue [89]. The B19 NS1 protein causes the secretion of proinflammatory cytokines, which could contribute to the arthritis and inflammatory and autoimmune disorders associated with the infection [90, 91]. Furthermore, peptides derived from VP2 may be pathogenic in B19 arthropathy as they can induce cross-reactive autoantibodies against human keratin, collagen, and cardiolipin [92].

Autoimmune disorders

Apart from RA, B19 infection has been associated with the onset of numerous autoimmune disorders including systemic lupus erythematosus (SLE) [93, 94], other connective tissue diseases and systemic vasculitides. Although a few cases of erosive RA and SLE have been associated with B19 infection, the virus is probably an extremely rare cause of these diseases. Systemic vasculitides including for example Henoch-Schönlein pupura [95], periarteritis nodosa [96], and giant cell arteritis [97] can occur after acute B19 infection. The role of B19 in these disorders is not clear and in some cases the infection may be a pure coincidence and in other cases it can be a triggering or even a rare aetiological factor. In any case, the infection is an important differential diagnosis for several autoimmune disorders both in clinical presentation and in immunological tests. In conclusion, in individuals with immunogenetic predisposing factors B19 may indeed be capable of causing at least arthropathy associated with severe, long-lasting morbidity.

Many viral infections including B19 induce the production of autoantibodies. Although these responses are normally of short duration they can create diagnostic difficulties. As reviewed by Meyer [98], B19 can, for example, induce antibodies to double-stranded DNA, anti-nuclear soluble antigens, cardiolipin and rheumatoid factor. The autoantibody production most likely results from both polyclonal stimulation of immune responses and production of polyspecific anti-B19 antibodies [92].

Patients with increased red cell turnover

In patients with either decreased production or increased loss of erythrocytes, there is a potential risk that B19 infection leads to TAC. When the red cell turnover is increased, the suppression of the BM caused by B19 leads to a severe drop in the haemoglobin level that can be fatal. This was first described in patients with haemolytic anaemia, but can occur in patients with a wide range of disorders, including thalassemia, hereditary spherocytosis, sickle-cell anaemia, malaria and even iron deficiency and haemorrhage [70, 99–104]. In addition to a cessation of the erythroid production, other blood cell lineages can be affected with clinically significant thrombocytopenia, neutropenia or pancytopenia as the result (Fig. 3) [105–107].

Figure 3.

 B19 binds to immature erythroblasts thereby arresting production of mature erythropoietic cells. Following acute infection, the reticulocyte count in peripheral blood is zero and if the patients have an underlying disorder with pathologic red cell survival, the number of erythocytes may fall dramatically in peripheral blood. The pathogenesis of thrombocytopenia is thought to be explained by the cytotoxicity of the NS1 protein [238].

Once the immune response clears the infection, the red cell production resumes and eventually normalizes followed by lifelong immunity in most cases. However, the aplastic crisis can cause severe and even fatal anaemia resulting in congestive heart failure, cerebrovascular events, and acute splenic sequestration [108].

Other B19 associated disorders

B19 has a cardiotropic potential as P-ag is expressed by myocytes and B19 DNA has indeed been detected in heart tissue [24, 43, 109]. Cases of sometimes fatal myocarditis and heart failure in both children and adults have been reported [110–115].

Other B19-associated disorders include hepatitis [116–118], transient erythroblastopenia of childhood [119], neutropenia [120], trombocytopenia [121], Kawasaki disease [122], Gloves-and-sock syndrome [123], neurological disease including meningitis and encephalitis [124, 125], fibromyalgia and chronic fatigue syndrome [126, 127]. Several other manifestations of B19 infection have been reported and have been reviewed in detail by Heegaard and Brown [128], but the evidence for a clear causality, as stated above, is sometimes scarce. It must be pointed out that apart from BM which is the primary site of viral replication, B19 DNA is found in tissues like synovia [83, 129], liver [130] and in skin [131] in both B19-associated clinical disorders and in healthy controls. This emphasize that caution should be taken in drawing conclusions about the aetiology of B19 in rheumatic and other disorders.

B19 infection in immunocompromised individuals

The clinical picture of acute and persistent B19 infection in the immunocompromised host differs significantly from immunocompetent subjects. In the absence of an efficient humoral and/or cellular immune response, the infection can cause persistent BM suppression manifested by chronic anaemia but not supposedly immune-mediated symptoms such as rash and arthralgia [132]. Predisposing conditions include congenital immunodeficiencies, leukaemia, lymphoma, myelodysplastic syndrome, BM and solid organ transplantation, chemotherapy, and infection with human immunodeficiency virus [50, 51, 132–142].

About 5% of adult patients and 10% of children undergoing chemotherapy for haematological malignancies are persistently infected with the virus resulting in severe and even lethal cytopenias whereas about 1–2% of organ and stem cell transplanted patients have life-threatening complications caused by parvovirus infection [45, 50]. Cases of parvovirus-associated pancytopenias or isolated ‘penias’ of different haematological cell lineages can be misinterpreted as relapse of the underlying malignant disorder or of other microbial infections, graft failure or drug toxicity. In Fig. 4, a case of B19 infection in a child suffering from acute lymphatic leukaemia is shown. In a group of children with acute leukaemia, the number of days of unwanted treatment interruptions was significantly higher in parvovirus infected than in uninfected individuals which is associated with poor prognosis (K. Broliden, unpublished observation). Early diagnosis of parvovirus infection is therefore of extreme importance and the infection can be treated with either blood transfusion or intravenous immunoglobulin.

Figure 4.

 The clinical and laboratory outcome of a 10-year-old girl with acute lymphocytic leukaemia and B19 infection, adapted from Broliden et al. [45]. The infection was not diagnosed until the end of December and thus the B19 diagnostic parameters were performed in retrospect. During maintenance chemotherapy the patient developed a rash on her cheeks in April. The rash reappeared two times during the observation time. During the ongoing B19 viraemia she had several periods of severe cytopenias that required blood transfusions. A relapse of the leukaemia was suspected and a new bone marrow aspirate was performed in December. At this time the B19 diagnosis was finally suspected and upon additional blood transfusions and three weekly periods of interruptions of the chemotherapy (November–December) the infection spontaneously resolved. It can be speculated that early diagnosis already at time of the first rash and cytopenic period in April followed by IVIG treatment could have led to immediate elimination of the B19 infection and avoidance of the critical course of infection. It should also be noted that serology is unreliable in immunodeficient patients as she did not develop IgG and IgM until the end of the infection when the immune system recovered following treatment interruption.

Many immunodeficiencies affect the production of neutralizing antibodies against the virus resulting in persistent infections associated with chronic anaemia [141, 143]. However, conditions or chemotherapies leading to deficient cellular immune responses may also result in persistent infections. The contribution of the cellular immune response for elimination of B19 may thus explain why IVIG therapy does not always clear infection in some cases and why chronic infection is seen in the presence of neutralizing antibodies without viral clearance. Yet another case demonstrating that neutralizing antibodies may be complemented by a cellular immune response was an AIDS patient with persistent B19 infection who showed an initial remission of B19 infection in the absence of a specific antibody response [144].

B19 persistence in immunocompetent patients

In immunocompromised individuals, B19 is known to cause persistent infection with potentially severe and chronic anaemia as the result. In addition, B19 seems to be capable of persisting in a subset of apparently immunocompetent individuals. B19 is thus reported to be present in about 2% of BM samples from healthy individuals [31]. The persistence in these individuals is, however, not only seemingly perpetual but also associated with various long-lasting symptoms such as fatigue, fever, arthralgia, and myalgia [145]. The symptom complex is sometimes similar to the chronic fatigue syndrome, and B19 has been proposed to play a role in this disease [127, 146, 147]. Whereas no specific sequence variations in the viral genome [3] or abnormal humoral B19 epitope specificities [148] or lack of functional neutralizing antibodies [145] could explain the persistence, a selective defect in the cellular immune response was seen in immunocompetent subjects together with an altered cytokine profile (K. Broliden, unpublished data) [149]. However, the role of this aberrant cellular immune response in relation to clinical symptoms or establishment of the persistent infection has not been proved.

B19 and pregnancy: vertical transmission and foetal hydrops

The association between foetal B19 infection and the development of non-immune foetal hydrops was first proposed by Brown et al., and it is estimated that about 15–20% of cases of non-immune hydrops fetalis are caused by B19 [150–152]. About 50% of pregnant women are susceptible to B19 infection, and maternal infection is reported to occur in a few percentage of pregnancies [153, 154]. There is approximately a 30% risk of vertical transmission to the foetus, and the over-all risk of an abnormal outcome after maternal infection is estimated to 5–10% [49, 151, 155–158]. Transmission over the placenta is reportedly most likely to occur in the first or second trimester, as placental P-ag, which may be necessary for transmission, becomes less frequent with increasing gestational age [23]. The infection causes anaemia, hypoalbuminaemia, inflammation of the liver and possible myocarditis, leading to cardiac failure and the development of foetal hydrops (Fig. 5) [159]. The condition may resolve but can also result in foetal death, with a mean time span between maternal infection and foetal symptoms of six weeks, but it may be several months in rare cases [151, 160–162].

Figure 5.

 Vertical transmission of B19 from a primary infected mother may cause foetal infection. Pathogenic mechanisms include development of acute anaemia upon infection of foetal haematopoietic cells. In early pregnancy haematopoiesis is seen in the liver and in later pregnancy this shifts to the bone marrow. The anaemia may resolve spontaneously or proceed by causing cardiac failure and development of hydrops fetalis and in rare cases foetal death. The virus may also cause myocarditis and heart arrest by direct infection of myocardial tissue. Modified from Anderson and Young [239].

Foetal B19 infection may also be asymptomatic, and there are several observations of infants born healthy despite evidence of intrauterine infection diagnosed by the presence of IgM in umbilical cord blood [163]. Malformations as a consequence of intrauterine B19 infection have been reported in a few isolated cases [164–166], but has not been concluded to be a common feature of this infection [49, 167].

B19 and pregnancy: intrauterine foetal death (IUFD)

There has been a prevailing apprehension that foetal demise caused by B19 infection in the third trimester is extremely rare or even nonexistent, but B19 is a common cause of IUFD also in late gestation, but without foetal hydrops in a majority of cases [168–170]. By investigating cases of IUFD during a 6-year period we found B19 DNA by PCR in 14% representing both second and third trimester cases.

Table 1 summarizes the different pictures of B19-associated IUFD that have emerged from our own studies. In the first trimester B19 DNA was found in 3% of cases of spontaneous abortion and an etiological link could not be proved [171]. In the second trimester, a serologically evident recent infection could be demonstrated in maternal sera. The foetuses were normally hydropic and B19 DNA was detected by PCR in foetal tissues and placenta and histopathological investigation often confirmed the diagnosis [168, 169]. This picture of second trimester IUFD cases is often reported in studies selecting for hydrops or serologically evident infection [172]. On the other hand, a partly revised clinical picture was proposed by our studies on B19 infection in late gestation [170]. The foetuses in third trimester IUFD associated with B19 infection were rarely hydropic and recent maternal infection was not commonly evident by the detection of B19-specific IgM. Maternal IgG may be present, or seroconversion may occur months later following IUFD [168]. The diagnosis was not readily established by histopathological methods but viral DNA was detected in foetal tissues and placenta.

Table 1.   Clinical complications associated with B19 infection during pregnancy
 Trimester
FirstSecondThird
  1. The table summarizes a generalized picture of more than 150 cases of spontaneous abortions and IUFDs representing different time-points (trimesters) during pregnancy. The studies were performed by a nested B19 PCR technique for detection of qualitative PCR in placental and/or foetal tissue [168–171].

Clinical foetal complicationSpontaneous abortionHydrops fetalis and/ or foetal deathFoetal death
Frequency of B19-DNA positivity in placenta and/or foetal tissue3%12%7%
Maternal symptomsNoneNone or erythema infectiosumNone
Maternal B19-IgM reactivityNegativePositiveNegative

It is interesting to speculate in the mechanisms underlying these different pictures at various time of gestation. The proposed pathogenic mechanism in foetal infection is summarized in Fig. 5 as mentioned above. In the second trimester, P-ag is present on the trophoblast layer in the placenta to allow the vertical transmission of B19 to the foetus from the infected mother [23]. The haematopoiesis is at this time located in the liver and is extremely active to increase the erythrocyte cell mass 34-fold to match the raised demand from the growing foetus. At the same time, the lifespan of the red blood cells is decreased to 45–70 days, making the foetus very vulnerable to any pause in the haematopoietic production [43]. The destruction of late erythroid precursors, caused by the B19 infection, leads to severe anaemia that in combination with the hepatic inflammation and possibly myocarditis due to B19 infection of cardiac myocytes, results in heart failure and the development of hydrops fetalis. In contrast, in the third trimester, the haematopoiesis migrates to the BM, the need for a quickly increasing red blood cell mass is relieved, and the lifespan of the erythrocytes is normalized. The cellular receptor P-ag is virtually nonpresent in the third trimester [23]. These factors act in concert to make the development of anaemia and subsequent heart failure and hydrops less likely. A low-grade persistent foetal and/or maternal infection may also cause IUFD at a later time-point, and in the case of placental dysfunction even without foetal infection. Degenerative lesions have been shown in the placenta due to the inflammatory response to B19 infection [109, 159, 173, 174]. In the foetus, myocarditis may be the cause of death caused by dysrhythmia or cardiac arrest without the development of anaemia or hydrops [159, 175]. Indeed, investigation of endomyocardial biopsy specimens revealed B19-associated inflammatory changes in 15% of cases of peripartum cardiomyopathy [176].

Immune responses in B19 infection

B19 is regarded to show a ‘hit-and-run’ mode of infection, which in the normal host is believed to quickly resolve with successful eradication of the virus. Prolonged detection of B19 DNA peripherally, in BM and in other compartments is now reported by several investigators, indicating that it may take longer to eradicate the virus than previously thought [80, 177–180]. Viral and host properties have thus been explored to explain this failure to eventually clear the virus.

Immune responses to acute infection

The humoral immune response is thought to be the most important in infections with short replication cycles, characterized by quick lysis of the infected cells and release of free virions readily exposed to antibodies that can bind to them and facilitate their destruction [181]. On the other hand, nonlytic and persistent viruses such as human hepatitis B virus (HBV), are less exposed to the humoral immune system and the host is therefore more dependent on the cellular immune response to localize and kill infected cells [182, 183].

The humoral immune response has been the focus of most studies on B19 and immunity, whereas the cellular response and the CD8+ T cell response in particular, has been detected and investigated only recently [184]. Being a virus that shares characteristics both with classically lytic resolving viruses and nonlytic and persistent viruses, it is not surprising that both the humoral and the cellular immune systems are mounted to battle B19.

It was first reported that no cellular immune responses were mounted in B19 infection, but CD4+ T cell proliferative responses directed to VP1 and VP2 have now been shown in seropositive individuals [185–189]. Analogous to the humoral response, CD4+ T cell responses to NS1 may be associated with acute and persistent arthropathy [190]. In contrast, our studies of the CD8+ T-cell response have revealed a predominance of NS1-directed responses in acute and remote infection whereas a skewing of the response towards VP2 was seen in persistent infection [184].

In 2001, Tolfvenstam et al. [184] defined the first B19-specific CD8+ T-cell epitope in an individual with remote infection, providing the first evidence of an existent cytotoxic cellular response to this infection. In another report [191], this finding was explored by following individuals with documented primary B19 infection during the acute phase and a thorough phenotypic analysis was later performed on the CD8+ T cells [192]. The CD8+ T-cell responses were mounted against three to five epitopes per individual with no sign of changing specificities over time, as for example seen in infection with the latent cytomegalovirus virus (CMV) [193]. The finding of predominantly NS1 specificities in acutely infected patients contrasts what is known about the humoral response to B19, where the neutralizing epitopes have been shown to be located mainly in the structural proteins [194–196]. The most unexpected finding was the lack of rapid contraction of the expanded CD8+ T-cell clones thought to be the hallmark of resolving infections of the ‘hit-and-run’ type [197, 198]. Sustained responses were shown that in most cases peaked after 1 year and were still detectable after 2 years (Fig. 6). Maintenance of the responses seems unlikely in the absence of antigenic drive, which means that B19, although not a classically persistent virus, manage to persist in the body for an extended period of time; and B19 may thus be more of a ‘hit-and-hang’ type of infecting virus. Indeed, we detected peripheral B19 DNA by PCR for over 1 year in some of the patients, one of whom was still DNA positive in the last sample collected after a little more than 2 years. It is plausible that B19 persists even longer in the BM, although undetectable in peripheral blood [145].

Figure 6.

 B19-specific CD8+ T-cell responses in relation to B19 viraemia in a healthy immunocompetent individual who acquired acute B19 infection with the onset of symptoms (rash, arthritis and fever) at time-point zero. B19 IgG and B19 IgM were present at onset of symptoms and the B19 IgM response disappeared after a few months. The B19-specific CD8+ response was measured in an ELISpot assay (gamma-interferon secretion) by using overlapping B19-specific peptides covering the whole B19 genome, adapted from Norbeck et al. [191]. Thus, this patient showed sustained levels of cells with three different specificities against the NS-1 protein (as represented by the blue, green and red line, respectively). No response was seen against the capsid proteins VP1 or VP2. The CD8+ T-cell response was subsequently confirmed by tetramer staining and the phenotypic markers were followed over time by multicolour staining using flow cytometry [192]. The corresponding viral titres are also shown (black line) [180]. The Y-axis indicates number of spot-forming cells per 106 PBMC.

Immune responses in persistently infected individuals

Recent data thus suggested that there is a skewing of the CD8+ T-cell responses in persistently infected individuals to the structural proteins when compared with acutely infected and healthy individuals [149]. This is the first time a discrepancy in the immune response was shown between healthy and persistently B19-infected individuals, and the lack of an efficient NS1 response may allow the virus to establish persistence. However, the other way around should be considered, namely that the persistence and the continuous antigenic stimulation results in exhaustion of the NS1 response, a phenomenon reported to occur in persistent infection [199]. The failed NS1 response may in turn favour the expansion of VP-specific CD8+ T-cell clones. It has been reported that clones of CD8+ T cells with high antigenic sensitivity are activated first, but succumb due to exhaustion if they for some reason fail to control the infection with high viral load as a result [200]. In this situation clones with lower sensitivity, possibly represented by the VP-specific CD8+ T cells in our study, have a better chance to avoid exhaustion but may not achieve viral clearance. In persistent HBV infection, low T-cell numbers is reported and is possibly the result of immunologic exhaustion [201]. The situation in persistent B19 infection may thus resemble that of HBV infection, and the increased T-cell activity seen after interferon treatment of HBV infection, suggests that a similar approach might prove beneficial also in B19-persistent infection [202]. Recently, Corcoran et al. [203] showed persistent infection in a child with acute lymphatic leukaemia despite the treatment with intravenous immunoglobulin. Interestingly, resolution of the infection was associated with the simultaneous strengthening of antigen-specific B-cell memory against B19 VP2 and diminution in the memory response against B19 NS1.

Diagnosis

Modern diagnostics of B19 infection usually include measurement of B19 IgG and IgM antibodies in blood and B19 DNA in blood or tissue samples by PCR. Morphologically, BM aspirates show no mature erythroid precursors and with characteristic giant pronormoblasts at time of acute infection. Immunohistochemistry may also be very specific and a complement to the more sensitive PCR assay in cases of placental or foetal infection.

Serology

Specific IgM antibodies directed to VP2 are present after 10–12 days after infection and usually disappear within 3–4 months, even though they may occasionally remain detectable longer [204]. The maturation and shift to IgG occurs shortly after the advent of IgM, and these high-avidity antibodies are believed to mediate lifelong immunity, with slowly decreasing titres boosted by subsequent encounters with B19. IgA antibodies are also detectable for a short period and may play a protective role in the respiratory tract [205]. In addition, long-term B19-specific IgE antibodies have been shown, but with unclear biological function [206]. The IgG is directed to both VP1 and VP2 with the majority of linear epitopes located in the VP1ur and the junction between VP1 and VP2, and these specificities are reported to represent the most effective immune response [194]. However, others have shown that IgG directed to VP2 is maintained even when IgG directed to linear epitopes within VP1 is lost, indicating immunodominance for VP2 [187, 195, 196]. Either way, the humoral responses seem to be directed to the structural proteins in normal infection, with persistence of IgG antibodies directed to conformational epitopes. Antibodies directed to NS1 have been described to be a feature only of acute and persistent infection [207–209], but this has not been confirmed in other studies [210–212].

Caution should be made when interpreting serology in immunodeficient individuals and in pregnant women. Due to their immune status they are not always able to mount an antibody response to pathogens at all. Furthermore, blood products or treatment with immunoglobulins may yield false-positive B19 IgG responses. Supplementary serological assays are sometimes needed for an accurate diagnosis and timing of maternal infection during pregnancy, which has recently been reviewed by de Jong et al. [213] and Enders et al. [214].

Polymerase chain reaction

Serology must in many cases be complemented by PCR analysis of B19 DNA. In immunocompromised patients a positive PCR test in blood indicates ongoing acute or persistent infection whereas a positive PCR test in BM may indicate either acute or remote infection as about 2% of healthy individuals with remote infection are PCR positive [31]. In cases of intrauterine foetal complications it can be very useful to analyse amniotic fluid or cord blood for viral DNA or RNA by PCR for differential diagnostic purposes including B19 [215–218]. To learn more about the clinical significance of viral load in foetal infection is of high priority as therapeutic intervention can be necessary in severe cases.

Detection of B19 DNA can thus be detected by PCR in serum, BM and other tissues for diagnostic purposes. Whilst B19 DNA may be positive also in tissues of healthy individuals as previously mentioned, the development of a quantitative PCR has been helpful in determining the viral load and should be used routinely. This also allow differentiation of human parvovirus variants [219, 220]. In healthy individuals with acute B19 infection, viral titres as high as 1012 geq mL−1 are detectable in blood [61]. However, a marked drop is seen at time of immunoglobulin production and onset of symptoms and titres remain at about 103 to 105 for months and even a few years following acute infection [180, 221]. Future studies will hopefully determine the concentrations of viral load in different tissues and clinical settings to guide the clinicians in therapeutic choices.

Treatment and prophylaxis

There is no specific antiviral drug against B19 infection but a number of alternative options to eliminate the virus can be recommended. The choice of treatment of B19 infection must however take host factors into account as the virus yields different pathogenesis in different risk groups of patients depending on underlying diseases and immunodeficiency status (Table 2).

Table 2.   Parvovirus B19-infection pathogenesis in different risk groups
Risk groupProposed pathogenesisClinical and laboratory signsTreatment
  1. The clinical and laboratory signs of B19 infection and corresponding therapeutic strategies is dependent on host factors and underlying clinical disorders. For example, the pathogenesis in the B19-infected foetus is a result of the physiologically higher red blood cell turnover and the relative immunodeficiency of the foetus. It must be pointed out that the infection resolves spontaneously in many cases in all risk groups and treatment is therefore only given to severe cases.

Immunocompetent individualsImmune complex depositionErythema infectiosum, arthropathyAntiinflammatory drugs
Patients with increased turnover of red blood cellsReduced erythropoeisisTransient aplastic crisisBlood transfusion
Immunodeficient patientsDeficient humoral and/or cellular immune responseChronic anemia or cytopeniaIVIG
FetusesDeficient humoral and/or cellular immune response
Reduced erythropoiesis
Hydrops fetalis, IUFD, anemia, cardiac failureBlood transfusion, IVIG

Immunocompetent hosts

There is no specific antiviral drug against B19 and the infection does not normally need treatment in the immunocompetent host. Nonsteroidal anti-inflammatory drugs are sometimes useful for pronounced symptoms such as arthralgia.

Transient aplastic crisis

Transient aplastic crisis usually requires hospitalization and erythrocyte transfusions, but has good prognosis if treated correctly with restored haematopoiesis within 1–10 days [103]. In a study including 62 patients with sickle-cell anaemia and B19-induced TAC, 87% required transfusion therapy and 63% were hospitalized; one patient died before the initiation of transfusion therapy [222].

Immunosuppressed patients

In immunosuppressed patients lacking neutralizing antibodies, IVIG has proved to be useful for treatment of persistent B19 infection. Administration of 0.4 g kg−1 × 5 days or 1 g kg−1 × 3 days induces an increase in reticulocyte count with an accompanied raise in the haemoglobulin level, and is often curative in that B19 is cleared from the body [58, 137, 142, 223].

Persistent infection in immunocompetent individuals

Persistent B19 infection in apparently immunocompetent individuals who already possess neutralizing antibodies does not respond well to IVIG treatment, but it may induce a transient remission [145]. In patients subject to chemotherapy or otherwise iatrogenically immunosuppressed, a persistent B19 infection usually resolves upon cessation of the therapy [224, 225].

Pregnancy

If B19 infection is confirmed in the pregnant woman, monitoring of the foetus by weekly ultrasound examinations are usually recommended. It must be emphasized though that foetal death may occur several months postmaternal infection and without foetal hydrops, or even in the absence of laboratory or clinical signs of maternal infection [160, 168, 226]. If hydrops and/or anaemia is diagnosed by Doppler ultrasound and cordocentesis, intrauterine erythrocyte transfusions have been shown to reduce the mortality rate from about 50–18% [227–231]. Maternally administrated IVIG or intrauterine therapy using B19 IgG-rich high titre gamma-globulin has also been tried but needs further evaluation [232, 233].

Vaccine development

A candidate vaccine has been tested consisting of 25% VP1 and 75% VP2, that with adjuvant elicits a strong humoral response, and is well tolerated [234, 235]. The vaccine is optimized with a humoral response in mind, which clearly has a well-documented role in B19 infection. However, in some situations the infection is not controlled despite the presence of neutralizing antibodies, and evidence of the importance of the cellular immune responses is growing. Therefore, cellular immune responses should be thought of when discussing responsiveness to a future B19 vaccine. Our findings of a predominant NS1-specific CD8+ T-cell response in normal infection and the aberrant CD8+ T-cell response in persistent infection may warrant the inclusion of NS1 epitopes using T-cell vaccine techniques. Unfortunately, commercial interest rather than lack of efficacy and safety has limited the development of a B19 vaccine [236]. The vaccine would primarily be intended for certain risk groups of severe B19 infection. For example it could be used to prevent TAC in patients with sickle-cell disease and pure red cell anaemia (PRCA) in immunodeficient patients. Whether seronegative women of childbearing age should be included is a matter of controversy. The risk of fatal outcome following maternal primary infection may be too low to warrant a generalized vaccination programme.

Conclusions and future perspectives

B19 infection causes a wide range of symptoms with the most severe clinical outcome in foetuses and immunosuppressed individuals. The infection can be both acute as well as establish persistency. Although BM is the major target organ for virus replication, the persistent infections seen in some individuals for years and even decades indicate that the virus may reside in additional immunoprotected compartments. The recently launched quantitative PCR techniques for measurement of B19 DNA titres have significantly improved the diagnostic repertoire as serology and qualitative PCR is insufficient in some cases. For example, increase in viral load could be monitored prior to relapse of anaemia in an AIDS patient with persistent B19 infection [237]. Likewise, the possibility of monitoring transplanted patients for B19 viral load with the aim of initiating preemptive therapy may be a future strategy to combat viral complications in this group of specific immunosuppressed patients. This strategy has been successful in the case of CMV infections in BM-transplanted patients. Furthermore, by performing quantitative PCR in amniotic fluid, maternal sera and cord blood we could correlate viral load to clinical outcome, in which case the quantitative PCR could serve as a diagnostic and prognostic tool, as well as be of guidance for therapeutic intervention. However, we still need to learn more about how to interpret quantitative PCR results in blood and tissue samples in relation to the immune status of the host.

B19 elicits a strong multi-specific CD8+ T-cell response in the acute phase of the infection that is sustained for up to 2 years. The specificity of the response does not change during the course of infection, and the response is probably antigen-driven as B19 DNA is also durably detectable by PCR in peripheral blood indicative of continued replication. This finding, unexpected in a virus typically associated with ‘hit-and-run’ infection, reveals a novel pattern of immune reactivity and mode of infection for which the paraphrase ‘hit-and-hang’ may be more suitable. In contrast to the humoral and CD4+ T-cell responses, the majority of the CD8+ T cell responses are directed to epitopes within NS1 in normal infection. Based on the recent definition of novel CD8+ T-cell epitopes, multimeric major histocompatibility complexes can be used for the detailed study of B19-specific CD8+ T cells with regard to frequency, phenotype and function. In this way, the maturation of the CD8+ T-cell response in different clinical manifestations of B19 infection can be assessed. Such investigations could provide further characterization of the concept of persistent versus normal viral clearance patterns, and hopefully provide knowledge about the underlying mechanisms for the unsuccessful clearance despite the presence of neutralizing antibodies and specific CD8+ T cells. Furthermore, the frequency, phenotype and function of specific CD8+ T cells, peripherally and locally in the placenta, will possibly reveal some new clues as to what determines the outcome in foetal infection.

In summary, measurement of quantitative viral titres and increased knowledge about the immune response to the virus are new developments to study B19 pathogenesis with the aim of improving the medical care of infected patients.

Conflict of interest statement

The authors have no conflict of interest.

Acknowledgements

We hereby acknowledge the Swedish Children Cancer Foundation, the Swedish Cancer Society, the Swedish Medical Research Council and the Tobias Foundation.

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