Clinical impact & pathogenic mechanisms of human parvovirus B19: A multiorgan disease inflictor incognito

Introduction

Human parvovirus B19 (B19V) infections are known to be associated with numerous clinical manifestations, which often culminate in sinister sequels; however, unfortunately, B19V infections remain greatly under-recognized and human sufferings continue incognito. B19V belongs to the genus Erythrovirus in the family Parvoviridae and is the smallest DNA virus (5.5 Kb). Although discovered in 19751, the clinical diseases caused by B19V were recognized much later (1981-1987) beginning with transient aplastic crisis in patients with haemolytic anaemia, erythema infectiosum (fifth disease), arthropathy and non-immune hydrops foetalis2345. B19V is an obligate human pathogenic virus6, and its clinical spectrum has gradually increased over decades789101112. Further, B19V has gained the status of an expanding13 and an emerging virus14. Still, B19V infections could not gain much clinical importance since most of B19V infections remain asymptomatic or self-limiting6. Thus, most clinical infections due to B19V go undiagnosed.

Transmission of B19V infection is largely through respiratory droplet, transfusion of B19V viraemic blood or blood components or transplacentally6. Droplet infection is the most common mode of transmission of B19V where it at first multiplies in the throat and then results in viraemia with very high titres of B19V67. Laboratory diagnosis of acute B19V infections is usually made by detecting B19V-specific immunoglobulin G (IgM) antibodies in the serum by ELISA and/or B19V DNA in serum or bone marrow aspirate67810. In case of infected tissues, B19V can be detected by in situ hybridization but commonly by polymerase chain reaction (PCR) or real-time PCR which is more specific though costly and technically demanding. Other modalities are electron microscopy to demonstrate B19V virions in serum since acute B19V infections result in high titre viraemia (up to 10¹²/ml)6; further, cytomorphology of bone marrow aspirate may show giant pronormoblast measuring 25-32 μm with large eosinophilic nuclear inclusion bodies, cytoplasmic vacuolization and dog ear projections called ‘Lantern cells’10 that provide presumptive diagnosis of B19V infection. There is no specific treatment of B19V infections; however, infusions of intravenous IgG (IVIG)15 which contains sufficient B19V neutralizing antibodies help in clearance of virus, but being very costly is neither recommended by clinicians nor is affordable by patients in developing countries specially. Cumulative reasons culminated in slackness in considering B19V infections as a possible cause of illness in patients as well as in investigating and even if found in treating for B19V infections. Thus, an unaccounted percentage of patients silently suffers from B19V infections.

Approach to unveil clinical manifestations further

To unveil various clinical manifestations of B19V infection, suspected and even unknown but pathogenically rational cases were investigated. Children with juvenile chronic arthropathy (n=69) now known as juvenile idiopathic arthropathy, were studied and B19V infection was observed in 27 per cent children16. Next, B19V-induced clinical cases ending fatally with pure red cell aplasia (PRCA), severe anaemia and thrombocytopaenia with hepatitis in a child and haemophagocytic syndrome in an infant were reported171819. Further three novel clinical associations of B19V were reported, namely B19V-induced pure amegakaryocytic thrombocytopaenia in a nine month old male infant (got cured by IVIG treatment)20, myositis21 as a complication of erythema infectiosum in a nine year old female child and a series of eight cases with non-occlusive ischaemic gangrene of stomach or bowel including four cases having extensive gangrene of either entire ileum or jejunum to right colon who died post-operatively due to short gut syndrome (mortality 50%)22.

Further, to conduct sero-epidemiological studies, a large sample size was required and limiting factors were high cost of commercial ELISA kits and non-availability of PCR, a few decades ago. Hence, ELISA was developed in our laboratory using cloned, baculovirus expressed and purified B19V VP1 and VP2 proteins as antigens23. To further detect early viraemic cases, detection of B19V DNA was required; hence, in-house DNA extraction from serum and then, PCR and nested-PCR were developed and standardized172324. Seroepidemiology and B19V susceptibility of general population to acquire B19V infection were determined by estimating B19V-specific IgG antibodies to B19V capsid proteins VP1 and VP2 by in-house ELISA in 1000 voluntary blood donors (patient's relatives) and it came out to be 39.9 per cent23 similar to a study from Japan25. This indicates that about 60 per cent of Indian population (now about 1.25 billion) is non-immune. Seroprevalence of B19V in different age-groups in our country has been reported in 5 per cent children less than 10 yr, in 13 per cent young adults of 10-20 yr and in 44 per cent of aged 20-60 years26. These data show that a major proportion of population, especially children and young adults, are susceptible and prone to acquire to B19V infection.

A study on foeto-pathogenic association of B19V was then done on 372 women, and B19V infection was found in 19.8 per cent of 116 women with recurrent spontaneous abortions, 11 per cent among 136 pregnant women and 5 per cent among 120 non-pregnant women27. In another study, 60 high-risk pregnant women with bad obstetric history and having polyhydramnios, oligohydramnios or intrauterine growth retardation were examined and B19V infection was found in 13.6 per cent28. In a study on 35 cases of paediatric haematological malignancies, B19V infection was seen in 17.1 per cent [5 acute lymphoblastic leukaemia (ALL), 1 Non-Hodgkin's lymphoma (NHL)] children while two children also had B19V DNA in serum besides giant pronormoblasts (lantern cells)29 in the bone marrow. Another study on 90 children with beta-thalassaemia major who were multi-transfused had high B19V seropositivity in 81 per cent and high anti-B19V IgM positivity of 41 per cent besides transmission occurred through B19V-infected donor units30.

Novel clinical manifestations of B19V by PCR were further analyzed by real-time PCR31 which comprised amegakaryocytic thrombocytopenia, non-occlusive ischaemic gangrene of stomach and bowel and on B19V-induced myositis which has been reported further3233. Another finding was of a novel ‘oncolytic property’ in children with leukaemia mostly ALL based on mortality of 17.1 per cent among B19V-uninfected groupin comparison to none (0%) in B19V-infected group besides finding unexplained prolonged remission in a child with chronic myeloid leukaemia which supported our hypothesis that B19V might have an oncolytic property3435. Some of the articles published by our group are summarized in Table I.

Other investigators also began to report cases of B19V infection from India and B19V infections were reported in 20 per cent (n=50) in patients with haematologic disorders with aggravation of anaemia3738. Others found B19V genotypes 1 and 3 in patients with solid and haematological cancers3940, and B19V infection in a prolonged anaemia in a case with dilated cardiomyopathy (DCM)41.

Multiorgan afflictions by human parvovirus B19 (B19V) and its pathogenic mechanisms

From four types of clinical infection recognized earlier beginning with TAC, erythema infectiosum, arthropathy and non-immune hydrops foetalis now multiorgan afflictions by B19V (Table II) and its pathogenic features are being elaborated beginning with heart (vide infra).

Pathogenicity and virus host-cell interactions

B19V is known to have tropism for human erythroid progenitors17 due to the presence of a globoside (Gb4Cer) receptor for B19V110111 which is also present in non-erythroid tissues such as endothelial cells and foetal hepatocytes, placental trophoblastic cells and some megakaryocytes cells68. Two co-receptors of B19V, namely α5β1 integrin112, a fibronectin and another Ku80 autoantigen113 help in exteriorisation of B19V.

Toxic effects are exerted by several B19V viral proteins on various types of human cells. For instance, B19V infects bone marrow and multiplies causing interference in erythropoiesis (about 55% inhibition) due to lytic or direct cytopathic effect which is mediated by VP2 protein of B19V. B19V inhibits colony forming unit-erythroid (cfu-E) and blast forming unit-erythroid (bfu-E) colonies of erythroid series cells in the bone marrow resulting in sudden drop of haemoglobin6667. Further interaction with Fas ligand114 through interferon-gamma induces apoptosis by caspase activation and this culminates in severe anaemia, PRCA, myelodysplasia17, thrombocytopaenia18 sometimes neutropaenia and pancytopaenia111115.

Persistent infection by B19V in erythroid cells as well as in the non-erythroid68 lineage cells and other body tissues such as myocardial endothelial cells is another important virulence mechanism of B19V in the causation of multi-tissue and multiorgan diseases. Low-level expression of viral gene has been observed in some cells with persistent infection and B19V capsid mRNA or proteins have been detected in the bone marrow, synovial, heart, liver, colon, lymphoid, thyroid and testicular tissues with different disease outcomes116117. Since vascular endothelial cells line all blood vessels which in turn supply blood to all organs through arteries, arterioles and capillaries and possess receptor for B19V, these get infected by B19V and induce multi-systemic vasculitis, necrotizing vasculitis, endothelialitis and micro-angiopathy118119120 and cause various diseases. To study the mechanisms underlying B19V infection and B19V DNA replication, many types of cell culture systems, including permissive and semipermissive erythroid lineage cells besides non-permissive cells, such as human embryonic kidney 293 or the endothelial cells of the myocardium, have been utilized121. B19V uptake into endothelial cells may get facilitated due to antibody-mediated enhancement through complement factor C1q122.

Molecular mechanisms of pathogenicity include VP1u and NS1 proteins of B19V which cause cellular cytotoxicity and apoptosis by caspase 3 and 9 pathways or immune-mediated molecular mimicry with several known human auto antigens such as collagen II, cardiolipin, keratin, myelin basic protein and platelet membrane glycoprotein IIb and IIIa, which are presented as self-antigens to T-lymphocytes; further, B19V VP1u protein with phospholipase activity may cleave host DNA116117. In addition, inflammatory signalling may be modulated through B19V NS1 protein due to activation of STAT3/PIAS3 in human endothelial cells123.

Evidences from animal parvovirus infections

Organ involvement by B19V has also been reported in animals by animal parvoviruses such as infection by canine parvovirus type-2 which has been reported to cause massive necrotizing MC in a mongrel puppy137 or enteritis causing death of a wild wolf138. A study in cats and dogs infected with animal parvoviruses showed intestinal lesions characteristic of feline or canine parvovirus infection with the detection of parvoviral DNA and antigen in the intestines139.

Time to realize the threat and attempts towards prevention

Organs or tissue detection of B19V DNA is often missed owing to cryptic140 sites of infection or due to unknown tissue distribution of B19V or being in low copies numbers of virions. Similar is the case of anti-B19V IgM antibodies which may not be detected in many of immunocompromised patients infected with B19V owing to failure in mounting of antibodies in sufficient quantities andtitresremain low. These leads to false-negative results owing to which a good proportion of actually B19V infected cases go undiagnosed causing underestimation of clinical impact of B19V infection in the population globally.

Now almost 40 years since its discovery disease burden due to B19V infection and associated mortality has not been determined largely because of lack of large series studies and vast majority being case reports. There is a need to recognize B19V infection as a sinister virus and for its prevention, there are no specific antiviral agents available in the present time. Recombinant B19V vaccine was developed in early nineties comprising VP1 with VP2 proteins and found immunogenic in animal141. Later double-blind, randomized, phase 1 clinical trial on B19V seronegative adults showed the vaccine as safe as well as immunogenic142. A phase II clinical trial which was underway has been terminated (https://clinicaltrials.gov/ct2/show/NCT00379938), under the belief that it may not be commercially feasible. There are issues on toxigenicity which remain to be solved, and meanwhile, drugs like cidofovir are being tested143.

Due to lack of efficient adaptation to cellular cultures and absence of animal models, the Koch's postulate remains unproven in the causation of a wide spectrum of clinical diseases due to parvovirus B19V10; however, with advances in molecular techniques and direct demonstration of viral genomes in infected tissues, an alternative method proposed is ‘molecular Koch's postulates’144. The research in B19V infections should be continued and B19V should gain its full recognition as an important pathogenic virus of public health importance globally145.

Conclusion

Nearly half of adult population of the world irrespective of age, sex and geographic location has been found to be B19V seronegative hence is prone to acquire B19V infection. Earlier, B19V was known to exert great tropism for erythroid progenitors in the bone marrow as these possess Gb4Cer receptors and thought to be restricted in causing haematological diseases only. Now, owing to recent recognition of multiple GSL receptors and co-receptors of B19V which are distributed widely on host tissues, explains multi-tissue or multiorgan diseases that may be caused by B19V. B19V has been reported to infect non-erythroid cells as well as vascular endothelial cells and produces a wide spectrum of clinical infections. Cellular pathogenicity is further substantiated by B19V causing endothelialitis, vasculitic and complement-mediated injuries. B19V-specific proteins especially NS1, VP2 and VP1u, molecular mimicry and immune enhancement besides potential to cause persistent infection points more to words a causal role of B19V in multiorgan diseases rather than a casual detection.