Antibodies to parvovirus B19 non-structural protein are associated with chronic but not acute arthritis following B19 infection

J. R. Kerr and V. S. Cunniffe

Departments of Medical Microbiology and Virology, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 Note added in proof
 
Objective. To determine the incidence and significance of antibodies to the parvovirus B19 non-structural (NS1) protein in B19-infected persons during acute infection and convalescence.

Methods. The B19 NS1 protein was expressed in SF9 cells using the baculovirus expression system and was used to prepare immunofluorescence slides. These were used in a fluorescent antibody test to determine anti-B19 NS1 IgG in a well-characterized cohort of 53 persons at the time of acute B19 infection and again after a follow-up period of 26–85 months. Results were examined for statistical significance by the use of Fisher's exact test.

Results. NS1 antibodies were detected in five of 32 persons with acute B19 infection (four with arthritis) and 10 of 53 persons with past B19 infection (six with chronic arthritis and two with chronic arthritis and chronic fatigue syndrome). Regarding the correlation of NS1 antibodies and arthritis, at the time of acute infection four of 24 persons with arthritis had NS1 antibodies detected compared with one of eight persons with any other symptoms (P = 1). During convalescence, eight of 20 persons with chronic arthritis had NS1 antibodies compared with two of 33 with symptoms of any other category (all except one were asymptomatic) (P = 0.007). All 10 patients with NS1 antibodies during convalescence had arthritis during acute infection, which persisted in eight persons until the time of follow-up.

Conclusion. Antibodies to parvovirus B19 NS1 protein are associated with chronic but not with acute arthritis after B19 infection.

KEY WORDS: B19 parvovirus, Antibodies, Arthritis, Non-structural protein, NS1 protein.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 Note added in proof
 
Human parvovirus B19 (B19) is the aetiological agent of the rash illness erythema infectiosum, which may be complicated by arthralgia and arthritis [1]. B19 infection in patients with shortened red cell survival has been associated with temporary cessation of erythropoiesis and life-threatening anaemia. B19 infection during pregnancy has been associated with fetal B19 infection, hydrops fetalis and death. Persistent B19 infections have been reported most frequently in persons with underlying immunodeficiency [1]. B19 infection has also been associated with arthropathy following acute infection, which may be present for months to years [1]. A recent report provides compelling evidence for a causative role for B19 virus in rheumatoid arthritis (RA) [2], although findings of other groups are conflicting [3]. Other diseases which have been associated with B19 infection are cardiac disease, hepatobiliary tract disease, neurological syndromes, ocular disease, renal disease, lower respiratory tract disease and chronic fatigue syndrome (CFS).

The parvovirus B19 genome consists of a linear, single-stranded DNA of ~5.6 kb, encoding the two capsid proteins VP1 and VP2 from the right side and the non-structural proteins (NS) from the left side [4]. Although the function of the major non-structural protein, NS1, is poorly characterized, it is known to be cytotoxic, an activity which may be mediated by its DNA-nicking and helicase activities [4]. NS1 also trans-activates the human interleukin-6 gene [5].

The humoral immune response to the B19 capsid proteins is commonly used for the diagnosis and determination of susceptibility to B19 infection. However, much less is known of the antibody response to the NS1 protein and its significance. Initially it was reported that, in addition to antibodies to B19 capsid proteins, patients with B19-associated arthritis and virus persistence also developed antibodies to NS1 [, ]. However, further work suggested that the presence of anti-NS1 antibodies does not correlate with unusual clinical outcome or chronicity [810]. To learn more about the incidence and significance of anti-NS1 antibodies in B19 infection, we expressed the B19 NS1 protein in SF9 cells using the baculovirus expression system, and we used an immunofluorescence assay to examine sera for antibodies to NS1 in 53 persons during acute and convalescent infection with various clinical outcomes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
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Serum
Human anti-NS1 positive control serum from a pregnant woman with B19 infection and documented anti-NS1 antibodies was kindly provided by S. Modrow (Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, Regensburg, Germany). For anti-NS1 antibody studies, acute and convalescent sera from 53 persons with known B19 infection and various clinical outcomes were examined (Tables 1Go and 2Go); details are published elsewhere [, 12]. These patients represent a group of acutely infected persons who were followed over 26–85 months (mean 57 months) to determine the clinical outcome. At the time of acute infection, NS1 IgG assay was omitted in 21 patients as there was insufficient serum available; thus, the serum of only 32 patients was tested at the time of acute infection.


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TABLE 1. Clinical details and B19 markers in 53 B19-infected persons at acute infection [, 12]

 

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TABLE 2. Clinical details and B19 markers in 53 B19-infected persons at convalescence [, 12]

 

Baculovirus expression of recombinant NS1 protein
Recombinant Autographica california nuclear polyhedrosis virus with the Stu NS1 nucleotide sequence inserted just downstream of the polyhedrin promoter was kindly provided by B. J. Cohen (Virus Reference Division, Central Public Health Laboratory, Public Health Laboratory Service, Colindale, London, UK) [13]. This was used to infect 3-day-old, freshly subcultured SF9 cells grown in monolayer at a multiplicity of infection of 10 in Grace's medium supplemented with fetal bovine serum (10%), yeast hydrolysate, lactalbumin hydrolysate and glutamine (Invitrogen, Leek, The Netherlands). At 72 h post-infection, medium was removed, cells were dislodged from the monolayer, washed, counted and spotted onto immunofluorescence slide wells. Expression of B19 NS1 was confirmed using positive control anti-NS1 serum by immunofluorescence (see below).

Immunofluorescence
Serum samples were diluted 1:100 in sterile phosphate-buffered saline (PBS) and used to stain both infected and uninfected SF9 cells for 30 min at 37°C in a humidified atmosphere. After thorough washing, goat anti-human IgG fluorescein isothiocyanate (FITC) conjugate (Sigma, UK) was added at its working dilution of 1:32 for 30 min at 37°C. After further washing, slides were mounted and viewed on a fluorescence microscope with a filter of emission waveband at 515 nm (Leitz, Switzerland). Positive control anti-NS1 serum and PBS (negative control) were used throughout. Sera showing the characteristic pattern of positive fluorescence in infected but not uninfected cells were designated positive, and the results were semiquantitated as follows: +/–, weakly positive; +, positive; + +, strongly positive.

Statistical analysis
Statistical analysis was performed using Fisher's exact test with Yates' correction for cell values less than 10.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Tables 1Go and 2Go show clinical details and B19 markers including NS1 antibodies for 53 B19-infected patients during acute infection and convalescence, respectively. Five of 32 patients tested during acute infection (Table 1Go) and 10 of 53 patients during convalescence (Table 2Go) had serum NS1 antibodies detected. Of the five persons who had NS1 antibodies during acute B19 infection, four had arthritis and one had a rash; during convalescence, two of these four still had arthritis and NS1 antibodies; the others were negative for NS1 antibodies, although one had persistent arthritis. Therefore, the presence of NS1 antibodies at onset was poorly predictive of NS1 antibodies at follow-up. Of the 10 persons testing positive for NS1 antibodies at convalescence, six had arthritis, two had arthritis and CFS and two were asymptomatic; at onset of infection two had been positive for these antibodies, four had been negative, and in four serum was not available for testing. Of the 10 persons positive for NS1 antibodies during convalescence, all had arthritis at acute infection, which persisted without remission in eight persons until the time of follow-up.

Table 3Go shows the results of statistical analysis using Fisher's exact test on the incidence of NS1 antibodies in acute (five of 32) versus convalescent (10 of 53) B19 infection (P = 0.93), acute B19 infection with arthritis (four of 24) vs acute B19 infection with any other symptom category (one of eight) (P = 1), and in convalescent B19 infection with B19 arthritis (eight of 20) vs convalescent B19 infection with any other B19-related symptom category (two of 33) (P = 0.007).


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TABLE 3. Statistical analysis of the incidence of NS1 antibodies in patients with acute and convalescent B19 infection, with and without B19-related arthritis

 


    Discussion
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 Materials and methods
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The B19 non-structural protein (NS1) is known to be cytotoxic to eukaryotic [4] and prokaryotic [6] cells. However, this does not present a problem in the baculovirus expression system as expression from the baculovirus polyhedrin promoter is very late, occurring after the production of infectious particles. In addition, polyhedrin, which is the natural gene product, eventually kills the cells. Therefore, the baculovirus expression system is suitable for the expression of such potentially toxic proteins [13].

Von Poblotzki et al. [6] were the first to describe NS1 antibodies, and reported their association with acute B19 arthropathy. NS1 antibodies were detected in 0 of 9 persons not previously infected with B19, 0 of 14 persons with past B19 infection (including five with RA), 0 of 4 cases of erythema infectiosum, and 3 of 3 cases of acute B19-related arthritis. Von Poblotzki et al. [7] also reported NS1 antibodies in three patients with persistent B19 infection associated with recurrent granulocytic aplasia, autoimmune haemolytic anaemia associated with B-cell leukaemia, and pancytopenia, respectively. However, subsequent reports demonstrated NS1 antibody production in approximately equal proportions in asymptomatic and symptomatic cases, casting doubt on their association with arthritis [810]. However, NS1 antibodies were shown to occur more frequently with past than with recent infection [810].

In the present study, considering the 53 B19-infected persons at follow-up, NS1 antibodies were detected in eight of 20 persons with arthritis and two of 33 persons with symptoms of any other category (all except one were asymptomatic) (Yates-corrected {chi}2 value = 7.28; P = 0.007). Therefore, in the setting of documented B19-associated arthritis, NS1 antibodies correlated with chronic but not acute arthritis. One reason why this correlation has not previously been documented [6, 9, 10] may be that, although cases of acute B19 arthritis were examined (IgM-positive), cases of chronic B19 arthritis (in which the onset of B19 infection coincided with the onset of arthritis which then persisted to chronicity) were not examined. This is important since many cases of aetiologically unrelated arthritis will frequently and coincidentally be B19-seropositive.

Searle et al. [8] reported that NS1 antibodies may take 6 weeks or longer to develop; however, in the present study, NS1 antibodies were detected 2 (patient 36), 23 (patient 53) and 30 (patient 18) days after the onset of infection, respectively (Table 1Go); in the other two cases of recent infection, the interval between onset of infection and serum sampling was not known.

In the present study, seven patients had persistent B19 infection (B19 viraemia) several years after acute infection; this was symptomatic in three cases (patients 40, 50 and 51; Table 2Go) and asymptomatic in four (patients 24, 39, 41 and 46; Table 2Go) [11]; clinical manifestations of the symptomatic cases were arthritis (patient 40), arthritis and CFS (patient 51), and chronic haemolytic anaemia (patient 50). However, of these seven cases, only one had detectable NS1 antibodies: patient 51, who had arthritis and CFS.

It is not clear why only 15–30% of B19-infected persons produce NS1 antibodies. In acute B19 infection, the virus targets erythroblasts, causing apoptosis in these cells, which is induced by NS1. However, in RA-associated B19, the virus would seem to be present over an extended period in lymphocytes, macrophages and follicular dendritic cells [2]. NS1 production is known to be increased in non-permissive cells [4] and it has been shown that NS1 antibodies can neutralize the virus [14]. Although B19 NS1 protein has not been described as part of infectious particles, the NS1 protein of minute virus of mice (MVM) is consistently associated with the genome, is transiently located on the virion surface and is therefore available for immune recognition [15]. The possibility that the B19 NS1 protein may be partly or temporarily associated with infectious B19 virions at low concentration cannot be excluded [14]. In this event, NS1 antibodies may interfere with virus attachment or NS1 function, and thus influence the pathogenesis of B19 infection.


    Notes
 
Correspondence to: J. R. Kerr. Back

Submitted 20 October 1999; revised version accepted 15 February 2000.
    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 Note added in proof
 

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    Note added in proof
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 Note added in proof
 
Further evidence for the correlation of NS1 antibodies with severe courses of B19 infection is provided by Hemauer et al. [16].