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Genetic variants of parvovirus B19 identified in the United Kingdom: Implications for diagnostic testing
Journal of Clinical Virology 36 (2006) 152–155
Short communication
Genetic variants of parvovirus B19 identified in the United Kingdom: Implications for diagnostic testing B.J. Cohen, J. Gandhi, J.P. Clewley ∗ Virus Reference Department, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5EQ, United Kingdom Received 10 January 2006; accepted 10 January 2006
Abstract Background: Discrepant results in diagnostic parvovirus B19 PCR assays have been observed with strains showing nucleotide sequence variation. Objectives and study design: To perform phylogenetic analysis on two parvovirus B19 strains that gave discrepant PCR results. Results: One strain was found to be genotype 2; the second strain was genotype 3. Conclusions: Parvovirus B19 genotypes 2 and 3 strains were identified in diagnostic samples of UK origin following the investigation of discrepant PCR results. More structured investigations are needed to estimate the prevalence of these variants. In the meantime, diagnostic PCR results should be interpreted cautiously when they are at variance with serological testing. Manufacturers of PCR kits for the detection of B19 sequences will need to consider re-designing their primers. © 2006 Elsevier B.V. All rights reserved. Keywords: Parvovirus B19 variants; Genotype 2 A6; Genotype 3 V9; Phylogeny
1. Introduction Human parvovirus B19 is classified within the erythrovirus genus of the family Parvoviridae, subfamily Parvovirinae (Fauquet et al., 2005). Early studies showed only limited (up to 4.8% in the structural proteins) genetic variation amongst parvovirus B19 strains (Erdman et al., 1996; Gallinella et al., 1995). However, more recent studies have reported variants with more than 10% sequence divergence compared to reference B19 strains. The first to be described, V9, showed 11–14% sequence divergence to 24 B19 strains (Nguyen et al., 1999). Subsequently, LaLi and related strains were found to be 10.8% divergent from reference B19 strains and 8.6% divergent from V9 in the protein coding region of the genome (Hokynar et al., 2002). In addition, the A6 strain was reported to have a 98% sequence similarity to LaLi (Nguyen et al., 2002). Servant et al. (2002) proposed that parvovirus B19 strains could be grouped into three genotypes: genotype 1 (reference B19 strains); genotype 2 (LaLi and A6) and genotype 3 (V9). ∗
Corresponding author. Tel.: +44 20 8327 6245; fax: +44 20 8327 6019. E-mail address: [email protected] (J.P. Clewley).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.01.011
There is evidence that genetic variation of parvovirus B19 has some temporal and geographical significance (Mori et al., 1987; Umene and Nunoue, 1995) and it has been shown recently that West Africa is an endemic region for V9 (Candotti et al., 2004). There is no evidence so far that any of the variants have distinctive pathological manifestations but, as they may escape detection in commercial PCR assays, they are of diagnostic significance (Hokynar et al., 2004; Braham et al., 2004; Baylis et al., 2004). We have now compared the sequence of PCR NS gene products from two variant parvovirus B19 strains that were not detected in commercial PCR assays with those of reference B19 strains, the recently described human parvoviruses, bocavirus (Allander et al., 2005) and PARV4 virus (Jones et al., 2005) and other parvoviruses.
2. Materials and methods 2.1. Patients and samples Patient SJ: Renal transplant, male, 33 years presenting with anaemia in May 2000 (Braham et al., 2004). Three sera
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
153
Table 1 Results of parvovirus B19 DNA and antibody assays on patient SJ Sample date
Parvovirus B19 DNA Roche LC PCR
26-05-2000 21-06-2000b 16-08-2000 21-08-2000 a b
Negative Negative Negative Negative
Nested PCR
B19 IgM in-house ELISA
B19 IgG Biotrin ELISA
Negative 0.2a Negative 0.2a Negative 0.2a Negative 0.2a
Negative 0.1a Not tested Negative 0.2a Negative 0.1a
Dot blot hybridisation
1st round
2nd round
Positive Positive Negative Negative
Positive Positive Negative Negative
Positive Positive Negative Negative
Test/cut-off ratio. This sample was a plasma (referred to as SJ in Fig. 1); all other samples were sera.
and one plasma sample collected during May–August 2000 were available for testing (Table 1). Patient AQ: Chronic renal failure, female 51 years presenting with anaemia and red cell aplasia in February 2005. Three sera collected during March 2005 were available for testing (Table 2). Positive controls: Two reference B19 strains, Nan and Stu, collected previously (Braham et al., 2004; Hicks et al., 1996).
Briefly, sequences were aligned against representative parvovirus sequences from the NCBI database using Clustal W and were then analysed with Paup. The optimal model of evolution was found using Modeltest.
3. Results 3.1. Results for patient SJ
2.2. Parvovirus B19 DNA and antibody detection Parvovirus B19 DNA was detected by dot blot hybridisation assay (Hicks et al., 1998), in-house nested PCR (Clewley, 1993), Roche LightCycler (LC) parvovirus B19 quantification kit (Roche Diagnostics, Lewes, Sussex, UK) or RealArtTM Parvo B19 LC PCR kit (Artus GmbH, Hamburg, Germany). Parvovirus B19 IgM was detected by inhouse ELISA (Brown et al., 1989) or by commercial ELISA (Biotrin, Dublin, Ireland) and parvovirus B19 IgG detected by commercial ELISA (Biotrin). 2.3. Sequencing and phylogenetic analysis A 511-nucleotide region of the parvovirus B19 NS gene was amplified using primers ‘I’ and ‘F’ (Clewley, 1993). PCR products were excised from a 2% agarose gel and purified with Geneclean (Anachem, Luton, UK) or QIAquick (Qiagen, Crawley, UK) kits. DNA was sequenced with Applied Biosystems kits (Warrington, UK) and sent to DBS Genomics (Durham University) for gel electrophoresis. Phylogenetic analysis was performed as described previously (McCormack and Clewley, 2002; McCormack et al., 2002).
Samples from this patient were first investigated during an evaluation of the Roche LC parvovirus B19 kit (Braham et al., 2004). In this study, two of 228 samples from the UK, both from the same patient, showed 8% sequence divergence compared to reference B19 genotype 1 strains (Braham et al., 2004). Phylogenetic analysis of the strain B19-SJ (Fig. 1) from these samples showed that it aligned with genotype 2 strains, with only 0.5% divergence over the small region of NS that was sequenced. This strain was positive when re-tested with the Artus B19 PCR kit. It is of interest to note that, although this patient subsequently cleared his B19 infection, no antibodies were detected in followup sera using standard parvovirus B19 antibody assays (Table 1). 3.2. Results for patient AQ Phylogenetic analysis of the parvovirus strain B19-AQ (Fig. 1) from this patient showed that it aligned most closely with genotype 3 strains, with about 3% divergence from V9. The strain was not detected during routine diagnostic testing with the Artus B19 LC kit, which was in use in the laboratory
Table 2 Results of parvovirus B19 DNA and antibody assays on patient AQ Sample date
Parvovirus B19 DNA Artus LC PCR
11-03-2005 29-03-2005 30-03-2005 a b
Negative Negative Negative
B19 IgM Nested PCR 1st round
2nd round
Negative Negative Negativeb
Positive Negative Negativeb
Test/cut-off ratio. Fifty microlitre extracted rather than 100 ul.
B19 IgG Biotrin ELISA
In-house ELISA
Biotrin ELISA
Positive 8.2a Positive 6.1a Positive 4.6a
Positive 13.0a Not tested Positive 9.2a
Positive 10.0a Positive 8.7a Positive 7.4a
154
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
levels of B19 IgM and IgG detectable by standard assays (Table 2).
4. Discussion
Fig. 1. Phylogenetic comparison of B19 partial NS gene sequences with other parvoviruses. The B19 clade is shown enlarged eight-fold compared to the rest of the tree because the separation of the B19 genotypes is hard to see when compared with more diverse parvovirus sequences. Sequences homologous with the 511 nucleotide PCR product from the NS gene of B19-SJ, B19-AQ, B19-Nan and B19-Stu (accession number Z68146) were downloaded from the NCBI nucleotide database. These were genotype 2 (G2) B19 sequences A6 1 and 2 (accession numbers AY064475 and AY064476) and LaLi (accession number AY044266); genotype 3 sequence V9 (accession number AJ24937); PARV4 (accession number NC 007018); simian parvovirus, SPV (accession number U06629); minute virus of canines, MVC (accession number NC 004442); bovine parvovirus, BPV (accession number NC 001540); human bocavirus, HboV (accession number DQ000495); Chipmunk parvovirus, ChiPV (accession number CPU86868); pig-tailed and long-tailed macaque parvoviruses, PTMPV and LTMPV (accession numbers AF221123 and AF221122); adeno-associated viruses 1 to 8 (accession numbers NC 002077, AF043303, NC 001729, NC 001829, NC 006152, AF028704, NC 006260 and NC 006261). The best fitting model of substitution found with Paup and Modeltest was the General Time Reversible with a gamma distribution shape parameter of 1.6. The trees shown were found by maximum likelihood searching with bootstrap values (out of 1000) from a congruent distance tree. The scale bars, indicated by 0.1, are in nucleotide substitutions per site and allow the B19 clade to be compared with the other parvoviruses.
between December 2003 and March 2005. During this time 1411 samples were examined and only one sample with the PCR reactivity displayed by B19-AQ was recognised. The patient in whom this strain was detected developed high
Commercial B19 PCR kits are suitable for the detection of parvovirus B19 genotype 1 strains, which are the dominant strains circulating in Europe (Hokynar et al., 2004; Servant et al., 2002). During an evaluation of the Roche B19 LC assay, however, Braham et al. (2004) found that one virus strain with a variant sequence was not detected by this assay. We have now shown that this strain was a genotype 2 strain. This is consistent with evaluations by Hokynar et al. (2004) and Baylis et al. (2004) who also found that the Roche LC assay failed to detect genotype 2 strains. Baylis et al. (2004) and Hokynar et al. (2004), however, both reported that genotype 2 strains were detected by the Artus B19 PCR kit, which we have now confirmed with the strain from patient SJ in this study. This patient failed to make detectable B19 antibodies. As serological cross-reaction between genotypes 1 and 2 has recently been demonstrated (Bl¨umel et al., 2005), this failure is more likely due to immunosuppression than antigenic difference. With respect to genotype 3 strains, both Baylis et al. (2004) and Hokynar et al. (2004) reported that they were not detected by the Roche kit. With the Artus kit, however, we detected a representative genotype 3 strain in the form of plasmid D91.1 (data not shown; plasmid kindly supplied by Servant et al. (2002)). Hokynar et al. (2004) also reported that the Artus kit detected D91.1 but failed to detect a genotype 3 strain in a serum sample containing a V9 isolate. We have also found that the Artus kit failed to detect a genotype 3 strain (patient AQ, Table 2). The viral load in this patient was low since it was detected only after second round amplification in the in-house nested PCR and no virus particles could be seen by electron microscopy (Hazel Appleton, Personal communication). These findings are consistent with the report by Baylis et al. (2004) that the Artus kit had a reduced sensitivity for the detection of genotype 3 strains. We have now demonstrated genotypes 2 and 3 among diagnostic samples in the UK, although the incidence appears to be low; one genotype 2 strain was found in 228 B19 samples and one genotype 3 strain was found in 1411 diagnostic samples. This may be an underestimate as current commercial B19 PCR kits may fail to detect these strains. The failure of PCR assays to detect divergent B19 strains is a consequence of designing primers based on the first strains to be characterised, which were all genotype 1. Therefore, the primers used in diagnostic PCR assays for B19 sequences should be re-evaluated in the light of our results. Although the discovery of B19 variants came as a surprise to researchers accustomed to thinking of B19 as a relatively highly conserved virus, there is, in fact, only a modest amount of genetic divergence among the three genotypes. This can be seen in the phylogenetic tree presented in Fig. 1, where the B19 clade has to be magnified (8×) to show the differences
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
between the three genotypes. These differences become less significant when B19 sequences are compared to other parvovirus sequences, as shown in Fig. 1 (compare the scale bar for the whole tree with that for the B19 clade). Primers chosen on the basis of conserved regions in a multiple sequence alignment of the three B19 genotypes should be capable of amplifying all B19 genomes, and should also allow detection of new sequence variants. As this happens it may be that the distinction between B19 genotypes breaks down and B19 becomes thought of once again as a single, slightly heterogeneous, group.
Acknowledgement We thank David W.G. Brown for advice and encouragement.
References Allander T, Tammi MT, Erikson M, Bjerkner A, Tiveljung-Lindell A, Anderson B. Cloning of human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 2005;102:12891–6. Baylis SA, Shah N, Minor PD. Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma. J Virol Methods 2004;121:7–16. Braham S, Gandhi J, Beard S, Cohen B. Evaluation of the Roche LightCycler parvovirus B19 quantification kit for the diagnosis of parvovirus B19 infections. J Clin Virol 2004;31:5–10. Brown KE, Buckley MM, Cohen BJ, Samuel D. An amplified ELISA for the detection of parvovirus B19 IgM using monoclonal antibody to FITC. J Virol Methods 1989;26:189–98. Bl¨umel J, Eis-H¨ubinger AM, St¨uhler A, B¨onsch C, Gessner M, L¨ower J. Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells. J Virol 2005;79:14197–206. Candotti D, Etiz N, Parsyan A, Allain J-P. Identification and characterisation of persistent human erythrovirus infection in blood donor samples. J Virol 2004;78:12169–78. Clewley JP. Polymerase chain reaction detection of parvovirus B19. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic molecular microbiology. Principles and applications. Washington (DC): American Society for Microbiology; 1993. p. 367–73.
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Erdman DD, Durigon EL, Wang Q-Y, Anderson LJ. Genetic diversity of human parvovirus B19: sequence analysis of the VP1/VP2 gene from multiple sites. J Gen Virol 1996;77:2767–74. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball L, editors. Virus taxonomy, VIIIth report of the ICTV. London: Elsevier/Academic Press; 2005. Gallinella G, Venturoli S, Gentilomi G, Musiani M, Zerbini M. Extent of sequence variability in a genomic region coding for capsid proteins of parvovirus B19. Arch Virol 1995;140:1119–25. Hicks KA, Cubel RCN, Cohen BJ, Clewley JP. Sequence analysis of a parvovirus B19 isolate and baculovirus expression of the non-structural protein. Arch Virol 1996;141:1319–27. Hicks KE, Beard S, Cohen BJ, Clewley JP. Diagnosis of parvovirus B19 DNA by dot-blot hybridisation. In: Stephenson JR, Warnes A, editors. Molecular medicine, diagnostic virology protocols, vol. 12. Totowa (NJ): Humana Press Inc.; 1998. p. 173–88. Hokynar K, S¨oderlund-Venermo M, Pesonen M, Ranki A, Kiviluoto O, Partio EK, et al. A new parvovirus genotype persistent in human skin. Virology 2002;302:224–8. Hokynar K, Norja P, Laitinen H, Palom¨aki P, Garbarg-Chenon A, Ranki A, et al. Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests. J Clin Microbiol 2004;42:2013–9. Jones MS, Kapoor A, Lukashov VV, Simmonds P, Hecht F, Delwart E. New DNA viruses identified in patients with acute viral infection syndrome. J Virol 2005;79:8230–6. McCormack GP, Clewley JP. The application of molecular phylogenetics to the analysis of viral genome diversity and evolution. Rev Med Virol 2002;12:1–20. McCormack GP, Glynn JR, Crampin AC, Sibande F, Mulawa D, Bliss L, Broadbent P, Abarca K, Ponnighaus JM, Fine PEM, Clewley JP. Early evolution of the human immunodeficiency virus type 1 subtype C epidemic in rural Malawi. J Virol 2002;76:12890–9. Mori J, Beattie P, Melton DW, Cohen BJ, Clewley JP. Structure and mapping of human parvovirus B19. J Gen Virol 1987;68:2797–806. Nguyen QT, Sifer C, Schneider V, Allaume X, Servant A, Bernaudin F, et al. Novel human erythrovirus associated with transient aplastic anemia. J Clin Microbiol 1999;37:2483–7. Nguyen QT, Wong S, Heegaard ED, Brown KE. Identification and characterisation of a second novel human erythrovirus variant, A6. Virology 2002;301:374–80. Servant A, Laperche S, Lallemand F, Marinho V, De Saint Maur G, Merinat JF, Garbarg-Chenon A. Genetic diversity within human erythroviruses: identification of three genotypes. J Virol 2002;76:9124–34. Umene K, Nunoue T. A new genotype of human parvovirus B19 present in sera of patients with encephalopathy. J Gen Virol 1995;76:2645–51.
Short communication
Genetic variants of parvovirus B19 identified in the United Kingdom: Implications for diagnostic testing B.J. Cohen, J. Gandhi, J.P. Clewley ∗ Virus Reference Department, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5EQ, United Kingdom Received 10 January 2006; accepted 10 January 2006
Abstract Background: Discrepant results in diagnostic parvovirus B19 PCR assays have been observed with strains showing nucleotide sequence variation. Objectives and study design: To perform phylogenetic analysis on two parvovirus B19 strains that gave discrepant PCR results. Results: One strain was found to be genotype 2; the second strain was genotype 3. Conclusions: Parvovirus B19 genotypes 2 and 3 strains were identified in diagnostic samples of UK origin following the investigation of discrepant PCR results. More structured investigations are needed to estimate the prevalence of these variants. In the meantime, diagnostic PCR results should be interpreted cautiously when they are at variance with serological testing. Manufacturers of PCR kits for the detection of B19 sequences will need to consider re-designing their primers. © 2006 Elsevier B.V. All rights reserved. Keywords: Parvovirus B19 variants; Genotype 2 A6; Genotype 3 V9; Phylogeny
1. Introduction Human parvovirus B19 is classified within the erythrovirus genus of the family Parvoviridae, subfamily Parvovirinae (Fauquet et al., 2005). Early studies showed only limited (up to 4.8% in the structural proteins) genetic variation amongst parvovirus B19 strains (Erdman et al., 1996; Gallinella et al., 1995). However, more recent studies have reported variants with more than 10% sequence divergence compared to reference B19 strains. The first to be described, V9, showed 11–14% sequence divergence to 24 B19 strains (Nguyen et al., 1999). Subsequently, LaLi and related strains were found to be 10.8% divergent from reference B19 strains and 8.6% divergent from V9 in the protein coding region of the genome (Hokynar et al., 2002). In addition, the A6 strain was reported to have a 98% sequence similarity to LaLi (Nguyen et al., 2002). Servant et al. (2002) proposed that parvovirus B19 strains could be grouped into three genotypes: genotype 1 (reference B19 strains); genotype 2 (LaLi and A6) and genotype 3 (V9). ∗
Corresponding author. Tel.: +44 20 8327 6245; fax: +44 20 8327 6019. E-mail address: [email protected] (J.P. Clewley).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.01.011
There is evidence that genetic variation of parvovirus B19 has some temporal and geographical significance (Mori et al., 1987; Umene and Nunoue, 1995) and it has been shown recently that West Africa is an endemic region for V9 (Candotti et al., 2004). There is no evidence so far that any of the variants have distinctive pathological manifestations but, as they may escape detection in commercial PCR assays, they are of diagnostic significance (Hokynar et al., 2004; Braham et al., 2004; Baylis et al., 2004). We have now compared the sequence of PCR NS gene products from two variant parvovirus B19 strains that were not detected in commercial PCR assays with those of reference B19 strains, the recently described human parvoviruses, bocavirus (Allander et al., 2005) and PARV4 virus (Jones et al., 2005) and other parvoviruses.
2. Materials and methods 2.1. Patients and samples Patient SJ: Renal transplant, male, 33 years presenting with anaemia in May 2000 (Braham et al., 2004). Three sera
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
153
Table 1 Results of parvovirus B19 DNA and antibody assays on patient SJ Sample date
Parvovirus B19 DNA Roche LC PCR
26-05-2000 21-06-2000b 16-08-2000 21-08-2000 a b
Negative Negative Negative Negative
Nested PCR
B19 IgM in-house ELISA
B19 IgG Biotrin ELISA
Negative 0.2a Negative 0.2a Negative 0.2a Negative 0.2a
Negative 0.1a Not tested Negative 0.2a Negative 0.1a
Dot blot hybridisation
1st round
2nd round
Positive Positive Negative Negative
Positive Positive Negative Negative
Positive Positive Negative Negative
Test/cut-off ratio. This sample was a plasma (referred to as SJ in Fig. 1); all other samples were sera.
and one plasma sample collected during May–August 2000 were available for testing (Table 1). Patient AQ: Chronic renal failure, female 51 years presenting with anaemia and red cell aplasia in February 2005. Three sera collected during March 2005 were available for testing (Table 2). Positive controls: Two reference B19 strains, Nan and Stu, collected previously (Braham et al., 2004; Hicks et al., 1996).
Briefly, sequences were aligned against representative parvovirus sequences from the NCBI database using Clustal W and were then analysed with Paup. The optimal model of evolution was found using Modeltest.
3. Results 3.1. Results for patient SJ
2.2. Parvovirus B19 DNA and antibody detection Parvovirus B19 DNA was detected by dot blot hybridisation assay (Hicks et al., 1998), in-house nested PCR (Clewley, 1993), Roche LightCycler (LC) parvovirus B19 quantification kit (Roche Diagnostics, Lewes, Sussex, UK) or RealArtTM Parvo B19 LC PCR kit (Artus GmbH, Hamburg, Germany). Parvovirus B19 IgM was detected by inhouse ELISA (Brown et al., 1989) or by commercial ELISA (Biotrin, Dublin, Ireland) and parvovirus B19 IgG detected by commercial ELISA (Biotrin). 2.3. Sequencing and phylogenetic analysis A 511-nucleotide region of the parvovirus B19 NS gene was amplified using primers ‘I’ and ‘F’ (Clewley, 1993). PCR products were excised from a 2% agarose gel and purified with Geneclean (Anachem, Luton, UK) or QIAquick (Qiagen, Crawley, UK) kits. DNA was sequenced with Applied Biosystems kits (Warrington, UK) and sent to DBS Genomics (Durham University) for gel electrophoresis. Phylogenetic analysis was performed as described previously (McCormack and Clewley, 2002; McCormack et al., 2002).
Samples from this patient were first investigated during an evaluation of the Roche LC parvovirus B19 kit (Braham et al., 2004). In this study, two of 228 samples from the UK, both from the same patient, showed 8% sequence divergence compared to reference B19 genotype 1 strains (Braham et al., 2004). Phylogenetic analysis of the strain B19-SJ (Fig. 1) from these samples showed that it aligned with genotype 2 strains, with only 0.5% divergence over the small region of NS that was sequenced. This strain was positive when re-tested with the Artus B19 PCR kit. It is of interest to note that, although this patient subsequently cleared his B19 infection, no antibodies were detected in followup sera using standard parvovirus B19 antibody assays (Table 1). 3.2. Results for patient AQ Phylogenetic analysis of the parvovirus strain B19-AQ (Fig. 1) from this patient showed that it aligned most closely with genotype 3 strains, with about 3% divergence from V9. The strain was not detected during routine diagnostic testing with the Artus B19 LC kit, which was in use in the laboratory
Table 2 Results of parvovirus B19 DNA and antibody assays on patient AQ Sample date
Parvovirus B19 DNA Artus LC PCR
11-03-2005 29-03-2005 30-03-2005 a b
Negative Negative Negative
B19 IgM Nested PCR 1st round
2nd round
Negative Negative Negativeb
Positive Negative Negativeb
Test/cut-off ratio. Fifty microlitre extracted rather than 100 ul.
B19 IgG Biotrin ELISA
In-house ELISA
Biotrin ELISA
Positive 8.2a Positive 6.1a Positive 4.6a
Positive 13.0a Not tested Positive 9.2a
Positive 10.0a Positive 8.7a Positive 7.4a
154
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
levels of B19 IgM and IgG detectable by standard assays (Table 2).
4. Discussion
Fig. 1. Phylogenetic comparison of B19 partial NS gene sequences with other parvoviruses. The B19 clade is shown enlarged eight-fold compared to the rest of the tree because the separation of the B19 genotypes is hard to see when compared with more diverse parvovirus sequences. Sequences homologous with the 511 nucleotide PCR product from the NS gene of B19-SJ, B19-AQ, B19-Nan and B19-Stu (accession number Z68146) were downloaded from the NCBI nucleotide database. These were genotype 2 (G2) B19 sequences A6 1 and 2 (accession numbers AY064475 and AY064476) and LaLi (accession number AY044266); genotype 3 sequence V9 (accession number AJ24937); PARV4 (accession number NC 007018); simian parvovirus, SPV (accession number U06629); minute virus of canines, MVC (accession number NC 004442); bovine parvovirus, BPV (accession number NC 001540); human bocavirus, HboV (accession number DQ000495); Chipmunk parvovirus, ChiPV (accession number CPU86868); pig-tailed and long-tailed macaque parvoviruses, PTMPV and LTMPV (accession numbers AF221123 and AF221122); adeno-associated viruses 1 to 8 (accession numbers NC 002077, AF043303, NC 001729, NC 001829, NC 006152, AF028704, NC 006260 and NC 006261). The best fitting model of substitution found with Paup and Modeltest was the General Time Reversible with a gamma distribution shape parameter of 1.6. The trees shown were found by maximum likelihood searching with bootstrap values (out of 1000) from a congruent distance tree. The scale bars, indicated by 0.1, are in nucleotide substitutions per site and allow the B19 clade to be compared with the other parvoviruses.
between December 2003 and March 2005. During this time 1411 samples were examined and only one sample with the PCR reactivity displayed by B19-AQ was recognised. The patient in whom this strain was detected developed high
Commercial B19 PCR kits are suitable for the detection of parvovirus B19 genotype 1 strains, which are the dominant strains circulating in Europe (Hokynar et al., 2004; Servant et al., 2002). During an evaluation of the Roche B19 LC assay, however, Braham et al. (2004) found that one virus strain with a variant sequence was not detected by this assay. We have now shown that this strain was a genotype 2 strain. This is consistent with evaluations by Hokynar et al. (2004) and Baylis et al. (2004) who also found that the Roche LC assay failed to detect genotype 2 strains. Baylis et al. (2004) and Hokynar et al. (2004), however, both reported that genotype 2 strains were detected by the Artus B19 PCR kit, which we have now confirmed with the strain from patient SJ in this study. This patient failed to make detectable B19 antibodies. As serological cross-reaction between genotypes 1 and 2 has recently been demonstrated (Bl¨umel et al., 2005), this failure is more likely due to immunosuppression than antigenic difference. With respect to genotype 3 strains, both Baylis et al. (2004) and Hokynar et al. (2004) reported that they were not detected by the Roche kit. With the Artus kit, however, we detected a representative genotype 3 strain in the form of plasmid D91.1 (data not shown; plasmid kindly supplied by Servant et al. (2002)). Hokynar et al. (2004) also reported that the Artus kit detected D91.1 but failed to detect a genotype 3 strain in a serum sample containing a V9 isolate. We have also found that the Artus kit failed to detect a genotype 3 strain (patient AQ, Table 2). The viral load in this patient was low since it was detected only after second round amplification in the in-house nested PCR and no virus particles could be seen by electron microscopy (Hazel Appleton, Personal communication). These findings are consistent with the report by Baylis et al. (2004) that the Artus kit had a reduced sensitivity for the detection of genotype 3 strains. We have now demonstrated genotypes 2 and 3 among diagnostic samples in the UK, although the incidence appears to be low; one genotype 2 strain was found in 228 B19 samples and one genotype 3 strain was found in 1411 diagnostic samples. This may be an underestimate as current commercial B19 PCR kits may fail to detect these strains. The failure of PCR assays to detect divergent B19 strains is a consequence of designing primers based on the first strains to be characterised, which were all genotype 1. Therefore, the primers used in diagnostic PCR assays for B19 sequences should be re-evaluated in the light of our results. Although the discovery of B19 variants came as a surprise to researchers accustomed to thinking of B19 as a relatively highly conserved virus, there is, in fact, only a modest amount of genetic divergence among the three genotypes. This can be seen in the phylogenetic tree presented in Fig. 1, where the B19 clade has to be magnified (8×) to show the differences
B.J. Cohen et al. / Journal of Clinical Virology 36 (2006) 152–155
between the three genotypes. These differences become less significant when B19 sequences are compared to other parvovirus sequences, as shown in Fig. 1 (compare the scale bar for the whole tree with that for the B19 clade). Primers chosen on the basis of conserved regions in a multiple sequence alignment of the three B19 genotypes should be capable of amplifying all B19 genomes, and should also allow detection of new sequence variants. As this happens it may be that the distinction between B19 genotypes breaks down and B19 becomes thought of once again as a single, slightly heterogeneous, group.
Acknowledgement We thank David W.G. Brown for advice and encouragement.
References Allander T, Tammi MT, Erikson M, Bjerkner A, Tiveljung-Lindell A, Anderson B. Cloning of human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 2005;102:12891–6. Baylis SA, Shah N, Minor PD. Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma. J Virol Methods 2004;121:7–16. Braham S, Gandhi J, Beard S, Cohen B. Evaluation of the Roche LightCycler parvovirus B19 quantification kit for the diagnosis of parvovirus B19 infections. J Clin Virol 2004;31:5–10. Brown KE, Buckley MM, Cohen BJ, Samuel D. An amplified ELISA for the detection of parvovirus B19 IgM using monoclonal antibody to FITC. J Virol Methods 1989;26:189–98. Bl¨umel J, Eis-H¨ubinger AM, St¨uhler A, B¨onsch C, Gessner M, L¨ower J. Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells. J Virol 2005;79:14197–206. Candotti D, Etiz N, Parsyan A, Allain J-P. Identification and characterisation of persistent human erythrovirus infection in blood donor samples. J Virol 2004;78:12169–78. Clewley JP. Polymerase chain reaction detection of parvovirus B19. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic molecular microbiology. Principles and applications. Washington (DC): American Society for Microbiology; 1993. p. 367–73.
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