| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Division of Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK
2 Blood Systems Research Institute, San Francisco, CA 94118, USA
3 University of California, San Francisco, CA 94118, USA
4 Department of Virology, University College London Hospital, The Windeyer Building, 46 Cleveland Street, London W1T 4JF, UK
5 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105AZ Amsterdam, The Netherlands
6 Laboratory of Immunochemistry, D. I. Ivanovsky Institute of Virology, 123098 Moscow, Russia
Correspondence
Sally A. Baylis
sbaylis{at}nibsc.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the PARV4 and PARV5 sequences determined in this study are DQ873386–DQ873391.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). This individual presented with symptoms of acute viral infection, including fatigue, vomiting, diarrhoea, sore throat, neck stiffness and joint pains, and was also co-infected with hepatitis B virus. Nothing is yet known about the role of PARV4 in human disease. DNA sequence analysis of the PARV4 genome identified two open reading frames, ORF1 and ORF2, with significant similarity to the non-structural and capsid proteins of parvoviruses, respectively. Phylogenetic analysis showed that PARV4 was distinct from other known animal or human parvoviruses (Jones et al., 2005).
The prototype human parvovirus B19 (B19V) is a frequent contaminant of plasma pools used in the manufacture of plasma-derived medicinal products (reviewed by Brown et al., 2001; Laub & Strengers, 2002). PARV4, and a related variant virus termed PARV5, have been identified in approximately 4–5 % of these manufacturing pools (Fryer et al., 2006, 2007b). PARV4 and PARV5 were found to share 92 % nucleotide identity over a 178 bp region of ORF1. In the present study, more extensive sequence analysis has been performed on PARV4 and PARV5 strains identified in recent and older manufacturing plasma pool samples (Fryer et al., 2006, 2007b). Strand-specific probes were used to analyse the packaging of the PARV4 genome.
Nucleic acid was extracted from 1 ml volumes of manufacturing plasma pools, sourced from Europe and North America, as described previously (Baylis et al., 2004). Samples were screened for the presence of PARV4 and PARV5 DNA by using primers to ORF1 and/or ORF2, as described previously (Fryer et al., 2006, 2007b), and nearly full-length PARV4 and PARV5 sequences determined in positive plasma pools. To avoid sequencing on different templates, two approximately 2.5–2.8 kb overlapping cDNA fragments spanning the ORF1 and ORF2 coding regions were amplified from extracted plasma samples. The 5' PARV4/5 fragment (nt 142–2977, GenBank accession no. AY622943) was amplified by using primers PARV4/5seq1 (5'-CGGTCCCGCGAAAATTACGTATT-3') and PVORF2R (Fryer et al., 2007b), whilst the 3' fragment (nt 2710–5247 of GenBank accession no. AY622943) was amplified by using primers PVORF2F (Fryer et al., 2007b) and PARV4/5seq21 (5'-CGCGAAAATTGCGTATTTCCGCT-3'). The reaction mixture consisted of 1x Phusion HF Buffer (Finnzymes OY), 200 µmol each dNTP l–1, 10 pmol each primer, 1 U proofreading Phusion Hot Start DNA Polymerase (Finnzymes OY) and 5 µl extracted DNA in a final volume of 50 µl. Thermal cycling was performed as follows: 98 °C for 10 s, followed by 45 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 3 min. PCR products were purified, cloned into the pT7 Blue vector (Novagen) and both strands were sequenced as described previously (Fryer et al., 2006). Sequences were assembled and ORFs were mapped by using the GCG software package, version 10.2 (University of Wisconsin). Comparisons of deduced protein sequences were performed by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Phylogenetic analysis of nearly full-length PARV4 and PARV5 genomes was performed alongside full genome sequences of other members of the subfamily Parvovirinae (Lukashov & Goudsmit, 2001). Sequences were aligned by using CLUSTAL_W (Chenna et al., 2003), and a neighbour-joining tree (nucleotide distance with Jukes–Cantor correction, pairwise gap deletion) with bootstrap resampling (100 replicates) was constructed by using MEGA3 software (Kumar et al., 2004). Bootscan analysis was performed by using the SimPlot software, version 3.5.1 (Lole et al., 1999).
For analysis of the encapsidation pattern of PARV4 virions, DNA was extracted from a high-titre PARV4-positive individual plasma donation and negative plasma controls as described above. This plasma sample was also positive for hepatitis C virus (HCV) RNA and was identified by a routine screening process to eliminate HCV-positive donations prior to the pooling of plasma for fractionation. Positive and negative plasmid controls were also tested. The PARV4-positive plasmid control contained a 1683 bp insert of PARV4 ORF2 (nt 1705–3387, GenBank accession no. AY622943). The PARV4-negative plasmid control contained a 1471 bp insert of PARV4 ORF1 (nt 603–2073 of AY622943). The PARV4 fragments were cloned into the vector pT7 Blue (Novagen) as described previously (Fryer et al., 2006). All DNA samples were denatured with 0.25 M NaOH at 37 °C for 15 min prior to spotting onto a Hybond XL membrane (GE Healthcare). Nucleic acids were UV cross-linked. Membranes were pre-hybridized with Rapid-hyb buffer (GE Healthcare) in accordance with the manufacturer's instructions. Strand-specific probes were prepared by 5'-end labelling of positive- and negative-sense oligonucleotides in ORF2 of PARV4 (PVORF2F and PVORF2R; Fryer et al., 2007b) by using [
-32P]ATP and T4 polynucleotide kinase (TaKaRa) and used in accordance with the manufacturer's instructions. Replicate filters were hybridized at 42 °C with either the positive- or negative-sense probes. Following hybridization, filters were washed at 42 °C with 0.1x SSC, 0.1 % SDS, in accordance with the manufacturer's instructions (GE Healthcare). Blots were quantified by using the InstantImager (Perkin Elmer) electronic autoradiography system.
Nearly full-length sequences (GenBank accession numbers indicated), comprising ORF1 and ORF2, were determined for the following strains of PARV4: BR10749-4 (DQ873386), BR11955-4 (DQ873388), A23-4 (DQ873389) and C51-4 (DQ873387); and for PARV5, BR10627-5 (DQ873390) and C25-5 (DQ873391). Strains BR10749-4 and BR10627-5 were identified in our preliminary study of manufacturing plasma pools (Fryer et al., 2006), whilst the other strains were identified in further screening studies of plasma pools (Fryer et al., 2007b). Strains BR10749-4, BR11955-4 and BR10627-5 were from plasma samples obtained between 2004 and 2005, whereas A23-4, C51-4 and C25-5 date from 1990–1993.
As with B19V, PARV4 and PARV5 genomes comprise two main ORFs: ORF1 (1992 nt, encoding 664 aa) and ORF2 (2745 nt, encoding 915 aa). However, unlike B19V, the two ORFs do not overlap and are separated by 103 and 106 nt for PARV4 and PARV5, respectively. By BLASTP analysis, PARV4 and PARV5 ORF1-encoded proteins are homologous to the non-structural protein of parvoviruses, NS1, showing greatest amino acid identity with NS1 of hamster (H-1) and goose parvoviruses (50 and 43 % identity, respectively). The amino acid sequence contains parvovirus-conserved motifs associated with rolling-circle replication [xuHuHuuux (aa 97–99) and uxxYuxxKxx (aa 157–161); Ding et al., 2002] and ATPase [A site GxxxxGK(T/S) (aa 334–341) and B site uuuu(D/E)(D/E) (aa 374–379); Astell et al., 1987; Ding et al., 2002]. The ORF2-encoded proteins are homologous to the viral capsid protein of parvoviruses, VP1, sharing approximately 45 and 41 % amino acid identity with VP1 of hamster parvovirus (H-1) and B19V-Au genotype 1, respectively. The amino acid sequence contains phospholipase A2 motifs [YxGxG (aa 224–228) and HDxxY (aa 247–251], required for parvovirus infectivity (Zádori et al., 2001). These motifs are completely conserved between PARV4 and PARV5.
Pairwise comparisons of each ORF of PARV4 and PARV5 strains indicate that ORF2 is more conserved than ORF1 (Table 1), with most nucleotide differences limited to synonymous substitutions (91 % of nucleotide substitutions in ORF2 are synonymous, compared with 89 % in ORF1). Consequently, the amino acid sequences are more conserved, particularly for ORF2, suggesting that if PARV4 and PARV5 represent two distinct genotypes, they might be expected to represent a single serotype. This would be analogous to B19V genotypes 1–3, where serological cross-reactivity has been demonstrated in vitro by using clinical sera against a genotype 2 virus isolate (Blümel et al., 2005) and baculovirus-expressed genotype 3 virus capsid antigens (Parsyan et al., 2006). PARV4 sequences share approximately 98–100 % nucleotide identity with the original PARV4 isolate (GenBank accession no. AY622943), whilst PARV5 sequences share approximately 91–94 % nucleotide identity with the original PARV4 sequence (Table 1). Sequence analysis indicates that there are two subgroups of PARV4, with a nucleotide divergence of 2 % between strains BR11955-4 and A23-4, and the prototype PARV4 sequence (GenBank accession no. AY622943). Overall, PARV4 and PARV5 strains share approximately 92 % nucleotide identity, similar to the level observed between B19V genotypes 1–3 (Servant et al., 2002; Gallinella et al., 2003). Comparison of recent with archived strains within both PARV4 subgroups and PARV5 shows little evidence for evolutionary change over 10–15 years (99.8 % nucleotide identity over an approx. 4800–5000 bp region between recent and archived strains). This is in contrast to current evidence suggesting that carnivore and B19V parvoviruses exhibit a high rate of genetic change (Shackelton et al., 2005; Shackelton & Holmes, 2006).
|
; GenBank accession no. AY622943), nt 128–244 and 5151–5267 respectively correspond to incomplete 5' and 3' ITRs, possibly forming part of the stem structure of the hairpins, with nt 1–128 at the 5' end forming part of a loop structure (an axis of symmetry lies between nt 6 and 7). Incomplete ITRs obtained in this study are distinct between PARV4 and PARV5 (97–98 % nucleotide identity) and are slightly shorter than those of the original PARV4 sequence (GenBank accession no. AY622943), reflecting the length of the sequences obtained. Further sequencing of the ITRs has proved difficult, possibly due to the presence of repeated heptanucleotide sequences (CTTCCGG) identified in the hairpin stems of the original PARV4 sequence (GenBank accession no. AY622943), which are similar to those observed in the ITRs of goose and muscovy duck parvoviruses (Zádori et al., 1995).
Phylogenetic analysis of PARV4 and PARV5 with other members of the subfamily Parvovirinae indicates that they are equidistant from human/primate autonomous parvoviruses and the adeno-associated (AAV)/avian viruses (Fig. 1a). Based upon this nearly full-genome analysis, PARV4 and PARV5 are related most closely to porcine parvovirus 2 (PPV-2), a virus identified in swine sera from Myanmar (Hijikata et al., 2001). Recombination analysis of the PARV4, PARV5 and PPV2 sequences indicates that these viruses do not appear to be recombinants of other known viruses (data not shown). Similarly, there was no evidence for recombination between PARV4 and PARV5, with the clustering pattern of all PARV4 and PARV5 strains being the same regardless of whether full-length sequences or individual ORFs were analysed. At the protein level, however, ORFs 1 and 2 of PARV4 and PARV5 showed closest amino acid identity to the non-structural and capsid proteins of hamster parvovirus H-1, a virus causing significant morbidity and mortality in neonatal hamsters (Besselsen et al., 1999). Phylogenetic analysis again indicates that there are two subgroups of PARV4 (also indicated in Table 1
); strains BR10749-4 and C51-4 cluster with the original PARV4 sequence (GenBank accession no. AY622943), whereas BR11955-4 and A23-4 represent a different subgroup. Phylogenetic analysis of non-synonymous substitutions also demonstrated that PARV4 and PARV5 sequences formed two separate clusters with bootstrap values of 98–100 % (data not shown). Nucleotide similarity plots along nearly full-length PARV4 and PARV5 sequences revealed a highly conserved region (>99 % nucleotide identity) between nt 2955 and 3420 (GenBank accession no. AY622943), located at the 5' end of ORF2 (Fig. 1b). The phospholipase A2 motifs, required for parvovirus infectivity, are located here. Based upon PARV4 and PARV5 sequence alignments of this region, we have designed a consensus real-time TaqMan PCR to quantify both PARV4 and PARV5 sequences (Fryer et al., 2007b).
|
) and PARV4 ORF2 control plasmid DNA (Fig. 2, plasmid concentrations indicated), but not to PARV4 ORF1 control plasmid DNA (data not shown) or to human genomic DNA present in the plasma pools, even when levels were of the order of 106 copies ml–1 (sample B, Fig. 2). When the number of counts binding to the PARV4-positive plasma DNA sample was determined and compared with the respective plasmid control samples, it was found that the positive and negative strands were present in similar amounts. This packaging pattern is not characteristic of all parvovirus genera (Cotmore & Tattersall, 2006).
|
, 2007; Hokynar et al., 2004). In some cases, assays fail to detect the newly identified genotypes 2 and 3 of B19V, with consequent issues in terms of clinical diagnosis and viral load determinations in the plasma-fractionation setting, where it is a regulatory requirement to limit levels of B19V (Baylis et al., 2004). The ability to design assays for PARV4 and PARV5 will assist in future studies to determine the role of these viruses in human disease. Nothing is yet known of the pathology associated with these viruses or whether PARV4 and PARV5 cause subclinical infections. However, PARV4 and PARV5 have been identified in plasma from healthy blood donors (Fryer et al., 2006, 2007b) with an increased incidence in febrile patients, including IVDUs and homosexual men (Fryer et al., 2007b), and in HCV-infected individuals (including IVDUs) (Fryer et al., 2007a). Phylogenetic analysis performed in this study shows that PARV4 and PARV5 cluster separately from each other, and could thus be considered as two genotypes of the same virus. We therefore propose the use of a single virus name, PARV4, comprising genotypes 1 and 2 (previously termed PARV5).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Astell, C. R., Mol, C. D. & Anderson, W. F. (1987). Structural and functional homology of parvovirus and papovavirus polypeptides. J Gen Virol 68, 885–893.
Baylis, S. A., Shah, N. & Minor, P. D. (2004). Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma. J Virol Methods 121, 7–16.[CrossRef][Medline]
Baylis, S. A., Fryer, J. F. & Grabarczyk, P. (2007). Effects of probe binding mutations in an assay designed to detect parvovirus B19: implications for the quantitation of different virus genotypes. J Virol Methods 139, 97–99.[CrossRef][Medline]
Besselsen, D. G., Gibson, S. V., Besch-Williford, C. L., Purdy, G. A., Knowles, R. L., Wagner, J. E., Pintel, D. J., Franklin, C. L., Hook, R. R., Jr & other authors (1999). Natural and experimentally induced infection of Syrian hamsters with a newly recognized parvovirus. Lab Anim Sci 49, 308–312.[Medline]
Blümel, J., Eis-Hübinger, A. M., Stühler, A., Bönsch, C., Gessner, M. & Löwer, J. (2005). Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells. J Virol 79, 14197–14206.
Brown, K. E., Young, N. S., Alving, B. M. & Barbosa, L. H. (2001). Parvovirus B19: implications for transfusion medicine. Summary of a workshop. Transfusion 41, 130–135.[CrossRef][Medline]
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. (2003). Multiple sequence alignment with the CLUSTAL series of programs. Nucleic Acids Res 31, 3497–3500.
Cotmore, S. F. & Tattersall, P. (2006). Structure and organization of the viral genome. In Parvoviruses, pp. 73–94. Edited by J. R. Kerr, S. F. Cotmore, M. E. Bloom, R. M. Linden & C. R. Parrish. London: Hodder Arnold.
Ding, C., Urabe, M., Bergoin, M. & Kotin, R. M. (2002). Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1. J Virol 76, 338–345.
Fryer, J. F., Kapoor, A., Minor, P. D., Delwart, E. & Baylis, S. A. (2006). Novel parvovirus and related variant in human plasma. Emerg Infect Dis 12, 151–154.[Medline]
Fryer, J. F., Lucas, S. B., Padley, D. & Baylis, S. A. (2007a). Parvoviruses PARV4/5 in hepatitis C virus–infected patient. Emerg Infect Dis 13, 175–176.[Medline]
Fryer, J. F., Delwart, E., Hecht, F. M., Bernardin, F., Jones, M. S., Shah, N. & Baylis, S. A. (2007b). Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals. Transfusion 47, 1054–1061.[CrossRef][Medline]
Gallinella, G., Venturoli, S., Manaresi, E., Musiani, M. & Zerbini, M. (2003). B19 virus genome diversity: epidemiological and clinical correlations. J Clin Virol 28, 1–13.[CrossRef][Medline]
Hijikata, M., Abe, K., Win, K. M., Shimizu, Y. K., Keicho, N. & Yoshikura, H. (2001). Identification of new parvovirus DNA sequence in swine sera from Myanmar. Jpn J Infect Dis 54, 244–245.[Medline]
Hokynar, K., Söderlund-Venermo, M., Pesonen, M., Ranki, A., Kiviluoto, O., Partio, E. K. & Hedman, K. (2002). A new parvovirus genotype persistent in human skin. Virology 302, 224–228.[CrossRef][Medline]
Hokynar, K., Norja, P., Laitinen, H., Palomäki, P., Garbarg-Chenon, A., Ranki, A., Hedman, K. & Söderlund-Venermo, M. (2004). Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests. J Clin Microbiol 42, 2013–2019.
Jones, M. S., Kapoor, A., Lukashov, V. V., Simmonds, P., Hecht, F. & Delwart, E. (2005). New DNA viruses identified in patients with acute viral infection syndrome. J Virol 79, 8230–8236.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Laub, R. & Strengers, P. (2002). Parvoviruses and blood products. Pathol Biol 50, 339–348.[CrossRef][Medline]
Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type I genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73, 152–160.
Lukashov, V. V. & Goudsmit, J. (2001). Evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses. J Virol 75, 2729–2740.
Nguyen, Q. T., Sifer, C., Schneider, V., Allaume, X., Servant, A., Bernaudin, F., Auguste, V. & Garbarg-Chenon, A. (1999). Novel human erythrovirus associated with transient aplastic anemia. J Clin Microbiol 37, 2483–2487.
Nguyen, Q. T., Wong, S., Heegaard, E. D. & Brown, K. E. (2002). Identification and characterization of a second novel human erythrovirus variant, A6. Virology 301, 374–380.[CrossRef][Medline]
Parsyan, A., Kerr, S., Owusu-Ofori, S., Elliott, G. & Allain, J. P. (2006). Reactivity of genotype-specific recombinant proteins of human erythrovirus B19 with plasmas from areas where genotype 1 or 3 is endemic. J Clin Microbiol 44, 1367–1375.
Servant, A., Laperche, S., Lallemand, F., Marinho, V., De Saint Maur, G., Meritet, J. F. & Garbarg-Chenon, A. (2002). Genetic diversity within human erythroviruses: identification of three genotypes. J Virol 76, 9124–9134.
Shackelton, L. A. & Holmes, E. C. (2006). Phylogenetic evidence for the rapid evolution of human B19 erythrovirus. J Virol 80, 3666–3669.
Shackelton, L. A., Parrish, C. R., Truyen, U. & Holmes, E. C. (2005). High rate of viral evolution associated with the emergence of carnivore parvovirus. Proc Natl Acad Sci U S A 102, 379–384.
Shade, R. O., Blundell, M. C., Cotmore, S. F., Tattersall, P. & Astell, C. R. (1986). Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. J Virol 58, 921–936.
Tattersall, P., Bergoin, M., Bloom, M. E., Brown, K. E., Linden, R. M., Muzyczka, N., Parrish, C. R. & Tijssen, P. (2005). Family Parvoviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 353–369. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego: Elsevier Academic Press.
Zádori, Z., Stefancsik, R., Rauch, T. & Kisary, J. (1995). Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2. Virology 212, 562–573.[CrossRef][Medline]
Zádori, Z., Szelei, J., Lacoste, M. C., Li, Y., Gariépy, S., Raymond, P., Allaire, M., Nabi, I. R. & Tijssen, P. (2001). A viral phospholipase A2 is required for parvovirus infectivity. Dev Cell 1, 291–302.[CrossRef][Medline]
Received 4 October 2006;
accepted 30 April 2007.
This article has been cited by other articles:
|
|
 |
|
  P. Simmonds, J. Douglas, G. Bestetti, E. Longhi, S. Antinori, C. Parravicini, and M. Corbellino A third genotype of the human parvovirus PARV4 in sub-Saharan Africa J. Gen. Virol., September 1, 2008; 89(9): 2299 - 2302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. P. Lau, P. C. Y. Woo, H. Tse, C. T. Y. Fu, W.-K. Au, X.-C. Chen, H.-W. Tsoi, T. H. F. Tsang, J. S. Y. Chan, D. N. C. Tsang, et al. Identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4 J. Gen. Virol., August 1, 2008; 89(8): 1840 - 1848. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
