Parvoviruses

Peter Tattersall, Yale University, New Haven, Connecticut, USA Susan F Cotmore, Yale University, New Haven, Connecticut, USA
Parvoviruses are amongst the smallest known animal viruses, consisting of a singlestranded DNA genome of approximately 5 kb, encapsidated in an icosahedral protein shell built from 60 copies of a single polypeptide. Viral DNA is replicated by a unique, unidirectional, leading-strand specific mechanism dubbed rolling hairpin replication, which depends critically upon a virally-coded nickase, and the sequential folding and unfolding of small palindromic terminal sequences.

Secondary article
Article Contents
. Introduction . Classification . Structure . Replication . Epidemiology . Pathogenesis . Use as Genetic Vectors

Introduction
Viruses from the family Parvoviridae are small, nonenveloped, icosahedral particles, 1828 nm in diameter. Infectious particles (called virions) contain a single copy of the linear, nonpermuted, single-stranded deoxyribonucleic acid (DNA) genome, 46 kb long, whose palindromic terminal sequences are capable of folding back on themselves to form small hairpin duplexes. These terminal hairpins play a critical role in the viral replication strategy. The Parvoviridae are major pathogens of insects and domestic animals, but only one virus, B-19 virus, the causative agent of fth disease, has so far been identied as a human pathogen. Several members are essentially apathogenic, and are currently gaining recognition as successful vectors for the delivery of vaccines or therapeutic genes. (see Virus taxonomy.) (see Parvovirus infections in humans.) Similarities of genome structure, organization and coding sequence throughout the family suggest that they all replicate their DNA via a unique rolling hairpin mechanism (see Figure 3), which is a linear adaptation of the more common rolling circle mechanism. In this process the palindromic viral telomeres are sequentially unfolded, copied and refolded, to allow a single replication fork to shuttle back and forth along the linear genome, creating palindromic duplex multimers, which are subsequently resolved to monomeric lengths prior to displacement and packaging of progeny single strands. (see DNA plant and animal virus replication.) (see Telomeres.) Parvoviral genomes typically contain just two gene cassettes: one encodes the nonstructural or Rep proteins essential for viral gene expression and DNA replication; and the other encodes an overlapping set of capsid polypeptides. This limited coding potential renders them highly dependent on the synthetic machinery of their host cell, which accordingly they have become masters at subverting. Members of one genus, Dependovirus (also known as the adeno-associated viruses or AAVs), have overcome their limited gene repertoire by resorting to a pattern of latent infection, only switching to a productive

life cycle when their host cell is coinfected with a helper adenovirus or herpesvirus. The helper virus reprogrammes the hosts cell cycle for its own use, but is rapidly overtaken by the resident parvovirus. In contrast, the helperindependent or autonomously replicating viruses from the genus Parvovirus, which are the major focus of this article, have evolved to use the synthetic machinery of cells of particular dierentiated phenotypes. Control of such cell-type specicity can occur at the level of cell entry, but is also frequently manifested at later stages in the viral life cycle. (see DNA virus genomes.) (see Adeno-associated viruses.) (see Herpesviruses (human).) (see Hepatitis delta virus.) Viral dependence on its host cell is rendered all the more extreme because the viral transcriptional promoters are embedded in single-stranded DNA, and cannot operate until that DNA is rendered duplex, a process that generally requires DNA replication. In consequence, virions characteristically remain quiescent in the newly infected host cell until it enters S phase as a result of its own cell cycle programme. At this time, the synthetic machinery provided for cellular DNA replication converts the viral genome into a transcriptionally active duplex. Unlike other DNA viruses, parvoviruses do not encode gene products that can coerce resting cells to enter S phase, and, probably as a consequence of this, they are universally nontumorigenic and are often oncosuppressive. (see DNA replication.)

Classification
Members of the family Parvoviridae are divided into two subfamilies, as summarized in Table 1, depending upon whether they infect vertebrates (mammals and birds, the Parvovirinae) or invertebrates (arthropods, the Densovirinae). Each subfamily is currently subdivided into three genera, grouped according to their genetic relatedness and life style (Berns et al., 1995). (see Virus taxonomy.)

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Table 1 Classication of the family Parvoviridae Subfamily Parvovirinae Genus Dependovirus Erythrovirus Parvovirus Densovirus Contravirus (also called Brevidensovirus) Iteravirus Type species Adeno-associated virus 2 (AAV2) B19 virus Minute virus of mice (MVM) Jujonia coenia densovirus (JcDNV) Aedes aegypti densovirus (AaDNV) Bombyx mori densovirus (BmDNV)

Densovirinae

The subfamily Parvovirinae

This subfamily contains the genera Dependovirus, Erythrovirus, and Parvovirus. Dependoviruses are a closely related group, which generally depend, for vegetative replication, upon coinfection with an adenovirus or herpesvirus. In the absence of their helper, these viruses can integrate into the host genome both randomly and sitespecically into a small region of human chromosome 19q13-qter. The dependoviruses have transcriptional promoters at 5, 19 and 40 map units, have terminal repeat sequences rather than unique termini, and package DNA strands of both senses in equal numbers in separate particles. There are six known human serotypes, of which the best studied is AAV2, and some less well characterized isolates from birds, dogs, cows, sheep and horses. The genetic strategy of Dependoviruses is described in detail elsewhere in this Encyclopedia. (see Adeno-associated viruses.) First recognized in the 1980s, Erythrovirus is a relatively new taxonomic group, which contains closely related, helper-independent viruses that have exquisite tissue specicity for cells of the erythroid lineage. The type species, human B19 virus, and the closely related Simian parvovirus (SPV), Pig-tail macaque parvovirus (PtPV) and Rhesus macaque parvovirus (RhPV) package equal numbers of positive and negative-sense DNA strands in separate particles, have relatively large inverted terminal repeats, and appear to use a single promoter at map unit 6. These viruses access their two gene cassettes by dierential splicing and the use of two alternate polyadenylation sites. An additional, closely-related but less well-characterized species has recently been isolated from the Manchurian chipmunk (Tamias sibiricus asiaticus). B19 is discussed in detail elsewhere in this Encyclopedia. In contrast to the above genera, the genus Parvovirus contains a somewhat disparate group of helper-independent viruses of major importance in veterinary medicine but, surprisingly, to date contains no known viruses of humans. The type species, Minute virus of mice (MVM), packages a single negative-sense DNA strand with hairpins that dier in both sequence and structure at the two ends of the genome. MVM is one of a group of closely related but serologically distinct parvoviruses, listed in Table 2, which
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predominantly infect rodents. In contrast, Feline panleukopenia virus (FPV), Canine parvovirus (CPV), Mink enteritis virus (MEV) and Racoon parvovirus (RPV) are important pathogenic members of a single distinct serotype. Members of this serotype, together with Porcine parvovirus (PPV), are quite closely related to the rodent viruses by DNA sequence. There are several other, less obviously related species currently included in the genus, but these should be reclassied as separate genera. As shown in Table 2, they include Bovine parvovirus (BPV), Minute virus of canines (MVC), and Aleutian mink disease virus (AMDV). Members of one additional outlying serotype, containing the goose and Muscovy and Barbary duck parvoviruses, are more closely related by DNA sequence and genome structure to dependoviruses than to the other parvoviruses. All Parvovirus species have two overlapping transcription units with promoters at 4 and 38 map units from the 3 (left-hand) end, and a single polyadenylation site at around map unit 95. Most package 4 90% negative strand, but some package positive strands, in separate virions, in variable amounts up to 50%.

The subfamily Densovirinae

This currently includes at least 30 distinct virus species, divided into three genera, Densovirus, Contravirus (or Brevidensovirus) and Iteravirus. They infect arthropods from at least four orders of Insecta, namely Lepidoptera (butteries), Diptera (ies), Orthoptera (crickets), Dictyoptera (cockroaches) and, possibly, Odonata (dragonies), and one order of Crustacea (Decapoda) infecting shrimps and possibly crabs. Although molecular analysis is proceeding rapidly (Tijssen and Bergoin, 1995), there remain many viruses in this subfamily that have not been characterized well enough to be assigned to one of the three recognized genera. Recombinant densoviruses are also being explored as potential agents of biological control. Members of the genus Densovirus are currently only known to infect lepidopterans. This is the only ambisense group of parvoviruses, encoding nonstructural proteins on one strand from a promoter at 7 map units from the viral

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Table 2 Members of the genus Parvovirus Species (serotype) The rodent group Mice minute virus Prototype strain Immunosuppressive strain Mouse parvovirus LuIII virusa H-1 virusb Rat virus (Kilham rat virus or H-3 virus) Rat parvovirus The feline panleukopenia complex Feline panleukopenia virusc Canine parvovirusc Raccoon parvovirusc Mink enteritis virusc The porcine group Porcine parvovirus The AAV-related group Goose parvovirusd Muscovy duck parvovirusd Distantly related members Aleutian mink disease virusd Bovine Parvovirusd Canines minute virusd Unassigned Lapine parvovirus
a b

Acronym MMVp MMVi MPV LuIIIV H-1PV RV (KRV/H-3PV) RV-1a FPV CPV RPV MEV PPV GPV MDPV AMDV BPV CMV LPV

Accession # J02275 M12032 U12469 M81888 X01457 U79033 AF036710 M75728 NC_001539 M24005 J02275 NC_001718 NC_001701 U22967 NC_001662 M14363 Not available Not available

Host species unknown. Infects rats and hamsters. c All viruses cross-neutralize. d Distantly related to rodent, porcine and feline groups, probably separate genera.

left-hand end and capsid proteins from the complementary strand using a promoter 9 map units in from the viral righthand end. Virions package, into separate particles, equal numbers of both strands, each of which is around 6 kb long with inverted terminal repeats. Under conditions that do not favour replication, some members have been found to integrate their genomes into host chromosomal DNA. In addition to the type species, Junonia coenia densovirus (JcDNV), members include Galleria mellonella densovirus (GmDNV), Mythimna loreyi densovirus (MlDNV), Culex pipiens densovirus (CpDNV), Acheta domestica densovirus (AdDNV), Diatraea saccharalis densovirus (DsDNV) and, probably, Agraulis vanillae densovirus (AvDNV). (see Viral genome.)

The Contravirus species, which are currently only known to infect Aedes mosquitoes and shrimp, have genomes which, at just over 4 kb, are signicantly smaller than are other members of the family. These viruses package negative-sense strands predominantly (  90%) and have unique termini. An alternative name, Brevidensovirus, has recently been proposed for this genus to reect the unusually small size of their genomes and to avoid the suggestion, implicit in the old name, that they are ambisense (Tijssen and Bergoin, 1995). The type species is Aedes aegypti densovirus (AaDNV), while other members include Aedes albopictus densovirus (AlDNV), and Hypodermal haematopoietic necrosis virus (IHHNV) of shrimp.
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The genus Iteravirus currently comprises two species. The type species, Bombyx mori densovirus (BmDNV), infects the silkworm Bombyx mori. This virus has inverted terminal repeats, two overlapping transcription units, and encapsidates both positive-and negative-sense strands. It is of economic signicance in Asia, where it is responsible for huge losses in the silk industry. The second Iteravirus, Casphalia extranea densovirus (CeDNV), infects the oil palm pest Casphalia extranea. The remainder of this article will focus, almost exclusively, on the properties of members of the genus Parvovirus. Some other members of the Parvoviridae family are the subjects of separate reviews in this Encyclopedia.

and up the adjacent edge of the threefold spike. Empty viral particles, generated in large numbers in many parvoviral infections, exhibit essentially identical surface features. In virions, however, some of the single-stranded DNA also displays icosahedral symmetry, so that about a third of the genome can be visualized, by crystallography, within the particle shell. This DNA is oriented with its bases pointing outwards, forming a number of proteinbase hydrogen bonds with the inner surface of the capsid shell. A single molecule of the viral nonstructural protein NS1 is located on the outside of newly released virions, covalently attached to the extreme 5 terminus of the genome by a 24 nucleotide, single-stranded tether sequence, which projects through the capsid shell. (see Virus structure.) (see Viral membrane, envelope and capsid.) (see Macromolecular structure determination by X-ray crystallography.)

Structure
The virion
Viral genomes are encapsidated in roughly spherical protein capsids of 1828 nm diameter, in which 60 copies of the coat protein are related by T 5 1 icosahedral symmetry. All capsid proteins share a C-terminal sequence of approximately 60 kDa, which makes up the protein shell of the virion. N-terminal extensions of various lengths are present on some of these polypeptides, so that mature particles contain 24 distinct polypeptide species of between 6083 kDa (designated VP1, VP2, etc.), incorporated in precise molar proportions. The three-dimensional structures of parvoviruses CPV, FPV and MVM, and the densovirus GmDNV have been determined to near atomic resolution by X-ray crystallography, revealing an eightstranded antiparallel b-barrel structure in which the bstrands are connected by elaborate and highly variable loops, which make up most of the viral surface. The disposition and function of the various N-terminal peptide domains are less well understood. As seen in Figure 1 (Agbandje-McKenna et al., 1998), the most striking surface feature of MVM is a hollow cylindrical structure, which surrounds each icosahedral vefold axis, forming a in diameter) that connects the central cavity of pore ( 4 8 A the virion with the particle exterior. In full virions, each pore is occupied by a glycine-rich sequence from a single VP2 molecule, positioned so that about 25 N-terminal amino acids are externalized. On the outer virion surface these cylindrical structures are themselves encircled by ) canyon-like depressions of unknown deep ( 5 15-A function. Other surface structures include a prominent long and 70 A in diameter, protruding from spike,  22 A the icosahedral threefold axes, and a deep depression at the twofold axes. Two neutralizing antibody-binding sites have been mapped to the shoulders of the threefold spike, and sequences that determine viral host range and oligosaccharide recognition lie in the twofold depression
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The genome
Infecting particles contain a single copy of the linear, nonpermuted, monopartite DNA genome, in which a relatively long single-stranded coding region (  5 kb) is bracketed by short (121421-nucleotide) palindromic terminal sequences, capable of folding into hairpin duplexes. Although some parvoviruses encapsidate DNA strands of either polarity, others, such as MVM, selectively package strands that are negative sense with regard to transcription. The duplex viral telomeres, enlarged 20-fold relative to the rest of the genome in Figure 2 to make them conspicuous, are critical for both DNA replication and encapsidation. They are in many ways the hallmarks of the parvoviral genome, but they vary markedly in size and structure between the various species and genera. (see DNA virus genomes.) (see Telomeres.) As mentioned previously, parvoviral genomes are organized into two separate gene cassettes. By convention, transcription is represented as proceeding left to right, placing the 3 end of the predominantly negative-sense packaged strand at the left side of the genetic map, as shown in Figure 2 for MVM. The left cassette gives rise to a small number of nonstructural proteins, which are involved in gene regulation and DNA replication, while the right cassette programmes synthesis of the overlapping set of capsid proteins. Although parvoviruses may encode several smaller accessory nonstructural proteins, such as the NS2 species depicted in the MVM map, only NS1 is absolutely required for replication in all cell types (NS2 functions are reviewed in Bodendorf et al., 1999). NS1 is an adenosine triphosphate (ATP)-dependent, site-specic DNA-binding protein with helicase activity, which initiates DNA replication at specic viral origin sequences by introducing a singlestrand nick, thus providing a base-paired 3 nucleotide to serve as a primer for successive rounds of strand displacement DNA synthesis (reviewed in Cotmore and

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Figure 1 Depth-cued image of minute virus of mice virion, constructed from the atomic model, viewed down a fivefold axis, showing the cylindrical pore that penetrates to the inside of the particle at each of these 12 vertices. The red triangle denotes the crystallographic asymmetric unit, the smallest repeating segment of the structure. Reprinted from Agbandje-McKenna et al. (1998). Copyright 1998, with permission from Elsevier Science.

Tattersall, 1996). During nicking, NS1 becomes covalently attached to the 5 nucleotide at the nick site. All replicons that use rolling circle or rolling hairpin replication mechanisms encode their own initiator proteins, and two protein motifs in NS1, representing a putative metal ion coordination site and the active site tyrosine of the nickase, can be traced from the Parvoviridae to their prokaryotic cousins. MVM NS1 is an 83-kDa nuclear phosphoprotein that self-associates in the presence of ATP to form an oligomeric complex, capable of binding to a consensus DNA motif found both in the viral origins and at many other locations throughout the genome. However, it is only able to nick the DNA at the viral origins, where it encounters the appropriate consensus nick sequence and specic host-derived accessory proteins (Christensen et al., 1997; Cotmore et al., 2000). (see Protein motifs for DNA binding.) Parvoviral initiators have evolved into highly pleiotropic proteins, playing multiple roles in the viral life cycle.

They act as potent transactivators of viral gene transcription, binding to their recognition sequences in each viral promoter and activating transcription through acidic Cterminal domains. In MVM, NS1 binding sites are reiterated so frequently that any sequence of 100 bp or more contains a site, and some carry multiple tandem and inverted reiterations. This suggests that NS1 is likely to play a signicant role in viral chromatin structure and progeny strand packaging.

Replication
Early events
Relatively little is known about viral entry or the mechanisms involved in delivering the viral genome to
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P4

P38

NS1

AAAAA

NS2Y

AAAAA

NS2P

AAAAA

NS2L

AAAAA

NS3

AAAAA

VP1

AAAAA

VP2

AAAAA

Frame 1

Frame 2

Frame 3

Figure 2 The coding strategy of the minute virus of mice genome, showing the positions of the two promoters, P4 and P38, within the negative-strand viral DNA molecule, with its terminal hairpins, displayed above the mRNA splicing strategy. The coloured boxes indicate the reading frame that encodes each segment of viral polypeptide. NS2 occurs in three forms, differing in their C-terminal hexapeptide sequences. NS3 is the putative product of readthrough of the single terminator codon of NS2P, which generates a 90 amino acid C-terminal extension, conserved throughout the rodent parvoviruses.

the host nucleus for replication. MVM receptors on murine broblasts are sialoglycoproteins, present at about 5x105 copies per cell, but binding is neuraminidase-sensitive, indicating a critical role for specic oligosaccharide sidechains in this interaction. In contrast, CPV/FPV appear to recognize species-specic protein domains in transferrin receptor molecules, so that infectious entry of these viruses remains insensitive to neuraminidase (J. S. L. Parker and C. R. Parrish, personal communication). Penetration by both MVM and CPV then proceeds via coated pits and acidied endosomes, and can be blocked by drugs that inhibit endosomal acidication, such as balomycin, for several hours after the virion has been internalized from the cell surface. This suggests that infectious entry may occur via a late endosomal/lysosomal compartment. Since these are rich in proteases and nucleases, such exposure would explain why genomes loose their covalently-linked 5 NS1 molecules, and the nucleotide tether sequence, before arrival in the nucleus, leaving a heterogeneous, but basepaired, 5 sequence. Exactly where, or how, the viruses penetrate the endosomal bilayer remains open to con6

jecture, but infectivity can be blocked by the intracytoplasmic injection of various antibodies directed against structural determinants in the capsid, indicating that there must be an essential, capsid-associated, cytoplasmic phase (Vihinen-Ranta et al., 2000). (see Viral replication.) (see DNA plant and animal virus replication.) (see Virus host cell interaction.) (see Viral host cell receptors.) (see Clathrin-coated vesicles and receptor-mediated endocytosis.) Parvoviral particles are very stable and are small enough to be transported through the nuclear pore, so that viral DNA may remain encapsidated until it is inside the nucleus. N-terminal sequences specic to the VP1 capsid protein carry karyopherin-a binding sequences, which probably serve to trac particles to the nuclear pore. The VP1 N-terminus also contains a consensus phospholipase 2 active site motif, which is highly-conserved among the Parvoviridae and appears to be essential for infectivity, although its role and site of action remain to be elucidated (Z. Zadori and P. Tijssen, personal communication). While VP1 N-terminal sequences are sequestered within mature virions before entry, they can become accessible at the

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R (i)

R (ii)

R (iii) r

(iv) r

L R

r (vi)

L R R

L R

(vii)

(v)

R r

L r l (viii) r

L l (ix)

L r l

R r

l L R

Figure 3 Rolling hairpin replication. The sequence of the parental parvoviral genome is shown in blue, and those of progeny genomes are shown in yellow. The green circle represents NS1, which nicks the covalently-continuous monomer and remains attached to its 5 end. DNA, which is newly synthesized in each step, is indicated by the black bar with an arrow at its 3 end. L and R depict the palindromic sequences at each terminus, with their complements represented by l and r, respectively. Step (ix) produces a tetramer in which there are three progeny genomes, in addition to the parental sequence. These genomic sequences overlap and are distributed throughout the molecule on alternate strands.

particle surface following conformational transitions, inducible in vitro by heating. Such transitions externalize the VP1 N-terminus and the viral DNA, while leaving the DNA still rmly attached to the otherwise intact particle via its left-end hairpin. Anti-VP1 specic antibodies injected into the cytoplasm block infectivity, suggesting that a similar transition may accompany entry into this cellular compartment in vivo. Docking of intact particles at the nuclear pore, and/or their translocation into the nucleus, has yet to be demonstrated for members of the Parvovirinae during natural infection. However, this is a likely route since labelled CPV virions injected into the cytoplasm eventually accumulate in the nucleus after a protracted (  6 h) lag period (Vihinen-Ranta et al., 2000). (see Nuclear–cytoplasmic transport.) (see Nuclear pores: methods for preparation.) Parvoviruses are unable to induce resting cells to enter S phase, and it is not until infected cells enter S phase in their own cell cycle that viral transcription initiates. Since the viral promoters are packaged as single-strand DNA, synthesis of the complementary strand necessarily precedes viral gene expression, and likely represents the rst S phase-dependent step in the viral life cycle. Initial transcription also depends upon the availability of transcription factor E2F to activate the MVM P4 promoter, so that viral transcription is optimized for expression during S phase. As the early gene products

accumulate, host cell DNA synthesis is terminated and progression through the cell cycle is suspended, but the molecular mechanisms underlying these reactions have yet to be established. (see Cell cycle.)

DNA replication
Parvoviral DNA is replicated through a series of duplex, concatemeric intermediates by the rolling hairpin mechanism depicted in Figure 3. The replication fork is aphidicolinsensitive, requires proliferating cell nuclear antigen (PCNA), is unidirectional and results in the synthesis of a single, continuous DNA strand, indicating that replication is probably mediated by polymerase d and its accessory proteins. In step (i) the base-paired 3 nucleotide of left-end hairpin is used by a host polymerase to prime conversion of virion DNA to the rst duplex intermediate. This generates a monomer length duplex molecule in which the two strands are covalently continuous at the viral left-end telomere. Synthesis of this intermediate precedes viral gene expression. Since the cellular replication fork is unable to displace and copy the right-end hairpin sequence, the 3 end of the new DNA strand is ligated to the 5 end of the hairpin by a host ligase, creating a covalently continuous duplex molecule (step ii). Replication beyond this point requires expression of NS1, which carries out a hairpin transfer
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reaction, in which it nicks the ligated strand, as illustrated in step (iii), in a reaction that, in vitro, requires the host chromosomal high mobility group 1 protein (HMG1). The replication fork now unfolds and copies the hairpin, thus replacing the original sequence of the terminus with its inverted complement (step iv). Since the terminal sequences are imperfect palindromes, and this inversion occurs with every round of replication, progeny genomes comprise equal numbers of each terminal orientation, dubbed ip and op. This hairpin transfer reaction occurs only at the MVM right-end, because of the dierent structural and cofactor requirements needed to activate the NS1 nickase at left-end termini, as discussed below. When it occurs on the rst monomer formed after uncoating, it regenerates the tether sequence, lost during entry, now attached to a newly synthesized NS1 molecule. (see DNA replication.) (see Eukaryotic DNA polymerases.) (see Eukaryotic replication fork.) Extended-form right-end termini are melted out and reformed into hairpin rabbit ear structures in a process facilitated by the direct binding of NS1 to sequences in the terminus (step v). This allows the newly synthesized DNA to create the base-paired hairpin structures needed to prime synthesis of additional linear sequences (step vi). The result of rolling hairpin synthesis is the accumulation of palindromic duplex dimeric (step vii) and tetrameric (step ix) concatemers, in the latter of which alternating unit length genomes are fused in left-end:left-end and rightend:right-end orientations. Individual genomic monomer duplexes are then excised from these concatemers by a process called junction resolution (reviewed in Cotmore and Tattersall, 1996). (see Base pairing in DNA: unusual patterns.) (see DNA: natural single-stranded.) During resolution, left-end sequences are replicated and separated asymmetrically, so that new termini are created in a single sequence orientation, ip, rather than the alternative, ip and op, orientations generated from the right-hand end. Although several steps in this resolution process remain to be elucidated, the early steps have been dissected for MVM. The 121-base left-end telomere of MVM has internal palindromic sequences of unknown function, which form the ears of the hairpin, and a mismatch bubble in the hairpin stem where a trinucleotide opposes a dinucleotide. During rolling hairpin synthesis, the hairpin is replicated to form the left-end:left-end junction, which links monomer genomes in the dimer intermediate (Figure 3, steps viiix), in which these asymmetric nucleotides segregate to opposite sides of the junction. It is this duplex junction structure, rather than a single hairpin terminus, that contains the replication origin required for copying and separating left-end sequences. The junction contains one candidate nick site for NS1 on each arm of the palindrome, located on opposite strands. However, only the origin on the arm carrying the bubble dinucleotide has the precise spatial organization required for NS1 to be activated for nicking. This is because, at the
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left-end origin, activation requires a cellular protein, parvoviral initiation factor (PIF), to bind the viral DNA in exact juxtaposition to NS1. This interaction occurs only across the dinucleotide, and not the trinucleotide, where no activation occurs. (see Eukaryotic replication origins and initiation of DNA replication.) The replication fork initiating at this origin is unidirectional and supports synthesis of a single continuous DNA strand. How the resulting sequences are rearranged to allow the nal resolution of two separate telomeres remains uncertain, but available evidence suggests that an obligate cruciform intermediate is formed, which could theoretically be resolved by cellular recombinases or by NS1, acting as a nickingjoining enzyme, to eect the asymmetric resolution observed. Why such a complex mechanism is employed to preserve the single, ip sequence orientation of the viral left-hand hairpin is, as yet, unclear.

Packaging
Late in infection, viral capsid gene expression predominates because NS1 is able to strongly transactivate the P38 promoter. VP1 and VP2 are synthesized in approximately the ratio required for capsid assembly, and partially assembled capsid subunits, probably in the form of trimers, are translocated into the nucleus. Here they are assembled into empty capsids, which are believed to be the precursors of full virions. Some aspect of the continued synthesis and assembly of the capsid precursors requires the small nonstructural polypeptide NS2, at least in MVM infections of murine cells. As assembled capsids accumulate, progeny single-strand DNA synthesis begins to predominate over duplex DNA amplication. How this switch is controlled is unknown, but progeny synthesis is entirely dependent upon the availability of preformed capsids, and all single-strand monomeric DNA in the cell appears to be encapsidated. However, since packaging requires ongoing viral DNA synthesis, it is not simply a process of melting out and sequestering preformed DNA strands. Infections are generally lytic, and cells remain actively synthesizing viral DNA until lysis or apoptosis occurs about 1620 h after entry into S phase, resulting in the release of progeny virions. However, virions appear to be able to exit the cell before general lysis occurs, and this phenomenon also requires NS2, even in cells in which NS2 is not required for capsid assembly (Cotmore et al., 1997). (see Apoptosis: molecular mechanisms.)

Epidemiology
In natural populations parvoviral infections are generally widely and rapidly disseminated. This is in part due to high level viraemias and ecient oral and/or faecal transmission

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routes, and in part to the fact that infectious particles are exceptionally rugged. Thus, when CPV, a new virus of the feline panleukopenia serotype, emerged as a pathogen of dogs in 1978, it spread through most of the world within a few weeks, most probably because it remained fully viable in faecal material, contaminating the clothes and shoes of international airline passengers. Exactly how long the shed parvoviral virions retain viability in nature is uncertain, but they are very resistant to most normal ambient temperature ranges, and in the laboratory they are very stable; for example, at 8<C the half-life for MVM infectivity is more than 9 months. Statistics for virus distribution in wild animal populations are not readily available. Many, such as MPV and H1, are known to be widespread in wild rodents, while in human populations in Europe and North America about 60% of adults are seropositive for B19, while 80% are seropositive for AAV. Infection is generally acute and self-limiting, with transient viraemias that last for 710 days while the host seroconverts. However, some viruses, such as Rat virus (RV), persist despite high neutralizing antibody titres, so that infected individuals may routinely shed virus for many months. Fetal and neonatal animals can be eectively protected during this highly susceptible period by maternal immunity, so that vaccination of the entire population against teratogenic viruses is a routine procedure in veterinary medicine.

Although most adult tissues are mitotically quiescent compared to those of the fetus, some, such as gut epithelium and the lymphopoietic system, contain large numbers of cycling cells. A small subset of autonomous parvoviruses, namely the feline panleukopenia complex, can cause fatal disease in adult animals involving extensive destruction of gut epithelium and reticuloendothelial cells. As discussed below, however, most parvoviruses exhibit variable degrees of tissue specicity, which restrict their replication in these available tissues. AMDV is unusual in that it gives rise to dierent pathologies in young and adult mink, causing acute pneumonia in mink kits, but a persistent infection in adults, involving antigen-presenting cells of the immune system, that results in immune complex-mediated glomerular nephritis.

Tissue and species specificity

Lytic growth is modulated by developmentally regulated factors operating in the host at the cellular level. Dierences in pathogenic potential exist not only between virus serotypes, but between virus strains of the same serotype, mediated in the case of MVM by a region of the viral capsid called the allotropic determinant. This determinant involves a small group of amino acids that map near the surface of the virion, grouped along the edge of the spike at the threefold symmetry axis and down into the depression at the twofold axis. Here a coordinated change in just a few amino acid residues can change the tissue or species specicity of a virus. Thus, for example, mutating one or two amino acids in the capsid of the lymphotropic murine virus MVMi allows it to extend, or even switch, its host range, gaining the ability to replicate productively in murine broblasts. Among members of the FPV/CPV group, a similar cluster of surface amino acid changes control canine and feline host ranges, determining the ability of the virus to replicate in canine cells in culture and to infect dogs in vivo. Genetically and pathogenically, distinct strains of PPV exhibit allotropic specicity that is determined in a similar way and controls their ability to replicate in particular porcine cell lines in vitro. Permissivity to AMDV replication has been similarly linked to a cluster of capsid residues. In each of these cases, infection of the wrong host cell is profoundly restricted at an unidentied step that occurs after virus internalization from the cell surface but before the onset of transcription and DNA amplication. The molecular basis for these dierences in tropism is still uncertain, and may perhaps vary somewhat between viruses. For FPV and CPV, recent developments suggest that host range is, at least in part, determined by the ability of the viruses to bind transferrin receptor molecules of the appropriate host species (J. S. L. Parker and C. R. Parrish, personal communication). This suggests that, for these viruses, the critical step is likely to involve receptor9

Pathogenesis
Disease spectra
Many of the Parvovirinae have coevolved with their host to such an extent that infection usually remains subclinical. Thus, for example, B19 in most people gives rise to little more than an innocuous rash-like illness, while infection with AAV is essentially asymptomatic. Members of the autonomously replicating Parvovirinae are dependent upon cellular functions expressed transiently during the S phase of the cell cycle, but since they cannot induce resting cells to enter S phase, productive viral replication is restricted to proliferating cells, both in vitro and in vivo. As lytic parasites of dividing cell populations, these viruses are potentially teratogenic agents, causing fetal and neonatal abnormalities by destroying specic cell populations that are rapidly proliferating during the normal course of development. These same tissues are often quiescent, and therefore resistant, in the adult. Indeed, the rst autonomous parvoviruses were discovered because of their teratogenic potential when injected into pregnant laboratory rodent hosts. (see Parvovirus infections in humans.) (see Neonatal infectious disease.) (see Cytopathic eects of viruses.)

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mediated internalization of the virus in a way that selectively leads to its successful translocation across the endosomal bilayer. In contrast to the case of CPV, cells lacking transferrin receptors are fully able to internalize both strains of MVM into endosomes, and to support productive replication. However, a similar mechanism, whereby MVM can be internalized into endosomes on a variety of receptors, but can only penetrate the bilayer successfully from a particular receptor species or type, remains possible. Some precedent for this type of restriction has been documented for the small plaque viruses of polyoma, but restriction on the parvovirus scale, whereby most of the bound virus would have to be presented on incompetent receptors, has yet to be described. Alternatively, MVM host range restriction could involve a selective interaction between the virion and cell typespecic host molecules that mediate nuclear entry, viral uncoating or the setting up of the initial viral transcription templates. (see Virus host cell interaction.) (see Polyomaviruses.)

promoter upregulation is known to be controlled by two separate transcription factor binding sites upstream of its TATA box, one of which binds CREB/ATF family members while the other is specic for members of the Ets family. Transformation-induced modulation of normal cell cycle control would thus appear to be capable of promoting viral replication, and pathogenicity, by impinging upon several dierent processes in the viral life cycle. (see Parvovirus B19 culture.) (see Polyomaviruses.) (see Oncogenes.) (see Cell cycle: regulation by cyclins.)

Use as Genetic Vectors

Members of the autonomously replicating Parvovirinae have been explored for use as gene therapy vectors using two alternate packaging strategies, which produce vectors with dierent characteristics and potential areas of usefulness. In one strategy the transgene is sandwiched between the terminal hairpins of the virus, and the viral nonstructural and structural genes are supplied in trans. This complete coding sequence replacement eectively packages the transgene in the parvovirus coat in the absence of other viral genes. Technical advantages of this strategy are that it cuts down on homology between vector and helper plasmid, and thus reduces the generation of replication competent virus, and it allows the transgene to be expressed under the control of a promoter of choice. Unlike dependoviruses, members of the parvovirus genus have yet to be shown to integrate into host chromosomes, but some are known to persist for long periods of time in the infected animal. (see Human gene therapy.) (see Gene delivery by viruses.) Complete coding sequence replacement vectors are not able to replicate in transduced cells, and do not take advantage of several unique aspects of parvoviral infection, such as their inherent oncotropism. Moreover, since they only mimic the very earliest stages of viral infection, they fail to exploit the complex interaction of the virus with host cell defences. For this reason, a second type of vector, in which only the capsid gene is replaced by a transgene, while the vector itself encodes the viral nonstructural genes, has been explored. Such constructs are generally replicated and packaged more eciently than complete coding sequence replacement vectors, but have more limited coding potential, as packaging is restricted to DNA strands of approximately the same size (5 kb) as the wild-type viral genome. Such viruses are thus, theoretically, better suited to serve as vectors for gene therapy in applications where: (1) transient high-level expression from an episomal viral genome in a replicating subpopulation of target cells is required; or (2) persistent, low-level reexpression of the transgene over a protracted period is an advantage, but where long-term stable transgene expression in all target

Oncotropism
The anity of parvoviruses for dividing cells is also reected in their ability to interfere with, and suppress, another type of rapid cellular proliferation in their hosts, namely, neoplastic disease. In particular, infection with autonomous parvoviruses has been shown to suppress tumour formation by a large number of viruses and carcinogens. For example, RV suppresses leukaemia induction in rats by Moloney leukaemia virus, and H1 infection of hamsters suppresses tumour formation by both adenovirus and dimethylbenzanthracene. (see Oncogenic viruses.) Despite their apparent lack of natural infectivity for humans, rodent parvoviruses can grow in many human cell lines in vitro, and this is especially true if the cells are transformed by agents such as gamma rays, nitroquinolines or Simian virus 40 (SV40). Extensive studies over the past two decades, focusing on MVM, have dened in vitro analogues of this oncotropism, and have established it as a cell-intrinsic, rather than immune-mediated, phenomenon. Sensitization to parvovirus-mediated cell killing has been demonstrated following transformation of rodent and human broblasts, human keratinocytes and human mammary epithelium. Transforming agents used have varied from activated oncogenes, such as h-ras and v-src, to c-myc and papovaviral T antigens. The biochemical basis for such sensitization is complex, and can vary from system to system. In some cells it manifests as a dramatic, transformation-associated increase in viral DNA amplication, while in others it can present as transcriptional upregulation of the P4 promoter, combined with a signicant lowering of the threshold concentration for viral nonstructural protein toxicity in the cell. In rastransformed cells, a signicant proportion of this P4
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Parvoviruses

cells is not required. In the rst scenario, transient highlevel transgene expression is being explored for tumourspecic immunotherapy, in which immune costimulatory molecules and/or cytokine expression is targeted to tumour cells, or strategies in which the expression of toxin(s) or suicide genes is required. In the latter case, a bystander eect from toxin or suicide gene expression can often be observed, either reducing tumour burden or stimulating natural tumour-specic immunity by providing a critical mass of dying cells for ecient antigen presentation on scavenging phagocytes. (see Virology.) Since parvoviruses are the only family of DNA viruses that have no tumorigenic members, and that can indeed be markedly oncosuppressive under some circumstances, such vectors are likely to be relatively safe. Nonintegrative vectors, in particular, pose little risk for insertional mutagenesis in the host chromosome, while in vectors of the capsid-replacement type, the rst viral gene expressed is the antiproliferative viral nonstructural protein, NS1. This inhibits further cell cycle progression and renders tumorigenic transformation of the infected cell even less probable. Viruses, such as MVM, that encapsidate strands of a single sense, cannot self-anneal inside the cell to support transgene expression before viral DNA replication, providing an additional safety feature that restricts transgene expression to actively dividing host cells. Their potential utility in a clinical setting, especially in developing countries, is considerably greater than that of many other viral vectors, because they package their genomes in such extremely rugged virions, and so should require little or no sophisticated equipment for maintenance as viable stocks. The incidence of positive antibody titre to rodent parvoviruses in human populations is extremely low, indicating that these viruses lack the ability to infect humans by any natural route. However, it is well established that rodent parvoviruses, including MVM, can grow in many human cell lines in vitro, and direct parenteral inoculation of H-1 virus into human patients has been shown to result in transient viraemia without observable morbidity. Recombinant versions of these viruses may therefore provide ideal vectors for potent, high level transgene expression in circumstances where targeted transient expression is desired and cell death is either desired or

inconsequential. The most likely scenarios for their eective use are thus in the elds of cancer immunotherapy and vaccination. (see Tumours: immunotherapy.) (see Cancer therapy, the future.)

References
Agbandje-McKenna M, Llamas-Saiz AL, Wang F, Tattersall P and Rossmann MG (1998) Functional implications of the structure of the murine parvovirus, minute virus of mice. Structure 6: 13691381. Berns KI, Bergoin M, Bloom M et al. (1995) Parvoviridae. Archives of Virology Supplement 10: 169178. Bodendorf U, Cziepluch C, Jauniaux JC, Rommelaere J and Salome N (1999) Nuclear export factor CRM1 interacts with nonstructural proteins NS2 from parvovirus minute virus of mice. Journal of Virology 73: 77697779. Christensen J, Cotmore SF and Tattersall P (1997) A novel site-specic DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication. Journal of Virology 71: 1405 1416. Cotmore SF and Tattersall P (1996) Chapter 28: Parvovirus DNA replication. In: DePamphilis M (ed.) DNA Replication in Eukaryotic Cells, pp. 799813. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Cotmore SF, DAbramo AM Jr, Carbonell LF, Bratton J and Tattersall P (1997) The NS2 polypeptide of parvovirus MVM is required for capsid assembly in murine cells. Virology 231: 267280. Cotmore SF, Christensen J and Tattersall P (2000) Two widely spaced initiator binding-sites create an HMG1-dependent parvoviral rollinghairpin replication origin. Journal of Virology 74: 13321341. Tijssen P and Bergoin M (1995) Densonucleosis viruses constitute an increasingly diversied subfamily among the parvoviruses. Seminars in Virology 6: 347355. Vihinen-Ranta M, Yuan W and Parrish CR (2000) Cytoplasmic tracking of the canine parvovirus capsid and its role in infection and nuclear transport. Journal of Virology 74: 48534859.

Further Reading
Abstracts of the VIIIth Parvovirus Workshop, June 28July 2 (2000). Mont-Tremblant, Quebec, Canada. Infectious Disease Review 2: 135 177. Corsini J, Afanasiev B, Maxwell IH and Carlson JO (1996) Autonomous parvovirus and densovirus gene vectors. Advances in Virus Research 47: 303351. Parrish CR (ed.) (1995) Autonomous animal parvoviruses. Seminars in Virology 6 (4).

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