Bull Fac. Agric., Cairo Univ., 53(2002): 689 - 706.

" Review article"

MOLECULAR DlVERSITY OF INVERTEDRATEPARVOVIRUSES (Densovirinae)

(Received: 8.5.2002)

Dy

R. EI-Mergawy, G. Fédière, M. EI-Sheikh, M. Bergoin*d P T·· **an • IJssen

Center of Vir%gy, Institut de Recherche pour /e dévelopement (IRD),Facu/ty ofAgriculture, Cairo University, Giza, Egypt, *Laboratoire

de Pathologie Comparée, Université de Montpellier II, 34095Montpellier cédex 5, France and **INRS-Institut Armand-li'rappier,

Université du Québec, Lava/, Canada H7VIB 7.

ABSTRACT

Parvoviruses are defined by having a linear, single-strandedDNA genome. They infect both vertebrates and invertebrates. Thoseoriginating from invertebrates are called densoviruses and constitutethe Densovirinae subfamily within the Parvoviridae.

The study of different biological, biophysical, biochemicalproperties is reviewed. In particular, we focused on the greatdiversity oftheir genomic organization and emphasized the genomicbasis for further classification of the different species of thissubfamily.

A better understanding of these viruses could provide themeans towards a more judicious use of densoviruses in biologicalcontrol of insect pests.

Key words: densovirus, densovirinae, parvoviridae, mo/ecu/ardiversity, virus taxonomy.

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1. INTRODUCTION

Ali small non-enveloped icosahedral viruses that contain alinear, single-stranded DNA genome are c1assitïed within the familyParvoviridae. The parvoviruses of invertebrates, isolated fromseveral species of Arthropoda, in particular Decapoda (shrimps) andInsecta (mainly lepidopteran insects), form the Densovirinaesubfamily (Tijssen and Bergoin, 1995; Bergoin and Tijssen, 2000).In view of the large number of species in these animal orders, it isexpected that the real number of densoviruses is very large and thatthe currently known do not ref1ect the final distribution andproperties of these viruses. Members ofthis group were originallycalled Densonucleosis viruses (DNVs) to describe the characteristichistopathologic symptoms, i.e., hypertrophied and densely-stained(Feulgen) nuclei of infected cells in permissive larvae (Amargier etal., 1965). The name was subsequently shortened to Densovirus forail invertebrate parvoviruses. Currently, this subfamily consists ofthree genera: Densovirus, Brevidensovirus and Iteravirus (Bergoinand Tijssen, 1998).

DNVs are identitïed by a two-Ietter abbreviation ofthe hostname, such as GmDNV for the DNV from Galleria mellone/la, orby a three-Ietter abbreviation (one-letter abbreviation of the genusand two-letter abbreviation of the species) from insects that wouldhave the same two-Ietter abbreviation, such as the DNVs fromLymantaria dispar (LdiDNV) and Leucorrhinia dubia (LduDNV).

Ali DNVs are characterized by an autonomous replication andseparate packaging of either of the complementary, single-strandedDNA strands. The structure and organization of densovirus genomesare more varied than those of the vertebrate parvoviruses. Althoughoriginally densoviruses were c1assified according to biological,biophysicaJ, biochemical or serologicaJ properties, currentclassifications are based on the organization and expression strategyofthe viral genome.

1.1. Biological properties of DNVs1.1.1. Geographical Distribution

Densoviruses are likely to be widely distributed among ail insectspecies ('fable 1). The first DNV was isolated in France in 1964 from

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Table Cl): Distribution of Densoviruses.Host Name Country of Year Ref.

isolation*InsectsLepidopteraAgraulis vaniUae AvDNV United Kingdom 1980 Kelly et al,

Bombyxmori BmDNV-l Japan 1975 ShimizuBombyxmori BmDNV-2 Japan 1983 Seki & lwashitaCasphalia extranea CeDNV Côte d'Ivoire 1981 Fédière et al.,Diatraea saccharalis DsDNV Guad eloupe 1977b Meynadier et al,Euxoa auxilliaris EaDNV United States 1973 SutterGalleria mel/onella GmDNV "'rance 1964 Meynadier et al,

Junonia coenia JeDNV United Kingdom 1972 Rivers & Longworth

Lymantria dispar(cellline) LdtDNV France 1982 GrignonMythimna loreyi MIDNV Egypt 1995 Fédière et aL,

Pieris rapae PrDNV China 1981 Sun et al,

Pseudoplusia inc/udens PiDNV United States 1985 Chao et al,

Sibine fusca SjDNV Colombia 1977a Meynadier et al,

DictyopteraPeriplaneta fuliginosa PjDNV Japan 1979 Suto

DipteraAedes aegypti AaeDNV Soviet Union 1973 Lebedeva et al,Aedes albopictus (ceilline) AalDNV France 1993 Jousset et aL,

Aedes pseu dDseute/laris (cellline) ApDNV Venezuela 1980 Gorziglia et a/.,

Cu/ex pipiens CpDNV France 1998 BaquerizoHaemagogus equinus (cellline) HeDNV United States 1995 O'Neill et al,

Simu/ium vittatum SvDNV llnited States 1976 FedericiToxorhynehites amboinensis (cellline) TaDNV United States 1995 O'Neill et aL,

HemipteraPlanocoeeus citr; PeDNV llSA 2001 MyLoetaL,OdonataLeuco"hinia dubia LduDNV Sweden 1979 CharpentierOrthoDteraAcheta dDmesticus AdDNV France 1977c Meynadier et al,*CrustaceaDecapodaCarcinus mediterraneus CmDNV France 1988 Mari&BonamiMacrobrachium rosenbergü MrDNV Malaisia 1990 Anderson et aL,Penaeus merguiensis PmeDNV Singapour 1985 Lightner&Redman.Penaeus monodon PmoDNV Philippin 1985 Lightner&Redman.Penaeus orientalis PoDNV China 1985 Lightner&Redman..~enaeus semisulCJltus PseDNV Koweit 1985 Lightner&Redman.Penaeus sty/irostris PstDNV Hawü 1989 Lu et al,

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larvae of the greater wax moth Galleria mellonella (Meynadier et al.,1964). Subsequently, other members of this Densovirinae wereisolated from insect orders Lepidoptera, Dictyoptera, Diptera,Odonata, Hemiptera, and Orthoptera, as weil as from Crustacea andDecapoda ail over the world thus providing evidence ofthe ubiquityofDNVs in the phylum of Arthropoda.

1.1.2. Host rangeAs reviewed by Fédière (1996) and Bergoin and Tijssen

(1998) investigations on the host range of DNVs indicate aconsiderable variation. The GmDNV, CeDNV and AdDNV have ahost range apparently restricted to their original hosts (Giran, 1966;Jousset et al., 1986; Fédière et al., 1986). ln contrast, other DNVs,also isolated from lepidoptera, have a broader host range. TheJcDNV can replicate in Aglais urticae, Bombyx mori, Chrysodeixischalcites, Lymantria dispar, Mamestra brassicae, Mamestraoleracea, Scotia ipsilon, Spodoptera exigua, Spodoptera littoralisbut not in G. mellonella (Rivers & Longworth, 1972; Diallo, 1978).Similary, the MlDNV is infectious for Chilo agamemnon, G.mellonella, Ostrinia nubilalis, Pectinophora gossypiella, Sesamiacretica, S. littoralis, S. exigua, Agrotis ipsilon, Agrotis segetum,Agrotis spinifera, Autographa gamma, Phthorimaea opercullella, B.mori and Helicoverpa armigera (Fédière et al., 1999; El-Mergawy etal., 2002). According to their sequences and genome organization,the MlDNV is very closely related to GmDNV (95% identity)(Fédière et al., 1998). The close relationship is interesting since theirtropism differs greatly, GmDNV being monospecific on its host,whereas MlDNV is polyspecific and infects a large number oflepidoptera pests. The striking differences in tropism related to theshort sequence differences offered an ideal system to study thesesequence-function relationships and the allotropie determinants(Tijssen et al., 1998). The host range of EaDNV extends toPseudaletia unipuncta and Heliothis zea (Sutter, 1973). The hostrange of the PjDNV was shown to extend to at least four otherspecies of the genus Periplaneta: P. americana, P. australasiae, P.brunnea and P. japonica (Suto, 1979). The host range of DNVsinfecting mosquitoes extends to different species. Larvae of A.

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albopictus, Aedes cantans, Aedes caspius, Aedes geniculatus, Aedesvexans, Culex pipiens and Culiseta annulata are ail suceptible to peros infection with AaeDNV (Lebedeva et al., 1973). The AaLDNVisolated from a chronically infectcd cellline of the C6/36 clone ofA.albopictus proved to be very pathogenic for A. aegypti and A.metallicus larvae (Barreau et al., 1996). In the case of BmDNV-1,there is no information concerning its cross-infectivity to otherinsects except between B. mori and the pyralid, Glyphodes pyloalis,infecting the mulberry plantations of sericultural farms (Watanabe,1981). Of practical interest for sericulture was the demonstration thatthe susceptibility to DNV infections varied from one strain ofsilkworm to another and that resistant strains could be selected.Among the economically important silkworm strains, severals aresuceptible to BmDNV-2. Almost aIl strains susceptible for BmDNV­1 are resistant to BmDNV-2 and reciprocally, strains resistant toBmDNV-l are sensitive to BmDNV-2 (Seki, 1984). The mode ofinheritance of the resistance to BmDNV infections has beeninvestigated and it was established that for each virus thenonsusceptibility is genetically controlled by a recessive gene that isnot sex linked. A practical aspect ofthis result was to recommendthe rearing of silkworm strains homozygous for the nonsusceptiblegene, in order to avoid DNV epizootics in sericu!turaJ farms (Seki,1984). Finally, it is worth mentioning that despite their highvirulence for their insect hosts, DNVs do not appear to be able toreplicate in vertebrates or marnais, including humans. No pathogeniceffect was detectcd following inoculation of GmDNV, .JcDNV,CeDNV, and .MlDNV to mice or rabbits for production of antisera(Fédière, 1996; Giran, 1966). Similary, no replication ofAaLDNVcould be detected in monkey MA-1 04 and SGM ceIls and in humanHela ce\ls (Jousset et al., 1993).

1.1.3. SymptomsThe densoviruses cause fatal diseases to thcir hosts. The

symptoms of GmDNV infections are similar to those of AvDNV(Agraulis vanillae) and MlDNV infections (Amargier et al., 1965;Fédière, 1996; and Chao et al., 1985). Generally the first symptomsare anorexia and lethargy fol1owed by flaccidity and the inhibition of

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moulting and metamorphosis. During the infection, larvae becomewhitish and progressively paralyzed, followed by a slow melanization(Vago et al., 1966). The cockroach P. fuliginosa infected with thePjDNV displays very characteristic symptoms. Prior to the death, thehind legs are paralyzed and their movements uncoordinated.lnterestingly, fema1es are particulary affected by this DNV (Suto,1979). The abdomen is swollen with an hypertrophied fat body,coJored milky white in contrast to the brownish-white tissues observedin an uninfected cockroach. More than halfofthe infected cockroachdevelop uicers in the hindgut by a process of accumulation ofhemocytes around injured hindgut epithelial ceUs (Suto et al., 1979).Sorne other DNVs produce tumor lesions in the intestine of theirhosts. Typical tumors were observed in heavily infected slugcaterpillar, pests of oil palm, S. fusca and C. extranea (Meynadier etal., 1977a ; Fédière, 1983). The midgut epithelial cells of diseasedlarvae undergo intensive proliferation and the progressive thickeningand opacity of the gut wall screensoff the intestinal content. In thecase of C. extranea, the larval color changes from green to yeIJowishbrown and the transparent gut becomes opaque (Fédière, 1983). Thenymphs of the Swedish dragonfly L. dubia infected with LduDNVbecome sluggish and flaccid, but there is no other external sign ofthedisease (Charpentier, 1979). When silkworm larvae are infected per oswith BmDNV-1, they usually die after seven days showing bodyflaccidity as a major sign. The alimentai canal of the diseased larvae ispale yeIlow with Iittle internaI content (Shimizu, 1975). Mosquitolarvae infected with DNV exhibit symptoms of paralysis.lnterestingly, despite the lack of cytopathic effect in the mosquito cellculture, the DNVs isolated from celllines proved to be pathogenic formosquito larvae by per os infection (Jousset et al., 1993; O'Neill etal., 1995; Barreau et al., 1996; Buchatsky et al., 1987). When tirstinstar larvae ofA. aegypti were infected with AalDNV, the symptomsof the disease appeared at stage IV. Affected larvae lost their mobiJityand hung near to the water surface. Their bodies were distorted andcurved. They lost their pigmentation and exhibited a whitish color.These symptoms appeared one day before death (Barreau et al., 1996).

1.1.4. Histopatbology associated with DensovirusesMost DNVs known sa far are polytropic in tissue tropism. ln

AvDNV, DsDNV, GmDNV, MlDNV, PiDNV, AaeDNV and AalDNV

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infections, almost alliarvai tissues, Le., fat body, hypodermis, centralnervous system, silk gland, muscular membrane, tracheal cells,ma1pighian tubules, foregut, hindgut, hemocytes, ovaries and moltinggland, are susceptible, with the exception of the midgut epithelium(Kelly et al., 1980; Meynadier et al., "1977b; Chao et al., 1985;Lebedeva, 1973; Jousset et al., 1993; Amargier et al., 1965; Vago etal., 1966; Fédière, 1996; Kurstak et al., 1977). On the other hand,DNVs infecting B. mori, C. extranea and S. fusca multiplypredominantly in the columnar cells of midgut epithe1ium (Meynadieret al., 1977a; Fédière, 1996; Watanabe et al., 1976).

The histopathological aspects of DNVs infections arecharacteristic. The main 1esions occur in the nuclei of infected cells.The nuclei become greatly hypertrophied very rapidly and denselystained (eosinophilic) and Feulgen positive (Amargier et al., 1965). Inthe GmDNV infections, the tirst obvious pathological changes occurin cells of the fat body. A voluminous dense homogeneous structureappears in each of the infected nuclei. Later, ail cells becomeprogressively involved (Amargier et al., 1965). In the larval tissues ofAalDNV-infected A. aegypti, no alterations were observed at 2, 3 and4 days postinfection. Anomalies appeared at day 5 principally in ceIlsof the fat body. Later, the dense nuclei appeared in almost ail of thelarval tissues (Barreau et al., 1996). Histopathological studies on themidgut epithelium of the silkworm infected with BmDNV-l showedthat the infected nuclei were more than 2.5 times as large as normalnuclei. At the last stage of infection, the degenerated columnar cellswere 1iberated into midgut lumen (Seki & Iwashita, 1983). In the caseof BmDNV-2 a1most the same features were observed under lightmicroscope (lwashita & Chun, 1982).

1.1.5. U1trastructure of infected cellsUltrastructural studies have led to a comprehensive description

of the pathogenesis of DNV infection in larval tissues of G. mellonella(Vago et al., 1966; Garzon & Kurstak, 1976; Kawase et al., 1990).The tirst ultrastructura1 changes in GmDNV infections are observedboth in the cytoplasm and the nucleus. In the cytoplasm, during thetirst six hours postinfection, polyribosomes disappear and the numberof free ribosomes and the formation of microbody-Iike structuresarising from the accumulation of smal1, spherical particles of 17 to 20

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nm inside the vesicles, increased. This step could represent theaccumulation and transport of viral proteins to the nucleus. In thenucleus, the heterochromatin becomes very condensed and is localizedat the nuclear membrane. The nucleolus undergoes hypertrophy whichis accompanied by a segregation of its fibriIl ar and granularcomponents. The development of the granular portion coïncides withthe synthesis of double-strand DNA of the. replicative form.Simultaneously, a virogenic stroma appears in close vicinity to thenucleolus. As the infection progresses, the granular portion ofthenucleolus regresses in favour of the fibrillar portion. After one or twodays, the virions are assembled inside the virogenic stroma whichinvades the whole nucleus and leads to a nuclear hypertrophy. By dayfour or five, mature virions replace progressively the virogenic stromaand paracrystalline viral concentration takes place, pushing thechromatin and the nucleolus to the nuclear periphery. At the end ofinfection, the nuclei are so hypertrophied that the nuclear envelope isdisrupted, allowing the virions to accumulate in the cytoplasm andviral inclusions, often arranged in paracrystalline arrays that can bethen observed. Similar ultrastructural changes of nuclei infected withother DNVs have been observed (Meyriadier et al., 1977c; Sutter,1973; Suto, 1979; Fédière, 1983; Barreau et al., 1996). In severalDNV-infected insects, the formation of cytoplasmic paracrystallinevirion arrays occurs prior to or without destruction of the nuclearmembrane (Chao et al., 1985; Charpentier, 1979; Diallo, 1978).Althoutgh both DNV-1 and DNV-2 multiply in the nucleus ofcolumnar cell, diffèrence in the ultrastructural studies of infected cellsis obvious when the sections are observed in the electron microscope.On the contrary of GmDNV and BmDNV-1, the virogenic stroma ofceIl infected with BmDNV-2 is less electron-dense than thesurrounding nuclear matrix and occupies most of the nucleus.Discretesites where virions replicate in linear array appear early in infection.These increase in size with each round ofmultiplication until theyeventually fuse (Watanabe & Kurihara, 1988).

1.2. Biophysical and biochemical propertiesAs reviewed by Kurstak et al., (1977) and Tijssen and Bergoin

(1995), DNVsare small (20-25) nm in diameter and thenonenveloped, icosahedral particle contains a linear molecule of

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single- stranded DNA with a size between 4 and 6 Kb. The capsidconsists of 2 to 4 structural polypeptides, generally between 40 and100 kda.

1.3. Molecular characterization of tbe genomcIt is possible to separate the molecular analysis of virus

genomes into two types of approaches: (1) Physical analysis ofstructure and nucleotide sequence (essentially performed in vitro). (2)Genetic analysis of the structure-function relationships of the intactvirus genome and its individual genetic elements (usually involvinganalysis of the virus phenotype in vivo).

As Densoviruses constitute an increasingly diversifiedsubfamily based on the structure and the organization oftheir genome,a new taxonomie structure was proposed (Bergoin and Tijssen, 1998).This subfamily has been subdivided into three genera which can beclustered into five types, three belonging to genus Densovirus and twobelonging to genus lteravirus and Brevidensovirus (Table 2). Asshown in Table (2), the classification was based on:-The size of the genome.-The presence or absence of the inverted terminal repeats.-The size of the extremities.-Genomic organization, ambisense or monosense.-Models of repl ication.-Transcription and translation.

The genome of DNVs contains two sets ofORfs (open readingframes) coding for NS (nonstructural proteins) and VP (structuralproteins), respectively. The great variation in the organization and sizeof these ORFs indicates also a great diversity in their mode ofexpression. Ail the members of the genus Densovirus (Fig. 1) sharethe property of having the NS and VP genes equally distributed in the5' halvcs of the two complementary strands. As exemplified byGmDNV, those infecting Lepidoptera (subgroup A) thus far exhibitan identical organization of their genome. Three ORFs coding forthree NS proteins are present on one strand, the left-most ORF (nearthe 5' extremity) being in frame with, and separated from the largestNS 0 RF (NSI) by on Iy a TAA stop codon, codes for NS3. Thesequence of the third ORF (NS2) is overlapping that of the largest

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Table (2): Classification of Densoviruses.

Genus Densovirus

SubgroupA:(Lepidoptera)

Subgroup B:(Orthoptera)

Subgroup C:(Diptera)

Genus Iteravirus

Genus Brevidensovirus

- Large genome: 5.5-6.0 kb- Large ITRs, both with "flip"/"flop" orientations- Ambisense genomic organization.

- Genome size: 6 kb- ITRs over 500 nt• Four structural proteins (47, 53, 58 and 89 kDa)-Representatives: GmDNV: Gal/eria mellonel/a, JcDNV:Junonia coenia, DsDNV: DÜllrea saccharalis, PiDNV:Pseudoplusia includens, MIDNV: Mythimna loreyi.

- Genome size: 5.5 kb- ITRs shorter than in subgroup A- Four structural proteins (41.5,55,88 and 120 kDa forAdDNV, 48, 52,61 and 76 kDa for PjDNV)- Distinctive properties: splicing within VP mRNA-Representatives: AdDNV: Acheta domesticus, PjDNV:

Periplaneta fuliginosa.

- Genome size: 6kb- ITRs over 300 nt- Four structural proteins (12,57,64 and 90k Da)- Distinctive properties: probable splicing in NS mRNA

Representative:CpDNV:Culex pipinus.

- Genome size: 5 kb- ITRs of about 250 nt, both with "flip''r'flop'' orientations- Monosense genomic organization-Structural proteins: two doublets (49-54 and 74-82 kDa)-Representatives: BmDNV: Bombyx mari, CeDNV:Casphalia extranea.

- Genome size: 4 kb- No ITRs, but terminal hairpins- Monosense genomic organization-Structural proteins: one doublet (38-40 kDa) andsometimes an additional protein at 53 kDa (AaIDNV)-Representatives: AaeDNV: Aedes aegypti,

AalDNV: Aedes albopictus.

o 1000 2000

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3000 4000 5000 600()

Genus Densovirus

JcDNVa ! 52b ~

(6 Kb)~

5' c ITR ITR 3'. 3 ' \'fI d S'TTR ITR

~ ~ ef

Genus Iteravirus CeDNV(5Kb)

ab

NS2 yp

5 ' C __.....__.-.Iu.w....-- ""'- ___

~

ITR

3'

Genus Brevidensovirus 1 AaIDNVI(4 Kb)

a . '..2b

~----:-:-:----~~-----'.....&...----'5 ' C .....:...I..-. ---l. _ 3'

Fig. (] ) : Overall organization of the genomes of insectparvoviruses (Densovirinae).

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ORF and starts 4 nucleotides downstream of the initiation codon ofNS1. A single large ORf codes for the four structural polypeptides onthe complementary strand. In the subgroup B, containing AdDNV,PcDNV and PjDNV, the genomes differ from that of subgroup A bythe presence of two overlapping ORFs on one strand, each codingvery Iikely for a single NS protein, and by the VP gene coding for thefour structural polypeptides on the complemetary strand which is splitinto two reading frames. The CpDNV is so far the only representativeof stlbgroup C. On one strand of its genome two overlapping genescode probably for two NS proteins. The coding sequence of each geneis split into two ORFs, SQ that four ORFs distributed in the threereading frames presumabJy participate to yield these NS proteins(Bergoin and Tijssen, 2000).

DNVs belonging to genera Iteravirus and Brevidensovirus (Fig.1) share :with the vertebrate Parvovirus a monosense organization oftheir genome, i.e., the sequences coding for NS and VP proteins arelocated in the left and the right halves of the same strand, respectively.In both Iteravirus and Brevidensovirus genera, two overlapping üRFsencode for two proteins with the typical motifs of the NS proteinswhereas the structural proteins originate from the right ORF, but theirsize, location and sequence differ considerably.

A shared characteristic of ail Parvoviruses is the presence ofself-priming hairpins at the ends ofthe genome. This enables a hastcell DNA polymerase to convert the single strand into a double strandand, subsequently, a cellular ligase to create covalently linking ofthenewly synthesized strand to the hairpin at the other end of the genome.This double stranded DNA cao then be transcribed by host cell RNApolymerase and be translated into NS protein required for replication(Bergoin and Tijssen, 2000).

CONCLUSIONThe molecular analysis of Densovirus genomes permits an

unequivocal identification of individual genera, species and strains,and is also predictive of the properties of previously unknown ornovel virus.

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El-Mergawy R., Fédière Gilles, El-Sheikh M., Bergoin M.,

Tijssen P. (2002)

Molecular diversity of invertebrate parvoviruses

(Densovirinae)

Bulletin of Faculty of Agriculture - University of Cairo, 53 (4),

689-706

ISSN 0526-8613