Genomic characterization of the unclassified bovine enteric virus Newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae

The pathogenic bovine enteric virus, Newbury agent-1 (Bo//Newbury1/1976/UK), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in CsCl and the presence of a single capsid protein but genomic sequence could not be obtained. In the present study, the complete genome sequence of Newbury1 was determined and classified Newbury1 in a new genus of the Caliciviridae. The Newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (ORFs). ORF1 encoded the non-structural and contiguous capsid proteins. ORF2 encoded a basic protein characteristic of the family Caliciviridae. Compared to the 4 recognized Caliciviridae genera, Norovirus, Sapovirus, Lagovirus and Vesivirus, Newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2C-helicase, 3C-protease, 3D-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized NB virus in these regions. By phylogenetic analyses, Newbury1 and NB viruses formed a distinct clade independent of the 4 recognized genera. However, amino acid identities showed that Newbury1 and the NB virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). Analyses of contemporary viruses showed that the two polymerase genotypes, Newbury1 and NB, were circulating in UK cattle and antibody to Newbury1-like viruses was common in cattle sera. The present study defined the existence of a new genus in the Caliciviridae that we propose be named Becovirus or Nabovirus to distinguish the new clade from bovine noroviruses.     

Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).     

Molecular characterization of Hop latent virus and phylogenetic relationships among viruses closely related to carlaviruses

Summary.  The complete nucleotide sequence of the hop latent virus (HpLV) genome was determined. The viral RNA genome is 8,612 nucleotides long, excluding the poly(A) tail, and contains six open reading frames (ORFs), which encode putative proteins of 224-kDa (ORF 1), 25-kDa (ORF 2), 11-kDa (ORF 3), 7-kDa (ORF 4), 34-kDa (ORF 5), and 12-kDa (ORF 6). ORF 5 encodes the coat protein as demonstrated by N-terminal sequencing of three proteolytic peptides derived from the virus particle. The genome organization of HpLV is similar to that of other species in the genus Carlavirus, and the overall sequence of HpLV is more similar to that of Potato virus M than to sequences of other carlaviruses reported to date. The amino acid sequences of the putative methyltransferase, RNA helicase, and RNA-dependent RNA polymerase encoded in ORF 1 and an ‘accessory’ helicase encoded in ORF 2 of the HpLV genome were compared with those of viruses in the ‘tymo’ lineage: the genera Carlavirus, Potexvirus, Allexivirus, Foveavirus, Trichovirus, Capillovirus, Vitivirus, and Tymovirus. The phylogenetic relationships among the viruses in these genera are discussed. This is the first molecular characterization of a carlavirus infecting hop plants. Accepted June 15, 2000 Received January 19, 2000     

Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.     

Complete sequence of the Pepino mosaic virus RNA genome

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.     

Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus

The complete genome sequence of a virus recently detected in switchgrass (Panicum virgatum) was determined and found to be closely related to that of maize rayado fino virus (MRFV), genus Marafivirus, family Tymoviridae. The genomic RNA is 6408 nucleotides long. It contains three predicted open reading frames (ORFs 1-3), encoding proteins of 227?kDa, 43.9?kDa, and 31.5?kDa, compared to two ORFs (1 and 2) for MRFV. The complete genome shares 76?% sequence identity with MRFV. The nucleotide sequence of ORF2 of this virus and the amino acid sequence of its encoded protein are 49?% and 77?% identical, respectively, to those of MRFV. The virus-encoded polyprotein and capsid protein aa sequences are 83?% and 74-80?% identical, respectively, to those of MRFV. Although closely related to MRFV, the amino acid sequence of its capsid protein (CP) forms a clade that is separate from that of MRFV. Based on the International Committee on Taxonomy of Viruses (ICTV) sequence-related criteria for delineation of species within the genus Marafivirus, the virus qualifies as a member of a new species, and the name Switchgrass mosaic virus (SwMV) is proposed.     

Sequence analysis of a reovirus isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae) and its parasitoid wasp Phobocampe tempestiva (Hymenoptera: Ichneumonidae)

A reovirus was isolated from Operophtera brumata (ObRV) and its parasitoid wasp Phobocampe tempestiva. Each of the 10 dsRNA genome segments of ObRV was sequenced and shown to contain a single open reading frame (ORF). Conserved motifs ([+ve] 5'-AAATAAA ...(G)/(T)AGGTT-3') were found at the termini of each segment, with the exception of Seg-6 and Seg-8, where the 5' termini were 5'-AACAAA...-3'. The putative proteins encoded by each segment were compared with those of other members of the family Reoviridae. Phylogenetic comparisons to published sequences for the RNA-dependent RNA polymerase genes from other reoviruses indicated that ObRV is most closely related to members of the genus Cypovirus. However, unlike the cypoviruses, ObRV has a double-layered capsid structure. When the protein encoded by ObRV Seg-10 was expressed (by inserting the open reading frame into a baculovirus expression vector) no 'occlusion bodies' were observed in the recombinant baculovirus infected insect cell cultures. This suggests that unlike the cypoviruses, Seg-10 of ObRV does not contain a polyhedrin gene. Further phylogenetic comparisons also identified relationships between Seg-2 and Seg-10 of ObRV, and genes of Diadromus pulchellus Idnoreovirus 1 (DpIRV1), suggesting that ObRV represents a new species from the genus Idnoreovirus.     

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.     

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus

Summary.  The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae. Accepted September 23, 1999     

A novel member of the family Hepeviridae from cutthroat trout (Oncorhynchus clarkii)

Beginning in 1988, the Chinook salmon embryo (CHSE-214) cell line was used to isolate a novel virus from spawning adult trout in the state of California, USA. Termed the cutthroat trout (Oncorhynchus clarkii) virus (CTV), the small, round virus was not associated with disease, but was subsequently found to be present in an increasing number of trout populations in the western USA, likely by a combination of improved surveillance activities and the shipment of infected eggs to new locations. Here, we report that the full length genome of the 1988 Heenan Lake isolate of CTV consisted of 7269 nucleotides of positive-sense, single-stranded RNA beginning with a 5' untranslated region (UTR), followed by three open reading frames (ORFs), a 3' UTR and ending in a polyA tail. The genome of CTV was similar in size and organization to that of Hepatitis E virus (HEV) with which it shared the highest nucleotide and amino acid sequence identities. Similar to the genomes of human, rodent or avian hepeviruses, ORF 1 encoded a large, non-structural polyprotein that included conserved methyltransferase, protease, helicase and polymerase domains, while ORF 2 encoded the structural capsid protein and ORF 3 the phosphoprotein. Together, our data indicated that CTV was clearly a member of the family Hepeviridae, although the level of amino acid sequence identity with the ORFs of mammalian or avian hepeviruses (13-27%) may be sufficiently low to warrant the creation of a novel genus. We also performed a phylogenetic analysis using a 262nt region within ORF 1 for 63 isolates of CTV obtained from seven species of trout reared in various geographic locations in the western USA. While the sequences fell into two genetic clades, the overall nucleotide diversity was low (less than 8.4%) and many isolates differed by only 1-2 nucleotides, suggesting an epidemiological link. Finally, we showed that CTV was able to form persistently infected cultures of the CHSE-214 cell line that may have use in research on the biology or treatment of hepevirus infections of humans or other animals.     

Tepovirus, a novel genus in the family Betaflexiviridae

Tepovirus is a new monotypic genus of plant viruses typified by potato virus T (PVT), a virus with helically constructed filamentous particles that are 640 nm long, previously classified as unassigned species in the family Betaflexiviridae. Virions have a single-stranded positive-sense polyadenylated RNA genome that is 6.5 kb in size, and a single type of coat protein with a size of 24 kDa. The viral genome contains three slightly overlapping ORFs encoding, respectively, the replication-related proteins (ORF1), a putative movement protein of the 30 K type (ORF2) and the coat protein (ORF3). Its structure and organization (number and order of genes) resembles that of trichoviruses and of citrus leaf blotch virus (CLBV, genus Citrivirus) but has a smaller size. Besides potato, the primary host, PVT can experimentally infect herbaceous hosts by mechanical inoculation. No vector is known, and transmission is through propagating material (tubers), seeds and pollen. PVT has a number of biological, physical and molecular properties that differentiate it from betaflexiviruses with a 30K-type movement protein. It is phylogenetically distant from all these viruses, but least so from grapevine virus A (GVA), the type member of the genus Vitivirus, with which it groups in trees constructed using the sequences of all of the genes.     

The complete nucleotide sequence of Beet black scorch virus (BBSV), a new member of the genus <Emphasis Type="Italic">Necrovirus</Emphasis>

The complete nucleotide sequence of Beet black scorch virus (BBSV) was determined. The BBSV genome is composed of 3641 nucleotides and has similar organization with Tobacco necrosis virus D of 61% nucleotide identity. The 5'-proximal open reading frame (ORF) encodes a putative 23 kDa protein and a 82 kDa protein by reading-through of an amber termination codon. Three small ORFs located in the center of the genome may encode for a 4.2 kDa protein and two 7 kDa proteins. The 3'-proximal ORF encodes a 24.5 kDa protein equivalent in mass to the viral coat protein. Considering biological and molecular similarities with TNV, it is concluded that BBSV is a new member of the genus Necrovirus.     

A double-stranded RNA as the genome of a potential virus infecting <Emphasis Type="Italic">Vicia faba</Emphasis>

Preparations of double-stranded (ds) RNAs extracted from naturally infected Vicia faba Linn. growing in Hangzhou, Zhejiang Province, Eastern China displayed 3 dominant bands (FaR1, FaR2, and FaR3). FaR2 and FaR3 were found to be identical to the genomic dsRNAs of a recently reported Vicia cryptic virus (VCV). The positive strand of FaR1 contained two large open reading frames (ORFs), ORF1 and ORF2. The putative proteins encoded by these ORFs were found to have certain similarities to the putative capsid protein [ABO36237] and RNA-dependent RNA polymerase [ABC96788], respectively, of Tomato yellow stunt virus. Thus, FaR1 may represent the genome of a new dsRNA virus, which we have named Vicia cryptic virus M. The GenBank Accession numbers of the sequences reported in this paper are EU605883, EU605884, and EU371896.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus A

Sclerophthora macrospora virus A (SmV A) found in S. macrospora, the pathogenic fungus responsible for downy mildew of gramineous plants, is a small icosahedral virus containing three segments (RNAs 1, 2, and 3) of the positive-strand ssRNA genome. In the present study we report the complete nucleotide sequence of the SmV A genome. The viral genome RNA 1 consists of 2928 nucleotides (nt) and has two open reading frames (ORFs 1a and 1b). ORF 1a contains the motifs of RNA-directed RNA polymerase (RdRp). The function of ORF 1b is unknown. RNA 2 consists of 1981 nt and single ORF (ORF 2). ORF 2 encodes a capsid protein. RNA 3 consists of 977 nt but not any ORFs, suggesting it as a satellite RNA. The deduced amino acid sequence of ORF 1a shows some similarity to those of RdRp of certain positive-strand RNA viruses, especially to the members of the family Nodaviridae, and that of ORF 2 to CP of the members in the family Tombusviridae. The nucleotide sequence of RNA 3 shows a 40-nucleotide length of partial similarity to S. macrospora virus B (SmV B) RNA. The capsid of SmV A is composed of two capsid proteins, CP 1 (p43) and CP 2 (p39), both encoded in ORF 2. CP 2 is apparently derived from CP 1 via proteolytic cleavage at the N-terminus. The genome organization of SmV A is characteristic and distinct from those of other known fungal RNA viruses, including SmV B. These results suggest that SmV A should be classified into a new group of mycoviruses.     

A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae

The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks.     

Isolation and characterization of a new Vesivirus from rabbits

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus B

Sclerophthora macrospora Virus B (SmV B) found in S. macrospora, the pathogenic fungus responsible for downy mildew in gramineous plants, is a small icosahedral, monopartite virus containing a positive-strand ssRNA genome. In the present study, the complete nucleotide sequence of the SmV B genome was determined. The viral genome consists of 5533 nucleotides and has two large open reading frames (ORFs). ORF1 encodes a putative polyprotein containing the motifs of chymotrypsin-related serine protease and RNA-directed RNA polymerase. ORF2 encodes a capsid protein. The deduced amino acid sequence shows some similarity to those of certain positive-strand RNA viruses, but the genome organization is characteristic and distinct from those of other known fungal RNA viruses. These results suggest that SmV B should be classified into a new group of mycoviruses.     

Sequence of the genomic RNA of nudaurelia beta virus (Tetraviridae) defines a novel virus genome organization.

The monopartite genome of Nudaurelia beta virus, the type species of the Betatetravirus genus of the family Tetraviridae, consists of a single-stranded positive-sense RNA (ss+RNA) of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerases of ss+RNA viruses. The 3' proximal ORF of 1836 nucleotides, which encodes the 66-kDa capsid precursor protein, overlaps the replicase gene by more than 99% (1827 nucleotides) and is in the +1 reading frame relative to the replicase reading frame. This capsid precursor is expressed via a 2656-nucleotide subgenomic RNA. The 3' terminus of the genome can be folded into a tRNA-like secondary structure that has a valine anticodon; the tRNA-like structure lacks a pseudoknot in the aminoacyl stem, a feature common to both genera of tetraviruses. Comparison of the sequences of Nudaurelia beta virus and another member of the Tetraviridae, Helicoverpa armigera stunt virus, which is in the genus Omegatetravirus, shows identities of 31.6% for the replicase and 24.5% for the capsid protein. The viruses in the genera Betatetravirus and Omegatetravirus of the Tetraviridae are clearly related but show significant differences in their genome organization. It is concluded that the ancestral virus with a bipartite genome, as found in the genus Omegatetravirus, likely evolved from a virus with an unsegmented genome, as found in the genus Betatetravirus, through evolution of the subgenomic RNA into a separate genomic component, with the accompanying loss of the capsid gene from the longer genomic RNA.     

Grapevine virus A: nucleotide sequence, genome organization, and relationship in the Trichovirus genus

Summary. The 5′ terminal region of the genomic RNA of grapevine virus A (GVA), a tentative member of the Trichovirus genus, encompassing 5 466 nucleotides, was sequenced. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORF) were identified: ORF 1 that codes for a 194 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses, and ORF 2 that encodes a 19 kDa polypeptide with no significant homology with protein sequences from databases. This polypeptide, however, showed 44% similarity with the product expressed by a comparable ORF present in grapevine virus B (GVB). GVA genome had the same size and structural organization as that of GVB. It also had the same size of the genome of apple chlorotic leaf spot virus (ACLSV), the type species of the Trichovirus genus, but differed substantially in the number (5 versus 3), size, and order of genes. Differences existed also in the degree of sequence homology between polymerases, which did not cluster together in phylogenetic trees. Definitive (ACLSV, PVT) and tentative (GVA, GVB) trichovirus species differ molecularly, biologically and epidemiologically to an extent that warrants the taxonomic revision of the genus. Received June 19, 1996 Accepted September 17, 1996     

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses

Summary.  RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3′ poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5′-UTR and 3′-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses. Received December 7, 2001; accepted April 12, 2002 Published online June 21, 2002