A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization

We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5' proximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3' proximal ORF2 (nucleotides 4390-7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st-2nd) and late (3rd-4th) instars, worker pupae, workers, sexual pupae, alates ( male symbol and female symbol ), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavated from the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.     

A new positive-strand RNA virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5' RACE reactions, a 3' RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3' end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of > or =100 codons) and 5 minor ORFs (comprised of 50-99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a positive-strand RNA virus. Further evidence for this conclusion includes abolished RT-PCR amplification by RNase treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of positive-strand RNA viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaviruses, Picornaviridae, and plant small RNA viruses.     

Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses

The complete genome sequence of acute bee paralysis virus (ABPV) was determined. The 9470 nucleotide, polyadenylated RNA genome encoded two open reading frames (ORF1 and ORF2), which were separated by 184 nucleotides. The deduced amino acid sequence of the 5' ORF1 (nucleotides 605 to 6325) showed significant similarity to the RNA-dependent RNA polymerase, helicase, and protease domains of viruses from the picornavirus, comovirus, calicivirus, and sequivirus families, as well as to a novel group of insect-infecting RNA viruses. The 3' ORF2 (nucleotides 6509-9253) was proposed as encoding a capsid polyprotein with three major structural proteins (35, 33, and 24 kDa) and a minor protein (9.4 kDa). This was confirmed by N-terminal sequence analysis of two of these proteins. The overall genome structure of ABPV showed similarities to those of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus, and Himetobi P virus, which have been classified into a novel group of picorna-like insect-infecting RNA viruses called cricket paralysis-like viruses. It is suggested that ABPV belongs to the cricket paralysis-like viruses.     

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.     

Molecular characterisation of hepatopancreatic parvovirus (PmergDNV) from Australian Penaeus merguiensis

Hepatopancreatic parvovirus infection is associated with reduced growth rates of prawns during the juvenile stages and overt mortalities. Hepatopancreatic parvovirus was purified from Penaeus merguiensis from northern Queensland and a partial consensus sequence of 5.9 kb was obtained. Nucleotide comparisons revealed that the Australian isolate of HPV has a nucleotide similarity (87%) closer to HPVchin and the full sequence of HPV Penaeus monodon (PmDNV) (6321 bp) than to HPVsemi (83%). Three putative open reading frames were identified. The first open reading frame encoded a nonstructural protein (NS2) and shared an amino acid similarity of 86% with PmDNV. The second ORF overlapped the first open reading frame and shared 93% and 26% amino acid similarity with PmDNV and PstDNV, respectively, and encoded NS1. The third ORF encoded the viral structural protein and shared an amino acid similarity of 73% with the capsid protein of PmDNV and HPVchin. The phylogeny suggests that the Australian HPV isolate is closely related to the Korean HPVchin isolate than to the Indian HPVsemi and Thai PmDNV isolates. HPV strains may be following the phylogenetic relationship of penaeid prawn hosts rather than their geography.     

Genetic, biochemical, and structural characterization of a new densovirus isolated from a chronically infected Aedes albopictus C6/36 cell line

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-A resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.     

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.     

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.     

Complete nucleotide sequence of the Japanese encephalitis virus genome RNA

The complete nucleotide sequence of the Japanese encephalitis virus (JEV) genome RNA was determined. The JEV genome contains 10,976 nucleotides and encodes a single long open reading frame (ORF) of 10,296 nucleotides corresponding to 3432 amino acid residues. This long polypeptide is thought to be cleaved into three structural proteins and several nonstructural proteins of the virus. The genetic location of the three structural proteins was determined by comparing the deduced amino acid sequence from the nucleotide sequence with the N-terminal amino acid sequences that were determined from the three purified structural proteins. The C-terminal region of the ORF may encode a RNA-dependent RNA polymerase which has significant sequence homology with those of other RNA viruses.     

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.     

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.     

Structure of the gene encoding the M1 protein of sonchus yellow net virus

The gene encoding the M1 protein of sonchus yellow net virus (SYNV), a plant rhabdovirus, has been sequenced and identified by Western blot analysis of SYNV proteins using antibodies directed against a fusion protein derived from a portion of the sequenced gene. The M1 gene is positioned between nucleotides 4039 and 5109 relative to the 3' end of the viral RNA and is the fourth gene from the 3' end of the genome. The 1071-nucleotide (nt) M1 gene lies between a putative nonstructural gene of unknown function and the gene encoding the glycoprotein and is bordered on either side by the same GG intergenic dinucleotide that separates other genes in the SYNV genome. The M1 mRNA (scRNA 6) consists of a 71-nt untranslated region at the 5' terminus followed by an 858-nt open reading frame (ORF) capable of encoding a protein with a calculated molecular weight of 31,779. The amino acid sequence deduced from this ORF is not highly homologous to those of other rhabdovirus matrix proteins, but has some localized regions of similarity. The UGA codon that terminates the M1 ORF is followed by a 3' untranslated region of 142 nt. The viral RNA (minus-sense) sequence corresponding to the extreme 3' end of the mRNA contains a 9-nt tract (3'-AUUGUUUUU-5') that is identical to the sequences at the termini of other SYNV genes.     

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.     

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.     

Morphology of temperate bacteriophage SfV and characterisation of the DNA packaging and capsid genes: the structural genes evolved from two different phage families

The entire genome of SfV, a temperate serotype-converting bacteriophage of Shigella flexneri, has recently been sequenced (Allison, G.E., Angeles, D., Tran-Dinh, N., Verma, N.K. 2002, J. Bacteriol. 184, 1974-1987). Based on the sequence analysis, we further characterised the SfV virion structure and morphogenesis. Electron microscopy indicated that SfV belongs to the Myoviridae morphology family. Analysis of the proteins encoded by orf1, orf2, and orf3 revealed that they were homologous to small and large terminase subunits, and portal proteins, respectively; the protein encoded by orf5 showed homology to capsid proteins. Western immunoblot of the phage with anti-SfV sera revealed two antigenic proteins, and the N-terminal amino acid sequence of the 32-kDa protein corresponded to amino acids 116 to 125 of the ORF5 protein, suggesting that the capsid may be processed. Functional analysis of orf4 showed that it encodes the phage capsid protease. The proteins encoded by orfs1, 2, 3, 4, and 5 are homologous to similar proteins in the Siphoviridae phage family of both gram-positive and gram-negative origin. The capsid and morphogenesis genes are upstream and adjacent to the genes encoding Myoviridae (Mu-like) tail proteins. The organisation of the structural genes of SfV is therefore unique as the head and tail genes originate from different morphology groups.     

Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America

Many different viruses that reduce virulence and alter the phenotype to varying extents have been identified in the chestnut blight fungus Cryphonectria parasitica. Most viruses identified in this fungus fall within the Hypoviridae family of positive-sense RNA viruses, which contains one genus and four species. Different species predominate in different geographic locations in chestnut-growing areas around the world. In this paper, we describe the genome organization and some variants of Cryphonectria hypovirus 4 (CHV-4), the species most commonly found in eastern North America. CHV-4 is distinguished from other hypoviruses by having little effect on fungal virulence and colony morphology. The 9.1-kb genome of strain CHV-4/SR2 is the smallest of any member of the family characterized to date. Like the recently characterized species CHV-3, a single ORF was predicted from deduced translations of CHV-4/SR2. Sequence analysis revealed the presence of a putative glucosyltransferase domain in both CHV-4 and in CHV-3, but no such homolog was detected in the more thoroughly examined CHV-1 or in CHV-2. Alignments with 8 other CHV-4 isolates from different regions of eastern North America revealed sequence diversity within the species and the likelihood that RNA recombination has led to this diversity.     

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5′ trailer sequence, completing the sequence of the 14 900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249 766 Da. The 70 nt BEFV 5′ trailer region displays partial terminal complementarity with the 3′ leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3′ leader region. The 47 nt 5′ trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3′ leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I–VI, conserved among RNA polymerase proteins of single-stranded (−) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (−) RNA viruses.     

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

Novel double-stranded RNAs (∼ 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3’-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.     

A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase

Double-stranded RNA representing four distinct electrophoretic patterns was found in a screen of Phytophthora infestans isolates. Two dsRNAs that always appeared together were sequenced. RNA 1, which was 3160 nt plus a poly (A) tail, contained a single deduced ORF with the potential to encode a polyprotein of 977 aa with motifs characteristic of supergroup I viral RdRps. The 2776 nt, polyadenylated RNA2 contained an ORF with a potential to encode a polyprotein of 847 aa including a possible trypsin-like serine protease, and a second putative ORF of unknown function. An alternative form of RNA2, in which a 19-nt stretch was replaced by a 9-nt sequence, was detected in 4 of 17 clones sequenced. Based on genome structure and phylogenetic analysis, this virus did not fit into any known virus family and we tentatively named it Phytophthora infestans RNA virus 1 (PiRV-1).