Identification and molecular characterization of a single-stranded circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1

A putative circular single-stranded DNA (ssDNA) virus was recovered from Hypericum japonicum collected in Vietnam. The viral isolate was tentatively named Hypericum japonicum-associated circular DNA virus (HJasCV). HJasCV shares 58.7-65.4% nucleotide sequence identity with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and SsHADV-1-like viruses. Like this group of viruses, the genome of HJasCV (2 200 nt) has two large ORFs, one in the virion-sense and the other in the complementary-sense DNA. The proteins encoded in the virion-sense and complementary-sense ORFs share 39-46 % and 45-67 % amino acid sequence identity with the putative capsid and replication-associated proteins (Reps), respectively, of SsHADV-1 and SsHADV-1-like viruses. The putative Rep of HJasCV contains all of the motifs related to rolling-circle replication. Its 111-bp intergenic region (IR) contains a hairpin structure with a geminivirus-like nonanucleotide sequence, TAATGTTAT, at the apex of the loop. Phylogenetic analysis revealed that HJasCV forms a monophyletic clade with SsHADV-1 and SsHADV-1-like viruses.     

Discovery of a novel circular single-stranded DNA virus from porcine faeces

A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.     

Complete nucleotide sequence of rose yellow vein virus, a member of the family Caulimoviridae having a novel genome organization

This report describes the complete nucleotide sequence and genome organization of rose yellow vein virus (RYVV), a proposed new member of the family Caulimoviridae. The RYVV genome is 9314 bp in size and contains eight open reading frames (ORFs). ORFs 1, 2, and 3 have 22–38 % amino acid sequence similarity to known members of the family Caulimoviridae. The remaining ORFs have no significant amino acid sequence similarity to known viruses. Based on differences in its genome organization, its low sequence similarity to known members of the family Caulimoviridae, and the results of phylogenetic analysis, RYVV appears to be a distinct new member of this family.     

Novel myco-like DNA viruses discovered in the faecal matter of various animals

A wide variety of novel single-stranded DNA (ssDNA) viruses have been found in faecal matter of chimpanzees, cows, rodents, bats, badgers, foxes and pigs over the last few years. Using a combination of rolling circle amplification coupled with restriction enzyme digests based approach as well as a next generation sequencing informed approach, we have recovered fourteen full genomes of ssDNA viruses which exhibit genomic features described for members of the recently proposed gemycircularvirus group from a wide variety of mammal and bird faecal samples across New Zealand. The fourteen novel ssDNA viruses (2122–2290 nt) encode two major proteins, a replication associated protein (Rep) and a capsid protein (Cp) which are bi-directionally transcribed. Interestingly, the Rep of these novel viruses are similar to gemycircularviruses detected in insects, cassava leaves, and badger faecal matter, the novel viruses share sequence similarities with the mycovirus sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and Rep-like sequences found in fungal genomes. Pairwise sequence similarities between the 14 novel genomes with other related viral isolates (gemycircularviruses) indicated that they share greater than 55.8% genome-wide identity. Additionally, they share between 55% and 59% pairwise identity with putative novel ssDNA virus genomes recently isolated from sewage baminivirus, niminivirus and nephavirus. Based on the similarities to SsHADV-1 and Rep-like sequences found in fungal genomes, these novel gemycircularviruses may infect fungi.     

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.     

Molecular characterisation of a novel cassava associated circular ssDNA virus

The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus' likely origin and actual host species.     

DNA-binding specificity determinants of replication proteins encoded by eukaryotic ssDNA viruses are adjacent to widely separated RCR conserved motifs

Eukaryotic ssDNA viruses encode a rolling-circle replication (RCR) initiation protein, Rep, which binds to iterated DNA elements functioning as essential elements for virus-specific replication. By using the iterons of all known circoviruses, nanoviruses and nanovirus-like satellites as heuristic devices, we have identified certain amino acid residues that presumably determine the DNA-binding specificity of their Rep proteins. These putative “specificity determinants” (SPDs) cluster in two discrete protein regions, which are adjacent to distinct conserved motifs. A comparable distribution of SPDs was uncovered in the Rep protein of geminiviruses. Modeling of the tertiary structure of diverse Rep proteins showed that SPD regions interact to form a small β-sheet element that has been proposed to be critical for high-affinity DNA-binding of Rep. Our findings indicate that eukaryotic circular ssDNA viruses have a common ancestor and suggest that SPDs present in replication initiators from a huge variety of viral and plasmid RCR systems are associated with the same conserved motifs.     

Sequence comparison of the right end of fowl adenovirus genomes

The nucleotide sequence at the right end of the genomes of fowl adenoviruses (FAdVs) representing species groups C (FAdV-4 CA and FAdV-10 C-2B), D (FAdV-2 CA), and E (FAdV-8 CA) was determined and analyzed and compared to FAdV-1 (species group A virus) and FAdV-9 A-2A (species group B virus). High nucleotide sequence identities and amino acid identities (74–94%) were found among viruses in the same species group. Homologues to known open reading frames (ORFs) of the right end of the reported FAdV genomes, such as lipase and Gam-1, were also found in all analyzed viral genomes. Homologues to a few FAdV ORFs were found in viruses other than FAdVs as well. Several novel FAdV ORFs were also found. Most identified ORFs have unknown function and thus further studies are necessary to establish their importance in virus replication. ORF content within the right end of the FAdV genomes varied among FAdVs, while the gene order and orientation of shared ORFs are conserved between different FAdVs. Amalia Garceac and Juan Carlos Corredor share equally with first authorship. The sequences reported in this work have been deposited in GenBank with accession numbers EF458160-EF458163.     

Complete genome sequence of a novel potato virus S strain infecting Solanum phureja in Colombia

Potato virus S (PVS) (genus Carlavirus, family Betaflexiviridae) is one of the most prevalent viruses in potato crops (Solanum tuberosum and S. phureja) around the world, causing reductions in crop yields between 10 and 20 %. Symptoms of PVS infection may include leaf mottling, rugosity of leaves, deepening of the veins and reductions in crop yields between 10 and 20 %. Virions are flexuous rods of 610-710 nm with a positive-sense ssRNA genome of approximately 8500 nt comprising six ORFs, a 5′CAP and a 3′poly-A tail. PVS has been classified into two groups: PVS^O (Ordinary) and PVS^A (Andean). PVSA induces severe symptoms in infected plants, such as premature senescence and defoliation, and is more efficiently transmitted by aphids than PVS^O. To date, only five PVS genomes have been completely sequenced, including those of three PVS^O and two PVS^A strains. Currently, there are no reports of complete PVS genome sequences from Andean South America. In this work, we present the complete genomic sequence of a novel PVS strain infecting S. phureja that is clearly distinct from currently known PVS isolates.     

Two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis

The application of viral metagenomic techniques and a series of PCRs in a human fecal sample enabled the detection of two novel circular unisense DNA viral genomes with 92% nucleotide similarity. The viruses were tentatively named circo-like virus-Brazil (CLV-BR) strains hs1 and hs2 and have genome lengths of 2526 and 2533 nucleotides, respectively. Four major open reading frames (ORFs) were identified in each of the genomes, and differences between the two genomes were primarily observed in ORF 2. Only ORF 3 showed significant amino acid similarities to a putative rolling circle replication initiator protein (Rep), although with low identity (36%). Our phylogenetic analysis, based on the Rep protein, demonstrated that the CLV-BRs do not cluster with members of the Circoviridae, Nanoviridae or Geminiviridae families and are more closely related to circo-like genomes previously identified in reclaimed water and feces of a wild rodent and of a bat. The CLV-BRs are members of a putative new family of circular Rep-encoding ssDNA viruses. Electron microscopy revealed icosahedral (∼23 nm) structures, likely reflecting the novel viruses, and rod-shaped viral particles (∼65–460 × 21 × 10 nm in length, diameter, and axial canal, respectively). Circo-like viruses have been detected in stool samples from humans and other mammals (bats, rodents, chimpanzees and bovines), cerebrospinal fluid and sera from humans, as well as samples from many other sources, e.g., insects, meat and the environment. Further studies are needed to classify all novel circular DNA viruses and elucidate their hosts, pathogenicity and evolutionary history.     

Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea

Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem–loop structure between 5′ ends and 3′ ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem–loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.     

Common Elements of Spiroplasma Plectroviruses Revealed by Nucleotide Sequence of SVTS2

DNA of SpV1-like spiroplasma plectroviruses (rods with single-stranded circular DNA) is scattered in the genome of the phytopathogen Spiroplasma citri and has significant consequences for evolution of the S. citri genome. We determined the complete nucleotide sequence of SVTS2, a SpV1-like virus of S. melliferum, a honeybee pathogen, to ascertain, by comparison with S. citri SpV1 viruses (GenBank U28974 and X51344), the defining features of this important group. The 6,824 nt DNA contains nine ORFs homologous to ORFs of S. citri SpV1 viruses and five ORFs unique to SVTS2. The predicted amino acid sequences of the homologous ORFs were 17–38% identical to those of their S. citri counterparts. The SVTS2 predicted ORF 1 product (Mr 47,031) was considerably smaller than those of known S. citri SpV1 viruses. Also, in contrast to those viruses, SVTS2 lacked an ORF with recognizable similarity to a transposase. ORF 2 of all three viruses had a homologue among the products of genes of MVL-1, a virus of Acholeplasma laidlawii, another plectrovirus. The results suggest that, at most, only slightly more than half of SpV1 genomes consists of genes shared by all spiroplasma viruses of the group.     

Detection and sequence analysis of two novel co-infecting double-strand RNA mycoviruses in Ustilaginoidea virens

Four novel double-stranded RNA molecules, named dsRNA 1 (5124 bp), dsRNA 2(1711 bp), dsRNA 3 (1423 bp) and dsRNA 4 (855 bp), were detected in strain HNHS-1 of Ustilaginoidea virens, the causal agent of rice false smut disease. Sequence analysis showed that the dsRNA1 contains two overlapping open reading frames (ORF) potentially encoding proteins with modest levels of sequence similarity to the coat protein (CP) and putative RNA-dependent RNA polymerase (RdRp), respectively, of viruses of the family Totiviridae. The deduced gene product of the ORF encoded by dsRNA2 is homologous to putative RdRp of viruses in the family Partitiviridae; the ORF encoded by dsRNA3 shares some similarity to a hypothetical protein with unknown function. It is noteworthy that the dsRNA4 lacked integrated ORFs. Isomeric viral particles of about 40 nm in diameter were observed by transmission electron microscopy in a mycelium tissue preparation of strain HNHS-1-R1, a single-spore subculture of strain HNHS-1 containing only the dsRNA1 segment. Phylogenetic analysis and examination of the organization of the two putative RdRp sequences both indicated that there are at least two novel virus species present in strain HNHS-1. We named the two novel viruses Ustilaginoidea virens RNA virus 2 and Ustilaginoidea virens partitivirus 4, respectively.     

Molecular detection and genomic characterization of Torque teno sus virus 1 and 2 from domestic pigs in central China

In the present study, Torque teno sus viruses (TTSuVs) were detected in tissue and blood samples obtained from domestic pigs in central China, and complete genomes of TTSuVs were characterized. A total of three tissue samples (3/20, 15 %) from post-weaning multisystemic wasting syndrome-affected pigs and 30 blood samples (30/40, 75 %) from healthy pigs were positive for Torque teno sus virus 1 (TTSuV1) and/or 2 (TTSuV2). Two TTSuV strains (TTV1Hn54 and TTV2Hn93) comprising 2,794 and 2,875 nucleotides, respectively, each had four open reading frames (ORFs) and the untranslated region with TATA box and GC-rich region. Genomic sequence of TTV2Hn93 strain was unique in length compared with other TTSuV2 genomic sequences. Interestingly, three rolling-circle replication (RCR) motif-IIIs (Y_XXK) which were located at amino acid (aa) position 166–169, 328–331, and 379–382, respectively, were found in the ORF1 of TTV1Hn54. Two RCR motif-IIIs (Y_XXK) at the aa position 105–108 and 480–483 respectively, were also identified in the ORF1 of TTV2Hn93. Phylogenetic tree based on complete genomes showed that TTV1Hn54 strain was designated into type TTSuV1b and had a slight high sequence identity of 91 % with the Canada strain (JQ120664). TTV2Hn93 strain was classified into subtype TTSuV2d and shared the highest identity (97 %) with the Spain strain (GU570207).     

Identification and genomic analysis of two duck-origin Tembusu virus strains in southern China

Egg-laying duck flocks in Guangdong province, southern China, have been suffering a widely spreading infectious disease with abrupt egg drops and death since the winter of 2010. However, the causative pathogen was not known. We obtained two independent virus isolates named FS and JM from the diseased layer duck flocks and identified them as duck-origin Tembusu virus by PCR detection, sequencing the entire length of the open reading frames (ORFs). The two isolates FS and JM shared high sequence similarity to the isolates of duck-origin Tembusu virus that was first emerged in eastern China in April 2010. Blast analysis shows that the whole ORF sequences of FS and JM have the highest similarity (>99 %) to BYD-1(the first reported duck-origin Tembusu virus) and JS804 (the first reported goose-origin Tembusu virus), indicating that the full-length genomes were highly conserved in waterfowl-origin Tembusu viruses. The present study suggests that duck-origin Tembusu viruses have spread fast from eastern China to southern China, causing widely spreading infections. The high conservation of duck-origin Tembusu virus strains provides the genomic basis for choosing the strains for vaccine preparation for better protection against this new virus infection.     

Genomic characterization of swine caliciviruses representing a new genus of Caliciviridae

This study reports the molecular characterization of novel caliciviruses, the St-Valérien-like viruses, which were isolated from pig feces in the province of Quebec, Canada between 2005 and 2007. The genomes of St-Valérien-like viruses contain 6409 nucleotides and include two main open reading frames (ORFs). ORF1 encodes the non structural (NS) polyprotein and the major capsid protein (VP1) while ORF2 encodes the putative basic minor capsid protein. Typical conserved amino acid motifs predict a gene order reminiscent of calicivirus genomes. Phylogenetic, pairwise homology, and distance analyses performed on complete genomic sequences and partial amino acid sequences from the NTPase, polymerase, and major capsid protein segregated the St-Valérien-like viruses in a unique cluster sharing a common root with the Tulane virus and the noroviruses. Based on the genomic analyses presented, the St-Valérien-like viruses are members of a new genus of Caliciviridae for which we propose the name Valovirus.     

Molecular characterisation of novel mitoviruses associated with Sclerotinia sclerotiorum

Seven putative mitoviral genomes, representing four species from three Sclerotinia sclerotiorum isolates, were fully sequenced. The genome lengths ranged from 2438 to 2815 nucleotides. The RNA-dependent RNA polymerase (RdRp) of one genome shared high amino acid (aa) sequence identity (98.5 %) with the previously described Sclerotinia sclerotiorum mitovirus 2 (SsMV2/NZ1) and was provisionally assigned the name SsMV2/14563. The RdRps of three of the genomes with closest aa sequence identity of 78.8-79.3 % to Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL1) were provisionally considered to represent a new species, and the corresponding virus was named Sclerotinia sclerotiorum mitovirus 5 (SsMV5/11691, SsMV5/14563 and SsMV5/Lu471). The remaining two novel genomes, for which the viruses were provisionally named Sclerotinia sclerotiorum mitovirus 6 (SsMV6/14563 and SsMV6/Lu471) and Sclerotinia sclerotiorum mitovirus 7 (SsMV7/Lu471), showed closest aa sequence identities to Sclerotinia sclerotiorum mitovirus 3 (SsMV3/NZ1; 57.5-57.8 %) and Cryphonectria cubensis mitovirus 1a (CcMV1a; 32 %), respectively. The RdRp proteins of all seven genomes contained the conserved aa sequence motifs (I-IV) previously reported for mitoviruses, and their 5′ and 3′ untranslated regions (UTRs) have the potential to fold into stem-loop secondary structures.     

Genetic diversity and host range studies of turnip curly top virus

Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterised as infecting turnips in Iran. The genome of TCTV shares <68 % pairwise identity with other geminiviruses and has a genome organisation similar to that of curtoviruses and topocuvirus. The replication-associated protein (Rep) bears the highest similarity to curtovirus Reps (48.5–69.0 %); however, in the case of the capsid protein (CP), the extent of similarity is only 39.5–44.5 %. We constructed an agroinfectious clone of TCTV and undertook host range studies on ten plant species; in three species (turnip, sugar beet and cowpea), we detected infection which presents curly top symptoms in turnip and sugar beet. The efficiency of TCTV infection in agroinoculated turnip plants was 71.7 %, and the infection was successfully transmitted to 80 % of the healthy turnip plants used in the insect transmission studies by Circulifer haematoceps under greenhouse conditions. We also determined the genome sequence of 14 new TCTV isolates from southern Iran isolated from turnips. We observed ~13 % diversity amongst all the TCTV isolates and found evidence of recombination in the CP- and Rep-coding regions of the genomes.     

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.     

A plasmid of phytoplasma encodes a unique replication protein having both plasmid- and virus-like domains: clue to viral ancestry or result of virus/plasmid recombination?

The genomes of most prokaryotic and eukaryotic single-stranded (ss) DNA viruses, and some prokaryotic plasmids such as pLS1, commonly replicate via a rolling circle replication (RCR) strategy, and thus the viruses are hypothesized to have evolved from the plasmids, although evidence for this view is sparse. We have sequenced a circular plasmid of 3933 nt, pOYW, obtained from onion yellows phytoplasma (OY-W), a cell-wall-less, unculturable prokaryote that inhabits the cytoplasm of both plant and insect cells. pOYW contains five open reading frames (ORFs) on the same strand and apparently replicates by an RCR mechanism. Its rep gene (ORF5) encodes a unique protein, pOYW-Rep, with an unprecedented structure. The N-terminal region of pOYW-Rep has similarities to the RCR initiator protein (Rep) of pLS1 family plasmids but, unlike the Rep of other plasmids, its C-terminal region was unexpectedly similar to the helicase domain of the replication-associated proteins (Rap) of eukaryotic viruses, especially circoviruses (ssDNA viruses of vertebrates). The pOYW-Rep was specifically detected in OY-W-infected plant phloem cells, suggesting that it is a functional protein. We suggest that an ancestral phytoplasma plasmid pOYW may have acquired a helicase domain from host phytoplasmal DNA, entered the surrounding eukaryotic cytoplasm, and subsequently evolved into an ancestral eukaryotic ssDNA virus. Alternatively, a pOYW ancestor could have obtained the helicase domain by recombination with a virus: this would be the first example of recombination between plasmids and viruses.