Amino Acid Sequence Of ORF Research Articles

Detection and molecular phylogenetic analysis of chicken astrovirus in Saga prefecture, Japan.

We detected three chicken astrovirus strains in 4-day-old broiler chickens with high mortality and visceral gout and one strain from 150-day-old hens without clinical symptoms in Saga prefecture, Japan. Phylogenetic analysis based on ORF2 amino acid sequences revealed that the strains from the visceral gout cases belonged to subgroup Bi, and the strain from hens without clinical symptoms belonged to subgroup Aiii. Our study showed that CAstV had infiltrated into Saga prefecture, Japan. This is the first report of CAstV in Japan.

Viral metagenomics reveals two novel anelloviruses in feces of experimental rats

BackgroundRodents are widely distributed and are the natural reservoirs of a diverse group of zoonotic viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. Rodents are commonly used in animal testing, particularly mice and rats. Experimental rats are important animal models, and a history of pathogenic infections in these animals will directly affect the animal trial results. The pathogenicity of Anellovirus (AV) remains poorly understood due to the lack of a suitable model cell line or animal to support the viral cycle. This study aimed to discover possible anelloviruses from the virome in feces of experimental rats by viral metagenomic technique.MethodsFecal samples were collected from 10 commercial SD rats and pooled into a sample pool and then subjected to libraries construction which was then sequenced on Illumina MiSeq platform. The sequenced reads were analyzed using viral metagenomic analysis pipeline and two novel anelloviruses (AVs) were identified from fecal sample of experimental rats. The prevalence of these two viruses was investigated by conventional PCR.ResultsThe complete genomic sequence of these two AVs were determined and fully characterized, with strain name ratane153-zj1 and ratane153-zj2. The circular genomes of ratane153-zj1 and ratane153-zj2 are 2785 nt and 1930 nt in length, respectively, and both include three ORFs. Ratane153-zj1 closely clustered with members within the genus Wawtorquevirus and formed a separate branch based on the phylogenetic tree constructed over the amino acid sequence of ORF1 of the two AVs identified in this study and other related AVs. While the complete amino acid sequences of ORF1 of ratane153-zj2 (nt 335 to 1390) had the highest sequence identity with an unclassified AV (GenBank No. ATY37438) from Chinchilla lanigera, and they clustered with one AV (GenBank No. QYD02305) belonging to the genus Etatorquevirus from Lynx rufus. Conventional PCR with two sets of specific primers designed based on the two genomes, respectively, showed that they were detectable at a low frequency in cohorts of experimental rats.ConclusionOur study expanded the genome diversity of AVs and provided genetic background information of viruses existed in experimental rats.

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

Co-infection of a novel fusagravirus and a partitivirus in a Korean isolate of Rosellinia necatrix KACC40168.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907bp and 1918bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Viral metagenomic identification of a novel anellovirus in blood sample of a child with atopic dermatitis.

Here, using viral metagenomics, a novel anellovirus with strain name HuAV-zj-ad1 was detected in blood sample from a child with atopic dermatitis. The complete genome sequence of HuAV-zj-ad1 was determined and fully characterized. The circular genome of HuAV-zj-ad1 is 2841 nt in length and includes four polyprotein ORFs. Phylogenetic analysis and pairwise sequence comparisons based on the amino acid sequences of ORF1, ORF2, ORF3, ORF4 indicated that HuAV-zj-ad1 belonged to a novel species within the genus Betatorquevirus. Polymerase chain reaction screening results showed this anellovirus was not present 50 blood samples from normal children. Whether this novel species of anellovirus has association with a certain disease needs further study.

The prevalence and genetic diversity of porcine circovirus types 2 and 3 in Northeast China from 2015 to 2018.

A total of 472 samples from domestic pigs collected in China from 2015 to 2018 were tested for the presence of porcine circovirus types 2 and 3 (PCV2 and PCV3, respectively) by conventional polymerase chain reaction analysis. The prevalence of PCV2, PCV3, and PCV2/3 co-infection was 50.0%, 13.3%, and 6.78%, respectively. The complete genomic sequences of 66 PCV2 isolates and four PCV3 isolates were determined. Based phylogenetic analysis, the PCV2 isolates were assigned to three genotypes, PCV2a, PCV2b, and PCV2d, representing 13.6% (9/66), 25.8% (17/66), and 60.6% (40/66) of the total, respectively. All four PCV3 isolates shared a high degree of similarity in their complete nucleotide sequences (98.8-99.8% identity) and ORF2 amino acid sequences (98.6-99.5% identity). These results indicate that all three PCV2 genotypes (PCV2a, PCV2b, and PCV2d) are present on pig farms and that PCV2d has become the predominant genotype. The predicted amino acid sequences of the four PCV3 isolates indicated that PCV3-CN-JL53/PCV3-CN-LN56, PCV3-CN-HLJ3, and PCV3-CN-0710, belonged to the genotypes PCV3a, PCV3b, and PCV3a-IM, respectively. In view of the great harm that PCV2 causes to the pig industry, the epidemic trend of PCV3 should continue to be closely monitored. This study provides information about the prevalence, genetic diversity, and molecular epidemiology of PCV2 and PCV3 in China from 2015 to 2018.

Detection and genetic characterization of Mamastrovirus 5 from Brazilian dogs.

Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype ‘a’ closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease.

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Full-genome sequence analysis of an uncommon norovirus genotype, GII.21, from South Korea.

Noroviruses (NoVs) are major causal agents of acute gastroenteritis in humans. NoV GII.4 is the predominant genotype globally. However, uncommon and minor types of NoVs are consistently detected and some have been shown to dominate over GII.4. Therefore, the prevalence of dominant and uncommon NoVs makes the identification of these viruses important for the prediction and prevention of pandemics. In this study, the full-genome sequence of a NoV (strain JW) detected in Korea was extensively characterized. The full-length genome was 7510 nucleotides long, and phylogenetic analysis based on the whole-genome sequences, including open reading frame (ORF)1, ORF2, and ORF3, indicated that it belonged to the GII.21 genotype. Strain JW showed maximum identity with strain YO284; however, comparison of the amino acid sequence of ORF2, which functions as an antigen, showed substitutions in several amino acids. GII.21 is not a prevalent epidemiological agent of acute gastroenteritis in humans, but it is consistently found in gastroenteritis patients from several countries. The present study provides the first full-genome sequence analysis of NoV GII.21 isolated from a patient in Korea. Our findings provide not only valuable genome information but also data for epidemiology studies, epidemic prevention, and vaccine development strategies.

Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China.

Astroviruses are the principal causative agents of gastroenteritis in humans and have been associated with diarrhea in other mammals as well as birds. However, astroviral infection of animals had been poorly studied. In the present study, 211 rectal swabs collected from cattle and water buffalo calves with mild to severe diarrhea were tested for bovine astrovirus (BAstV) by RT-PCR. Results: 92/211 (43.6%) samples were positive for BAstV, at a rate of 46.10% (71/154) in cattle and 36.84% (21/57) in water buffalo. Phylogenetic analysis based on the partial and full-length of 25 ORF2 amino acid sequences obtained in this study classified the Guangxi BAstVs isolates into five subgroups under the genus of Mamastrovirus, genotype MAstV33, which suggested that the water buffalo was a new host of this genogroup that previously included only cattle and roe deer. Despite the origin of the host, the Guangxi BAstV isolates were closely related to the BAstV Hong Kong isolates (B18/HK and B76-2/HK), but highly divergent from the BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the U.S.A. Nucleotide sequence-based characterization of the ORF1b/ORF2 junction and corresponding overlapping regions showed distinctive properties, which may be common to BAstVs. Our results suggested that cattle and water buffalo are prone to infection of closely related astroviruses, which probably evolved from the same ancestor. The current study described astroviruses in water buffalo for the first time and is thus far among the largest epidemiological investigations of BAstV infection in cattle conducted in China.

Phylogenetic analysis of 32 porcine circovirus type 2 isolates from Shandong, China.

Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome (PMWS), can be divided into distinct genotypes. Thirty-two PCV2 isolates obtained made from pigs in Shandong Province between 2005 and 2013. Complete genome sequences were obtained in three replicates for each virus isolate, and the sequences were submitted to the NCBI database. The ORF1-encoded amino acid sequences had 98.4%-100% identity among the 32 isolates, and there were no significant differences among the three potential glycosylation positions: aa 23-25 (NPS), aa 256-258 (NQT) and aa 286-288 (NAT). The amino acid sequences of ORF2 had 88%-100% identity among the 32 isolates and the potential glycosylation position in the cap protein, aa 143-145 (NYS), had no variation. Phylogenetic analysis revealed that the PCV2b/1C genetic lineage was prevalent in swine populations in Shandong Province. It also suggested that selection pressure has made the PCV2 isolates more genetically distant from current vaccine strains.

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca2+ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.

Sequence analysis of ORFs 5, 6 and 7 of equine arteritis virus during persistent infection of the stallion—A 7-year study

Nucleotide and amino acid sequences of ORFs 5, 6 and 7 of EAV during persistent infection in the stallion of the Malopolska breed were analysed in the study. A total of 11 blood and semen samples were collected between 2004 and 2011. The titre of specific EAV antibodies in this carrier stallion was maintained at a high level throughout the study and was equal approximately 1:128. The sequence analysis of ORF5 showed 16 variable sites including 12 with synonymous substitutions and 4 with non-synonymous substitutions. The degree of nucleotide sequence identity among the strains ranged from 98.92% to 100%, whereas amino acid homology ranged from 98.06% to 100%. Ten substitutions were identified including 7 with synonymous mutations and 3 with non-synonymous mutations in ORF6. The degree of similarities among the strains ranged from 94.55 to 100% and from 98.41% to 100% at the level of nucleotide and amino acid sequence, respectively. Only a single point mutation at position 255 of ORF7 (99.6% identity) was found in nucleotide sequences of these strains. Phylogenetic analysis showed that all strains present in the semen of this carrier stallion created a separate cluster of "quasi-species" within the second European subgroup of EAV.

The nucleotide sequence and genome organization of Plasmopara halstedii virus

BackgroundOnly very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking.MethodsViral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis.ResultsThe complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2) were established. RNA1 consisted of 2793 nucleotides (nt) exclusive its 3' poly(A) tract and a single open-reading frame (ORF1) of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR) of 18 nt and a 3' untranslated region (3' UTR) of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp) and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A) and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A) tract and a second ORF (ORF2) of 1128 nt. ORF2 coded for the single viral coat protein (CP) and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb) and RNA2 (ca. 1.4 kb) were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl.ConclusionsThe results showed the presence of a single and new virus type in different P. halstedii isolates. Insignificant viral sequence variation indicated that the virus did not account for differences in pathogenicity of the oomycete P. halstedii.

Molecular cloning of apoptosis-inducing Pierisin-like proteins, from two species of white butterfly, Pieris melete and Aporia crataegi

Pierisin-1, present in cabbage butterfly, Pieris rapae, induces apoptosis against various kinds of cancer cell lines. Another cabbage butterfly, Pieris brassicae, also has an apoptosis-inducing protein, Pierisin-2. These proteins exhibit DNA ADP-ribosylating activity. Pierisin-like proteins are found to be distributed in subtribes Pierina, Aporiina and Appiadina. In this study, we performed the cDNA cloning of Pierisin-like proteins designated Pierisin-3 from gray-veined white, Pieris melete, and Pierisin-4 from black-veined white, Aporia crataegi. The nucleotide sequences of Pierisin-3 and -4 encode an 850 and an 858 amino acid protein, respectively. The partial peptide sequences of Pierisin-3 and -4 purified from pupae were identical to the deduced amino acid sequence of ORF. The deduced amino acid sequence revealed that Pierisin-3 is 93% similar to Pierisin-1 and Pierisin-4 is 64%. Pierisin-3 and -4 synthesized in vitro with the rabbit reticulocyte lysate exhibited apoptosis-inducing activity against human cervical carcinoma HeLa and human gastric carcinoma TMK-1 cells. Site-directed mutagenesis at a glutamic acid residue comprising the NAD-binding site resulted in a significant decrease in cytotoxicity of both proteins. Moreover, the proteins incubated with calf thymus DNA and beta-NAD resulted in the formation of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine, as in the case of Pierisin-1 and -2. These findings could provide useful information for understanding the importance of apoptosis-inducing ability and molecular evolution of Pierisin-like proteins in family Pieridae.

Molecular Characterization of a dsRNA Mycovirus, Fusarium graminearum Virus-DK21, which Is Phylogenetically Related to Hypoviruses but Has a Genome Organization and Gene Expression Strategy Resembling Those of Plant Potex-like Viruses

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,621 [corrected] nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and four [corrected] putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 4 [corrected] revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.

Characterization of a cryptic plasmid pSFKW33 from Shewanella sp. 33B

A cryptic plasmid pSFKW33 from psychrotrophic bacterium Shewanella sp. 33B, an isolate from the Gulf of Gdansk (the Baltic Sea), was sequenced and characterized. It is an 8021 bp circular molecule with 38% GC content, which shows a distinctive nucleotide sequence without homology to other known plasmids. The nucleotide sequence analysis predicts eight open reading frames. The deduced amino acid sequence of ORF-1 shared significant similarity with the phage integrase protein from Pseudomonas putida GB-1. The ORF2 product showed some similarity with the hypothetical protein Shal_1786 from Shewanella halifaxensis HAW-EB4. The ORF-3 product revealed high amino acid sequence homology with the Escherichia coli replication initiation protein similar to pi protein of R6K plasmid. Five repeat regions (three perfect 22-bp repeats and two imperfect motifs), a putative ribosome binding site, and −10 and −35 promotor sequences were identified upstream of the ORF-3 ( rep). The rep module is very similar to several theta replicating iteron family plasmids, suggesting that pSFKW33 replication follows the same course. The ORF-5 product revealed significant identity with the entry exclusion protein 1 of plasmid pIS2 from enteropathogenic strain E. coli 0111:H. The ORF-6 product showed significant identity with the putative transcriptional regulator protein from Shewanella benthica KT99 belonging to HTH-XRE-family protein. The ORF-8 encoded protein that showed some similarity with the hypothetical protein KT99_00146 from Shewanella benthica KT99. The ORF-4 and ORF-7 encoded putative proteins of 189 and 118 amino acid residues, respectively, with no homologies to any known proteins. The absolute copy number of pSFKW33 was estimated to be five copies per chromosome by real-time PCR.

Analysis of the entire genomes of fifteen torque teno midi virus variants classifiable into a third group of genus Anellovirus

Recently, we identified a novel human virus with a circular DNA genome of 3.2 kb, tentatively designated as torque teno midi virus (TTMDV), with a genomic organization resembling those of torque teno virus (TTV) of 3.8-3.9 kb and torque teno mini virus (TTMV) of 2.8-2.9 kb. To investigate the extent of genomic variability of TTMDV genomes, the full-length sequence was determined for 15 TTMDV isolates obtained from viremic individuals in Japan. The 15 TTMDV isolates comprised 3175-3230 bases and shared 67.0-90.3% identities with each other, and were only 68.4-73.0% identical to the 3 reported TTMDV isolates over the entire genome. TTMDV possessed a genomic organization with four open reading frames (ORF1-ORF4) with characteristic sequence motifs and stem and loop structures with high GC content, similar to TTV and TTMV. The total of 18 TTMDV genomes differed by up to 60.7% from each other in the amino acid sequence of ORF1 (658-677 amino acids), but segregated phylogenetically into the same cluster, which was distantly related to the TTVs and TTMVs. These results indicate that TTMDV with a circular DNA genome of 3.2 kb, has an extremely high degree of genomic variability, and is classifiable into a third group in the genus Anellovirus.

Comparative analysis of the plasmids from two isolates of “ Candidatus Phytoplasma australiense”

Two plasmids from the plant-pathogenic mollicute “ Candidatus Phytoplasma australiense” were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607 bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635 bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of “ Ca. P. australiense.” The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.

Complete nucleotide sequence and genome organization of a single-stranded RNA virus infecting the marine fungoid protist Schizochytrium sp.

The complete nucleotide sequence of the genomic RNA of a marine fungoid protist-infecting virus (Schizochytrium single-stranded RNA virus; SssRNAV) has been determined. The viral RNA is single-stranded with a positive sense and is 9,018 nt in length [excluding the 3' poly(A) tail]. It contains two long open reading frames (ORFs), which are separated by an intergenic region of 92 nt. The 5' ORF (ORF1) is preceded by an untranslated leader sequence of 554 nt. The 3' large ORF (ORF2) and an additional ORF (ORF3) overlap ORF2 by 431 nt and are followed by an untranslated region of 70 nt [excluding the 3' poly(A) tail]. The deduced amino acid sequences of ORF1 and ORF2 products show similarity to non-structural and structural proteins of dicistroviruses, respectively. However, Northern blot analysis suggests that SssRNAV synthesizes subgenomic RNAs to translate ORF2 and ORF3, showing that the translation mechanism of downstream ORFs is distinct from that of dicistroviruses. Furthermore, although considerable similarities were detected by using a blast genome database search, phylogenetic analysis based on both the nucleotide and amino acid sequences of the putative RNA-dependent RNA polymerase (RdRp) and the RNA helicase suggests that SssRNAV is phylogenetically distinct from other virus families. Therefore, it is concluded that SssRNAV is not a member of any currently defined virus family and belongs to a novel, unrecognized virus group.