Strawberry necrotic shock virus is a distinct virus and not a strain of <Emphasis Type="Italic">Tobacco streak virus</Emphasis>

Summary. Fragaria (strawberry) and Rubus species (blackberry, wild blackberry, red raspberry and black raspberry) were thought to be infected with distinct isolates of Tobacco streak virus (TSV). Employing serology and nucleic acid hybridization it has been shown that these isolates form a cluster distinct from other strains of TSV. In this study we have cloned and sequenced the complete RNA 3 of an isolate of TSV from strawberry (Fragaria) as well as the coat protein (CP) gene of 14 additional isolates of TSV originating from Fragaria and Rubus species. Our data suggest that the isolates of TSV that infect Fragaria and Rubus belong to a distinct virus for which we propose the name Strawberry necrotic shock virus (SNSV). The RNA 3 of SNSV contains 2248 nucleotides, 43 more than the type isolate of TSV from white clover (TSV-WC), with a CP gene that is 669 nucleotides long, in contrast to the 714–7 nucleotides of the TSV CP sequences found in the database. The movement protein gene of SNSV is 897 nucleotides in length, 27 more than that of the TSV-WC isolate of TSV. The CP genes of the 15 Fragaria and Rubus isolates that we studied form two distinct phylogenetic clusters that share about 95% amino acid sequence identity, while they only share 60–65% amino acid sequence identity with TSV-WC.     

A quick fuse and the emergence of Taura syndrome virus

Over the last two decades, Taura syndrome virus (TSV) has emerged as a major pathogen in penaeid shrimp aquaculture and has caused substantial economic loss. The disease was first discovered in Ecuador in 1991, and the virus is now globally distributed with the greatest concentration of infections in the Americas and Southeast Asia. To determine the evolutionary history of this virus, we constructed a phylogeny containing 83 TSV isolates from 16 countries sampled over a 16-year period. This phylogeny was inferred using a relaxed molecular clock in a Bayesian Markov chain Monte Carlo framework. We found phylogenetic evidence that the TSV epidemic did indeed originate in the Americas sometime around 1991 (1988–1993). We estimated the TSV nucleotide substitution rate at 2.37 × 10^− 3 (1.98 × 10^− 3 to 2.82 × 10^− 3) substitutions/site/year within capsid gene 2. In addition, the phylogeny was able to independently corroborate many of the suspected routes of TSV transmission around the world. Finally, we asked whether TSV emergence in new geographic locations operates under a quick fuse (i.e. rapid appearance of widespread disease). Using a relaxed molecular clock, we determined that TSV is almost always discovered within a year of entering a new region. This suggests that current monitoring programs are effective at detecting novel TSV outbreaks.     

Sequence analysis of CP and HC-Pro genes of Turnip mosaic virus isolates from China

Fourteen isolates of Turnip mosaic virus (TuMV) were obtained from the leaves of diseased cruciferous plants in China. According host tests, the isolates were classified into B-host and BR-host group. The nucleotide sequences of the coat protein (CP) and helper component proteinase (HC-Pro) genes of the isolates were determined. The CP genes consisted of 864 nucleotides encoding a polypeptide of 288 amino acids. The HC-Pro genes comprised 1374 nucleotides encoding a polypeptide of 458 amino acids. The genes CP and HC-Pro of the 14 isolates shared nucleotide sequence identities ranging from 89.2 to 99.5% and 79.1 to 99.9%, respectively. Amino acid sequence identities of CP and HC-Pro proteins ranged from 95.1 to 100% and 94.8 to 99.8%, respectively. Phylogenetic tree based on the CP gene indicated that 13 of the 14 TuMV isolates belonged to the world-B group, while the remaining isolate ZJ1 belonged to the basal-BR group. The phylogenetic tree based on the HC-Pro gene was similar to that of CP gene with the exception of the isolate JX that clustered with the Asian-BR group. Our results were consistent with the previous results demonstrating that a majority of the isolates collected from Brassica spp. belonged to the world-B group. Key words: Turnip mosaic virus; HC-Pro gene; CP gene; phylogenetic relationship.     

Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates

Tomato spotted wilt virus (TSWV) infects numerous host plants and has three genome segments, called L, M and S. Here, we report the complete genome sequences of three Korean TSWV isolates (TSWV-1 to -3) infecting tomato and pepper plants. Although the nucleotide sequence of TSWV-1 genome isolated from tomato is very different from those of TSWV-2 and TSWV-3 isolated from pepper, the deduced amino acid sequences of the five TSWV genes are highly conserved among all three TSWV isolates. In phylogenetic analysis, deduced RdRp protein sequences of TSWV-2 and TSWV-3 were clustered together with two previously reported isolates from Japan and Korea, while TSWV-1 grouped together with a Hawaiian isolate. A phylogenetic tree based on N protein sequences, however, revealed four distinct groups of TSWV isolates, and all three Korean isolates belonged to group II, together with many other isolates, mostly from Europe and Asia. Interestingly, most American isolates grouped together as group I. Together, these results suggested that these newly identified TSWV isolates might have originated from an Asian ancestor and undergone divergence upon infecting different host plants.     

Characterization of a Taura syndrome virus isolate originating from the 2004 Texas epizootic in cultured shrimp

A comprehensive investigation of the Taura syndrome virus (TSV) isolate that caused epizootics in shrimp farms in Texas in 2004 (Texas isolate) revealed that this virus was more virulent in laboratory bioassays than the TSV reference isolate, Hawaii 1994, causing severe symptom development and rapid mortality. Histopathology of moribund animals demonstrated epithelial necrosis within the stomach, appendages, general body cuticle and gills, and the surviving animals demonstrated moderate to numerous lymphoid organ spheroids. Purified virions showed icosahedral morphology, with a diameter of 31 nm. Comparative genome analysis showed that the Texas isolate is more closely related to TSV isolates from Thailand and China than to the Hawaii isolate. The predicted tertiary structures of the inhibition of apoptosis protein (IAP) and protease domains of the Texas isolate are very similar to those of the Hawaii isolate. However, the RNA-dependent RNA polymerase (RdRp) of the Texas isolate has significant structural differences from the Hawaii isolate due to point mutation(s) in the RdRp gene. Changes in the RdRp tertiary structure might contribute to the replication fidelity, virulence and ecological adaptability of the Texas isolate.     

Partial characterization of Maize rayado fino virus isolates from Ecuador: phylogenetic analysis supports a Central American origin of the virus

Maize rayado fino virus (MRFV) infects maize and appears to be restricted to, yet widespread in, the Americas. MRFV was previously unreported from Ecuador. Maize plants exhibiting symptoms of MRFV infection were collected at the Santa Catalina experiment station in Quito, Ecuador. RT-PCR reactions were performed on total RNA extracted from the symptomatic leaves using primers specific for the capsid protein (CP) gene and 3' non-translated region of MRFV and first strand cDNA as a template. Nucleotide sequence comparisons to previously sequenced MRFV isolates from other geographic regions revealed 88-91% sequence identity. Phylogenetic trees constructed using Maximum Likelihood, UPGMA, Minimal Evolution, Neighbor Joining, and Maximum Parsimony methods separated the MRFV isolates into four groups. These groups may represent geographic isolation generated by the mountainous chains of the American continent. Analysis of the sequences and the genetic distances among the different isolates suggests that MRFV may have originated in Mexico and/or Guatemala and from there it dispersed to the rest of the Americas.     

Genetic variability in the coat protein genes of Cymbidium mosaic virus isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Cymbidium mosaic virus (CymMV), which naturally infects orchids worldwide, was investigated. The CP genes of 55 CymMV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 223 aa. The CP sequences of the Korean isolates were compared with those of previously published CymMV isolates originating from different countries at both nt and aa levels. The Korean isolates shared 74.9–98.3 and 52.7–100% CP homology with CymMV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either grouping of viruses. In the deduced CymMV CP aa sequence, the C-terminal region was more divergent than the N-terminal. The phylogenetic tree analysis based on nt sequence diversity of CP genes of CymMV isolates supported the hypothesis that CymMV isolates were divided into two subgroups. However, these subgroups were not formed by phylogenetic tree analysis of CP aa sequences. There was no distinct correlation between geographical locations and specific sequence identity, while recombination analysis revealed that there were no intra-specific recombination events among CymMV isolates.     

Comparative study on genomes of two Japanese melon necrotic spot virus isolates

Nucleotide sequences of the genomes of two Japanese Melon necrotic spot virus (MNSV) isolates, NH and NK were determined. The open reading frames (ORFs) in both genomes encode five proteins: p29 (the pre-readthrough domain of p89), p89 (the readthrough domain of p89 identified as the putative RNA-dependent RNA polymerase), p14 (the pre-readthrough domain of p7A), p7A (the putative movement protein), and p42 (coat protein, CP). Nucleotide and amino acid sequence identities of the five proteins of NH and NK isolates were estimated at 97.4-99.5% and 97.7-100%, respectively. NK isolate but not NH isolate infected systemically leaves of Cucumis melo plants. When deduced amino acid sequences of p7A proteins of NH and NK isolates were compared, only one difference at position 16 (serine in NH isolate and isoleucine in NK isolate) was observed. p7A protein is considered the putative movement protein. The serine of p7A protein of NH isolates may be involved in systemic infection. In addition, phylogenetic relationships of genes based on nucleotide sequences revealed that NH and NK isolates might form a group, and S isolate, serologically different from NH and NK isolates, might represent a distinct isolate not belonging to this group.     

Nucleotide sequences of the coat protein and readthrough protein genes of the Chinese GAV isolate of barley yellow dwarf virus

The nucleotide sequences of the coat protein (CP) and readthrough protein (RTP) genes of the Chinese GAV isolate of Barley yellow dwarf virus (BYDV) were determined. The CP and RTP genes of GAV isolate comprised 600 and 1374 nucleotides, respectively. When the CP and RTP gene sequences of GAV isolate were compared with those of BYDV isolates MAV-PS1, P-PAV, NY-SGV and Cereal yellow dwarf virus RPV (CYDV-RPV), the highest similarity (97.2%) between the CP genes of GAV and MAV-PS1 isolates was observed, while the RTP genes of these two isolates shared a lower similarity (87.8%). The results of the alignment of the deduced amino acid sequences of RTP showed that the sequence diversity observed was located at the C terminus.     

High sequence conservation among <Emphasis Type="Italic">Odotoglossum ringspot virus</Emphasis> isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Odontoglossum ringspot virus (ORSV), which naturally infects orchids worldwide, was investigated. The CP genes of 48 ORSV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 158 aa. The CP sequences of the Korean isolates were compared at the nt and aa levels with those of the previously published ORSV isolates originating from different countries. The Korean isolates share 94.8–100% and 92.4–100% CP identity to ORSV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either sequence of the viruses. In the deduced aa sequence, the N-terminal region was more conserved than the C-terminal region in ORSV. The phylogenetic tree analysis and recombination analysis revealed that there was no distinct grouping between geographic locations and sequence identity, and nor distinct intra-specific recombination events among ORSV isolates.     

Comparison of nucleic and amino acid sequences and phylogenetic analysis of the GS protein of various equine arteritis virus isolates

The genetic variation in equine arteritis virus (EAV) G_S protein encoding gene was investigated. Nucleic and deduced amino acid sequences from eight different EAV isolates (one European, two American and five Canadian isolates) were compared with those of the Bucyrus reference strain. Nucleotide and amino acid sequence identities between these isolates and the Bucyrus reference strain ranged from 92.3 to 96.4%, and 93.2 to 95.5%, respectively. However, phylogenetic tree analysis and estimation of genetic distances based on the G_S protein encoding gene sequences showed that the European prototype Vienna strain, the American 87AR-A1 isolate and all other North American EAV isolates could be classified into three genetically divergent groups. Our results showed that the G_S protein-encoding gene can be subjected on the basis of phylogenetic analysis to genetic variation, as previously shown for the other three EAV structural protein (M, N and G_L)-encoding genes.     

Phylogenetic analysis of <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis> isolates from China

A survey detected Beet necrotic yellow vein virus (BNYVV) infection in six Chinese sugar-beet-growing regions. To study the diversity of virus isolates among the regions, nucleotide sequences of four proteins, namely CP, p25, p31, and p26, were determined and the amino acid sequences thus deduced were analyzed using sequence alignments and the phylogenetic method, respectively. Amino acid sequence analysis of CP revealed A-type isolates in Harbin, Hohhot, Baotou, Wuwei, and Jiuquan and B-type isolates in Hohhot and Changji; no Chinese isolate was found to cluster with European P-type isolates. Chinese p25 proteins clustered into three groups with seven tetrad motifs (positions 67–70). Of the seven, the tetrad ASHG has not been reported previously. Most Chinese p31 proteins clustered in p31-2 group, diverging from European p31 proteins. Isolates containing RNA 5 occurred in most of the investigated regions and were associated with both A-and B-types. Phylogenetic analyses of these four proteins showed complex patterns of genetic diversity among these Chinese isolates and indicated that the isolates of China and Japan were more closely related and may have a common origin. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers EF473089–EF473126.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two isolates (AY362547 and AY102034) from Thailand.     

Genetic diversity of the coat protein of olive latent virus 1 isolates

The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very few nucleotide sequence differences were observed among the isolates, olive isolates exhibited lower diversity (0.012). In addition, at position 158 (157 in C7 and G1S and 159 in G6) of the deduced aa sequences, an alanine residue was found to be conserved among the olive isolates. In citrus and tulip isolates, a threonine residue was present at position 158, whereas a valine was present at this same position in tomato isolates. Phylogenetic analysis indicated that OLV-1 isolates clustered in five groups according to original host. However, G6, originally recovered from olive but repeatedly inoculated and maintained in N. benthamiana plants for 8 years in our laboratory, was separated from other isolates. This may be attributable to adaptation to the experimental host over time. There was no correlation of phylogenetic grouping of isolates based on geographical location or year of collection. Strong negative selection may have contributed to the low diversity among the OLV-1 CP isolates.     

Molecular characterization of Czech and Chinese leek yellow stripe virus isolates from garlic

Using an RT-PCR specific for nuclear inclusion b (NIb) and coat protein (CP) genes of Leek yellow stripe virus (LYSV) we detected two Czech (LYSV-5CZ and LYSV-22CZ) and one Chinese (LYSV-16) isolate of LYSV in garlic plants. The RT-PCR products were cloned and sequenced. Phylogenetic analysis based on deduced amino acid sequence of NIb-CP region placed the Czech isolates in the group I and the Chinese isolate in the group II of LYSV.     

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.     

Biological and molecular variability among geographically diverse isolates of sweet potato virus 2

Summary. Sweet potato virus 2 (SPV2) is a tentative member of the genus Potyvirus, family Potyviridae. In addition to the type isolate of SPV2 recently characterised in greater detail, twelve additional isolates of this virus were obtained from sweet potato clones originating from China, Portugal, South Africa and Zambia. Sequences of the coat protein (CP) gene and 3′ non-translated region (NTR) were determined. Comparisons of the CP gene sequences of these isolates revealed nucleotide and amino acid sequence identities ranging from 81 to 99% and from 86 to 99%, respectively. Phylogenetic analysis of sequences distinguished several groups, which partially correlated with the geographic origin of the isolates, and indicated that some isolates from South Africa and a Zambian isolate are most distinct both in CP and 3′NTR sequences. Host range studies of a selected number of isolates revealed some differences in test plant reactions, which appeared to correlate to some extent with the geographic origin and molecular distinctness of the SPV2 isolates. The results strongly suggest the occurrence of biologically and genetically diverse strains of SPV2.