Deduced Amino Acid Identity Research Articles

Initial Evidence That Gilthead Seabream (Sparus aurata L.) Is a Host for Lymphocystis Disease Virus Genotype I.

Simple SummaryNodular lesions were observed on the skin and fins of 95% of one and a half million juvenile gilthead seabreams cultured in Egypt, shortly after importation from Europe. We undertook a study to describe the clinical disease course, identify the causative agent, and investigate the origin of the causative agent. Preliminary diagnosis based on gross lesions and postmortem examination suggested lymphocystis disease caused by lymphocystis disease virus, Lymphocystivirus, Iridoviridae. Histopathological and ultrastructural pictures were typical of lymphocystis disease virus infections. Polymerase Chain Reaction followed by sequencing and phylogenetic analysis of the major capsid protein gene demonstrated the presence of lymphocystis disease virus genotype I, originally associated with lymphocystis disease in Northern European countries, with 99.7% and 100% nucleotide and deduced amino acid identity values, respectively. Lymphocystis disease virus genotype I has never been reported in this species or in the region. Regardless of whether it has maintained a previously undetected state of endemicity in Egypt or was introduced through importation or contamination of ship ballast water, the findings of this study add to existing knowledge about the lymphocystis disease’s ecology, and lymphocystis disease virus genotypes and their host range.Marine and brackish water aquacultures are rapidly expanding in the Mediterranean basin. In this context, Egypt recently received a shipment of a 1.5 million juvenile gilthead seabream (Sparus aurata L.) from European Mediterranean facility. Within a few weeks of their arrival, 95% of the imported fish developed nodules on their skin and fins that lasted for several months. This study was undertaken to describe the clinical disease course, to identify the causative agent, and to investigate its origin. Preliminary diagnosis based on gross lesions and postmortem examination suggested lymphocystis disease (LCD), caused by the lymphocystis disease virus (LCDV; genus Lymphocystivirus, family Iridoviridae). Histopathological and ultrastructural features were typical of LCDV infections. PCR followed by sequencing and phylogenetic analysis of a 306-bp fragment of the major capsid protein (MCP) gene demonstrated the presence of LCDV genotype I, originally associated with LCD in Northern European countries, with 99.7% and 100% nucleotide and deduced amino acid identity values, respectively. LCDV genotype I has neither been reported in this species nor in the region. Regardless of the source of infection, findings of this study add to existing knowledge about the ecology of LCDV genotype I and its host range.

Characterization of a not so new potexvirus from babaco (Vasconcellea x heilbornii).

A new member of the genus Potexvirus was fully sequenced and characterized. The virus was isolated from babaco (Vasconcellea x heilbornii), a natural hybrid native to Ecuador. The virus contains a 6,692 nt long genome organized in five open reading frames in an arrangement typical of other potexviruses. Sequence comparisons revealed close relatedness with Papaya mosaic virus (PapMV), Alternathera mosaic virus (AltMV) and Senna mosaic virus (SenMV), exhibiting nucleotide identities up to 67% for the polymerase (Pol) and 68% for the coat protein (CP), with deduced amino acid identities of 70% and 72% for the Pol and CP, respectively. The presence of an AlkB domain, in the polymerase region, was observed. Terminal nucleotide sequences were conserved across potexviruses with characteristic motifs and predicted secondary structures at the 3’ UTR. Although serologically undistinguishable from PapMV and AltMV, the new virus showed differences in host range and symptom induction. The name babaco mosaic virus is proposed for this newly characterized Potexvirus. The complete genome sequence of the new virus has been deposited in NCBI GenBank under accession number MF978248.

First report of Grapevine Pinot gris virus infecting grapevine in Brazil

Grapevine Pinot gris virus (GPGV) has been reported infecting grapevines (Vitis spp.) in several countries of the world. In this study, 19.5% of grapevine samples collected in Brazil and indexed for GPGV by RT-qPCR was infected. The complete coat protein and the partial replicase genes of two Brazilian isolates (CF-BR and ME-BR) were sequenced and exhibited very high nucleotide and deduced amino acid identities with many foreign characterized isolates of GPGV available in the GenBank. This is the first detection of GPGV infecting grapevines in Brazil.

Complete genome sequence of Streptococcus troglodytae TKU31 isolated from the oral cavity of a chimpanzee (Pan troglodytes).

Streptococcus troglodytae TKU31 was isolated from the oral cavity of a chimpanzee (Pan troglodytes) and was found to be the most closely related species of the mutans group streptococci to Streptococcus mutans. The complete sequence of TKU31 genome consists of a single circular chromosome that is 2,097,874 base pairs long and has a G + C content of 37.18%. It possesses 2082 coding sequences (CDSs), 65 tRNAs and five rRNA operons (15 rRNAs). Two clustered regularly interspaced short palindromic repeats, six insertion sequences and two predicted prophage elements were identified. The genome of TKU31 harbors some putative virulence associated genes, including gtfB, gtfC and gtfD genes encoding glucosyltransferase and gbpA, gbpB, gbpC and gbpD genes encoding glucan-binding cell wall-anchored protein. The deduced amino acid identity of the rhamnose-glucose polysaccharide F gene (rgpF), which is one of the serotype determinants, is 91% identical with that of S. mutans LJ23 (serotype k) strain. However, two other virulence-associated genes cnm and cbm, which encode the collagen-binding proteins, were not found in the TKU31 genome. The complete genome sequence of S. troglodytae TKU31 has been deposited at DDBJ/European Nucleotide Archive/GenBank under the accession no. AP014612.

Glutathione S-Transferase Genes Differently Expressed by Pathogen-Infection in Vitis flexuosa

Glutathione S-transferase (GST) genes from transcripts of Vitis flexuosa leaves infected with Elsinoe ampelina were characterized and analyzed for their expression using primers based on specific regions. Comparison of deduced amino acid sequences from GST transcripts of V. flexuosa showed that the score of the deduced amino acid identity ranged from 43.38% (VfGST26625 and VfGST774) to 6.67% (VfGST13892 and VfGST774). Primary and secondary structure analysis was performed using the ProtParam and Self-Optimized Prediction Method with Alignment software. A phylogenetic tree was constructed from the GST proteins by the neighbor joining method using MEGA 6.0 to investigate the relationship among VfGST, VvGST, and At GST proteins. To evaluate the differential expression pattern of GST genes by real-time polymerase chain reaction (PCR), primers specific to unique regions in each gene were obtained by alignment of the sequences. Real-time PCR revealed that GST genes were expressed differentially in the leaves of V. flexuosa infected with Botrytis cinerea, E. ampelina, and Rhizobium vitis. The expression of VfGST26625 was up-regulated, while that of others were down-regulated among five GSTs in all grapevine leaves inoculated with each pathogen. The results provided herein improve our understanding of defense responses to various pathogen attacks in grapevines.

Characterization of Panax ginseng UDP-Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rh1 in Metabolically Engineered Yeasts

Ginsenosides, the main pharmacologically active natural compounds in ginseng (Panax ginseng), are mostly the glycosylated products of protopanaxadiol (PPD) and protopanaxatriol (PPT). No uridine diphosphate glycosyltransferase (UGT), which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPg1, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside F1. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity (>84%) with UGTPg1. We demonstrate that UGTPg100 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rh1, and UGTPg101 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rg1 from F1. However, UGTPg102 and UGTPg103 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rh1 by introducing the genetically engineered PPT-producing pathway and UGTPg1 or UGTPg100. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

Isolation and complete nucleotide sequence of a Batai virus strain in Inner Mongolia, China.

BackgroundBatai virus (BATV) is a member of the Orthobunyavirus genus of the family Bunyaviridae, and a vector-borne pathogen. Genomic variations of BATV exist in different regions of the world, due to genetic reassortment. Whole-genome sequencing of any isolate is necessary for a phylogenetic analysis. In 1998, a BATV strain was isolated from an Anopheles philippines mosquito in Yunnan Province, China. This strain has not been found to infect any other host. We investigated BATV infection in cattle in Inner Mongolia, China and performed deep sequencing of the genome of the BATV isolate.FindingsNinety-five blood samples were collected from cattle in Inner Mongolia, China in 2012. The BATV infection rate was 2.1%. Previously, BATV strain NM/12 was isolated from two cattle in Inner Mongolia, China, and the whole genomic sequence of the strain has been available. We determined the complete genomic nucleotide sequences of the small (S), medium (M), and large (L) genome segments using bovine blood obtained in 2012, and the nucleotide homologies of these segments with those from GenBank were 88.7%-97%, 84%-95.4%, and 72.6%-95.8%, respectively. The deduced amino acid identities were 87.2-99.7%, 64.2-96.8%, and 81.1-98.6%. Phylogenetic analyses based on full-length genomic sequences indicated that the M and L segments, and a portion of the S segment, of NM/12 are most closely related to the BATV strains isolated in Asia. The S and M segments of NM/12 were independent of phylogenetic lineages. The L segment was the most closely related to Chittoor/IG-20217 (isolated in India), and distantly related to isolated strains in Italy. Nucleotide substitution rates in the nucleotide sequences that code for the nucleocapsid, envelope glycoprotein, and polymerase protein of NM/12 strain were 2.56%, 4.69%, and 4.21%, respectively, relative to the original strain of MM2222.ConclusionA novel BATV NM/12 strain from bovine serum collected in Inner Mongolia was isolated from cattle in China for the first time. Our findings elucidate the evolutionary status of the BATV NM/12 strain among different orthobunyavirus strains and may provide some clues to prevent the emergence of BATV infection in cattle and humans.

First Report of Potato Virus H on Solanum muricatum in China.

Solanum muricatum, commonly known as pepino, pepino dulce, or tree melon, is a perennial shrub well known for its attractive, sweet, flavorful fruits and is frequently cultivated as an annual. It has gained increasing popularity in China and is grown as a cash crop in many provinces. S. muricatum belongs to the family Solanaceae and is closely related to tomato, eggplant, and potato. In 2012, during a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. PVH is a recently discovered carlavirus reported from potato plants from Huhhot, Inner Mongolia Autonomous Region. Since then, it was found on potatoes in Yunnan, Hebei, Liaoning, Heilongjiang, and Xinjiang provinces. PVH induces mild symptoms with a slight leaf curl in systemic leaves, but most often it is almost symptomless or latent on potatoes (2). To confirm the presence of PVH on S. muricatum, surveys were conducted in 2012 and 2013 in Gansu, Yunnan, and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR (2), and extracted samples were probed by Western blotting with the specific polyclonal antiserum against PVH (2). Among the 50 plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6 kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair (PVHB1F 5'-TGATGGAATTTACAAAAAC-3' and PVHUR 5'-CTTATGCGCATCTATCAATC-3'), and then cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced (PVH-Pepino with GenBank Accession No. KF546312). Further sequence comparison showed that PVH-Pepino shared 91 to 98% nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN (HM584819 and JQ904630, respectively). PVH-Pepino shared deduced amino acid identity of 98 to 99% in CP gene with PVH-Ho and PVH-YN, respectively, but only shared 57 to 67% amino acid identities with other reported carlaviruses (1,2). Thus, latent infection of PVH on S. muricatum was confirmed. To our knowledge, this is the first report of S. muricatum as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepinos are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult. Although this finding does not show that PVH is economically important to pepino, this cannot be excluded in the presence of other viruses (2).

Isolation and characterization of multiple F-box genes linked to the S 9 - and S 10 -RNase in apple (Malus × domestica Borkh.)

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of a potential new VP4 genotype.

Swine fecal samples collected from seven farms were screened for group C rotaviruses (RVCs) using a reverse transcription-polymerase chain reaction assay. A total of 380 samples were tested and 19.5% were positive. Of the 128 samples collected in 2012, 23.5% from nursing piglets and 8.5% from weaned piglets were RVC positive, with a higher RVC frequency in diarrheic (28.4%) than in non-diarrheic (6.6%) piglets. Two strains (RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px) from two different farms were characterized genetically to gain information on virus diversity based on full length sequences of the inner capsid VP6, enterotoxin NSP4 and the outer capsid VP7 and VP4 (partial for RV0104) genes. The VP6 gene of the two strains showed high (99%) nucleotide identity to one another, 84-91% identity to other porcine RVCstrains and 81-82% identity to human and bovine RVC strains. The NSP4 gene analysis revealed that RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px strains were not closely related to each other (87% identity), but shared higher identity with prototype RVC/Pig-wt/USA/Cowden/1980/G1Px strain (93% and 89%, respectively) and were more distantly related to human strains (72-76% identity). The VP7 gene analysis indicated that the two strains were distantly related to one another (72% identity). RVC/Pig-wt/USA/RV0143/2012/G6Px was most closely related to porcine RVC G6 strains (82-86% identity), whereas RVC/Pig-wt/USA/RV0104/2011/G3PX was most closely related to porcine HF (G3) strain (94% identity). Analysis of the full length nucleotide sequence of the VP4 gene revealed that RVC/Pig-wt/USA/RV0143/2012/G6Px was distantly related to porcine (75%), bovine (74%) and human (70%) strains. The deduced amino acid identities (69.5-75.6%) of VP4 between RVC/Pig-wt/USA/RV0143/2012/G6Px and other RVCs were low; hence, we propose that this strain comprises a new VP4 genotype. Our results indicate high genetic heterogeneity in RVCs genes and the concurrent co-circulation of different genotypes at the same time. Our findings are useful for the development of more accurate diagnostic tools, for basic research to understand gene function and to provide information for RVC diversity germane to vaccine development.

Comparative sequence and phylogenetic analysis of open reading frame 30 (ORF30) of neuropathogenic and non-neuropathogenic EHV-1 strains from the united states and france

EHV-1 than EHV-4 with 96% and 88% deduced amino acid identities respectively. Like EHV-4, AHV-3 contained a D at the 752 amino acid position equivalent. Even among the most closely related equid alphaherpesviruses, the maintenance of EHV-1 with a DNA polymerase containing N752 is unique and suggests a relatively recent evolution. Despite the increasing number of outbreaks of neuropathogenic EHV-1 in the US and Europe in the last decade, this disease manifestation and associated SNP remain less common than EHV-1 abortions and detection of EHV-1 containing a DNA pol with N752. There may be many reasons for apparent fitness advantage of these viruses, including their relative transmissibility that may be greater from high titred aborted foetal tissue compared to levels of virus shed in the respiratory tract.

Anti-inflammatory drugs will decrease infection of endothelial cell with EHV-1

EHV-1 than EHV-4 with 96% and 88% deduced amino acid identities respectively. Like EHV-4, AHV-3 contained a D at the 752 amino acid position equivalent. Even among the most closely related equid alphaherpesviruses, the maintenance of EHV-1 with a DNA polymerase containing N752 is unique and suggests a relatively recent evolution. Despite the increasing number of outbreaks of neuropathogenic EHV-1 in the US and Europe in the last decade, this disease manifestation and associated SNP remain less common than EHV-1 abortions and detection of EHV-1 containing a DNA pol with N752. There may be many reasons for apparent fitness advantage of these viruses, including their relative transmissibility that may be greater from high titred aborted foetal tissue compared to levels of virus shed in the respiratory tract.

Characterization of the S-RNase genomic DNA allele sequence in Prunus speciosa and P. pseudocerasus

In this study, eight S-RNase alleles were isolated from two Prunus pseudocerasus and two Prunus speciosa accessions by PCR amplification from genomic DNA. Analysis of the amino acid sequences revealed five novel and three published S-alleles. These S-RNases share typical structural features with S-RNases from other Prunus species exhibiting gametophytic self-incompatibility. The deduced amino acid identities ranged from 60.8 to 75.6% among four S-RNase alleles in Prunus speciosa and ranged from 73 to 81.4% among four S-RNase alleles in Prunus pseudocerasus. The size of the first introns ranged from 197 to 341bp, and the size of second introns ranged from 81 to 1182bp. Sequence analysis demonstrated that the deduced amino acid identities, by comparison with other Prunus species, were often higher than those of intraspecific identities. Moreover, exceptionally high identities were found between Pspe-S1 and Pd-S28; between Pspe-S31 and Pm-S6; among Pspe-S51, Pa-S29 and Pweb-S7; and among Pps-S13, Psim-S4 and Ps-Sb, indicating that the S-RNase alleles evolved before Prunus species divergence. Interestingly, the similarities of the first and second introns were also high between the two S-RNase alleles, which range from 83.63 to 100% among the first introns and from 46.13 to 100% among the second introns. These information could not increase our knowledge on the S-alleles of Prunus species, but is available for molecular breeding of fruit trees, to avoid cross-incompatibility.

Identification and sequential analysis on rabies virus isolated from a donkey

To identify and analyze the genetic characteristics of nucleoprotein (N) and glycoprotein (G) genes of rabies virus (RABV) isolated from a donkey in Wuhan. N gene and G gene of the virus were compared with other representative street strains isolated around Hubei areas as well as the vaccine strains used in China and abroad. RABV in brain tissue of a donkey was detected by direct immunofluorescent method and then inoculated in suckling mice to observe the incidence of rabies. Brain samples of the donkey and infected suckling mice were detected by ELISA. The N gene and G gene fragment of the isolated RABV were amplified by RT-PCR and cloned into pMD18-T vector for sequencing and genetic analysis. RABVs were detected in both donkey brain and suckling mice brain samples. The N gene and G gene nucleotide homology of RABV isolated from the donkey with other representative street strains found around Hubei areas as well as vaccine strains used in China and abroad were 85.7% - 99.1% and 82.2% - 99.7%, and the deduced amino acid identity were 95.6% - 99.8% and 87.8% - 99.4%, respectively. Novel RABV was successfully identified and isolated from a donkey and showed close relationship to the representative street strains found around Hubei areas as well as vaccine strains used in China through genetic analysis.

Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China

Since late 2010, porcine epidemic diarrhea virus (PEDV) has been re-emerging in immunized swine herds with devastating impact in the Hebei province of China. Seven prevailing strains of PEDV were isolated from fecal samples out of piglets suffering from severe diarrhea. The M gene of the seven PEDV isolates encompasses an open reading frame of 681 nucleotides, encoding a protein of 226 amino acids. The seven PEDV isolates showed 99.4–99.9 % nucleotide sequence identity and 98.2–99.1 % deduced amino acid identity. When compared with other Chinese isolates and foreign isolates, the seven isolates showed high nucleotide identity with the Thailand isolate M-NIAH1005 (99.6–99.9 %) and Korea isolate PFF188 (99.7–100 %), but low identity with other Chinese isolates (96.6–99.1 %) and with the vaccine strain CV777 used in China (97.8–98.2 %). Phylogenetic analyses showed that all seven Chinese field isolates were grouped together in the same cluster. Although CV777 was also separated into the same cluster with the seven isolates, they were belonged to different sub-cluster. These results showed that the seven prevailing isolates in China are closely related phylogenetically to each other and have close relationships with the Korean strain PFF188 and Thailand strain M_NIAH1005. However, they differ genetically from other Chinese isolates and the vaccine strain CV777. Therefore, a more efficient vaccine strain should be chosen to prevent outbreaks of PEDV in China.

Detection and molecular chracterization of porcine type 3 orthoreoviruses circulating in South Korea

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0–98.1% and deduced amino acid identities of 96.4–98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198–204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.

Genetic and phylogenetic analysis of South Korean sacbrood virus isolates from infected honey bees (Apis cerana)

Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains. Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities. This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.

Molecular analysis of eight SFB alleles and a new SFB-like gene in Prunus pseudocerasus and Prunus speciosa

This study identified eight S-haplotype-specific F-box genes (SFB alleles) and one S-haplotype-specific F-box-like gene (SFB-like gene) from genomic DNA by PCR combined with cleaved amplified polymorphic sequence markers in Prunus pseudocerasus and Prunus speciosa. The unknown sequences of C-termini were obtained by thermal asymmetric interlaced PCR. The whole nucleotide sequences of these genes were submitted to the EMBL/GenBank database. The SFBs shared typical structural features with SFBs from other Prunus species exhibiting gametophytic self-incompatibility. The deduced amino acid identity ranged from 77.1% to 82.4% among the four PpsSFBs and from 70.4% to 80.2% among the four PspeSFBs. The typical structural features were also detected in the PpsFB, but the sequence polymorphism was lower. The nucleotide identities ranged from 71.3% to 90.3% among the eight introns of the SFBs, the length of these introns varied from 95 to 121 bp and showed few polymorphisms. The distance between these SFBs and the corresponding S-RNases (S-ribonucleases) varied from 33 to 956 bp. Moreover, sequence analysis showed that interspecific amino acid identities in comparison with some other Prunus species were often higher than intraspecific identities, similar to S-RNase alleles. In addition, a similarity comparison found that the deduced amino acid identities among SFB alleles were higher than among S-RNase alleles, and the similarity data showed that the relationships among SFB alleles differed among S-RNase alleles, suggesting that the S-RNase and SFB alleles were separated but correlated during the coevolutionary process.

Genetic analysis of two Taiwanese bluetongue viruses

BTV2/KM/2003 and BTV12/PT/2003 are the first identified bluetongue viruses in Taiwan. The prototype virus BTV2/KM/2003 was previously characterized in various respects as low virulent. In the present study, nucleotide sequences of the ten genome segments and their coding regions of the Taiwan strains were determined and analyzed. The two strains had >96.8% nucleotide and >97.9% deduced amino acid identities to each other, except for the VP2 genes. Their genome sequences, except for NS1 and VP2 genes, clustered overall in the Asian lineage, and were closely related to strains from China, India, Indonesia, and Japan. The phylogenetic trees and nucleotide identities of six BTV genes were suggestive of the geographical origin of the bluetongue virus strains analyzed, with a few exceptions. To examine which genes better distinguished strains from different origins (topography), the distribution of and the levels of differences in nucleotide identities were analyzed, revealing that VP3, NS2, and NS3 genes were more suitable for topotyping of BTVs. Analysis of ratios of non-synonymous/synonymous substitutions (dN/dS values) between putative ancestry and their descendant strains suggested that most BTV genes evolved under a negative selection, whereas the VP7 gene evolved under positive selection, and its non-synonymous substitutions accumulated more rapidly in strains from the Mediterranean region.

Isolation and characterization of three duplicated PISTILLATA genes in Brassica napus

Three coding region cDNAs of duplicated PISTILLATA-like (PI-like) MADS-box genes, BnPI-1, BnPI-2 and BnPI-3, were isolated from B. napus by RT-PCR. The sequence analysis showed that the three PI cDNAs possessed 627, 627 and 625 nucleotides, respectively, and their nucleotide sequences had 96.49-98.72% similarity. Due to a deletion of two nucleotides, the protein sequence in the downstream of the frameshift site was altered in BnPI-3. Therefore, there were only 171 amino acids coded by BnPI-3, while there were 208 ones coded by BnPI-1 or BnPI-2. The deduced amino acid identity between BnPI-1 and BnPI-2 was 97.6% and the amino acid sequence of BnPI-1 and BnPI-2 shared 72.6% identity with BnPI-3. The deduced amino acid sequences of the coded proteins indicated high homology with the members of the PI family of MADS-box proteins. RT-PCR analysis showed that BnPI transcription was only detectable in petals and stamens. The yeast two-hybrid assays results showed that the three BnPI proteins exhibited different dimerization affinities with three BnAP3. BnPI-1 and BnPI-2 could form strong heterodimers with BnAP3. The dimerization affinity of BnPI-1 with BnAP3-4 is the strongest in all the combinations, while the affinity of BnPI-3 with BnAP3-4 is the weakest. The dimerization affinity to BnAP3-4 of BnPI-1 is 3.5 times of that of BnPI-3. The distinguished weak interaction to AP3 of BnPI-3 is probably due to the loss of the PI motif. The divergences of sequence and affinity of protein interaction might reflect some functional divergence of the three PI genes in B. napus.