Nt Sequence Identity Research Articles

Genome characterization, infectivity assays of in vitro and in vivo infectious transcripts of soybean yellow mottle mosaic virus from India reveals a novel short mild genotype

Nucleotide sequence of a distinct soybean yellow mottle mosaic virusisolate from Vignaradiata (mungbean isolate, SYMMV-Mb) from India was determined and compared with othermembers of the family Tombusviridae. The complete monopartite single-stranded RNA genome of SYMMV-Mb consisted of 3974nt with six putative open reading frames and includes 5′ and 3′ untranslated regions of 35 and 254nt, respectively. SYMMV-Mb genome shared 75% nt sequence identity at complete genome level and 67–92% identity at all ORFs level with SYMMV Korean and USA isolates (soybean isolates) followed by CPMoV, whereas it shared very low identity with other tombusviridae members (5–41%). A full-length infectious cDNA clone of the SYMMV-Mb placed under the control of the T7 RNA polymerase and the CaMV35S promoters was generated and French bean plants on mechanical inoculation with in vitro RNA transcripts, p35SSYMMV-O4 plasmid and agroinoculation with p35SSYMMV-O4 showed symptoms typical of SYMMV-Mb infection. The infection was confirmed by DAC-ELISA, ISEM, RT-PCR and mechanical transmission to new plant species. Further testing of different plant species with agroinoculation of p35SSYMMV-O4 showed delay in symptoms but indistinguishable from mechanical sap inoculation and the infection was confirmed by DAC-ELISA, RT-PCR and mechanical transmission to new plants. The system developed here will be useful for further studies on pathogenecity, viral gene functions, plant-virus-vector interactions of SYMMV-Mb and to utilize it as a gene expression and silencing vector.

Complete genome sequence of bean leaf crumple virus, a novel begomovirus infecting common bean in Colombia.

A copy of the complete genome of a novel bipartite begomovirus infecting common bean (Phaseolus vulgaris L.) in Colombia was obtained by rolling-circle amplification (RCA), cloned, and sequenced. The virus is associated with leaf crumple symptoms and significant yield losses in Andean and Mesoamerican beans. Such symptoms have been reported increasingly in Colombia since at least 2002, and we detected the virus in leaf material collected since 2008. Sequence analysis showed that the virus is a member of a distinct species, sharing 81% and 76% nucleotide (nt) sequence identity (in DNA-A and DNA-B, respectively) to other begomoviruses infecting common bean in the Americas.The data obtained support the taxonomic status of this virus (putatively named 'bean leaf crumple virus', BLCrV) as a member of a novel species in the genus Begomovirus.

Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World

Suspected virus-like symptoms were observed in cacao plants in Trinidad during 1943, and the viruses associated with these symptoms were designated as strains A and B of cacao Trinidad virus (CTV). However, viral etiology has not been demonstrated for either phenotype. Total DNA was isolated from symptomatic cacao leaves exhibiting the CTV A and B phenotypes and subjected to Illumina HiSeq and Sanger DNA sequencing. Based on de novo assembly, two apparently full-length badnavirus genomes of 7,533 and 7,454 nucleotides (nt) were associated with CTV strain A and B, respectively. The Trinidad badnaviral genomes contained four open reading frames, three of which are characteristic of other known badnaviruses, and a fourth that is present in only some badnaviruses. Both badnaviral genomes harbored hallmark caulimovirus-like features, including a tRNAMet priming site, a TATA box, and a polyadenylation-like signal. Pairwise comparisons of the RT-RNase H region indicated that the Trinidad isolates share 57-71% nt sequence identity with other known badnaviruses. Based on the system for badnavirus species demarcation in which viruses with less than 80% nt sequence identity in the RT-RNase gene are considered members of separate species, these isolates represent two previously unidentified badnaviruses, herein named cacao mild mosaic virus and cacao yellow vein banding virus, making them the first cacao-infecting badnaviruses identified thus far in the Western Hemisphere.

Deep Sequencing Analysis of Apple Infecting Viruses in Korea.

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.

First molecular evidence of kobuviruses in goats in Italy.

By screening 139 rectal swabs collected from either asymptomatic or diarrhoeic goats in Italy, we identified kobuvirus RNA in eight samples (5.8%). Higher positivity rates were observed in diarrhoeic goats (6.5%, 3/46) than in asymptomatic animals (5.4%, 5/93), although the difference was not statistically significant. Based on the analysis of a portion of the 3D gene, four strains were found to share the highest nucleotide (nt) sequence identity with bovine kobuviruses (95.0-98.0%), which had been detected previously in calves in the UK and Korea. Interestingly, two strains were genetically related to the newly discovered caprine kobuviruses (83.0-97.0% nt sequence identity), which had been identified in black goats in Korea and in roe deer in Italy. Taken together, these findings demonstrate that kobuviruses are common enteric viruses of goats, although their clinical relevance remains to be investigated.

INCIDENCE AND DISTRIBUTION OF FIG BADNAVIRUS 1 AND MULBERRY BADNAVIRUS 1 ON MULBERRY TREES IN IRAN

Fig badnavirus 1 (FBV-1) and mulberry badnavirus-1 (MBV-1) are two closely related badnaviruses which were recently identified in fig and mulberry plants. Thirty-six fig and 27 mulberry orchards were surveyed to determine the presence and incidence of FBV-1 and MBV-1 in four provinces of Iran. A total of 81 fig and 70 mulberry asymptomatic and symptomatic leaf samples were collected in April to August 2015 and tested by PCR. Approximately 62% and 33% of the tested fig and mulberry samples, respectively, were infected with FBV-1. None of the fig samples was found to be infected with MBV-1. MBV-1 was detected in only 8.5% of the mulberry samples collected in two provinces. Mixed infections of FBV-1 with MBV-1 were detected in 7% of the samples. The presence of both viruses in asymptomatic and symptomatic trees was verified by PCR. Sequence analysis of the cloned movement protein (MP) gene of thirteen Iranian FBV-1 isolates showed 97-99% nucleotide (nt) sequence identity among the isolates and 98-99% nt sequence identity to the American reference isolate. The MP fragments of two Iranian MBV-1 isolates showed 98-100% nt sequence identity to the reference Lebanese MBV-1 isolate. In phylogenetic analyses, isolates of FBV-1 formed two distinct clades and grouped according to the host. To our knowledge, this is the first report of FBV-1 and MBV-1 infecting mulberry plants in Iran.

TOMATO YELLOW LEAF CURL YUNNAN VIRUS, A NEW BEGOMOVIRUS SPECIES ASSOCIATED WITH TOMATO YELLOW LEAF CURL DISEASE IN CHINA

Yellow leaf curl disease was observed in tomato (Solanum lycopersicum) plants in Yunnan Province, China. Analysis of the full-length viral DNA isolated from an infected tomato plant indicated that the viral genome contained 2759 nucleotides (nt) and had the typical genome structure of begomoviruses. Further analysis of the viral sequence showed that it shared the highest nt sequence identity (86.0%) with Tomato yellow leaf curl Vietnam virus (TYLCVNV) previously identified in Vietnam. The virus is therefore a new Begomovirus species for which the name Tomato yellow leaf curl Yunnan virus (TYLCYnV) has been proposed. The betasatellite was also obtained and showed the highest nt sequence identity (70.1%) with Tomato yellow leaf curl China betasatellite (TYLCCNB).

Complete genome sequence of a novel sub-subgenotype 2.1g isolate of classical swine fever virus from China.

Current subgenotype 2.1 isolates of classical swine fever virus (CSFV) play a dominant role in CSF outbreaks in China, and a novel sub-subgenotype 2.1g of CSFV was recently identified, but the complete genome sequence of this new sub-subgenotype has not been reported. In this study, complete genome of 2.1g isolate GD19/2011 collected from Guangdong province of China in 2011 was sequenced. It was found to be 12,298 nucleotides (nt) in length, including a 375-nt 5'UTR, a 11,697-nt opening reading frame (ORF), and a 227-nt 3'UTR. GD19/2011 shared 91.0-93.7% and 95.6-97.5% nt and amino acid sequence identity, respectively, with other subgenotype 2.1 isolates. The topology of a phylogenetic tree constructed based on complete genome sequences of GD19/2011 and other CSFV isolates was identical to that obtained with full-length E2 gene sequences, but it was significantly different from those obtained with the 5'UTR and core sequences. Serial passages of GD9/2011 in PK-15 cells generated a highly cell-adapted virus stock with an infectious titer of 10(7.8) TCID50/ml at the 12(th) passage in which two amino acid substitutions, S476R and N2494S, were observed in comparison with the complete polyprotein sequence of the original isolate from kidney tissue, GD19/2011. This is the first report of the complete genome sequence of a 2.1g isolate, and the GD19/2011 isolate will be useful for further analysis of the evolution and virulence of CSFV isolates.

Biological and molecular characterization of a highly divergent johnsongrass mosaic virus isolate from Pennisetum purpureum.

The complete genome sequence (9,865 nucleotides) of a highly divergent johnsongrass mosaic virus isolate (JGMV-CNPGL) was determined using Illumina sequencing. This isolate infected 10 genotypes of gramineous plants including maize. A comparative analysis of the complete genome showed 80% nucleotide (nt) sequence identity (86% amino acid (aa) sequence identity) to a johnsongrass mosaic virus isolate from Australia. The coat protein (CP) identity values, however, were lower than those for the whole genome (78% and 80% for nt and aa, respectively) and were close to the species demarcation values (77% nt and 80% aa). Unexpectedly, the amino-terminal portion of CP of JGMV-CNPGL showed only 38% sequence identity to other JGMV isolates. The biological implications of this sequence divergence remain to be elucidated.

First detection and molecular characterization of Nebovirus in Brazil.

Nebovirus is a new genus of viruses belonging to the Caliciviridae family recently characterized in cattle, and is associated with gastrointestinal disorders, such as diarrhoea, anorexia and intestinal lesions particularly in calves. The aim of this study was to investigate the prevalence of neboviruses in Brazilian cattle and analyse phylogenetically the virus strains detected. A prevalence of 4·8% of neboviruses in faecal samples from 62 head of cattle from different Brazilian states was detected. All positive animals were aged 96·0% nt (100% aa) sequence identity between the virus sequences in this study and >88·8% nt (>94·4% aa) identity with Newbury1/UK. Our results indicate, for the first time, the occurrence of neboviruses in Brazil as well as in South America, and the first Newbury1-like nebovirus found outside the UK.

Molecular characterization of two divergent variants of sugarcane bacilliform viruses infecting sugarcane in China

Sugarcane bacilliform viruses (SCBV; genus Badnavirus, family Caulimoviridae) are considered economically important pathogens of sugarcane, limiting the exchange of its germplasm worldwide. Similar to other badnaviruses, SCBV are genetically diverse and highly complex, hence the need for a thorough analysis of its genetic structure and diversity. In the present study, we report the molecular characterization of two new SCBV isolates (SCBV-CHN1 and SCBV-CHN2) from China and their genetic variation and phylogenetic relationship with 10 global SCBV isolates. The complete genomes of SCBV-CHN1 and SCBV-CHN2 were determined to be 7764 and 7629 nucleotides (nt) in size, respectively. Both isolates displayed a typical Badnavirus genome organization with three open reading frames (ORFs) but differed in their predicted sizes and putative scanning model for P2 (ORF2-encoded) and P3 (ORF3-encoded) protein translation. Phylogenetic analysis revealed the segregation of complete genomes, individual ORFs, and the RT/RNase H region of the 12 SCBV isolates (two from this study and 10 from the GenBank) into nine phylogroups. SCBV-CHN1 was more closely aligned with SCBV-BB in the SCBV-H clade with both isolates sharing 83 % nt sequence identity and low genetic distance (0.19), based on analysis of the RT/RNase H region. In contrast, SCBV-CHN2 clustered with SCBV-BT into a distinct clade designated as SCBV-G. Recombination analysis identified SCBV-CHN1 and SCBV-CHN2 as putative new recombinant variants arising from putative inter- and intra-specific recombination events. Our findings provide evidence for the presence of distinct genotypic variants of SCBV affecting sugarcane in China and contribute to the understanding of genetic diversity and evolution of sugarcane badnaviruses.

Molecular characterization of a first begomovirus associated with lentil (Lens culinaris) from India.

A disease of lentil with symptoms of distortion, mottling and chlorosis in the leaves, shortening of internodes and excessive branching was noticed in lentil at Kanpur, India, during 2012-2014. Results of polymerase chain reaction and reverse transcriptase polymerase chain reaction employed to detect suspected RNA and DNA viruses indicated involvement of a geminivirus, which was further characterized by sequencing of full genome amplified by rolling circle amplification. Analysis of full length DNA-A revealed 96.4-96.7% nucleotide similarity with bitter gourd yellow vein virus (BGYVV) isolates and tomato leaf curl New Delhi virus (ToLCNDV) isolate. As per the recent revision of begomovirus species demarcation criteria, if a new virus isolate shares ≥91% nt sequence identity with any other isolate of an existing species, it should be treated as an isolate of that species, even if it is <91% identical to all other isolates from that species. This made BGYVV an isolate of ToLCNDV and resulted in the de-recognizing of the BGGYV. Hence, the present virus has been named as a strain of ToLCNDV and designated as Tomato leaf curl New Delhi virus-Lentil-[India:Kanpur:Lentil:2014] with the acronym as ToLCNDV-Lentil-[IN:Knp:Len:14]. This is the first report of a begomovirus found associated with a disease in lentil from India.

Identification of a picornavirus from chickens with transmissible viral proventriculitis using metagenomic analysis.

Transmissible viral proventriculitis (TVP), an infectious disease in chickens, is responsible for economic losses in the commercial poultry industry. The major etiologic agent, however, is unknown. Using metagenomics, we compared the diversity of viruses present in proventriculus samples from flocks diagnosed with TVP to those of healthy flocks in South Korea between 2003 and 2012. Each sample had a mean of 21,538,726 sequence reads generated by high-throughput sequencing, with a mean length of 160 nt. Enrichment in viral sequences suggested that at least three viruses were present in each TVP sample. Although we could not determine a pathogen of TVP that matched the known morphology, picornavirus sequences were present in all five disease samples, suggesting an association with TVP. The five samples yielded 1,045-1,720 bp contigs with 81-84 % nt sequence identity to turkey hepatitis virus (accession number: HM751199). Whole-genome analysis indicated that the QIA01 strain of the novel picornavirus was similar to turkey hepatitis virus in the P2 and P3 regions (82.7 % nt and 95.5 % aa sequence identity), but different in the structural region and partial 2A peptides (56.2 % nt and 23.9 % aa sequence identity). In addition, the QIA01 virus was similar (87.0 % nt and 95.6 % aa sequence identity) to chicken megrivirus, recently detected in chickens with malabsorption syndrome in Hungary. Our results are useful for understanding the genetic diversity of avian picornaviruses and for classifying chicken megrivirus as a pathogen affecting the digestive tract of chickens.

Molecular characterisation of contagious ecthyma (ORF) virus from small ruminants

Contagious ecthyma (CE) or contagious pustular dermatitis (CPD) is an acute, contagious, debilitating and economically important zoonotic viral skin disease that affects domesticated as well as wild ruminants. In the present study, three different polymerase chain reaction (PCR) assays and nucleotide sequence analysis were employed successfully for the identification and characterisation of CE/CPD virus from nine [scabs (n = 8) and cell-culture adapted (n = 1)] field samples. PCR assays based on B2L gene encoding major envelope glycoprotein (based on the full length and partial length amplification) and GIF/IL-2 gene (GM-CSF and interleukin-2 inhibitory factors) were standardised. The B2L specific assays yielded amplicons of 1, 137 and 235 bp, whereas the IL-2 PCR yielded 408 bp products. The PCR amplicons were gel purified and sequenced using BigDye terminator chemistry to confirm specific amplification and to study the genetic variability among field isolates of ORF viruses. Sequencing studies demonstrated that both B2L and GIF/IL-2 genes were highly conserved with > 96 and 98% nt sequence identity, respectively. The ORF virus isolates in the present study demonstrated high relatedness with other Indian strains. Further, these studies have also shown that sequencing studies can be reliably used to identify the ORF virus isolates in clinical samples along with their genetic relatedness.

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: Predominance of P[8] genotype resembling human strains

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1–29.7%). This study revealed that age, management system and pig density significantly (p<0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811–1604nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4–91.3% and 95.1–96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6–100% to 92–100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6–91.7% and 89.3–96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

FIRST REPORT OF AGERATUM YELLOW VEIN VIRUS ASSOCIATED WITH A NEW BETASATELLITE INFECTING CARICA PAPAYA IN CHINA

A Carica papaya plant exhibiting downward curling of the leaves and yellow vein was observednear Sanya (Hainan Province, China). Total DNA was extracted from three symptomatic samples (P1, P2 and P3) for amplifying part of begomovirus DNA-A and DNA-B using the primer pair PA/PB and PBL1v2040/PCRc1 (Zhou et al., 2001; Rojas et al., 1993). No DNA-B was detected but a ca. 500 bp DNA-A fragment amplified from all samples was cloned, sequenced and shown to have 100% nucleotide (nt) identity. The primer pair BE1 (5’-GGTACGCCGACGGCTGAACTTC-3’) and BE2 (5’-CGTACCTTGGATGCGGGAGTTGAAATG-3’) was designed to amplify the full-length DNA-A from P3, which was determined to be 2,759 nt in length (GenBank accession No. KM051844) and showed a nt sequence identity (99%) with Ageratum yellow vein virus (AYVV) from Lycopersicon esculentum (KC810890). When alpha- and betasatellite molecules were looked for using the universal primer pairs DNA101/102, UN101/102 and Beta01/02 by PCR, respectively (Briddon et al., 2002; Bull et al., 2003), no alphasatellite was detected, but a betasatellite amplicon of ca. 1.3 kb was obtained from the three samples. The complete betasatellite sequence from P2 (1,350 nt, GenBank accession No. KJ642219), shares the highest nucleotide sequence identity (66.6%) with Tomato leaf curl China betasatellite (GenBank accession No. AJ704617), and appears to be a new satellite species, for whichthe name Papaya leaf curl China betasatellite [China: Hainan: 2014] is proposed. To our knowledge, this is the first report of AYVV associated with a new betasatellite infecting papaya and their first identification in China.

First Report of Watermelon chlorotic stunt virus Infecting Watermelon in Saudi Arabia.

In the Saudi Arabian deserts, watermelon [Citrullus lanatus (Thunb.)] is cultivated in the lowlands and wadis (washes) where water accumulates following rainfall, and where heat, salt, and drought stress are common constraints on production. During the spring of 2014, watermelon leaves exhibited yellowing and severe chlorotic mottling symptoms. The foliar symptoms were reminiscent of Watermelon chlorotic stunt virus (WmCSV), a bipartite begomovirus previously reported in several neighboring countries (1,3). Ten samples were collected from three farms in the Leith region, where 100% of the watermelon plants were symptomatic. Total nucleic acids were extracted from the symptomatic watermelon plants and were subjected to PCR using WmCSV DNA-A specific primers designed based on a complete genome sequence (GenBank Accession No. AJ012081), WmCSVF-3'-CGTGCTGTTGCCCCCACTGT-5' and WmCSVR-3'-CCTGCATATCTCGTGCCAGAATC-5' to obtain an expected size fragment of 1,111 bp located between the nucleotide (nt) coordinates 400-1510. The amplicons (one per sample) were cloned, and the DNA sequence was determined for each and used to search the NCBI database. The top hits for sequences obtained from all 10 samples were to WmSCV sequences, with shared nt identity values of 97 to 98%. To clone the full-length begomoviral DNA-A and DNA-B components, nucleic acids were subjected to rolling circle amplification (RCA) (2). The RCA products were cloned into the pGEM7 plasmid vector using the unique restriction sites, identified as HindIII for DNA-A, and as EcoRI for DNA-B, respectively. Ten DNA-A clones and two DNA-B component clones were sequenced. The DNA-A components ranged in size from 2,751 (KM066100) to 2,752 bp (KJ939448), whereas the DNA-B components were 2,757 bp in size (KJ939447). Analysis of the viral sequences from the DNA-A clones indicated they had the characteristics of a typical genome of a begomovirus DNA-A component that consist of a hairpin stem-loop structure in the intergenic region, two tandem copies of the iteron (TGGAGAC) located between the nt coordinates 2675 and 2680, 2682 and 2688 predicted to be involved in Rep binding, and six predicted genes encoding proteins with high sequence identity to those encoded by other WmCSV isolates. The 10 DNA-A component sequences were aligned with sequences for previously described WmCSV isolates available in GenBank using Muscle, followed by pairwise comparisons using SDT software (4). The analysis revealed that the cloned DNA-A components shared 99 to 100% nt sequence identity with each other, and 97 to 98% nt identity with WmCSV isolates reported from Yemen (AJ012081), Jordan (EU561237), Iran (AJ245652), and Sudan (AJ245650). Further, the WmCSV DNA-B from Saudi Arabia shared the highest nt identity with sequences from Yemen (AJ012082) at 96%, Iran (AJ245653) at 95%, and only 94% with DNA-B from both Sudan (AJ245651) and Jordan (EU561236). To our knowledge, this is the first report of WmCSV in Saudi Arabia. WmCSV poses a serious threat to the production of this highly valued crop in Saudi Arabia and considerably reduces crop yield (1).

Identification of Tomato yellow leaf curl virus Naturally Infecting Common Bean in Japan.

Tomato yellow leaf curl virus (TYLCV; family Geminiviridae, genus Begomovirus) is a devastating pathogen vectored by the whitefly Bemisia tabaci causing significant yield losses to tomato crops in Japan since 1998 (2). So far, there has been no report of this virus infecting common bean (Phaseolus vulgaris; family Fabaceae) in Japan. But recently, TYLCV has been reported from P. vulgaris in Spain and China (1,3). P. vulgaris is a vegetable crop commonly grown during spring to summer in Japan. In the course of a study to assess virus incidence on P. vulgaris exhibiting yellowing and thickening symptoms with 20 to 30% incidence, four symptomatic and two healthy samples were collected in August, 2012 from a single mix-cropping field (where P. vulgaris was cultivated together with tomato [Solanum lycopercisum] crop and a high B. tabaci population density was observed on plants) in Komae, Japan. Meanwhile, the vector for one crop can transmit the disease to the next crop very easily. To identify possible begomovirus present in symptomatic P. vulgaris plants, total nucleic acids were extracted from plants with and without symptoms using PhytoPure Plant DNA Extration Kit (GE Amersham Biosciences, UK) and begomovirus replication was confirmed from symptomatic leaves in 3 out of 4 plants when hybridized with a specific non-radioactive probe to the coat protein region of TYLCV (5) using a Biotin DNA Labelling Kit (Fermentas). To confirm the identity of the virus detected, leaf samples were further tested by PCR using the TYLCV specific detection primers (TYF/TYR) corresponding to the V2/CP region and a DNA fragment of an expected size was obtained from the samples positive for TYLCV in Southern hybridizations (4). The PCR products were sequenced directly and these sequences showed the highest nucleotide sequence identity >99% to the TYLCV isolate AB116630 reported from Japan. Rolling circle amplification (RCA) was done on all samples to produce concatamers and with Sac I restriction enzyme an amplicon of ~2.7 kb was obtained and cloned into PUC118 vector (Takara, Japan). Multiple clones were obtained showing the same restriction pattern with different restriction enzymes, and three of them were randomly selected and sequenced by Macrogen (Japan). Sequence analysis showed all three clones were identical and one sequence (J18.11) was submitted to GenBank. This sequence (GenBank Accession No. KJ585666) was 2,774 nt long and displayed the arrangement of four ORFs (AC1, AC2, AC3, and AC4 in complementary sense) and two ORFs (AV2 and AV1) on virion sense) typical of the genome of a begomovirus. This whole genome sequence was compared with those of other reported begomoviruses and exhibited greater than 99% nt sequence identity to a previously reported TYLC sequence (JN183876). Efforts to identify the presence of an additional begomovirus components or DNA satellites either by PCR and/or RCA were negative, suggesting that this is a monopartite begomovirus and an isolate of TYLCV. This is the first report of a natural infection of TYLCV in P. vulgaris in Japan. The presence of TYLCV on P. vulgaris could therefore represent a serious threat for this valuable crop in Japan. Thus, it is important to develop an effective way to control this virus to reduce the further losses.

Unraveling the mysteries of Middle East respiratory syndrome coronavirus.

Unraveling the mysteries of Middle East respiratory syndrome coronavirus.

Identification and molecular characterization of two begomoviruses from Pouzolzia zeylanica (L.) Benn. exhibiting yellow mosaic symptoms in adjacent regions of China and Vietnam.

Two monopartite begomoviruses were isolated from Pouzolzia zeylanica (L.) Benn. plants showing yellow mosaic symptoms in Gaoyao, Guangdong Province, China (GD1) and in Phu Tho, Vietnam (VN), respectively. A comparison of the complete genome sequence of GD1 (2,739 nucleotides [nt]) with VN (2,741 nt) indicated that they shared 86.2 % nt sequence identity. GD1 and VN shared the highest nucleotide sequence identity at 86.7 % and 91.4 % respectively, with isolate TY01 of pouzolzia golden mosaic virus (PGMV-TY01), another begomovirus isolated from P. zeylanica. Phylogenetic analysis revealed that GD1, VN, and PGMV-TY01 were members of a distinct begomovirus clade. Based on the ICTV guidelines for begomoviral species demarcation, GD1 belongs to a new begomovirus species, for which the name Pouzolzia yellow mosaic virus is proposed. Likewise, VN represents a previously unreported strain of PGMV. Recombination analysis predicted that VN was a recombinant between PGMV-TY01 and ageratum yellow vein China virus isolate G13 (AYVCNV-G13), and that PGMV-TY01 and VN were likely the parents of GD1 through recombination with allamanda leaf curl virus isolate G10 (AlLCV-G10), a begomovirus endemic to Guangdong Province of China.