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In this study, the focus was on the bioinformatics analysis of the coat protein (cp) gene of a specific Egyptian isolate of Zucchini yellow mosaic virus (ZYMV), which was identified based on both biological and molecular characteristics. The presence of ZYMV was confirmed in 27 squash samples using DAS-ELISA and RT-PCR techniques employing ZYU-F and ZYD1186-R primers that flank the cp gene of ZYMV. Transmission experiments involving mechanical and aphid transmission, specifically through Aphis gossypii, were carried out on various hosts, including Chenopodium amaranticolor, which served as an indicator necrotic local lesion host. Using light and electron microscopies, amorphous and cylindrical inclusions (pinwheels and scrolls) were observed, respectively. A partially purified virus preparation of ZYMV was obtained from infected squash leaves using a protocol involving polyethylene glycol purification and ultracentrifugation, revealing the presence of filamentous viral particles that were negatively stained with 2% uranyl acetate. Subsequently, the ZYMV-cp gene was amplified via RT-PCR for sequencing following the purification of PCR products from the agarose gel. The nucleotide sequences obtained, consisting of 831 base pairs, and encoding a 236 amino acid open reading frame, were deposited under the ID: LC778450.1 as the ZYMV-Shrouk-23 strain in GenBank. Bioinformatics analyses were conducted, comparing the Egyptian isolate to 25 overseas isolates of ZYMV. The resulting phylogenetic analysis dendrogram revealed a distinct clade with identities ranging from 91.94% to 99.16% based on nucleotide sequences and from 96.19% to 100.00% based on deduced amino acid sequences. [ABSTRACT FROM AUTHOR]

In July 2020, total RNA was extracted from passion fruit (Passiflora edulis) leaves showing virus symptoms such as chlorotic spots and vein banding in Haenam, South Korea. Cucumber mosaic virus (CMV)-HN2 was identified through reverse transcription polymerase chain reaction and sequencing analysis. To confirm the biological characteristics of the CMV infecting passion fruit, 10 indicator plants were inoculated with CMV-HN2, and the results showed a typical CMV symptoms. Phylogenetic analysis based on the amino acid of the coat protein (CP) of CMVs revealed that the CMV passion fruit isolates belonged to subgroup I, among which CMV-HN2 belonged to subgroup IA. Additionally, CMVs isolated from passion fruit in Korea have amino acid sequence variation between the subgroup. Among them, CMV-HN2 had four to eight amino acid differences in CP from other CMV isolates from passion fruit. These results confirm the presence of genetic diversity in the CPs of passion fruit CMV isolates.

Black gram is one of the most important short duration grain legume, which contributes significantly towards nutritional security and environmental sustainability. The virus specific primers confirms the presence of mungbean yellow mosaic India virus (MYMIV) in representative samples. A total of 27 cultivated and two wild species were found as highly resistant (HR) to MYMIV and validated through molecular markers. The start codon target (SCoT) markers analysis revealed that the SCoT loci, namely, SCoT‐4 (2200 bp), SCot‐9 (1150/ 1200 bp), SCoT‐15 (1150/1100 bp), SCoT‐16 (700 bp), SCoT‐24 (2500 bp), SCoT‐25 (700 bp), SCoT‐33 (900/1000 bp), and SCoT‐34 (600 bp), were found unique, able to distinguish HR and highly susceptible (HS) genotypes. Biochemical characterization and gene expression profiling revealed the higher expression of antioxidants and R‐genes just after pathogen inoculation indicated the activation of defence mechanism in both cultivated and its wild relatives, which modulates the resistant responses in cultivated and wild accessions. These information will be really helpful in accelerating resistance breeding in black gram. [ABSTRACT FROM AUTHOR]

The prevalence of apple chlorotic leaf spot virus (ACLSV) in different Rosaceae species, mainly from cultivated apple (Malus domestica), pear (Pyrus communis), plum (Prunus domestica), apricot (Prunus armeniaca) and wild plants of genus Amelanchier, Cotoneaster, Crataegus, Malus, Prunus and Sorbus were studied. ACLSV was found in cultivated M. domestica, P. communis, P. domestica and wild‐growing Malus, including M. sylvestris. Thirty‐four sequences of the ACLSV coat protein gene were obtained and compared with 372 homologous sequences from GenBank to evaluate the genetic diversity of the virus in Latvia and worldwide. The sequence identities varied from 79.6% to 98.4% at the nucleotide level and 84.5% to 100% at the amino acid level. Phylogenetic analyses clustered ACLSV pome fruit isolates into three genetic types but isolates from stone fruits into four phylogenetic groups. Three genetic types of ACLSV pome and stone fruit isolates were identified in Latvia. Two of them were previously characterized as type B6 and P205. The third genetic type was newly recognized among the stone fruit isolates and distinguished from other virus isolates by amino acid substitutions in three positions, Ser14, Asn23 and Leu72. Two natural recombinants, LV27 and NZ5, were detected among ACLSV isolates from Latvia, and recombination events were identified for the first time in wild‐growing Malus, confirming that recombination is a common process in the ACLSV population. [ABSTRACT FROM AUTHOR]

A survey of yellow dwarf viruses (YDVs) -associated diseases of wheat plants was performed for the first time in the Şırnak Province, Turkey. In plants wheat cultivated areas, symptoms such as diminished leaf size, dwarfing, yellowing, and reddening of leaves wheat plants were observed in cultivated areas. A total of 441 specimens were collected regardless of showing symptoms and assayed for Barley/Cereal Yellow Dwarf Viruses [B/CYDV] association by PCR tests using virus coat protein gene (CPG)-specific primer pairs. These assays demonstrated that 13.38% (55 samples) were positive to the occurrence of BYDV-PAV (9.73%) and CYDV-RPV (0.73%) for wheat viruses, but no BYDV-MAV, -SGV, and -RMV infections of BYDVs were found in PCR assays of collected samples. In addition, double infection of BYDV-PAV and CYDV-RPV was detected in 12 samples with a 2.91% infection rate. The CPG sequences of randomly selected two isolates were revealed using bacterial cloning and sequencing. Sequences obtained were deposited in GenBank under Accession numbers OL685734 and OL685736 for BYDV-PAV; and OL685735 and OL685737 for CYDV-RPV. BLASTn analysis of CPG sequences of four isolates shared high nucleotide homology with their intraspecies isolates, with 97.35-98.67% for two BYDV-PAV and 97.72-97.89 for two CYDV-RPV. The consensus tree constructed classified B/CYDV-associated wheat viruses with their phylogenetically closest individuals from Turkey and the world. This survey is the first report of BYDV-PAV and CYDV-RPV associated with wheat plants in Şırnak Province of Turkey. [ABSTRACT FROM AUTHOR]

The banana bract mosaic virus (BBrMV) is a major virus affecting bananas and plantains. Banana being propagated vegetatively, there arises a high risk of virus transmission through planting materials. Available molecular detection technique like the Reverse Transcriptase Polymerase Chain Reaction needs post-amplification sample handling, predisposing to sample cross contamination. A one-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) assay coupled with colorimetric detection was optimised for easy and quick detection of BBrMV in banana. The viral coat protein gene was amplified under isothermal conditions at 65 ºC. The RT-LAMP assay was optimised with respect to concentrations of MgSO4, dNTP, Bst polymerase enzyme and HNB dye. The total RNA purified from symptomatic samples was directly amplified under isothermal conditions by including 100 U M-MLV reverse transcriptase and 20 U RNasin® plus RNase inhibitor in the reaction. With the addition of 120 µM of Hydroxy Naphthol Blue (HNB) dye in the RT-LAMP reaction, the BBrMV-positive samples had a colour change from violet to sky blue after the reaction. The RT-LAMP assay detected BBrMV in 0.1 pg of total RNA isolated from symptomatic plants. Molecular characterisation of RT-LAMP products was done using restriction profiling and sequence analysis. The RT-LAMP assay was validated using field-collected banana leaf samples. The assay successfully detected the virus from symptomatic samples while the healthy samples showed no amplification. Samples sourced from banana plants with symptoms of banana bunchy top virus, banana streak virus and cucumber mosaic virus tested negative in the RT-LAMP assay, thus ensuring the specificity of the assay. [ABSTRACT FROM AUTHOR]

This study was aimed to confirms the Banana Bunchy Top Virus (BBTV) infection of ten banana accessions from Indonesia through PCR assay using primer of BBTV coat protein (CP) gene, also the sequences characteristics and phylogeny. Preliminary morphological results showed five acessions were positively infected with slight to severe intensity symptoms i.e. Pisang Berlin, Candi, Billa, Morosebo, and Mas Kripik; and five accessions were symptomless i.e. M. acuminata var. rutilifes and M. balbisiana, Pisang Ebung, Madu and Moseng. However, PCR results qualitatively confirms that all accessions were positively infected. Two sizes of amplicons were identified. Pisang Candi isolate was showing ±500 bp amplicons, whereas the others were ±1000-1100 bp. The total aligned and selected BBTV CP sequences of 36 accessions (bananas and other host species) was 450 nt. It was considered highly conserved (85.55%), low G+C content (41%), and high genetic similarities (92.47 to 100%). Phylogenetic analysis delineates isolates into two large groups, i.e. the Asian group (East Asia and South-east Asia) and the South Pacific group (South Asia, Africa and the Pacific). The findings of this study are usefull for broader applicability of further banana breeding program particularly for mitigation, selection and evaluation of banana with BBTV resistance. [ABSTRACT FROM AUTHOR]

Lettuce mosaic virus (LMV) is one of the most important and destructive members of Potyviruses in the family Potyviridae that is transmitted by aphid and seed which cause economic damage and serious yield losses to different crops around the world. To explore phylogenetic relationships, population evolution, and the effect of selection forces on the complete coat protein (CP) gene of LMV isolates, 36 complete coat protein gene sequences retrieved from the GenBank database in several different countries in four continents: Central and East Asia (China, Taiwan, South Korea, and Turkey), Europe (France), Africa (Tunisia), and America (Chile and Brazil). The phylogenetic tree of LMV isolates was grouped into independent clades with the significance of FST values (>0.27). The ratio of dN/dS is calculated less than one and showed that the LMV-CP has been under negative selection. Statistical tests (Tajima’s D, Fu and Li’s D* and F*) were used to estimate non-significantly negative values for all clades and geographic populations except for the Brazilian population and Clade III in a phylogenetic group. The negatives values revealed that there is less polymorphism estimation. All geographic populations in four phylogroups of LMV seem to be at equilibrium because all neutrality test statistics were non-significant. The findings suggested that the dynamics of LMV molecular evolution may be dependent on mutation, recombination, and negative selection. No recombination events were observed in this part of the LMV genome. Therefore, this study provides the first time evolution and differentiation between populations of LMV isolates from around the world and suggested that they may be occurred by the transmission of the virus among lettuce plants by types of aphid species, migration in different geographical areas by infected seeds and plant material, and broad host range which seems that these events have played an important role in shaping the LMV population structure.

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Co-infection of bean yellow mosaic virus (BYMV) and cucumber mosaic virus (CMV) was confirmed in gladioli var. Lehenda Kyieva, Pamyat', Victoria Skvyrska by TEM, DAS-ELISA, and RT-PCR. The symptoms of co-infection were chlorotic streaks and mosaics on the leaves, flower colour breaking. The infectious nature of the symptoms was proven by biotesting method (Koch's postulates). Partial CP gene sequences of three gladiolus BYMV isolates BYMV-Gl-LeK-19 (MZ286969), GlMP-18 (MK416160), BYMV-Gl-Skv-20 (MZ286967) and three CMV isolates CMV-Gl-LeK-19 (MW847707), CMV-Gl-MP-19 (MW847708), CMV-Gl-Skv-20 (MW847710) were deposited to the NCBI GenBank. BYMV isolates are clustered with Monocot group, having identity with the isolates from this group of 94.8–99.4% nt and 95.3–100% aa. CMV isolates belong to subgroup IA and share with them identity of 81.2–99.3% nt and 82.5–97.6% aa. Analysis revealed high level of CP nucleotide sequence diversity for BYMV-Gl-LeK-19, BYMV-Gl-Skv-20 and CMV-Gl-Skv-20 as some identified substitutions were nonsynonymous. [ABSTRACT FROM AUTHOR]

Coat protein gene of potato virus X is a structural protein that plays a vital role in viral transmission and pathogenesis. RNA interference or post transcriptional gene silencing is a regulatory conserved mechanism that uses small interfering RNAs to control the expression of desired gene by inhibiting mRNA transcription. The present study aims to investigate the potential role of different siRNAs in down-regulating the mRNA expression of potato virus X in cell culture assays. Three specific siRNAs against CP-PVX gene were designed in which siRNA 3 significantly reduced the CP-PVX mRNA expression in HepG 2 cells upto 91.91% (12.5 fold) as compared to siRNA 1 and siRNA 2 where knockdown percentages were only 20 and 73.04%, respectively as revealed by realtime PCR. Maximum knockdown values were obtained at 100 nM concentration of siRNA 1 and 2, while for siRNA 3 promising knockdown was obtained at 50 nM concentration. For long lasting effect of siRNAs, short hairpin RNA corresponding to siRNA3 was designed and tested against CP-PVX which resulted in a similar pattern of inhibition on RNA expression of target gene as synthetic siRNA3. It was found that mRNA expression of targeted gene was reduced to 91% depicting strong RNAi response generated by shRNA. Our findings support the possibility of using consensus siRNA and shRNA-based approach as a promising strategy in effective inhibition of PVX in in-vitro assay. Down regulation of potato virus X through coat protein gene targeted siRNA is a potential way of inhibiting invading virus and to effectively stop its systemic spread. [ABSTRACT FROM AUTHOR]

A duplex PCR assay was standardized by optimizing PCR reaction constituents and cycles for the simultaneous detection of chickpea chlorotic dwarf virus (CpCDV) and a peanut witches' broom (PnWB) phytoplasma associated with the chickpea stunt disease. Coat protein gene and tuf gene specific primers for CpCDV and phytoplasmas were used. Different concentrations of the PCR components such as Taq polymerase, primers and PCR annealing temperature were standardized for the identification of the two agents by a duplex PCR assay. Expected amplicons of 590 bp for CpCDV and 1090 bp for phytoplasmas were consistently amplified from the symptomatic chickpea tissues. That resulted in equally efficient and sensitive in detecting single or mixed infection of CpCDV and PnWB phytoplasma in 148 symptomatic chickpea stunt samples collected in two states of India. The results indicate the robustness in the detection of pathogens present in chickpea showing stunt disease and for theoretical use in epidemiological studies that would help the appropriate disease management strategies. [ABSTRACT FROM AUTHOR]

A duplex PCR assay was standardized by optimizing PCR reaction constituents and cycles for the simultaneous detection of chickpea chlorotic dwarf virus (CpCDV) and a peanut witches' broom (PnWB) phytoplasma associated with the chickpea stunt disease. Coat protein gene and tuf gene specific primers for CpCDV and phytoplasmas were used. Different concentrations of the PCR components such as Taq polymerase, primers and PCR annealing temperature were standardized for the identification of the two agents by a duplex PCR assay. Expected amplicons of 590 bp for CpCDV and 1090 bp for phytoplasmas were consistently amplified from the symptomatic chickpea tissues. That resulted in equally efficient and sensitive in detecting single or mixed infection of CpCDV and PnWB phytoplasma in 148 symptomatic chickpea stunt samples collected in two states of India. The results indicate the robustness in the detection of pathogens present in chickpea showing stunt disease and for theoretical use in epidemiological studies that would help the appropriate disease management strategies. [ABSTRACT FROM AUTHOR]

Potato viruses were been identified in several countries around the world included Egypt. Potato virus X (PVX), is one of the common viruses infecting. potato crops. In the current study PVX isolated from naturally mixed viruses and identified based on biological characters; serologically and molecularly. The incidence of, PVY, PVX, PLRV, AMV, TYLCV were found single and mixing infection with 33.3, 37.1, 12.38, 12.38, 5.12 % frequency respectively out of 105 natural infected potato plants cv. Sponta. PVX was detected by distinct symptoms (severe mosaic, yellow blotch) and DAS-ELISA. It was isolated on Chenopodium.amaranticolor and Datora. stramonium L. It had wide host range, transmitted mechanically by syringe injection and Grafted tuber. The virus particles rod with length wide (475) nm and (12) nm. The virus stability was TIP expected at 800C for 10 min. DEP expected at 10-8 and LIVexpected at 7days. It was formed amorphous and crystal inclusion bodies in epidermal cells. PVX have antigenicity character that serological precipitation reactionwith DAS-ELISA. PVX infectivity revealed that 90%, disease severity 85% and high concentration in infected potato plants. The partial nucleotide sequence of CP gene was about 433 bp. The genetic distance with other recommended PVX strains registered in GenBank with 10 bar. The similarity values were recorded 100% with GU384726 1, 97.92 % with Accession no.MK587495, MF662526, KR605392, KR605379, KR605360 and 98.30% similarity with strain no.KJ631111. The predict numbers 144 amino acids were starting with Tyrosine. The genetic distance with other recommended PVX strains registered in GenBank with 0.20 bar It was recorded in gene bank with accession number MW 650651. [ABSTRACT FROM AUTHOR]

Cucumber mosaic virus (CMV) is one of the most widespread and harmful viruses infecting gladiolus plants worldwide. The aim of the study was to perform a phylogenetic analysis of two CMV isolates from gladioli grown in different regions of Ukraine. For the first time, 443 nt sequences of the capsid protein (CP) gene of gladiolus isolates CMV-Gl-Skv-20 (MW847710) and CMV-Gl-SkvP-20 (MW847714) from Kyiv and Poltava regions, respectively, were submitted to the NCBI GeneBank. Phylogenetic analysis showed that isolates clustered with different phylogenetic subgroups. CMV-Gl-Skv-20 belongs to subgroup IA, and has nucleotide (nt) sequence identity 81.9%-99.27% and amino acid (aa) identity 82.3%-97.6% with isolates from this group. The highest identity of the CMV-Gl-Skv-20 was found to be with Turkish CMVs from Rapistrum rugosum TUR83, TUR86 and Brassica TUR4 (98.8%-99.3% nt and 97,6% aa), as well as with Australian isolates Ny and 207 from tomato, and banana isolate Cameroon (98.9%-99% nt and 97,6% aa). CMV-Gl-SkvP-20 belongs to subgroup IB and shares 95.8%-100% nt and 96%-100% aa identity with the members of this subgroup. CMV-Gl-SkvP-20 has the highest identity with Ukrainian isolates from cucumber Ukr-1409 and Echinacea P-EP-Ukr-19 (99.5%-100% nt and 99.2-100% aa), Chinese pumpkin isolates ZBR, WHR, isolate lu-17-14 from sweet potato and SXCH from Bupleurum sp. (98.3-99% nt and 98.4%-99.2% aa), as well as with gladiolus South Korean isolate ABI (98.3% nt and 97.7% aa). The results support the fact that gladioli are affected by CMV not only through the corms, but by vector insects circulating in growing areas. [ABSTRACT FROM AUTHOR]

Sri Lankan cassava mosaic virus (SLCMV) is the principal causal agent of cassava mosaic disease in the Indian subcontinent. To gain resistance against the virus, the coat protein (CP) gene, namely the AV1 of SLCMV-Adivaram isolate, was cloned in either sense or antisense orientation under the Cauliflower mosaic virus 35S promoter, and transgenic Nicotiana benthamiana plants were obtained through Agrobacterium-mediated transformation. A total of eight T1 transgenic lines, four harboring the CP-sense construct and four harboring the CP-antisense construct were challenged with agro-infectious clones of SLCMV DNA-A and DNA-B. Based on symptom exhibition at 20 days post inoculation, 3 out of the 4 CP-sense transgenic lines and all 4 CP-antisense transgenic lines showed a high level of resistance against SLCMV. In addition, a delay in symptom initiation was observed in all the transgenic lines inoculated with a high viral load at agro-dilution 1:625 from an absorbance (A600) of 1. However, the resistance was more prominent at a lower viral load of 1:1000 agro-dilution. The viral titer was lower in the SLCMV-challenged transgenic lines compared to the non-transgenic N. benthamiana plants as confirmed by quantitative PCR and dot blot analysis. Furthermore, small RNA Northern blot analysis revealed lowered amounts of virus-specific small interfering RNAs in the resistant transgenic lines as compared to the non-transgenic plants upon SLCMV infection, which correlates to lower virus titers due to resistance against the virus.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of 'Candidatus Liberibacter asiaticus' (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of 'Ca. L. asiaticus' was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The 'Ca. L. asiaticus' isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of 'Ca. L. asiaticus' and CTV isolates and their association with citrus decline in Bhutan. [ABSTRACT FROM AUTHOR]

Tobacco streak virus (TSV) was detected from Anthurium samples collected from Anthurium variety Medioria Red from College of Horticulture, Bangalore, India grown under polyhouse cultivation. Sap inoculation of the samples expressing positive reaction through direct antigen coating-enzyme linked immunosorbant assay (DAC-ELISA) produced both localized and systemic infection. The localized infection produced circular necrotic lesions on the leaves of cowpea cv. C152 plants. The samples were also confirmed through dot blot immunibinding assay (DIBA), which showed positive reaction by producing circular, purple coloured spot against TSV specific antibody. Further molecular characterization of TSV through RT-PCR with coat protein gene specific primers produced amplicon size of 929 bp pertaining to RNA3 region. The basic local alignment search tool (BLAST) analysis of coat protein gene through National Centre for Biotechnology Information (NCBI) database had nucleotide and amino acid sequence similarity of 97.6 to 98.8 % and 94.9 to 97.4 %, respectively with known strains of TSV from India. Phylogenetic analysis of the nucleotide sequences Confirmed the Anthurium strain of TSV forms single subgroup with other crop of Indian isolates. This study provides important clues about spread of TSV. To the best of our knowledge it is the first detailed study of TSV infecting Anthurium. [ABSTRACT FROM AUTHOR]

Three isolates of Cucumber mosaic virus isolated from Commelina communis plants showing chlorosis and mosaic were collected in Chungju and Chuncheon, Korea. To confirm genetic variation of these three isolates (CMV-Co, CMV-Co2, and CMV-Co3), we performed PCR-RFLP and sequence analysis. Sequences of coat protein genes of CMV-C0, -Co2 and -Co3 were compared with CMV-Fny and showed 96.3%, 96.3%, and 95.9% similarities, respectively. In host reactions, three CMV-Co isolates induced systemic necrosis in Cucurbita pepo unlike CMV-Fny and CMV-Co, CMV-Co2 and CMV-Co3 observed differential symptoms responses in Physalis angulata and Nicotiana rustica. These results indicated that three isolates of CMV isolated from C. communis have genetic and biological variation.

Plant viruses cause serious disease of crop plants reducing both quality and quantity of final produce. Serological tests are used all over the world in laboratory and field based detection of plant viruses and they are of great indispensable importance in agricultural production; virus certification programs; agricultural quarantine and production of virus-free crops grown for processing or fresh market. Cross reaction between viruses and their strains antisera limits serological differentiation of viruses and their strains by enzyme-linked immunosorbent assay (ELISA). This study aims to characterize the antigenic property of Tomato mosaic virus ToMV coat protein by using some bioinformatic tools to analyze its gene. Therefore, new methods in antibody production could be used as equivalent to Mabs in its high specificity. ToMV isolate was confirmed by Transmission electron microscope and differential hosts and propagated on N. tabacum cv. Samson. Systemic infection was developed on N. tabacum cv. Samson and local infection on Datora metel; D. stramonium; N. glutinosa; Chenopodium amaranticolor; C. quinoa. ToMV was purified and used as immunizing agent for antiserum production. TEM showed rod shaped particles with 300 x 18 nm dimensions. The titer of produced antiserum was 1:1024 evaluated by microprecipitin test and indirect-ELISA. Coat protein gene was amplified by RT-PCR with expected size of (Approx. 500 bp). The PCR product was sequenced then the generated nucleotide sequence was translated into 160 amino acids. The amino acid sequence of Five B-cell epitopes, of 14 amino acid residues each, were predicted. Identifying Bcell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting B-cell epitopes are studied.

Control of insects in stored products is very important. Sitophilus granarius (Linnaeus, 1758) (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidea) are two important insect species that cause damage to stored products. The need for alternative drugs that can be used to control of pests in stored products is increasing day by day. In this study, the usability of plant extracts obtained from Achillea phrygia Boiss. et Bal. (Compositae), Prangos ferulacea (L.) Lindl. (Apiaceae) ve Salvia wiedemannii Boiss. (Lamiaceae) plants to control S. granarius and T. castaneum were investigated. At the end of 72 hours, the highest contact activity among the plants tested on S. garanarius showed P. ferulaceae hexane with 74% and A. biserrata methanol extract with 71%, respectively. The highest activity on T. castaneum was obtained by A. biserrata ethyl acetate extract at 42% after 72 hours. On the other hand, the highest repellent activity on S. granarius was obtained from P. ferulaceae ethyl acetate extract (52%) after 24 hours. All plant extracts showed high repellent effect on T. castaneum. [ABSTRACT FROM AUTHOR]

Rice tungro is one of the most economically damaging virus diseases in the Philippines, which is caused jointly by Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The disease causes a significant effect on rice propagation and cultivation. This study aimed to determine the level of genetic diversity of RTBV coat protein gene sequences collected from tungro-hotspot areas in the Philippines and infer their phylogenetic relationships. A total of 144 RTBV coat protein gene sequences were analyzed from six provinces with 21 municipalities representing Luzon, Visayas, and Mindanao. The highest percentage of RTBV detected was in Camarines Sur at 78% while Nueva Ecija had the least at 45%. Among the provinces, nucleotide diversity and number of segregating sites of RTBV were highest in Camarines Sur (π = 0.010797, S = 145) and lowest in Laguna (π = 0.006233, S = 67). Phylogenetic analysis revealed six divergent groups but did not completely reflect geographical origin while principal coordinate analysis (PCoA) resulted in three major groups: Laguna, Negros Occidental, and Nueva Ecija (Luzon and Visayas); and Camarines Sur and North Cotabato plus Isabela (Luzon and Mindanao). These results could infer genetic differentiation is relevant that disease severity variation could be observed including some breaking in resistance in some areas. There is a need to re-design breeding such as locationspecific breeding for RTBV-resistant rice varieties that would consider genetic variation within and between location. Other molecular approaches such as the use of next-generation sequencing could be recommended to support the findings from this study. [ABSTRACT FROM AUTHOR]

In order to study the current situation of Citrus psorosis virus (CPsV) in Tunisia, field surveys were conducted in commercial orchards in Cap Bon region and 575 citrus trees were sampled. As determined by DAS-ELISA, the CPsV incidence in Tunisia was high, and ranged between 20 and 47%, according to geographical location, citrus species and varieties. cDNA encompassing the full-length RNA3 from Tunisian isolates was synthesized by RT-PCR using specific primers and two isolates were cloned and sequenced. Full length sequences of the coat protein (CP) gene were obtained and deposited in GenBank. Based on CP gene sequences, phylogenetic clustering of isolates revealed two populations, one includes isolates from Spain, Morocco and New Zealand and the other includes those from Tunisia, Spain, Italy, Mexico and Egypt. Genetic haplotype network provided evidence of the existence of two distinct clusters with an ambiguous origin. Selective neutrality test was significantly positive, suggesting a balancing selection of CPsV CP gene.

Sugarcane mosaic virus (SCMV) is one of the most damaging viruses infecting sugarcane, maize and some other graminaceous species around the world. To investigate the genetic diversity of SCMV in Iran, the coat protein (CP) gene sequences of 23 SCMV isolates from different hosts were determined. The nucleotide sequence identity among Iranian isolates was more than 96%. They shared nucleotide identities of 75.5–99.9% with those of other SCMV isolates available in GenBank, the highest with the Egyptian isolate EGY7-1 (97.5–99.9%). The results of phylogenetic analysis suggested five divergent evolutionary lineages that did not completely reflect the geographical origin or host plant of the isolates. Population genetic analysis revealed greater between-group than within-group evolutionary divergence values, further supporting the results of the phylogenetic analysis. Our results indicated that natural selection might have contributed to the evolution of isolates belonging to the five identified SCMV groups, with infrequent genetic exchanges occurring between them. Phylogenetic analyses and the estimation of genetic distance indicated that Iranian isolates have low genetic diversity. No recombination was found in the CP cistron of Iranian isolates and the CP gene was under negative selection. These findings provide a comprehensive analysis of the population structure and driving forces for the evolution of SCMV with implications for global exchange of sugarcane germplasm. Gene flow, selection and somehow homologous recombination were found to be the important evolutionary factors shaping the genetic structure of SCMV populations.

Aim. Establishing the genetic diversity of Plum pox virus (PPV) in Ukraine. Methods. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), polymerase chain reaction with reverse transcription (RT-PCR), DNA sequencing and phylogenetic analysis. Results. The samples with visual symptoms of virus infection were collected from different regions of Ukraine and diagnosed using DAS-ELISA. The amplicons of the coat protein (CP) gene were obtained using RT-PCR and sequenced. The obtained viral cDNAs were analyzed using phylogenetic methods to elucidate the genetic diversity of PPV in different regions of Ukraine. The phylogenetic relationships between Ukrainian isolates were studied and compared to PPV isolates from neighboring countries. Conclusions. The PPV strains M and D circulate in Ukraine. For the first time, PPV was found in the Kharkiv region. The PPV isolates circulating in Ukraine are similar to the isolates from Germany, Turkey, Hungary, and Canada. The results can be used to predict the spread of the virus in different Ukrainian regions and in neighboring countries and to establish its origin and predict the development of possible epidemics. [ABSTRACT FROM AUTHOR]

In 2013, a chili pepper (Capsicum annuum cv. 'Favorite') sample displaying crinkled and mottled leaves was collected from a field in the Hunan Province, China. Small RNA (sRNA) sequencing identified the putative virus as pepper mottle virus (PepMoV), which was confirmed by RT-PCR analysis. Full length sequence of this virus strain (designated as HN) was obtained and shown to be typical of a potyvirus. Coat protein (CP) genes of nine PepMoV isolates obtained from south China were sequenced and they shared 89 to 99% sequence identity with PepMoV CP sequences retrieved from GenBank. Phylogenetic analysis, and the level of recombination, gene flow and genetic differention revealed that the PepMoV isolates of Chinese lineage clustered within the south-east group. PepMoV has now been detected in three different provinces of southern part of China. [ABSTRACT FROM AUTHOR]

Viruses assumed to belong to a new Potyvirus were detected in passionfruit plants with foliar mosaic, leaf curl and fruit malformation that were collected from Akita City, Akita Prefecture in 2013, Nago City, Okinawa Prefecture in 2014, and Satsuma Town and Yoron Island, Kagoshima Prefecture in 2015. The isolates of this virus species were not detected by ELISA and RT-PCR targeting East Asian Passiflora virus (EAPV)-AO (Amami Oshima strain), which induces woodiness disease of passionfruit in Japan. The isolates were designated PV-AK (Akita), PV-OK (Okinawa), PV-YO (Yoron), and PV-SA (Satsuma). In a host-range test of 13 cultivars of French bean, four were systemically infected with the PV isolates and two with EAPV-AO. Cowpea cv. Kurodane sanjaku was systemically infected with PV isolates and induced mosaic symptoms on non-inoculated leaves distant from the inoculated leaves but was immune to EAPV-AO. The complete genomic sequences of the PV isolates consisted of 9973 nucleotides (nt) excluding the poly(A) tail, and encoded 3217 amino acids of polyprotein, flanked by 129–130 nt of the 5′-noncoding region and 193 nt of the 3′-noncoding region. Whole-genome nucleotide and amino acid sequences indicated that they belong to identical species. The potyvirus with the highest whole-genome nucleotide identity to the PV isolates is watermelon mosaic virus, which shares 68.1% identity, versus 65.3% for EAPV-AO. On the basis of ICTV-naming regulations, these PV isolates belong to a new species in the Potyvirus genus, but are distantly related to EAPV. Here, we propose East Asian Passiflora distortion virus as the new species name. [ABSTRACT FROM AUTHOR]

Necrosis disease incited by tobacco streak virus (TSV) causes significant yield losses in sunflower in the tropics and sub-tropics. Genetic engineering of sunflower through deployment of the coat protein gene of TSV (TSV-CP) was attempted along with neomycin phosphotransferase gene (nptII) for selection of putative transformants on kanamycin. Split cotyledons from decoated mature seeds were used as target tissues for Agrobacterium tumefaciens-mediated transformation and putative transformed shoots were obtained with an average frequency of 3.3%. Presence of the introduced transgene was identified by PCR; expression was determined through RT-PCR, northern blot analysis and quantitative Real-time PCR (qRT-PCR); stable integration was ascertained through Southern blot analysis of plants in T0 to T4 generations. Out of 102 positive T0 events, 20 events were carried to T1 generation from which five events (CP-S-237, CP-S-247, CP-S-481, CP-S-648 and CP-S-753) were advanced till T4 generation. Challenge inoculations of plants of event No 481 showed resistance to necrosis disease and plants grew to maturity in TSV-CP transgenics while control plants (untransformed) showed mortality within 1-2 weeks following inoculation. Expression analysis of the TSV-CP and nptII genes in different tissues at flowering and seed setting stages revealed constitutive expression of the transgene till seed maturation. One event (No 481) was selected for transfer of the TSV-CP gene into agronomically superior genotypes. [ABSTRACT FROM AUTHOR]

【Objective】Cymbidium mosaic virus(CyMV) and Odntoglossum ringspot virus(ORSV) are the two main viruses which often infect Phalaenopsis severely and lead to huge economic losses to the industry. In presented paper, these two viruses were detected rapidly, which would provide a basis for breeding Phalaenopsis of resistance to both viruses of CyMV and ORSV. 【Method】The specific primers were designed based on CyMV coat protein(CP) gene and ORSV CP gene sequence which had been reported in NCBI, and antisense RNA technology was used in this study. 【Result】12 samples of Phalaenopsis by using designed specific primers were detected, and 25% of them contained the two viruses, and the cloning products were clear and no redundancy from the same PCR reaction. 125 bp fragment of CyMV CP gene and 164 bp fragment of ORSV CP gene from the leaf tissue of infected Phalaenopsis were obtained. The CP gene fragment of CyMV linked to the expression vector p CAMBIA1300 in 3'→5'orientation, and named p CAMBIA1300-CyMV which could obtain 125 bp fragment by PCR and restriction enzyme digestion. The CP gene fragment of ORSV linked to the constructed vector p CAMBIA1300-CyMV in 3'→5'orientation, and designated p CAMBIA1300-CyMV-ORSV which could obtain 289 bp fragment by PCR and restriction enzyme digestion. 【Conclusion】CyMV and ORSV by using the specific primers could be detected simultaneously in the same PCR reaction system, and the double-antisense expression vector containing two kinds of the virus CP genes was constructed successfully. [ABSTRACT FROM AUTHOR]

Plant viruses cause serious disease of crop plants reducing both quality and quantity of final produce. Serological tests are used all over the world in laboratory and field based detection of plant viruses and they are of great indispensable importance in agricultural production; virus certification programs; agricultural quarantine and production of virus-free crops grown for processing or fresh market. Cross reaction between viruses and their strains antisera limits serological differentiation of viruses and their strains by enzyme-linked immunosorbent assay (ELISA). This study aims to characterize the antigenic property of Tomato mosaic virus ToMV coat protein by using some bioinformatic tools to analyze its gene. Therefore, new methods in antibody production could be used as equivalent to Mabs in its high specificity. ToMV isolate was confirmed by Transmission electron microscope and differential hosts and propagated on N. tabacum cv. Samson. Systemic infection was developed on N. tabacum cv. Samson and local infection on Datora metel; D. stramonium; N. glutinosa; Chenopodium amaranticolor; C. quinoa. ToMV was purified and used as immunizing agent for antiserum production. TEM showed rod shaped particles with 300 x 18 nm dimensions. The titer of produced antiserum was 1:1024 evaluated by microprecipitin test and indirect-ELISA. Coat protein gene was amplified by RT-PCR with expected size of (Approx. 500 bp). The PCR product was sequenced then the generated nucleotide sequence was translated into 160 amino acids. The amino acid sequence of Five B-cell epitopes, of 14 amino acid residues each, were predicted. Identifying Bcell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting B-cell epitopes are studied. [ABSTRACT FROM AUTHOR]

Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84–99% to 81–100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

During 2014 a total of 67 pot marigold samples from five different localities in the Province in Vojvodina were collected and analysed for the presence of Cucumber mosaic virus (CMV) and Impatiens necrotic spot virus (INSV) using commercial double-antibody sandwich (DAS)-ELISA kits. CMV was detected serologically in all inspected localities in 67.16% collected samples. None of the analysed samples was positive for INSV. The virus was successfully mechanically transmitted to test plants including Chenopodium amaranticolor, C. quinoa, Datura stramonium, Nicotiana tabacum 'Samsun' and N. glutinosa, as well as pot marigold seedlings, confirming the infectious nature of the disease. The presence of CMV in pot marigold plants was further verified by RT-PCR and sequencing, using the specific primers CMV CPfwd/CMVCPrev that amplify coat protein (CP) gene. Phylogenetic analysis based on the CP gene sequences showed clustering of the selected isolates into three subgroups, IA, IB and II, and Serbian CMV isolates from pot marigold belong to subgroup II.