Generation and Characterization of Nanobodies Against Tomato Leaf Curl Sudan Virus.

Begomoviruses infect food, fiber, and vegetable crop plants, including tomato, potato, bean, cotton, cucumber, and pumpkin, and damage many economically important crop plants worldwide. Tomato leaf curl Sudan virus (ToLCSDV) is the most widespread tomato-infecting begomovirus in Saudi Arabia. Using phage display technology, this study isolated two camel-derived nanobodies against purified ToLCSDV virions from a library of antigen-binding fragments (VHH or nanobody) of heavy-chain antibodies built from an immunized camel. The isolated nanobodies also cross-reacted with purified tomato yellow leaf curl virus virions and showed significant enzyme-linked immunosorbent assay reactivity with extracts from plants with typical begomovirus infection symptoms. The results can pave the way to developing diagnostics for begomovirus detection, design, and characterization of novel nanomaterials based on virus-like particles, in addition to nanobody-mediated begomovirus resistance in economically important crops, such as tomato, potato, and cucumber.

Molecular characterization of a naturally occurring intraspecific recombinant begomovirus with close relatives widespread in southern Arabia.

BACKGROUND: Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed. FINDINGS: The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants. CONCLUSION: Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation.

The Oman strain of Tomato yellow leaf curl virus (TYLCV-OM) and its associated betasatellite, an isolate of Tomato leaf curl betasatellite (ToLCB), were previously reported from Oman. Here we report the isolation of a second, previously undescribed, begomovirus [Tomato leaf curl Oman virus (ToLCOMV)] and an alphasatellite from that same plant sample. This alphasatellite is closely related (90 % shared nucleotide identity) to an unusual DNA-2-type Ageratum yellow vein Singapore alphasatellite (AYVSGA), thus far identified only in Singapore. ToLCOMV was found to have a recombinant genome comprising sequences derived from two extant parents, TYLCV-OM, which is indigenous to Oman, and Papaya leaf curl virus from the Indian subcontinent. All possible combinations of ToLCOMV, TYLCV-OM, ToLCB and AYVSGA were used to agro-inoculate tomato and Nicotiana benthamiana. Infection with ToLCOMV yielded mild leaf-curl symptoms in both hosts; however, plants inoculated with TYLCV-OM developed more severe symptoms. Plants infected with ToLCB in the presence of either helper begomovirus resulted in more severe symptoms. Surprisingly, symptoms in N. benthamiana infected with the alphasatellite together with either of the helper viruses and the betasatellite were attenuated and betasatellite DNA accumulation was substantially reduced. However, in the latter plants no concomitant reduction in the accumulation of helper virus DNA was observed. This is the first example of an attenuation of begomovirus-betasatellite symptoms by this unusual class of alphasatellites. This observation suggests that some DNA-2 alphasatellites encode a pathogenicity determinant that may modulate begomovirus-betasatellite infection by reducing betasatellite DNA accumulation.

Third molar impaction in the Jazan Region: Evaluation of the prevalence and clinical presentation.

OBJECTIVE: To provide information on the prevalence and clinical features of impacted third molar teeth in the South-Western region of Saudi Arabia. MATERIAL AND METHODS: In this cross-sectional study, 1200 panoramic radiographs (50% males and 50% females) were retrieved from the electronic clinical records of patients at the College of Dentistry, Jazan University from December 2014 to December 2016, and impacted third molars were evaluated. Data on clinical and radiographic presentation were analyzed. RESULTS: Overall, there were 291 (24.3%) patients with impacted third molars among 1200 radiographs. The distribution of impacted third molars according to the number of impacted teeth was as follows: one impaction in 121 (41.6%); two impactions in 90 (30.9%); three impactions in 42 (14.4%); and four impactions in 38 (13.1%) patients. There was a high prevalence of all impaction types among females (54.5%). Maxillary vertical angulation was most common (50%) followed by mandibular mesioangular angulation (48.3%). The depth of impaction in maxillary teeth was higher than in mandibular teeth. Pain was uncommon (4.5% of patients). DISCUSSION: Clinically, vertical impaction in the maxilla was present in 50% of patients because of limited posterior space, and mesioangular angulation in the mandible was present in 48% of patients because of inadequate space between the ramus and the second molar. These findings are similar to other reports. Vertical impaction of the maxillary wisdom tooth is mostly related to the discrepancy between the mesiodistal size of the tooth crown and the limited retromolar space. CONCLUSION: Noiseless presentation of an impacted third molar requires raising the population's awareness about the need for diagnosis and treatment of the problem to avoid any further complications. The study can be to guide surgical procedures. This study documented the prevalence, pattern, and clinical features of impacted third molars in South Western region of Saudi Arabia.

Molecular characterization and phylogenetic relationships of Desmodium leaf distortion virus (DeLDV): a new begomovirus infecting Desmodium glabrum in Yucatan, Mexico.

The complete DNA-A component sequence of Desmodium leaf distortion virus (DeLDV, Begomovirus) isolated in Yucatan was determined to be 2569 nucleotides (nt) in length, and it was most closely related to Cotton leaf crumple virus-California (CLCrV-[Cal]), at 76%. The complete DNA-B component sequence was 2514 nt in length, and shared its highest nucleotide identity (60%) with Potato yellow mosaic Trinidad virus (PYMTV). Phylogenetic analyses group the DeLDV DNA-A component in the SLCV clade, whereas, the DeLDV DNA-B was grouped with the Abutilon mosaic virus clade, which also contains PYMV, suggesting that the DeLDV components have distinct evolutionary histories, possibly as the result of recombination and reassortment.

Minimal genomic variability in Merremia mosaic virus isolates endemic in Merremia spp and cultivated tomato in Puerto Rico.

Merremia mosaic virus (MerMV), a bipartite begomovirus, was identified for the first time as a pathogen of commercial tomato plantings. Infection of tomato by MerMV caused mild leaf curling and yellow foliar mosaic symptoms. Herein, the MerMV was identified in symptomatic Merremia quinquefolia and M. aegyptia (Convolvulaceae) plants exhibiting bright yellow or yellow-green foliar mosaic symptoms, respectively. The full-length begomoviral components were amplified from total DNA isolated from two wild species of Merremia and commercial tomato plants during 1991-1998. The DNA was subjected to rolling circle amplification, restriction digestion, and DNA sequencing. The resultant 19 and 26 apparently full-length DNA-A and DNA-B components were ~ 2557 and ~ 2492 bases, respectively. The 140-base common region was 97.9% identical between DNA-A and -B components, a predictive evidence for cognate DNA-A and -B components. Although the DNA-A components were highly conserved at 96-100%, the DNA-B components diverged at ~ 89 to 100%, respectively. The overall clonal genomic features strongly suggested that MerMV lineage has been under host-selection for some time, and only recently, has undergone a host-shift, putatively, from wild convolvulaceous species to tomato (Solanaceae). Phylogenetically, MerMV grouped with other bipartite begomoviruses indigenous to the Caribbean region, with MerMV DNA-A components forming three clusters, and the DNA-B components grouped in one clade. Both clades contained only one closet relative, an isolate of MerMV from Venezuela, MerMV-VE. Biolistic inoculation of M. quinquefolia and tomato seedlings with the DNA-A and -B components of PR68 and PR80 resulted in development of symptoms like those observed in naturally-infected species, respectively.

Retrospective analysis of biopsied oral and maxillofacial lesions in South-Western Saudi Arabia.

OBJECTIVES: To report the prevalence and types of biopsied oral and maxillofacial lesions (OMLs) in South-Western (Jazan Province) region, , Kingdom of Saudi Arabia (KSA). Methods: This retrospective study was based on the retrieval of clinicopathological data for a period of 6 years between January 2009 and December 2014. These  data were obtained between October 2014 and June 2015 from the histopathology records of King Fahad Central Hospital, Jazan, KSA, which is the only referral center for biopsy services. Results: Out of the 32149 biopsies received, 714 (2.2%) were OMLs. The age ranged from 0 (neonatal) to 100 years, with a mean age of 46.8±23.4 and a male-to-female ratio of 1:1.3. The tongue was the most common site for OMLs and for malignant neoplasms, in particular. The most common category was malignant neoplasm (38.7%), followed by inflammatory lesions (16.5%). Oral malignancies accounted for 15.8% of all malignancies. Oral squamous cell carcinoma (OSCC) (36.1%) was the most frequent type, followed by pyogenic granuloma and mucocele (7% each). Shammah-associated OSCC and epithelial dysplasia were twice as common in females. Conclusion: The number of non-malignant OMLs was much lower than expected in comparison to oral malignancies. This difference can likely be explained by the fact that the biopsies were taken only when malignancy was suspected. The higher rate of OSCC reported from this region is attributed to shammah usage. This study emphasizes the importance of biopsy services for all OMLs and the prevention of shammah use.

Retrospective analysis of benign orofacial tumors at a tertiary referral center in Saudi Arabia.

AIM: The aim of the present study was to analyze the prevalence of benign tumors of the orofacial region at a tertiary referral center in the south-western region of Saudi Arabia. METHODS: Cases from 2009 to 2014 were retrieved from October 2014 to June 2015 from the archives of the histopathology department of the center. Demographic and clinical details of the patients were recorded. RESULTS: Of the 714 oral and maxillofacial biopsy specimens, 78 (10.9%) were benign tumors. The mean age and range were 34.6±19.8 and 3-85 year, respectively. Sex distribution was equal. Most tumors were mesenchymal (34.6%), followed by epithelial (26.9%), odontogenic (20.5%), and salivary gland tumors (17.9%). Squamous cell papilloma (20.5%) was the most common, followed by pleomorphic adenoma (15.4%) and fibrous tumors (15.4%). CONCLUSIONS: The low prevalence of benign orofacial tumors found in this study indicates a lack of awareness of the importance of taking biopsy for such lesions. The information reported here emphasizes the need for biopsy investigation for all oral lesions to ascertain appropriate diagnosis.

Leaf curl diseases of two solanaceous species in Southwest Arabia are caused by a monopartite begomovirus evolutionarily most closely related to a species from the Nile Basin and unique suite of betasatellites.

The complete genome of 2780 bases was amplified using rolling circle amplification, and cloned, and sequenced for two distinct strains of the monopartite begomovirus Tomato leaf curl Sudan virus (ToLCSDV). The two strains shared 86-91% identity with the previously described ToLCSDV from the Nile Basin, and 90-91% identity with one another. One strain was cloned from symptomatic tomato plants from Tihamah (ToLCSDV-YE[YE:Tih:05]) while the other was cloned from symptomatic tobacco plants collected from Wadi Hadramaut (ToLCSDV-YE[YE:Had:89]). A distinct full-length betasatellite molecule (1352 bases) was cloned from the respective field-infected tomato and tobacco plants. Agro-inoculation of tomato and Nicotiana benthamiana plants with cloned partial tandem repeats of ToLCSDV-YE[YE:Tih11:05]) and the associated betasatellite, Tomato leaf curl Yemen betasatellite (ToLCYEB-[Tih:tom:137:05]), resulted in the reproduction of leaf curl disease symptoms in test plants like those observed in the field-infected plants. The betasatellite contributed to symptom severity in N. benthamiana test plants when it was co-inoculated with ToLCSDV-YE, compared to the milder symptoms that were observed in tobacco plants infected with the helper virus alone.

Phylogenetic analysis of Melon chlorotic leaf curl virus from Guatemala: another emergent species in the Squash leaf curl virus clade.

The genome of a new bipartite begomovirus Melon chlorotic leaf curl virus from Guatemala (MCLCuV-GT) was cloned and the genome sequence was determined. The virus causes distinct symptoms on melons that were not previously observed in melon crops in Guatemala or elsewhere. Phylogenetic analysis of MCLCuV-GT and begomoviruses infecting cucurbits and other host plant species indicated that its closest relative was MCLCuV from Costa Rica (MCLCuV-CR). The DNA-A components of two isolates shared 88.8% nucleotide identity, making them strains of the same species. Further, both MCLCuV-GT and MCLCuV-CR grouped with other Western Hemisphere cucurbit-infecting species in the SLCV-clade making them the most southerly cucurbit-infecting members of the clade to date. Although the common region of the cognate components of MCLCuV-GT and MCLCuV-CR, shared ∼96.3% nucleotide identity. While DNA-A and DNA-B components of MCLCuV-GT were less than 86% nucleotide identity with the respective DNA-A and DNA-B common regions of MCLCuV-CR. The late viral genes of the two strains shared the least nt identity (<88%) while their early genes shared the highest nt identity (>90%). The collective evidence suggests that these two strains of MCLCuV are evolutionarily divergent owing in part to recombination, but also due to the accumulation of a substantial number of mutations. In addition they are differentially host-adapted, as has been documented for other cucurbit-infecting, bean-adapted, species in the SLCV clade.

A divergent isolate of tomato yellow leaf curl virus from Oman with an associated DNA beta satellite: an evolutionary link between Asian and the Middle Eastern virus-satellite complexes.

Tomato is cultivated in the coastal region of Al-Batinah, in the Sultanate of Oman, during the winter season, to meet the high demand for fresh produce in the domestic market. In order to identify the causal agent of a widespread disease associated with infestations of the whitefly Bemisia tabaci (Genn.) leaves were collected from tomato plants showing symptoms characteristic of the disease in Al-Batinah during 2004 and 2005. Total nucleic acids were isolated from the tomato leaves and used as the template for Phi29 DNA polymerase amplification of begomoviral circular DNA. Putative full unit length begomoviral DNA multimers were digested with Nco I and cloned into the plasmid vector pGEM7Zf+. The complete nucleotide (nt) sequence was determined as 2,765 bases, indicative of a monopartite begomoviral genome. A comparison of the genome sequence for the seven field isolates examined, indicated that they shared 99% nt identity. The virus from Oman was most closely related to TYLCV-IR at 91% nt identity, a monopartite begomoviral species described previously from Iran. Based on the guidelines of the ICTV the Oman isolate has been designated TYLCV-Om and is considered an isolate of TYLCV-IR. A satellite DNA (satDNA beta), was amplified by polymerase chain reaction using degenerate primers and cloned, and the DNA sequence was determined. Analysis of the complete nt sequence of 1,371 bases indicated that the satDNA shared 88.5% similarity with its closest relatives, which are DNA beta molecules from tomato in Pakistan. This is the first report of a satDNA beta associated with the TYLCV species. The TYLCV-Om and associated satDNA, thus represent a begomovirus-complex at the Asian-Middle East crossroads that quiet uniquely share geographical and genetic hallmarks of both.

Molecular characterization and phylogenetic relationships of two new bipartite begomovirus infecting malvaceous plants in Yucatan, Mexico.

Sida acuta and Corchorus siliquosus plants showing yellow mosaic and yellow vein symptoms, respectively, were collected in the Yucatan Peninsula, Mexico. Total DNA was isolated from both plant species and used for the amplification, cloning, and sequencing of the Begomovirus genome. Nucleotide comparison of the complete DNA-A component isolated from S. acuta and C. siliquosus confirmed the presence of two distinct begomoviruses species. Based on phenotypic symptoms observed in infected field plants, the names Sida yellow mosaic Yucatan virus (SiYMYuV) and Corchorus yellow vein Yucatan virus (CoYVYuV) were proposed. The SiYMYuV DNA-A shared the highest nucleotide identity (86%) with the Okra yellow mosaic Mexico virus (OkYMMV). The complete DNA-B component shared the highest nucleotide identity (80%) with CoYVYuV. The CoYVYuV DNA-A shared the highest nucleotide identity (84%) with SiYMYuV. The 166-nt common region (CR) sequence for the DNA-A and DNA-B components of SiYMYuV shared a high nucleotide identity of 99%, and the 151 nt of CoYVYuV CR shared 95% of nucleotide identity. The organization and the iterated sequence of the putative AC1 binding site (located within the common region) of both isolates, were similar to that of the begomoviruses of the Western Hemisphere. Phylogenetic analyses placed the DNA-A and DNA-B of SiYMYuV and CoYVYuV in the clade containing the Abutilon mosaic virus (AbMV).

Preliminary identification and coat protein gene phylogenetic relationships of begomoviruses associated with native flora and cultivated plants from the Yucatan Peninsula of Mexico.

A number of native and cultivated eudicots in the Yucatan Peninsula of Mexico (YPM) exhibit symptoms associated with virus infection. Symptomatic leaves were collected and assessed for begomoviral detection using polymerase chain reaction (PCR), and universal primers that amplify a fragment of the coat protein gene (core Cp). Begomovirus were detected in nine native and seven cultivated species, representing seven eudicot families. DNA extracts from the 16 hosts were used for PCR amplification and sequencing of a fragment containing the coat protein (Cp) gene. The complete Cp sequence was used to establish provisional species identification. Results indicated that 13 distinct begomovirus species were represented. Among these, five potentially new begomovirus species were identified, for which we propose the names Anoda golden mosaic virus (AnGMV), Boerhavia yellow spot virus (BoYSV), Papaya golden mosaic virus (PaGMV), Desmodium leaf distortion virus (DeLDV), and Hibiscus variegation virus (HiVV). Five previously described begomoviral species were provisionally identified for the first time in the YPM; these include Euphorbia mosaic virus (EuMV), Melon chlorotic leaf curl virus (MCLCuV), Okra yellow mosaic Mexico virus (OkYMMV), Sida golden mosaic virus (SiGMV), and Tobacco apical stunt virus (TbASV). Additionally, viruses previously reported from this region, Bean golden yellow mosaic virus (BGYMV), Pepper golden mosaic virus (PepGMV), and Tomato mottle virus (ToMoV) were provisionally identified in cultivated hosts. Phylogenetic analysis provisionally placed all isolates from the YPM in a Western Hemisphere begomovirus clade.

Molecular analysis of Cotton leaf curl virus-Sudan reveals an evolutionary history of recombination.

Monopartite begomoviral DNAs (2761 bp) were cloned and sequenced from field cotton, okra, and Sida alba, from Gezira, and field okra from Shambat. Comparison of the four apparent full-length begomoviral DNAs revealed 99.3-99.5% shared nucleotide (nt) identity, indicating that they are the same viral species, hereafter, referred to as Cotton leaf curl virus-Sudan (CLCuV-SD). Host range studies revealed that the field okra isolate of CLCuV-SD was whitefly-transmissible from okra to okra, M. parviflora, and hollyhock, but not to cotton. In contrast, the cotton isolate of CLCuV-SD infected cotton and hollyhock, but not okra. The genome of CLCuV-SD encodes six open reading frames (ORFs), and was most closely related to other monopartite begomoviruses of the Eastern Hemisphere. CLCuV-SD shared highest nucleotide sequence identity (95.5%) with Okra enation virus (OkEV), but was distantly related (approximately 74% nt sequence identity) to begomoviruses isolated from cotton in Pakistan. While extensive genomic regions of CLCuV-SD and OkEV are highly conserved (approximately 99% nt identity), nt sequence identity of the V1 ORF encoding the coat protein was uncharacteristically low (87.9%), suggesting a history of recombination. An analysis conducted with Sawyer's GENECONV program support the recombination hypothesis, indicating that the V1 ORF and a small segment of the intergenic region of CLCuV-SD and OkEV were derived from other begomoviruses. As a BLAST analysis failed to identify a prospective extant source of either V1 ORF, the parental viruses serving as CP donors remain undiscovered or are extinct.

Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses.

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.