Twindemic Threats of Weeds Coinfected with Tomato Yellow Leaf Curl Virus and Tomato Spotted Wilt Virus as Viral Reservoirs in Tomato Greenhouses.

Tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV) are well-known examples of the begomovirus and orthotospovirus genera, respectively. These viruses cause significant economic damage to tomato crops worldwide. Weeds play an important role in the ongoing presence and spread of several plant viruses, such as TYLCV and TSWV, and are recognized as reservoirs for these infections. This work applies a comprehensive approach, encompassing field surveys and molecular techniques, to acquire an in-depth understanding of the interactions between viruses and their weed hosts. A total of 60 tomato samples exhibiting typical symptoms of TYLCV and TSWV were collected from a tomato greenhouse farm in Nonsan, South Korea. In addition, 130 samples of 16 different weed species in the immediate surroundings of the greenhouse were collected for viral detection. PCR and reverse transcription-PCR methodologies and specific primers for TYLCV and TSWV were used, which showed that 15 tomato samples were coinfected by both viruses. Interestingly, both viruses were also detected in perennial weeds, such as Rumex crispus, which highlights their function as viral reservoirs. Our study provides significant insights into the co-occurrence of TYLCV and TSWV in weed reservoirs, and their subsequent transmission under tomato greenhouse conditions. This project builds long-term strategies for integrated pest management to prevent and manage simultaneous virus outbreaks, known as twindemics, in agricultural systems.

An Effective Integrated Machine Learning Framework for Identifying Severity of Tomato Yellow Leaf Curl Virus and Their Experimental Validation.

Tomato yellow leaf curl virus (TYLCV) dispersed across different countries, specifically to subtropical regions, associated with more severe symptoms. Since TYLCV was first isolated in 1931, it has been a menace to tomato industrial production worldwide over the past century. Three groups were newly isolated from TYLCV-resistant tomatoes in 2022; however, their functions are unknown. The development of machine learning (ML)-based models using characterized sequences and evaluating blind predictions is one of the major challenges in interdisciplinary research. The purpose of this study was to develop an integrated computational framework for the accurate identification of symptoms (mild or severe) based on TYLCV sequences (isolated in Korea). For the development of the framework, we first extracted 11 different feature encodings and hybrid features from the training data and then explored 8 different classifiers and developed their respective prediction models by using randomized 10-fold cross-validation. Subsequently, we carried out a systematic evaluation of these 96 developed models and selected the top 90 models, whose predicted class labels were combined and considered as reduced features. On the basis of these features, a multilayer perceptron was applied and developed the final prediction model (IML-TYLCVs). We conducted blind prediction on 3 groups using IML-TYLCVs, and the results indicated that 2 groups were severe and 1 group was mild. Furthermore, we confirmed the prediction with virus-challenging experiments of tomato plant phenotypes using infectious clones from 3 groups. Plant virologists and plant breeding professionals can access the user-friendly online IML-TYLCVs web server at https://balalab-skku.org/IML-TYLCVs, which can guide them in developing new protection strategies for newly emerging viruses.

Emergence of Asian endemic begomoviruses as a pandemic threat.

Plant viruses are responsible for the most devastating and commercially significant plant diseases, especially in tropical and subtropical regions. The genus begomovirus is the largest one in the family Geminiviridae, with a single-stranded DNA genome, either monopartite or bipartite. Begomoviruses are transmitted by insect vectors, such as Bemisia tabaci. Begomoviruses are the major causative agents of diseases in agriculture globally. Because of their diversity and mode of evolution, they are thought to be geographic specific. The emerging begomoviruses are of serious concern due to their increasing host range and geographical expansion. Several begomoviruses of Asiatic origin have been reported in Europe, causing massive economic losses; insect-borne transmission of viruses is a critical factor in virus outbreaks in new geographical regions. This review highlights crucial information regarding Asia's four emerging and highly destructive begomoviruses. We also provided information regarding several less common but still potentially important pathogens of different crops. This information will aid possible direction of future studies in adopting preventive measures to combat these emerging viruses.

A Novel Approach of Antiviral Drugs Targeting Viral Genomes.

Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.

Enhancing neutralizing activity against influenza H1N1/PR8 by engineering a single-domain VL-M2 specific into a bivalent form.

Flu disease, with high mortality and morbidity, is caused by the influenza virus. Influenza infections are most effectively prevented through vaccination, but it requires annual reformulation due to the antigenic shift or drift of hemagglutinin and neuraminidase proteins. Increasing resistance to available anti-influenza drugs was also recently reported. The M2 surface protein of the influenza virus is an attractive target for universal vaccine development as it is highly conserved and multifunctional throughout the viral life cycle. This study aimed to discover a single-chain variable fragment (scFv) targeting the M2 protein of influenza A H1N1/PR8, showing neutralizing activity through plaque inhibition in virus replication. Several candidates were isolated using bio-panning, including scFv and single-domain VL target M2 protein, which was displayed on the yeast surface. The scFv/VL proteins were obtained with high yield and high purity through soluble expression in E. coli BL21 (DE3) pLysE strains. A single-domain VL-M2-specific antibody, NVLM10, exhibited the highest binding affinity to influenza virions and was engineered into a bivalent format (NVL2M10) to improve antigen binding. Both antibodies inhibited virus replication in a dose-dependent manner, determined using plaque reduction- and immunocytochemistry assays. Furthermore, bivalent anti-M2 single-domain VL antibodies significantly reduced the plaque number and viral HA protein intensity as well as viral genome (HA and NP) compared to the monovalent single-domain VL antibodies. This suggests that mono- or bivalent single-domain VL antibodies can exhibit neutralizing activity against influenza virus A, as determined through binding to virus particle activity.

Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea.

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

Nanovirus Disease Complexes: An Emerging Threat in the Modern Era.

Multipartite viruses package their genomic segments independently and mainly infect plants; few target animals. Nanoviridae is a family of multipartite single-stranded DNA plant viruses that individually encapsidate single-stranded DNAs of approximately 1 kb and transmit them through aphids without replication in the aphid vectors, thereby causing important diseases of leguminous crops and banana. Significant findings regarding nanoviruses have recently been made on important features, such as their multicellular way of life, the transmission of distinct encapsidated genome segments through the vector body, evolutionary ambiguities, mode of infection, host range and geographical distribution. This review deals with all the above-mentioned features in view of recent advances with special emphasis on the emergence of new species and recognition of new host range of nanoviruses and aims to shed light on the evolutionary linkages, the potentially devastating impact on the world economy, and the future challenges imposed by nanoviruses.

Rapid diagnosis of two marine viruses, red sea bream iridovirus and viral hemorrhagic septicemia virus by PCR combined with lateral flow assay.

Red sea bream iridovirus (RSIV) and Viral hemorrhagic septicemia virus (VHSV) are the most important viral marine pathogens in South Korea because RSIV and VHSV infect and cause high mortality rates in major fish species such as Paralichthys olivaceus and Sebastes schlegelii. These viruses can be transmitted both vertically and horizontally, and early diagnosis is imperative. In this research, RSIV and VHSV viral genomes are detected by PCR-lateral flow assay (LFA). PCR-LFA is sensitive, capable of detecting a viral genome at a concentration of 2-200 fg/µL. Development of this detection method is very meaningful because LFA is simple, requiring a minimum of personnel training to perform. Additionally, LFA requires less time than other detection methods and can be an immediate detection tool that is indispensable in preventing rapid viral spread.

Seed Transmission of Tomato Leaf Curl New Delhi Virus from Zucchini Squash in Italy.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting tomato cultivation on the Indian subcontinent. Recently, however, a new strain of the virus, named ToLCNDV-ES, has spread to Mediterranean countries such as Spain, Italy, and Tunisia, and occurred in Cucurbita crops, causing economic damage. Although ToLCNDV is spread by the sweet potato whitefly (Bemisia tabaci), like other begomoviruses, it has not been clear how ToLCNDV suddenly spread from the Indian subcontinent to the Mediterranean region. In 2017, ToLCNDV was diagnosed in young seedlings germinated naturally from fruits fallen in the prior year on a farm located in Giugliano in Campania, Naples, Italy, suggesting a possible role of the seeds in vertical transmission of the virus. Because sweet potato whiteflies were widespread naturally in that region, it was necessary to verify that in an artificial insect vector-free condition. Seeds were harvested from two ToLCNDV-infected zucchini squash cultivars in Naples in 2017 and 2018 to examine whether ToLCNDV can be transmitted from zucchini squash seeds to young plants. Viral DNA was amplified from these seeds and 1- to 3-week-old seedlings germinated from them with a ToLCNDV-specific primer set. According to PCR results, viral contamination was confirmed from all harvested seeds and dissemination was proven from 61.36% of tested seedling samples. Mechanical transmission from seed-borne virus-infected seedlings to healthy zucchini squash plants was also succesful, demonstrating that seedlings from ToLCNDV-infected seeds did act as inoculum. This is the first report demonstrating that ToLCNDV is a seed-transmissible virus in zucchini squash plants in Italy.

A novel huperzine A-producing endophytic fungus Fusarium sp. Rsp5.2 isolated from Huperzia serrate.

The isolation, identification and characterization of a novel huperzine A (HupA)-producing fungal strain Rsp5.2 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in Vietnam. The fifty-eight endophytic fungi were recovered from roots of natural H. serrata in Lao Cai province of northern Vietnam and screened for HupA-producing by thin-layer chromatography (TLC). The only one of the 58 strains, Rsp5.2, could produce HupA. The amount of HupA produced by Rsp5.2 was quantified to be 19.45 µg g-1 dried mycelium by high-performance liquid chromatography (HPLC). Acetylcholine esterase (AchE) inhibition (IC50) of the crude HupA extract from Rsp5.2 fermentation broth was 2.849 ± 0.0026 µg mL-1. The fungus was identified as Fusarium sp. Rsp5.2 by morphological characteristics and Internal Transcribed Spacer (ITS) sequences. This is the first report of Fusarium sp. as a HupA-producing endophyte isolated from H. serrata.

Antimicrobial resistance gene expression associated with multidrug resistant Salmonella spp. isolated from retail meat in Hanoi, Vietnam.

The purpose of this study was to further characterize the multi-antimicrobial resistance and antibiotic resistance gene expression associated with multi-drug resistance (MDR) in Salmonella spp. isolates from retail meats in Hanoi, Vietnam. A total of 14 Salmonella spp. belonging to 9 serotypes (e.g., Warragul, London, Derby, Indiana, Meleagridis, Give, Rissen, Assine, and Typhimurium) were tested for sensitivity to 8 antibiotics. Resistance to at least one antibiotic was shown in 13 strains (92.85%). The multiple antimicrobial resistances accounted for 64.29% of isolates (9/14). One hundred percent of MDR isolates possessed antibiotic resistant genes, in which 17, 16 and 11 genes were found in Salmonella (Salm) Typhimurium S360, S384, S181 respectively; 12 genes in each strain as Indiana, Warragul, and Meleagridis; 11 genes in Give, 8 genes in Derby and 6 genes in Rissen. Three antibiotic resistance genes (ssaQ, aadA, and gyrB) were present in all isolates, whereas Cephalosporin-resistant gene (e.g., CTX-M3-like) was not detected in any isolates. The results suggest that retail meats could constitute a source of human exposure to multi-drug resistant Salmonella and future research should focus on the impact of these MDR source on the human genome. [Int Microbiol 20(2): 85-93 (2017)].

Antimicrobial resistance gene expression associated with multidrug resistant Salmonella spp isolated from retail meat in Hanoi, Vietnam

The purpose of this study was to further characterize the multi-antimicrobial resistance and antibiotic resistance gene expression associated with multi-drug resistance (MDR) in Salmonella spp. isolates from retail meats in Hanoi, Vietnam. A total of 14 Salmonella spp. belonging to 9 serotypes (e.g., Warragul, London, Derby, Indiana, Meleagridis, Give, Rissen, Assine, and Typhimurium) were tested for sensitivity to 8 antibiotics. Resistance to at least one antibiotic was shown in 13 strains (92.85%). The multiple antimicrobial resistances accounted for 64.29% of isolates (9/14). One hundred percent of MDR isolates possessed antibiotic resistant genes, in which 17, 16 and 11 genes were found in Salmonella (Salm) Typhimurium S360, S384, S181 respectively; 12 genes in each strain as Indiana, Warragul, and Meleagridis; 11 genes in Give, 8 genes in Derby and 6 genes in Rissen. Three antibiotic resistance genes (ssaQ, aadA, and gyrB) were present in all isolates, whereas Cephalosporinresistant gene (e.g., CTX-M3-like) was not detected in any isolates. The results suggest that retail meats could constitute a source of human exposure to multi-drug resistant Salmonella and future research should focus on the impact of these MDR source on the human genome (AU)

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The complete mitochondrial genome sequence of the indigenous I pig (Sus scrofa) in Vietnam.

OBJECTIVE: The I pig is a long nurtured longstanding breed in Vietnam, and contains excellent indigenous genetic resources. However, after 1970s, I pig breeds have become a small population because of decreasing farming areas and increasing pressure from foreign breeds with a high growth rate. Thus, there is now the risk of the disappearance of the I pigs breed. The aim of this study was to focus on classifying and identifying the I pig genetic origin and supplying molecular makers for conservation activities. METHODS: This study sequenced the complete mitochondrial genome and used the sequencing result to analyze the phylogenetic relationship of I pig with Asian and European domestic pigs and wild boars. The full sequence was annotated and predicted the secondary tRNA. RESULTS: The total length of I pig mitochondrial genome (accession number KX094894) was 16,731 base pairs, comprised two rRNA (12S and 16S), 22 tRNA and 13 mRNA genes. The annotation structures were not different from other pig breeds. Some component indexes as AT content, GC, and AT skew were counted, in which AT content (60.09%) was smaller than other pigs. We built the phylogenetic trees from full sequence and D loop sequence using Bayesian method. The result showed that I pig, Banna mini, wild boar (WB) Vietnam and WB Hainan or WB Korea, WB Japan were a cluster. They were a group within the Asian clade distinct from Chinese pigs and other Asian breeds in both phylogenetic trees (0.0004 and 0.0057, respectively). CONCLUSION: These results were similar to previous phylogenic study in Vietnamese pig and showed the genetic distinctness of I pig with other Asian domestic pigs.

Potential effect of combined xenoestrogens during gestation stages on mouse offspring.

The synergistic effect of numerous environmental endocrine disrupting chemicals (EDCs) has raised research concern among researchers. To extend previous studies, the measured additional potential interactions among bisphenol A (BPA), 4-nonylphenol (NP), 4-tert octylphenol (OP) and isobutylparaben (IBP) in mouse model were observed. Pregnant Swiss-albino mice were treated with binary combined chemicals (5, 50 or 500 mg kg(-1) b.wt. day(-1)) from gestation day (GD) 1 to 21. Interestingly, maternal exposure to these EDCs caused fluctuation in GD time, live ratio, female/male ratio, body and organ weights of mouse offspring at postnatal day (PND) 1, 21, and 41 days. At most doses early reduced 0.85 to 1.87 GD compared to controls. Besides females/males ratio showed a significant difference in BPA+ OP, BPA+IBP groups. Female body weight at PND 21 and 41, showed a significant reduction at all combined levels, whereas male offspring showed reduction in weight at dose 50 mg kg(-1) b.wt. day(-1). The potential effects of synergic estrogenicity detected histopathological abnormities, such as ovary analysis revealed increase of corpora lutea, cystic follicles and an endometrial hypertrophy and morphometric changes in uteri measurement. Taken together, these results provided an additional insight into synergistic effects of EDCs toxicology on reproductive tracts.

A Simple, Single Triplex PCR of IS6110, IS1081, and 23S Ribosomal DNA Targets, Developed for Rapid Detection and Discrimination of Mycobacterium from Clinical Samples.

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/ discrimination of this bacterium from clinical samples.