Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

Avian influenza H5N1 in a great white pelican (Pelecanus onocrotalus), Mauritania 2022.

In February 2022, mortalities among great white pelicans (Pelecanus onocrotalus) were reported in the Parc National de Diawling, southwestern Mauritania. Samples were collected and processed, indicating the presence of high pathogenicity avian influenza subtype H5N1. A nearly complete genome was generated for one sample, revealing a high similarity [> 99.5% (H5) nucleotide sequence identity] with Clade 2.3.4.4b H5N1 identified in Europe in 2022.

Highly pathogenic avian influenza H5N1 virus outbreak among Cape cormorants (Phalacrocorax capensis) in Namibia, 2022.

In January 2022, significant mortality was observed among Cape cormorants (Phalacrocorax capensis) on the west coast of Namibia. Samples collected were shown to be positive for H5N1 avian influenza by multiplex RT-qPCR. Full genome analysis and phylogenetic analysis identified the viruses as belonging to clade 2.3.4.4b and that it clustered with similar viruses identified in Lesotho and Botswana in 2021. This is the first genomic characterization of H5N1 viruses in Namibia and has important implications for poultry disease management and wildlife conservation in the region.

Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b in Wild Birds and Poultry in Botswana.

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020-2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.

Low Dose Gamma Irradiation of Trypanosoma evansi Parasites Identifies Molecular Changes That Occur to Repair Radiation Damage and Gene Transcripts That May Be Involved in Establishing Disease in Mice Post-Irradiation.

The protozoan parasite Trypanosoma evansi is responsible for causing surra in a variety of mammalian hosts and is spread by many vectors over a wide geographical area making it an ideal target for irradiation as a tool to study the initial events that occur during infection. Parasites irradiated at the representative doses 100Gy, 140Gy, and 200Gy were used to inoculate BALB/c mice revealing that parasites irradiated at 200Gy were unable to establish disease in all mice. Cytokine analysis of mice inoculated with 200Gy of irradiated parasites showed significantly lower levels of interleukins when compared to mice inoculated with non-irradiated and 100Gy irradiated parasites. Irradiation also differentially affected the abundance of gene transcripts in a dose-dependent trend measured at 6- and 20-hours post-irradiation with 234, 325, and 484 gene transcripts affected 6 hours post-irradiation for 100Gy-, 140Gy- and 200Gy-irradiated parasites, respectively. At 20 hours post-irradiation, 422, 381, and 457 gene transcripts were affected by irradiation at 100Gy, 140Gy, and 200Gy, respectively. A gene ontology (GO) term analysis was carried out for the three representative doses at 6 hours and 20 hours post-irradiation revealing different processes occurring at 20 hours when compared to 6 hours for 100Gy irradiation. The top ten most significant processes had a negative Z score. These processes fall in significance at 140Gy and even further at 200Gy, revealing that they were least likely to occur at 200Gy, and thus may have been responsible for infection in mice by 100Gy and 140Gy irradiated parasites. When looking at 100Gy irradiated parasites 20 hours post-irradiation processes with a positive Z score, we identified genes that were involved in multiple processes and compared their fold change values at 6 hours and 20 hours. We present these genes as possibly necessary for repair from irradiation damage at 6 hours and suggestive of being involved in the establishment of disease in mice at 20 hours post-irradiation. A potential strategy using this information to develop a whole parasite vaccine is also postulated.

Highly pathogenic avian influenza (A/H5N1) virus outbreaks in Lesotho, May 2021.

In May 2021, Lesotho reported its first outbreak of highly pathogenic avian influenza (HPAI) to the OIE. Samples were collected from infected poultry and the virus was confirmed by molecular tests to be of the H5N1 subtype. Full genome sequencing and phylogenetic analysis revealed that the viruses belonged to clade 2.3.4.4b and showed high identity with A/H5N1 viruses identified in Nigeria and Senegal in early 2021. The identification of A/H5N1 HPAI in Lesotho has important implications for disease management and food security in the region.

Hyperthermia: A Potential Game-Changer in the Management of Cancers in Low-Middle-Income Group Countries.

Loco-regional hyperthermia at 40-44 °C is a multifaceted therapeutic modality with the distinct triple advantage of being a potent radiosensitizer, a chemosensitizer and an immunomodulator. Risk difference estimates from pairwise meta-analysis have shown that the local tumour control could be improved by 22.3% (p < 0.001), 22.1% (p < 0.001) and 25.5% (p < 0.001) in recurrent breast cancers, locally advanced cervix cancer (LACC) and locally advanced head and neck cancers, respectively by adding hyperthermia to radiotherapy over radiotherapy alone. Furthermore, thermochemoradiotherapy in LACC have shown to reduce the local failure rates by 10.1% (p = 0.03) and decrease deaths by 5.6% (95% CI: 0.6-11.8%) over chemoradiotherapy alone. As around one-third of the cancer cases in low-middle-income group countries belong to breast, cervix and head and neck regions, hyperthermia could be a potential game-changer and expected to augment the clinical outcomes of these patients in conjunction with radiotherapy and/or chemotherapy. Further, hyperthermia could also be a cost-effective therapeutic modality as the capital costs for setting up a hyperthermia facility is relatively low. Thus, the positive outcomes evident from various phase III randomized trials and meta-analysis with thermoradiotherapy or thermochemoradiotherapy justifies the integration of hyperthermia in the therapeutic armamentarium of clinical management of cancer, especially in low-middle-income group countries.

Isolation and Identification of a Highly Pathogenic Avian Influenza H5N6 Virus from Migratory Waterfowl in Western Mongolia.

In April 2020, two Whooper Swans (Cygnus cygnus) and one Swan Goose (Anser cygnoides) were found dead at three different locations in western Mongolia. Virus isolation from organs taken from the carcasses and full genome sequencing revealed that all three birds were positive for highly pathogenic H5N6 avian influenza virus (HPAIV) belonging to subclade 2.3.4.4h. Confirming similar reports from central Mongolia and western China, these findings have important implications for the monitoring, control, and management of HPAIVs in wild bird and commercial poultry populations in Mongolia.

Quantification of thermal dose in moderate clinical hyperthermia with radiotherapy: a relook using temperature-time area under the curve (AUC).

BACKGROUND: Thermal dose in clinical hyperthermia reported as cumulative equivalent minutes (CEM) at 43 °C (CEM43) and its variants are based on direct thermal cytotoxicity assuming Arrhenius 'break' at 43 °C. An alternative method centered on the actual time-temperature plot during each hyperthermia session and its prognostic feasibility is explored. METHODS AND MATERIALS: Patients with bladder cancer treated with weekly deep hyperthermia followed by radiotherapy were evaluated. From intravesical temperature (T) recordings obtained every 10 secs, the area under the curve (AUC) was computed for each session for T > 37 °C (AUC > 37 °C) and T ≥ 39 °C (AUC ≥ 39 °C). These along with CEM43, CEM43(>37 °C), CEM43(≥39 °C), Tmean, Tmin and Tmax were evaluated for bladder tumor control. RESULTS: Seventy-four hyperthermia sessions were delivered in 18 patients (median: 4 sessions/patient). Two patients failed in the bladder. For both individual and summated hyperthermia sessions, the Tmean, CEM43, CEM43(>37 °C), CEM43(≥39 °C), AUC > 37 °C and AUC ≥ 39 °C were significantly lower in patients who had a local relapse. Individual AUC ≥ 39 °C for patients with/without local bladder failure were 105.9 ± 58.3 °C-min and 177.9 ± 58.0 °C-min, respectively (p = 0.01). Corresponding summated AUC ≥ 39 °C were 423.7 ± 27.8 °C-min vs. 734.1 ± 194.6 °C-min (p < 0.001), respectively. The median AUC ≥ 39 °C for each hyperthermia session in patients with bladder tumor control was 190 °C-min. CONCLUSION: AUC ≥ 39 °C for each hyperthermia session represents the cumulative time-temperature distribution at clinically defined moderate hyperthermia in the range of 39 °C to 45 °C. It is a simple, mathematically computable parameter without any prior assumptions and appears to predict treatment outcome as evident from this study. However, its predictive ability as a thermal dose parameter merits further evaluation in a larger patient cohort.

Strategies to Maximize Available Resources With Minimum Cost Escalation for Improving Radiation Therapy Accessibility in the Post-Coronavirus Disease 2019 Era: An Analysis for Asia.

PURPOSE: There is widespread accord among economists that the corona virus disease 2019 (COVID-19) pandemic will have a severe negative effect on the global economy. Establishing new radiation therapy (RT) infrastructure may be significantly compromised in the post-COVID-19 era. Alternative strategies are needed to improve the existing RT accessibility without significant cost escalation. The outcomes of these approaches on RT availability have been examined for Asia. METHODS AND MATERIALS: The details of RT infrastructures in 2020 for 51 countries in Asia were obtained from the Directory of Radiotherapy Centers of the International Atomic Energy Agency. Using the International Atomic Energy Agency guidelines, the percent of RT accessibility and the additional requirements of teletherapy (TRT) units were computed for these countries. To maximize the utilization of the existing RT facilities, 5 options were evaluated, namely, hypofractionation RT (HFRT) alone, with/without 25% or 50% additional working hours. The effect of these strategies on the percent of RT access and additional TRT unit requirements to achieve 100% RT access were estimated. RESULTS: In 46 countries, 4617 TRT units are available. The mean percent of RT accessibility is 62.4% in 43 countries (TRT units = 4491) where the information on cancer incidence was also available, and these would need an additional 6474 TRT units for achieving 100% RT accessibility. By adopting HFRT alone, increasing the working hours by 25% alone, 25% with HFRT, 50% alone, and 50% with HFRT, the percent of RT access could improve to 74.9%, 78%, 90.5%, 93.7%, and 106.1%, respectively. Correspondingly, the need for additional TRT units would progressively decrease to 4646, 4284, 3073, 2820, and 1958 units. CONCLUSIONS: The economic slowdown in the post-COVID-19 period could severely impend establishment of new RT facilities. Thus, maximal utilization of the available RT infrastructure with minimum additional costs could be possible by adopting HFRT with or without increased working hours to improve the RT coverage.

TILLING: The Next Generation.

Gene space: the final frontier in plant functional genomics. These are the voyages of TILLING, the reverse-genetics strategy that sought to boldly go where no-one had gone before by combining high-density chemical mutagenesis with high-throughput mutation discovery. Its 18-year mission has been to explore new technologies such as next generation sequencing and to seek out new strategies like in silico databases of catalogued EMS-induced mutations from entire mutant plant populations. This chapter is a clip show highlighting key milestones in the development of TILLING. Use of different technologies for the discovery of induced mutations, establishment of TILLING in different plant species, what has been learned about the effect of chemical mutagens on the plant genome, development of exome capture sequencing in wheat, and a look to the future of reverse-genetics with targeted genome editing are discussed. Graphical Abstract.

Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low-coverage whole-genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar 'Novaria' and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar 'Williams'. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy-treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.

Deep Assessment of Genomic Diversity in Cassava for Herbicide Tolerance and Starch Biosynthesis.

Cassava is one of the most important food security crops in tropical countries, and a competitive resource for the starch, food, feed and ethanol industries. However, genomics research in this crop is much less developed compared to other economically important crops such as rice or maize. The International Center for Tropical Agriculture (CIAT) maintains the largest cassava germplasm collection in the world. Unfortunately, the genetic potential of this diversity for breeding programs remains underexploited due to the difficulties in phenotypic screening and lack of deep genomic information about the different accessions. A chromosome-level assembly of the cassava reference genome was released this year and only a handful of studies have been made, mainly to find quantitative trait loci (QTL) on breeding populations with limited variability. This work presents the results of pooled targeted resequencing of more than 1500 cassava accessions from the CIAT germplasm collection to obtain a dataset of more than 2000 variants within genes related to starch functional properties and herbicide tolerance. Results of twelve bioinformatic pipelines for variant detection in pooled samples were compared to ensure the quality of the variant calling process. Predictions of functional impact were performed using two separate methods to prioritize interesting variation for genotyping and cultivar selection. Targeted resequencing, either by pooled samples or by similar approaches such as Ecotilling or capture, emerges as a cost effective alternative to whole genome sequencing to identify interesting alleles of genes related to relevant traits within large germplasm collections.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Mitogen-activated protein kinase-regulated AZI1 - an attractive candidate for genetic engineering.

Mitogen-activated protein kinases and their targets have been in the limelight of plant stress research. Signaling pathways mediating the responses to multiple stresses deserve particular attention. In a recent study, we reported AZI1, a member of the lipid transfer protein family, to play a role in MPK3-mediated responses to salt stress in Arabidopsis thaliana. MPK3 controls AZI1 at the transcriptional and posttranslational level. The AZI1 protein has several properties that make it very attractive for genetic engineering. A model of multi-level control of AZI1 by MPK3 is proposed, and strategies toward optimizing AZI1 protein properties are briefly discussed.

Salt stress in Arabidopsis: lipid transfer protein AZI1 and its control by mitogen-activated protein kinase MPK3.

A plant's capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.