Viruses are the main agents causing emerging and re-emerging infectious diseases. It is therefore important to screen for and detect them and uncover the evolutionary processes that support their ability to jump species boundaries and establish themselves in new hosts. Metagenomic next-generation sequencing (mNGS) is a high-throughput, impartial technology that has enabled virologists to detect either known or novel, divergent viruses from clinical, animal, wildlife and environmental samples, with little a priori assumptions. mNGS is heavily dependent on bioinformatic analysis, with an emerging demand for integrated bioinformatic workflows. Here, we present Lazypipe 2, an updated mNGS pipeline with, as compared to Lazypipe1, significant improvements in code stability and transparency, with added functionality and support for new software components. We also present extensive benchmarking results, including evaluation of a novel canine simulated metagenome, precision and recall of virus detection at varying sequencing depth, and a low to extremely low proportion of viral genetic material. Additionally, we report accuracy of virus detection with two strategies: homology searches using nucleotide or amino acid sequences. We show that Lazypipe 2 with nucleotide-based annotation approaches near perfect detection for eukaryotic viruses and, in terms of accuracy, outperforms the compared pipelines. We also discuss the importance of homology searches with amino acid sequences for the detection of highly divergent novel viruses. Fast development in metagenomic Next-generation sequencing (mNGS) and analysis has enabled virologists to assess the true diversity of viruses in clinical, animal, wildlife and environmental samples. mNGS is a high-throughput, impartial technology with many advantages compared to established diagnostic methods for virus detection . mNGS caThere is a growing interest within virology in utilizing mNGS, specifically in the detection of viruses that cannot be cultured ,5. TheremNGS approaches are heavily dependent on bioinformatic analysis that processes raw sequence output by the NGS sequencer into metagenomic assemblies and into various reports on the micro-organisms’ nucleic acid presence and relative abundances in the analyzed samples. Generally, analysis of NGS data requires bioinformatic expertise, computational resources and, in many cases, installation and maintenance of large reference databases ,3,7. Thiwww.csc.fi, accessed on 21 December 2022) and has been successfully applied to detect and characterize a multitude of novel viral pathogens from a variety of different sample types, including arthropod vectors ?)|(canis lupus familiaris)”. This returned 21,083 unique dog-associated samples. We then selected from RefSeq genomes database (version 214) all bacterial accessions that were sequenced from dog-associated samples. We further pruned this list to include only unique taxon ids. This resulted in a collection of 195 accessions and 159 bacterial species. We also created a smaller collection that included only complete genomes from the above set (58 accession). Accessions and other details for both canine bacterial collections are available in To create the canine simulated metagenome we processed our viral, bacterial and host (GCF_000002285.5) sequences with ART , applyinWe compared Lazypipe 2 with minimap2 and SANSparallel aligners to Lazypipe1 with SANSparallel aligner, Kraken2, CZID and Genome Detective on the human simulated metagenome. Precision, recall and F1-score  for predicted virus and bacterial taxa are given in Lazypipe 2 with minimap2 aligner demonstrated the best accuracy for virus calling with recall at 95.2% and precision at 97.5%  had the overall best accuracy for bacterial calling . Notably, the high precision was achieved by pruning the last 20 percentiles from the result list. This had a large effect by significantly decreasing the false positive bacterial predictions compared to the default pruning of 5 percentiles (Lazypipe --ann sans with 39.1% precision). Lazypipe 2 with minimap2 aligner was second best  followed by Lazypipe1 --ann sans, IDseq, Lazypipe 2 --ann sans and Kraken2.To gain a better understanding of errors in virus detection, these were examined in more detail. We focused on erroneous calls for eukaryotic viruses reported by Lazypipe 2 with SANSparallel and minimap2 aligners . These rErroneous calls were due to a limited number of typical causes . DetaileFor endogenous retroviruses, false-negatives were caused by host-genome filtering which, in most cases, also removed the retroviral reads. These errors can be avoided by turning host-filtering off.For the aa-based annotation, common false positive errors were due to homologs in the aa space  and Toscana Phlebovirus  were identical within the assembled region.There were also cases of contigs having similar identity to both true and false positives (labelled as “Contig with high identity to FP”). For example Mopeia Lassa virus reassortant 29 and Lassa virus) as a single contig leading to a false negative error.There was a single case of mis-assembling two closely related viruses (Bifidobacterium pseudocatenulatum assembly (GCF_022496265.1). We hypothesize that this may represent an unclassified bacteriophage genome. For the dog5X-ba.comp5X-vi5X metagenome false positive was for the Human gammaherpesvirus 4  with just 18 reads. This originated from Corynebacterium amycolatum assembly (NZ_CP102778.1), which was added to RefSeq at a later time point (23 August 2022) than the compilation of our reference database (20 June 2022). There were fewer errors in virus recall from the canine metagenome compared to human metagenome, although this difference was minor. Additional errors for the human metagenome were due to included retroviruses, misclassification of two closely related phlebo-viruses and mis-assembly of Arenavirus and Mammarenavirus reads into the same contig.We benchmarked our pipeline on canine simulated metagenome with default options and minimap2 annotation. Pipeline demonstrated 100% recall of virus species for all variants of the canine metagenome except the lowest 1X coverage version, for which there was a single false negative . PipelinSimilar to benchmarking on the human simulated metagenome, retrieval of bacterial taxa was evaluated, ignoring the least abundant taxa that accounted for the last 5 percentiles of read distribution. The accuracy for bacteria was comparable to human metagenome results. Recall and precision for bacterial genera were relatively high: 85.9–96.9% and 95.3–96.9%, respectively .We evaluated read binning and genome coverage from Lazypipe 2 --ann minimap results for the canine simulated metagenome (dog5X-ba5X-vi5X). For each viral genome we compared read ids of the simulated reads and reads assigned by the pipeline. From these numbers, we estimated recall and precision for read taxonomic binning. For 64% of viral genomes, both recall and precision for read binning exceeded 99%, and for 85% of viral genomes these exceeded 80% .We also estimated horizontal coverage of viral genomes by the resulting assemblies. The pipeline created assemblies for 37 out of 39 unique virus taxids in the benchmark. The exceptions were the three canine parvovirus genomes that were all assembled to a single genome. For these 37 genomes, the median coverage by the simulated reads and Lazypipe assemblies was 97.7% (96.7–98.8%) and 92.3% (86.7–95.8%), respectively .We compared the execution time of Lazypipe 2 (with options --ann minimap and --ann sans) to Lazypipe1 (with option --ann sans), Kraken2 and Genome Detective. Benchmarking was carried out with human simulated metagenome  on LinuxThe reduction of disk footprint for the human simulated metagenome analysis with Lazypipe 2 versus Lazypipe1 was approximately two-fold.Metagenomic analysis pipelines are vital for global pathogen detection and monitoring. Here, we presented Lazypipe 2, an updated version of our mNGS pipeline with significant improvements in code ability, transparency and support for new software components. The previous version, Lazypipe, and now Lazypipe 2 have been used, contributing to virus discovery and demonstrating its potential for unbiased NGS-based studies ,25,26,27Benchmarking on simulated metagenomes demonstrated that assembling and taxonomic binning of contigs with minimap2 against a subset of NCBI nt is a highly accurate strategy for calling known viruses. For the human simulated metagenome Lazypipe 2 --ann minimap achieved 95.2% recall and 97.5% precision for viral species. For the canine simulated metagenome Lazypipe 2, --ann minimap had 100% recall and 97.2% precision. Notably, this high accuracy was sustained even with a heavy host and bacterial sequence background with viral reads spiked at just 1X coverage and constituting just 0.004% of the total NGS library. Additionally, we demonstrated that viral genomes assembled from viral reads spiked at 5X had good horizontal coverage (median at 92%). In addiction, recovery of the spiked reads for viral genomes was highly accurate  for two thirds of viral genomes and at high level (>80%) for 85% of viral genomes spiked at 5X.Annotating with SANSparallel (a homology search for aa sequences) had a slightly lower performance for calling known viruses from the human simulated metagenome. On this benchmark, Lazypipe 2 --ann sans showed 89.2% recall and 85.1% precision for viral species. For annotations based on aa sequences, most errors were failures to identify measurable orfs and misalignments of orfs to false positives. For nt-based annotations, misalignment to false positives were limited to just two cases, one due to mis-assembly and one due to identical genomic regions in closely related viruses. These observations support the choice of nt-based annotations for known viral targets with low divergence from reference sequences. Possible scenarios for applying nt-based annotations include surveillance of a list of known pathogens from various samples and diagnostics or research targeting known viruses with clinical samples.Annotations based on aa sequences have higher sensitivity for viruses with higher divergence from the reference ,17,21. WIdentification of known viruses with Lazypipe 2 --ann minimap approached perfect accuracy in detecting viruses from simulated metagenomes. The remaining errors were caused by filtering of endogenous retroviruses with the host reads and close homologs that were identical within the assembled region. Correct identification of endogenous retroviruses prior to host filtering is an important goal for future development. This study also left out benchmarking on real datasets, although we expect the performance to be at least at the level of Lazypipe1 (tested on a mock-virome dataset).Another important goal for future development is improving the detection of divergent novel viruses. This can be approached in several ways, for example, by integrating well established and highly sensitive techniques based on Hidden Markov Models for detecting protein homologs . SimilaMost of the currently existing mNGS pipelines only support short-read sequencing platforms but we are planning to work further on Lazypipe 2 to support long-read platforms  and make it more user friendly by developing a web interface for fairly self-explanatory results in Hypertext Markup Language. This will help scientists and clinicians with minimum bioinformatics skills to analyze their samples and gain insight from mNGS datasets for both known and novel pathogens.


Carissa macrocarpa) contains anthocyanins, cyanidin 3-O-β-sambubioside (Cy-3-Sa), and cyanidin 3-O-glucoside (Cy-3-G) that possess great bioactive properties. During in vitro gastrointestinal digestion, Cy-3-Sa and Cy-3-G are highly sensitive to pH changes and have low bioaccessibility rates of 7.9% and 22%, respectively. This study aimed to therefore use microencapsulation techniques to improve the bioaccessibility of Cy-3-Sa and Cy-3-G. The crude anthocyanin-rich extract was extracted from freeze-dried Natal plum fruit using ultrasonic-assisted ethanol extraction. The anthocyanin-rich extract was encapsulated using the ionic gelation method. Four distinct carrier agents, namely sodium alginate, pectin, xanthan gum and psyllium mucilage were used to form the wall materials. Encapsulation efficiency was highest for alginate/psyllium mucilage beads (93.67%), while alginate showed the least efficiency (86.80%). Scanning Electron Microscopy revealed a cracked and porous structure for the Natal plum extract and a continuous smooth structure for all the beads. Fourier transform infrared spectroscopy showed peaks at 3300 and 1610 cm−1, confirming the presence of polyphenols and polysaccharides in all beads. Thermal stability was higher for the alginate/psyllium mucilage beads and the observed thermal transitions were due to the bonds formed between the polymers and the polyphenols. Alginate beads combined with xanthan gum, pectin, and psyllium mucilage showed a prolonged release of anthocyanins compared to alginate in vitro alone. The highest anthocyanin bioaccessibility was obtained from alginate/psyllium mucilage beads (85.42 ± 1.03%). The results showed the effectiveness of alginate/psyllium mucilage beads in improving stability and in vitro anthocyanin release.Natal plum ( In the food industry, antioxidants and pigments produced from natural sources are valuable additives. The stability of antioxidants and pigments (anthocyanins) is affected by their low solubility, light, pH, oxygen, and temperature of the environment, and they are rapidly destroyed during gastrointestinal digestion, resulting in lower bioaccessibility and bioactivity . DesigniCarissa macrocarpa .In addition, at a lower pH, the flavylium cation formed enables the anthocyanin (pigments) to be highly soluble in water , and antCarissa macrocarpa  is an indigenous red berry fruit rich in anthocyanins, native to Natal, South Africa [O-β-sambubioside (Cy-3-Sa), cyanidin 3-O-pyranoside, and cyanidin 3-O-glucoside (Cy-3-g) [h Africa . Natal p(Cy-3-g) ,10. The (Cy-3-g)  demonstr(Cy-3-g) . The low(Cy-3-g) . Further(Cy-3-g) . This study aimed to investigate the encapsulation of Natal plum anthocyanin extract in different delivery systems . Encapsulation efficiency was used to assess the process’ efficiency, while polymer–polyphenol interactions, microstructure, and thermal stability were also assessed. The recovery, bioaccessibility, and release behavior of the anthocyanin extract in vitro was also assessed.Plantago psyllium husk, sodium alginate (pure (>99%) food grade, viscosity 15–25 cp), porcine bile, xanthan gum , citrus pectin with a high degree of esterification (53%), sodium acetate (≥99%), sodium citrate (99.5), sodium chloride (≥99%), potassium phosphate (≥98%), acetate buffer (pH 5.2 ± 0.1 (25 °C)), α-Amylase from porcine pancreas, potassium chloride (≥99%), pancreatin from porcine pancreas (8 × USP), pepsin , magnesium chloride , bovine bile , HCl , ferric chloride , ethanol (95%), calcium chloride (99%), ammonium carbonate , were procured from Sigma Aldrich . All other reagents used were of analytical grade purity. A commercially available, food grade g for 20 min at 4 °C using a Hermle Z326k, Hermle Labortechnik GmbH, Wehingen, Germany). The supernatant was collected. Alginate beads were made following a method described by Li et al. [w/v, 2 g/100 mL), alginate (2% w/v) in combination with xanthan gum , or pectin  or psyllium mucilage  was dispersed in distilled water and agitated until completely dispersed. The alginate mixture was dropped into a Natal plum extract solution (0.5% w/v) containing calcium chloride solution (5% w/v), using a syringe under manual control. Calcium chloride was used as a hardening solution. Beads were allowed to sit in the hardening solution for 10 min to ensure complete hardening. The filtrate was then recovered after the beads had been filtered through a Whatman #4 paper filter. After that, water was used to wash the beads. The schematic representation of the process is illustrated in Natal plum phenolic extract was produced using a previously described method by Ndou et al. . Natal pi et al. . AlginatThe total anthocyanin content of the beads was determined using a method reported by Aizpurua-Olaizola et al. . Beads sThe microstructure of freeze-dried Natal plum powder and alginate beads was examined using a JEOL Scanning Electron Detector microscope (SEM) with an Energy Dispersive X-ray, running at 3 kV . The sam−1 in nitrogen.The thermostability of the beads was analyzed using a method by Mendes et al. . To meas−1. A total of 32 scans were done and spectra resolution was maintained at 4 cm−1.Polymer–polyphenol interactions in anthocyanin-enriched freeze-dried beads were investigated using Fourier transform infrared spectroscopy (FT-IR), following a methodology proposed by Fathordoobady et al. . PotassiThe in vitro release properties of the Natal plum anthocyanins were done following a method by Marefati et al. . A totalGCB (TAC mg C3G g−1 DW)-was the phenolic compounds content in the gastric digesta and NDB (TAC mg C3G g−1 DW) was the anthocyanin content in the undigested beads.The SIB (TAC mg C3G g−1 DW) was the phenolic compound content in the intestinal digesta and NDB (TAC mg C3G g−1 DW) was the anthocyanin content in the undigested beads.The L* value, positive a* values represented the intensity of the red color.The color of the beads during in vitro digestion was measured using a Minolta CR-400 chromameter . The brightness was represented by the p < 0.05 were substantially different. The data were analyzed using the statistical application Minitab for Windows (2018).All of the analyses were done in triplicate and twice. Tukey’s multiple range test and analysis of variance (ANOVA) were used to assess whether the values of p < 0.05) impact on encapsulation efficiency. The best encapsulation efficiency was found in alginate/psyllium mucilage beads, followed by alginate/pectin, alginate/xanthan gum, and alginate. While encapsulation efficiencies are significantly different, alginate/pectin, alginate/xanthan gum and alginate/psyllium mucilage beads were still high, the encapsulation efficiency of alginate by itself (approximately 87%) can also be considered high. These findings are consistent with previous research on polymers encapsulated in alginate and pectin hydrogels [β-D-mannuronic acid and 1–4 linked α-L-guluronic residues, organized in a homogenous or heterogeneous pattern. The pKa is between 3.38–3.65. Xanthan gum is a polysaccharide consisting of a β-(1→4) d-glucose chain backbone, with a glucose molecule in every second position of the backbone substituted at C3, with a trisaccharide side chain consisting of β-d-mannose-(1→4)-β-d-glucuronic acid-(1→2)-α-d-mannose. The terminal mannose on the side chain is partially substituted with a pyruvate residue and acetylated at the C-6 position. Psyllium gum is a highly branched anionic glycoprotein with a high molecular weight of approximately 1500 kDa [The evaluation of encapsulation efficiency is a good way to see if the applied method can entrap active materials efficiently. As seen in ydrogels . The effydrogels . Alginat1500 kDa ,20. Conv1500 kDa . The pKa1500 kDa . Gum Ara1500 kDa .SEM images of freeze-dried Natal plum fruit and beads A–E allow−1 confirmed that the extract’s major constituent is anthocyanins. The wide peak at 3216 cm−1 proposed that O–H bonds were stretching vibrationally. This may be due to intramolecular and intermolecular bonding, as well as free hydroxyl groups. Alginate beads exhibited a bigger and a broader region for O–H stretching in the presence of Ca2+ ions. C–H bonds were lengthening, as shown by the peak at 2932 cm−1. The stretching vibration of C–O at 1706 cm−1 and the bending vibration of C–O–C groups at 1031 cm−1 indicated the existence of carbohydrates. The band at 1244 cm−1 matched the aromatic rings’ skeletal stretching vibration and the flavonoids’ C–O–C group [−1 (O-H symmetric stretching vibration) with others showing at 2900–2840 cm−1 , 675–870 cm−1 (C–H aromatic) and 1660 cm−1 (C=C aromatic), confirming the presence of phenolic compounds within the alginate beads [−1) were detected via several peaks that show increasing intensity on the alginate.FTIR was used to investigate the different spectra of Natal plum powder and anthocyanin-infused alginate-based beads to determine the possible molecular interactions between the elements of the four delivery systems . Distinc–C group . All of te beads . Further−1 absorption band in polyphenols. The deformation vibration of C–C bonds in phenolic groups adsorbs in the 1500–1400 cm−1 range [O-H in-plane deformation may potentially be responsible for the 1363 cm−1 range . The addAnthocyanin retention in foods has a significant impact on their sensory qualities, including flavor, taste, and appearance . During The bioavailability of anthocyanins in plasma after consuming a meal is limited , which i2+ ions associated with –COO– groups of alginates, which are primarily defined by polymannuronate sequences, with Na+ ions present in the saline solution at pH 6.8. [During the intestinal phase, the integrity of the alginate beads was maintained for the first 10 min, but, after that, the amount of anthocyanin extract released from the beads increased B. During pH 6.8. . Chain r pH 6.8. . Other mAn in vitro digestion model was used to calculate the anthocyanin released during digestion. The total anthocyanin content of the beads followed the same pattern in all of the digestion phases . The beaL* and a* parameters of samples from gastric and intestinal fluids after 120 min are presented in L* values indicated lower levels of whiteness in the undigested beads, which was supported by the * values that were on significantly higher levels, indicating a higher intensity of redness after 120 min. The color eluted by anthocyanin is pH-dependent [L*) compared to the gastric phase. The pH levels of the intestinal phase could be the reason for this behavior. The stability of anthocyanins is greatly reliant on pH, with neutral-alkaline conditions making them unstable [The ependent . At pH 1ependent . Hence, unstable . Further−1 confirmed that the extract’s major constituent is anthocyanins. Thermal stability analysis also revealed a positive effect of gelation on the physicochemical stability of the encapsulated Natal plum extract. Due to their increased swelling properties in bioactive compound-loaded beads at a higher pH, natural polymers significantly affect mechanical properties, porosity, and in vitro release control. As a result, combing alginate with other polysaccharides resulted in the delayed release of anthocyanins from the beads in vitro. The percentage of released anthocyanins to the gastric phase in alginate beads containing extract was only 18%, while samples containing pectin, xanthan gum, and psyllium mucilage had 16, 14, and 12%, respectively, and the beads released some of the entrapped anthocyanins (24–29%). Generally, alginate/psyllium beads can be used for food additives and for oral delivery of natal plum anthocyanins. The anthocyanin-rich phenolic extract from Natal plum fruit was encapsulated. Alginate/psyllium gum beads resulted in the most efficient encapsulation compared to beads containing alginate alone or combined with xanthan gum or pectin. The microstructural analysis showed that encapsulation led to a smooth structure with no cracks compared to the unencapsulated extract. FTIR analysis confirmed the presence of polyphenols and that the extract was successfully incorporated into the beads. Peaks at approximately 3300, 2932, 2160, 1717, 1610, 1426, 1244, and 1031 cm


In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue. Japanese encephalitis virus (JEV) is a single-stranded, positive-sense, RNA flavivirus endemic to tropical regions of South and Southeast Asia and is the most common cause of vaccine-preventable encephalitis in the Asia-Pacific region  and elevated protein of 0.96 g/L (reference range 0.15–0.45). No organisms were isolated. Results of CSF nucleic acid testing (NAT) were negative for Neisseria meningitidis, Streptococcus pneumoniae, herpes simplex virus types 1 and 2, enterovirus, varicella zoster virus, parechovirus, cytomegalovirus, and Epstein-Barr virus. CSF and serum samples tested negative for antineuronal antibodies (PCA-1/PCA-2/ANNA-1/ANNA-2/Ma1/Ma2/Amphiphysin/SOX-1/CRMP-5/Tr) and limbic encephalitis panel (anti-NMDA/CASPR-2/LGI-1/GABA-B/DPPX/IgLON5/AMPA-1/AMPA-2). Empiric treatment was changed to intravenous ceftriaxone, benzylpenicillin, and aciclovir. Magnetic resonance brain imaging showed an equivocal T2/FLAIR hyperintensity in the dorsal midbrain and pons with sparing of both thalami and basal ganglia (6 cells/L mononuclear cells), and results of repeat culture, NAT, and antineuronal/limbic encephalitis panels were negative. CSF protein remained elevated at 0.86 g/L.A man in his 70s sought care at a rural hospital after 3 days of fever and progressive confusion. Results septic screen, chest radiography and computed tomography brain scan were unremarkable. Despite empiric intravenous flucloxacillin and gentamicin, the patient experienced progressive neurologic deterioration and required intubation. Cerebrospinal fluid (CSF) testing revealed a lymphocyte predominant pleocytosis of 126 × 10 ganglia . ElectroDespite supportive management and broadening of antimicrobial therapy with meropenem, the patient showed no neurologic improvement and, after discussion with his family, ventilator support was withdrawn; he died 22 days after symptom onset. In the absence of a definitive diagnosis, a noncoronial limited brain autopsy was performed. Twenty-four brain tissue samples were sent for neuropathologic examination; 10 were sent for RNA-mNGS analysis . RetrospJEV was detected for the first time across southeastern Australia in 2022 and, as of January 5, 2023, a total of 45 human cases of JEV had been notified in Australia since January 1, 2021 (of which 14 were from NSW), including 7 deaths  testing accordingly when treating undifferentiated meningoencephalitis. The case also demonstrates the new incursion of a pathogen across a broad, previously nonendemic geographic area and into a largely nonimmune population. The origins remain unclear but, given the widespread geographic area of infected piggeries and human cases, JEV has likely been circulating undetected in wild birds, mosquitoes, and pigs for some time. Whole-genome sequencing has demonstrated that this outbreak is caused by genotype IV, previously thought to be restricted to Indonesia, Papua New Guinea, and the Tiwi Islands.Changes in vector distribution have been associated with changes in climate, destruction of natural habitats altering bird migratory patterns, agricultural practices, and periurban growth (


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.Plant viruses are responsible for the most devastating and commercially significant plant diseases, especially in tropical and subtropical regions. The genus  Bemisia tabaci; Geminiviridae have circular single-stranded DNA (ssDNA) genome encapsulated in a twinned icosahedral coat protein.Geminiviruses are the most destructive group of viruses infecting agricultural crops in the tropical and subtropical regions of the world . They arIn the nineteenth century, symptoms of geminivirus infection were first observed in plants in the tropical and subtropical regions . They seGeminiviridae proposed a new standard for species identification : (Nicotiana Benthamiana (The largest and most important genus of tabaci): . It is ptabaci): . The siztabaci): . They retabaci): . Begomovtabaci): . In OW atabaci): . Most NWtabaci): . They altabaci): . V3 prottabaci): . DNA-B itabaci): . Two protabaci): . Begomovtabaci): . Alphasatabaci): . The bettabaci): . Deltasatabaci): . NW bipathamiana . Figure Begomovirus infects a wide range of dicot and monocot plants and causes severe loss, exhibiting symptoms such as stunting, deformed growth, and decreased seed production in infected plants ; leaf crGeminiviridae in Europe is low  is the host of a large number of viruses. These viruses cause significant losses in fruit production and quality  and Israel (TYLCV-IL) are truly international TYLCD-causing entities . NumerouB. tabaci , Capsicum frutescens (chili), Piper nigrum (pepper), and Solanum melongena (eggplant). In its early days, its outbreak was restricted to Asian countries , Cucumis melo L (melon), and Cucumis sativus L (cucumber) crops in Southern Spain , Cotton leaf curl Kokhran virus (CLCuKoV), Cotton leaf curl Gezira virus (CLCuGV), Cotton leaf curl Burewala virus etc. . These cokra yellow crinkle virus, and ageratum leaf curl Cameroon alphasatellite. This study provides evidence for the interaction of begomoviruses with unrelated DNA satellites, which evolved new complexes of these begomoviruses in Asia. This may occur across continents, leading to the development of novel viruses (Asia and the Mediterranean regions suffer from leaf curling disease due to the begomoviruses and satellite DNA that make up a complex system . However viruses .Manihot esculenta (cassava) crop is highly influenced by CMD due to the activity of at least nine bipartite local African begomoviruses, called cassava mosaic geminiviruses -related viruses are another example of essential begomoviruses in Africa, Sri Lanka and India. Currently, the es CMGs; . This hies CMGs; . Since tes CMGs; .African cassava mosaic virus (ACMV) is the most common cassava mosaic begomovirus in Nigeria. Which spread by contaminated stem cuttings and whitefly vectors . AlthougEmerging begomoviruses pose a serious threat to agricultural production worldwide. These viruses can be new  or already known; however, they share the common characteristic of occupying and spreading within new niches . The facSeveral studies on the localization and movement of begomoviruses in host plant system have concluded that the virus is limited to cambium and phloem parenchyma cells of plants. Sometimes, they exit the phloem and enter the mesophyll parenchymatous tissue . One advIt is crucial to identify viral transmission methods, as it helps to understand the epidemiology and outbreak of viruses . The traTransovarial transmission of viruses from vector parents to offspring is important for their epidemiology. The majority of begomoviruses are limited to transmit only by insect vector but TYLCV have been found to show transovarial transmission. It was observed that TYLCV access into the reproductive organ of its vector was mostly determined by the developmental stage of the whitefly ovary, and that TYLCV transmission to offspring increased with whitefly adult age. The precise interaction between viral coat protein with whitefly vitellogenin was required for virus entry into the whitefly ovary .The global increase in the population of insect vectors and the rapid evolution of virus variants are also associated with the worldwide distribution of begomoviruses . The rolBemisia tabaci, is the key player in the global spread of begomoviruses. Its increased adaptability resulted in the introduction of many begomoviruses in previously unreported areas , and Papaya leaf curl virus (PaLCuV). These begomoviruses cause substantial economic losses in Asian countries and increase their host range through mutation and recombination. Brief descriptions of some emerging begomoviruses that are not widely known but very destructive are given below. Capsicum species, Solanum melongena, and Solanum lycopersicum. The spread of TYLCKaV can be reduced using simple procedures, such as isolating greenhouses for transplant production from outdoor sources of begomoviruses. The destruction of whitefly populations is another key management method that might be beneficial for controlling the spread of TYLCKaV  on Sumatra Island in 2005 , Cestrum nocturnum, Capsicum species, Cyamopsis tetragonoloba, Nicotiana tabacum, and S. lycopersicum (PaLCuV is a monopartite begomovirus and is transmissible through persicum . It causpersicum . SymptomHibiscus cannabinus (kenaf) worldwide  has a monopartite genome that contains a single molecule of circular ssDNA (DNA-A). Bemisia tabaci is responsible for its transmission. The major symptoms of TYLCTHV infection are leaf curling and yellowing  for the Exportation Support of Agricultural Products at the Animal and Plant Quarantine Agency of the Republic of Korea.JL was employed by company NongWoo Bio.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Panicovirus) is identified for the first time in Australia and in only the second country after the USA. A full-length genome sequence was obtained, which has 97% nucleotide sequence identity to that of the species exemplar isolate. Surveys for BGLV, utilising a newly designed universal panicovirus RT-PCR assay for diagnosis, demonstrated widespread infection by this virus in a broad variety of Bermuda grass cultivars  grown in both New South Wales and Queensland. The virus was also detected in Rhodes grass (Chloris gayana) and Kikuyu grass (Cenchrus clandestinus), which are both important pasture grasses in subtropical Australia, and the latter is also grown as turf. Furthermore, the Rhodes grass plant, which had strong mosaic symptoms, was also infected with sugarcane mosaic virus, warranting further investigations as to whether synergistic interactions occur between these two viruses.Bermuda grass latent virus (BGLV; genus The online version contains supplementary material available at 10.1007/s00705-022-05434-6. Cynodon dactylon and C. dactylon × Cynodon transvaalensis hybrids) is among the most widely planted turf grass species in warm temperate and subtropical regions of Australia [Panicovirus, family Tombusviridae [Stenotaphrum secundatum) ‘Floratam’ in central and southern Florida called viral lethal necrosis. Viral lethal necrosis is thought to be due to a synergistic interaction between a panicovirus [Potyvirus. When either virus is present as a single infection, the symptoms are mild and not reason for major concern [Bermuda grass  and Kikuyu grass (Cenchrus clandestinus). The complete genome sequence of an Australian isolate of BGLV is also presented.SCMV has recently been recorded as a pathogen of St. Augustine grass in Australia  but BGLVmatK) using primers matK472F and matK1248R [et al. [In February 2020, a weedy Bermuda grass plant with chlorotic mottling and spindle-shaped lesions on the leaves Fig. a, was obatK1248R , and the [et al. . Carbon-Panicum mosaic virus (GenBank U55002), Cocksfoot mild mosaic virus (GenBank EU081018), and Thin paspalum asymptomatic virus (GenBank JX848617), were aligned using the MUSCLE algorithm within the MEGA software package, and the aligned sequences were back-translated to give the DNA sequence alignment. Potential primer-binding sites with minimal degeneracy were identified using GPrime [c. 350 bp. RT-PCR primer annealing temperatures were optimised using Panicum mosaic virus (PMV) isolate 2349 [To test the hypothesis that the plant was infected with a panicovirus, reverse transcription PCR (RT-PCR) and amplicon sequencing were done. In the absence of a proper diagnostic control and adequate knowledge of sequence diversity, we chose to develop a universal panicovirus assay. RNA-dependent RNA polymerase (RdRp) amino acid sequences of the exemplar isolates of BGLV (GenBank KX758441) and members of the three officially recognized panicovirus species, ate 2349  from theTotal nucleic acid extracts (TNAEs) were prepared using a BioSprint 15 DNA Plant Kit  as per the manufacturer’s instructions but omitting the RNase A digestion step. RT-PCR assays were done using a MyTaq One-Step RT-PCR Kit  as per the manufacturer’s instructions. RT-PCR thermocycling conditions were 20 min at 45°C and 1 min at 95°C; followed by 40 cycles of 95°C for 10 s, 65°C for 10 s, and 72°C for 30 s; and finally, 2 min at 72°C. The amplicon was electrophoresed through a 1.5% (w/v) agarose gel and visualised on a UV transilluminator after staining with ethidium bromide. Virus isolate 5657 was RT-PCR positive. Amplicons were sequenced directly by Macrogen Inc., South Korea, and BLASTn and BLASTx search results showed that BGLV (GenBank KX758441) was the closest match, with 96.5% and 100% identity at the nt and amino acid (aa) levels, respectively.de novo using CLC Genomics Workbench v. 6.5 (QIAGEN) with automatic word size and bubble size and a minimum contig length of 700 nt. The number of contigs produced after de novo assembly was 239 and these were sorted by length and sequence homologies identified by doing a BLASTn search of the NCBI Nucleotide Database. One contig of 4,097 nt had a significant hit to BGLV but no other viruses were identified. The sequence of the exemplar isolate of BGLV (GenBank KX758441) was then used as a scaffold to map all reads, producing a contig of 4,044 nt, which was assembled from 78,263,232 reads, with an average depth of coverage of 1,580,041 sequence reads.To better characterise virus isolate 5657, high-throughput sequencing was done. Total RNA was extracted using a TRIzol Plus RNA Purification Kit  as per the manufacturer’s instructions. The total RNA extracts were submitted to the Australian Genome Research Facility  for library preparation and sequencing. Ribosomal RNA was removed using a TruSeq Stranded Total RNA with Ribo‐Zero Plant kit (Illumina) with 500 ng of input RNA. The library was sequenced as 100-bp paired-end reads on an Illumina NovaSeq platform with an SP 300 cycle kit. The sequences were paired, and adaptor and primer sequences were removed using the BBDuk Plugin for Geneious Prime v. 2021.0.1 . Trimming options included removal of reads with a Phred value less than 30 and shorter than 30 nt in length. One million reads, which were randomly sampled from 110,832,104 reads obtained after trimming for quality, were assembled Escherichia coli poly(A) polymerase . cDNA of the polyadenylated RNA was synthesised using Superscript IV reverse transcriptase  and an oligo (dT) primer (5ʹ CACGGATCCCGGG(T)17V 3ʹ) based on potyvirid primer 1 of Gibbs and Mackenzie [The 5ʹ terminus of the genome of isolate 5657 was determined using a 5ʹ/3ʹ RACE Kit, 2nd Generation  as per the manufacturer’s instructions and with the virus-specific, reverse primers listed in Supplementary Table S1. The 3ʹ end was confirmed by polyadenylating the RNA using The genome sequence of virus isolate 5657 (GenBank MZ671022) contains 4,047 nt and is 97% identical to the exemplar isolate of BGLV (GenBank KX758441). The 5ʹ UTR of virus isolate 5657 is 3 nt longer than that of the exemplar isolate, but otherwise, its genome architecture  is identical to that previously described , which iTo survey for BGLV, a collection of 23 accessions of Bermuda grass representing 16 cultivars was assembled from a variety of sources . The closest match (95.8% nt sequence identity over the entire length of the amplicon sequence) was to an SCMV isolate from St. Augustine grass found on a turf farm in New South Wales (GenBank MW026606). A universal panicovirus RT-PCR was also done, and again, the plant tested positive, with amplicon sequencing confirming infection by BGLV. Pairwise sequence comparisons showed that this virus isolate has 87.4% nt sequence identity to the exemplar isolate of BGLV and 96.7% identity to the virus isolate in ‘Gully Gold’ (isolate 5663) from New South Wales (Supplementary Table S2).Finally, a Rhodes grass plant  search method implemented in the RA×ML plugin in Geneious Prime. The GTR GAMMA nucleotide model was selected, with rapid bootstrapping of 1000 replicates, searches for the best-scoring ML tree algorithm, and starting from a random tree topology. The phylogenetic tree grouped all Australian isolates into the BGLV clade Fig. . The OxlIn this study, we report for the first time the presence of BGLV in Australia, which represents only the second record of this virus anywhere in the world after the USA. BGLV was also detected in two hybrid Bermuda grass cultivars, as well as Rhodes grass and Kikuyu grass, representing extensions to the natural host range of this virus. Given the relative ease with which BGLV was found in New South Wales and Queensland using the RT-PCR diagnostic tool described in this paper, it is likely that BGLV occurs throughout Australia and in many more countries around the world, wherever Bermuda grass occurs. Turf grass species are normally traded as vegetative propagules in order to maintain genetic purity and also because many cultivars have poor fertility. Hence, turf grasses probably represent an important pathway for the spread of BGLV, as well as a range of other grass-infecting viruses.Our finding of a severely diseased plant of Rhodes grass that was infected by both SCMV and BGLV raises the question of synergism between these two viruses, similar to that proposed to occur in St. Augustine grass in Florida, resulting in lethal viral necrosis . InvestiSupplementary file1 Figure S1 Genome organization of Bermuda grass latent virus isolate 5657 showing five predicted open reading frames (orange boxes) and the putative protein products (green boxes). RT = internal amber stop codon that can be read through. The scale is the number of nucleotides (PPTX 802 KB)Supplementary file2 (TXT 17 KB)Supplementary file3 (DOCX 32 KB)Supplementary file4 (XLSX 21 KB)Below is the link to the electronic supplementary material.


This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandiinifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts  or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants.Engineering plants to synthesize nitrogenase and assimilate atmospheric N Unfortu archaea .nitrogen fixation (nif) genes to the plant  corresponds to an α2β2-tetramer with two unique metalloclusters per αβ-dimer: iron-molybdenum cofactor (FeMo-co) and P-cluster. The [Fe8S7] P-cluster is responsible for the successive electron transfers from the [Fe4S4] cluster of NifH to the [MoFe7S9C-R-homocitrate] FeMo-co, the enzyme catalytic cofactor  radical enzyme that converts two [Fe4S4] cluster units into an [Fe8S9C] cluster called NifB-co  itself is the determining factor for cluster delivery and suggesting that engineering of nitrogenase in heterologous hosts can be divided in independent modules in which the end product from one module  can be transferred to another module , although the components within each module originate from different species. We then expressed and targeted the 30 NifB proteins to mitochondria or chloroplasts in Nicotiana benthamiana in two parallel screening and selection procedures. Three NifB proteins from methanogens were found soluble and accumulated abundantly either in chloroplasts or mitochondria. Importantly, selected NifB proteins supported in vitro FeMo-co synthesis using [Fe4S4] cluster substrates provided by the physiological donor NifU, proving that these identified variants were isolated as functional proteins from both plant organelles.Here, we have expressed 30 distinct in vivo activity and cross-compatibility of NifB variants with the rest of the A. vinelandii proteins required for FeMo-co synthesis, each one of the 30 nifB genes (A. vinelandii UW140 (ΔnifB) Nif– strain  (Cyanothece sp. strain PCC 7425 and Synechococcus sp.) did not support significant acetylene reduction under the conditions tested but restored diazotrophic growth when plates were incubated for longer time periods. Detection of the NifB protein variants by immunoblotting was unsuccessful when probing for the TS-tag, similarly to what we observed when expressing TS-NifHHt in A. vinelandii   and c. Tnelandii . HoweverW483 see  using a 10.1128/mbio.00268-22.1TEXT S1Text S1, PDF file, 4.7 MB.Supplemental Materials and Methods and Fig. S1 to S7. Download Copyright © 2022 Jiang et al.2022Jiang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00268-22.2TABLE S1A. vinelandii UW140 (ΔnifB). Download Table S1, PDF file, 0.1 MB.List of vectors used to complement Copyright © 2022 Jiang et al.2022Jiang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00268-22.3TABLE S2Table S2, XLSX file, 0.05 MB.Information about the NifB variants used in the library screening. Download Copyright © 2022 Jiang et al.2022Jiang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cyanobacteria phylum, 100% (4/4) from Euryarchaeota, 33% (1/3) from Firmicutes, 100% (1/1) from Chlorobi, 0% (0/1) Chloroflexi, and 54% (7/13) from Proteobacteria reverted the Nif– phenotype of UW140. There was no correlation between complementation ability and NifB protein sequences or phylogenic distances of the organisms from where the NifB proteins originated to A. vinelandii  of the NifB variants from the N. benthamiana leaves , Methanosarcina acetivorans (NifBMa), and Methanothermobacter thermautotrophicus (NifBMt) were soluble , and isolated for functional analysis using STAC  apo-NifDK  to render the NifB-co functional for FeMo-co synthesis. However, a very small proportion of NifB is normally isolated in this state primed for catalysis, and its [Fe4S4] clusters are first reconstituted in vitro by incubation with ferrous iron and sulfide in the presence of dithiothreitol (DTT) (NifB activity was assayed by the NifB-dependent po-NifDK . Activatocitrate . NifB muol (DTT) .4S4] clusters) to reconstitute the full complement of NifB [Fe4S4] clusters . NifU is the in vivo donor of [Fe4S4] clusters to NifB  and 750 (NifBMa) units after in vitro chemical reconstitution of the NifB [Fe4S4] clusters  (M. infernus (85°C) and M. thermautotrophicus (65°C)   compareds (65°C) , 50. MesA. vinelandii was difficult to validate , in vivo complementation by some NifB variants confirmed that expression was sufficient to support nitrogenase activity. Another limitation of our study is that the codon usage of the nifB genes was not optimized for A. vinelandii. Nevertheless, the number of NifB variants that could revert the Nif– phenotype in the in vivo ΔnifB complementation is encouraging for future efforts to create N2-fixing plants. The lack of sequence similarity between the A. vinelandii NifB protein and that of the complementing NifB variants suggested that, if NifB is functional and produces NifB-co, the cluster is transferred to NifX/NifEN without the need of constructive protein interactions. It also indicates that the A. vinelandii NifU scaffold protein serves [Fe4S4] cluster substrates to a great variety of NifB acceptor proteins. This result shows that the maturation of nitrogenase in a heterologous host can be divided in independent modules. Thus, rather than identifying a unique source (organism) for the full Nif pathway, Nif proteins showing superior solubility, stability and functionality can be selected from very distinct origins.Although expression of the NifB proteins in M. thermautotrophicus and M. infernus) and chloroplasts (M. acetivorans and M. infernus). NifB yields were similar for both organelles. Although the as-isolated proteins did not exhibit color typical of NifB with full complement of [Fe4S4] clusters, all as-isolated NifB proteins were active in vitro when reconstituted with [Fe4S4] clusters by their natural cluster donor NifU. This either indicates that cluster-loading in vivo is poor or that the activity of NifB in mitochondria and chloroplasts is high . Due to the small amount of protein isolated from tobacco, we could not perform electron paramagnetic resonance to determine the Fe-S cluster composition of as-isolated NifB. As the stability of the NifB-co product is low, further studies using NifB-co acceptor proteins  are needed to distinguish between these two possibilities. We have previously shown that NifH expressed in tobacco mitochondria was mainly isolated devoid of its [Fe4S4] cluster , the expression of functional NifB is crucial as it produces the NifB-co required for the synthesis of the active-site clusters FeMo-co, FeV-co, or FeFe-co. The identification of soluble and functional NifB variants expressed in planta is therefore crucial to the engineering of the nitrogenase cofactor biosynthesis, and hence the expression of active nitrogenase in plants.Irrespectively of the type of nitrogenase being engineered in a NTaxonomic IDs from selected organisms for the NifB library see  were retE. coli DH5α was used for cloning of plasmid libraries (see section below) and was grown in lysogenic broth (LB) at 37°C with appropriate antibiotics. A. tumefaciens GV3101 (pMP90) used for agroinfiltration experiments was grown in LB supplemented with rifampicin (25 μg/mL) and gentamicin (10 μg/mL) plus appropriate antibiotics for maintaining the binary plasmids at 28°C. A. vinelandii strains were cultivated in Burk’s modified medium (containing 28 mM ammonium acetate) or in Burk’s modified N-free medium at 30°C  of 0.01. At the day of infiltration, A. tumefaciens cultures (at an OD600 of ≥3.0) were transferred to induction medium containing 10 mM Mg2SO4, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 150 μM acetosyringone. Cells were induced at a final OD600 of 0.9 for 2 h and then used for infiltration into tobacco leaves using a syringe.A.vinelandii strains DJ (wild type) and UW140 (ΔnifB) have been described (nifB genes with codon usage optimized for expression in Saccharomyces cerevisiae) generated in this study for complementing UW140 strain are listed in A. vinelandii strains were grown in Burk medium at 30°C with shaking at 200 rpm. The procedure for A. vinelandii transformation has been described .For N. benthamiana plants were grown in a greenhouse under 16:8 h (light:dark) cycle and watered weekly with 1 g/L Sequestrene G100 (Syngenta). Young leaves of 4- to 5-week-old tobacco plants were infiltrated using a 1-mL syringe containing A. tumefaciens cells in induction medium. At the end of the dark phase 4 days after infiltration, leaves were sampled for immunoblot analysis or purification experiments.2. Leaf tissue was ground into fine powder using a lab vibration mill mixer (Qiagen Retsch MM300 TissueLyser). Total protein extracts were prepared by adding 2× Laemmli buffer to the leaf powder at a ratio of 2:1 (vol/wt) and heating for 10 min at 95°C. For soluble protein extracts, prechilled tobacco protein extraction buffer  for 1 h at 4°C. The supernatant was filtered through a 0.2-μm vacuum filter unit  to obtain the cell extract (CFE) that was loaded into a Macroprep Strep-Tactin (IBA lifesciences) affinity column by gravity. After loading the extract, the column was washed with 10 column volumes of wash buffer . Bound protein was eluted from the column by applying 3 column volumes of wash buffer supplemented with 5 mM desthiobiotin (IBA Life Sciences), concentrated using Amicon centrifugal filter units with a 10-kDa cutoff (Merck Millipore), and then snap-frozen and stored in liquid N2.NifB protein purifications were performed in anaerobic chambers (Coy System and MBraun) at <1 ppm Oescribed , 37. FroA. vinelandii purified protein preparation in rabbit. Antibodies for the detection of HA tag , GFP , and TS-tag  are commercially available. α-NifB, α-NifU, α-NifS, StrepMAB, and α-GFP were used at 1:3,000, 1:2,500, 1:2,500, 1:5,000 and 1:2,000 dilutions, respectively, in TBS-T supplemented with 5% bovine serum albumin (BSA) and 0.01% sodium azide. α-HA-HRP and Streptactin-HRP were used at 1:3,000 and 1:50,000 dilutions, respectively, in 2% skimmed milk in TBS-T. The secondary antibodies used for the detection of nonconjugated primary antibodies were α-rabbit-HRP  and α-mouse-HRP (for StrepMAB and α-GFP) at a 1:20,000 dilution.Antibodies used in this study and their dilutions for immunoblotting were as follows: antibodies detecting NifB, NifU and NifS were raised against For peptide mass fingerprinting analysis, Coomassie blue-stained protein bands were sliced with a scalpel blade and transferred to an Eppendorf tube with water. The service was provided by the Proteomic Unit from Universidad Complutense de Madrid.For NifB N-terminal determination, samples were prepared according to the instruction provided by the service . In short, 200 pmol of each protein variant was separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and stained with Coomassie blue R-250. The bands corresponding to NifB were excised with a clean scalpel, dried, and transferred to an Eppendorf tube for analysis.A. vinelandii NifU (pDB2174) and strep-tagged A. vinelandii NifS (pDB2123) were kindly donated by Dennis Dean (Virginia Polytechnic Institute and State University). The NifU purified from E. coli (designated NifUEc) was reconstituted in vitro following the protocol as previously described 2Fe(SO4)2 were added. The iron was added in three steps with 15-min separations until reaching a final concentration of 0.3 mM. The reconstitution mix was kept on ice for 3 h and then desalted using centrifugal units with a 10-kDa cutoff  to replace the reaction mixture with 100 mM MOPS (pH 7.5). The [Fe4S4] cluster reconstituted NifU  was stored in liquid N2 until used.Plasmids encoding strep-tagged escribed , 60 withAv, 1.0 μM tobacco purified NifBXx, 1.5 μM apo-NifENAv, 0.6 μM apo-NifDKAv, 17.5 μM Na2MoO4, 175 μM R-homocitrate, 9 μM holo-NifUEc, 125 μM SAM, 1 mg/mL BSA, and ATP-regenerating mixture  in 100 mM MOPS buffer (pH 7.5) for 1 h at 30°C. NifB-co-dependent FeMo-co synthesis assays were performed as positive controls for NifB activity. These assays were performed as described above but without holo-NifUEc and by replacing 1.0 μM NifB protein with 2.5 μM NifB-co.The NifB-dependent FeMo-co synthesis assays were performed under anaerobic conditions in 100-μL reaction mixtures containing 3.0 μM NifHAv and transferring the mixture to 9-mL rubber-stopped serum vials containing an Ar atmosphere and 500 μL of acetylene. The ARA was performed for 20 min at 30°C. After stopping the reactions by addition of 100 μL of 8 M NaOH, 50 μL of the gas phase was removed and injected into a GC-2014 gas chromatograph (Shimadzu) equipped with a Porapak N 80/100 column to measure ethylene formation.The activity of reconstituted NifDK was determined after FeMo-co synthesis and insertion by adding 500 μL of ATP-regenerating mixture and 2.0 μM NifHAccession numbers of genes used here are listed in 10.1128/mbio.00268-22.5TABLE S4Table S4, PDF file, 0.1 MB.List of modular pieces and primers used for the generation of transcriptional units to assemble multigenic vectors using MoClo cloning. Download Copyright © 2022 Jiang et al.2022Jiang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Due to the prevalence of COVID-19, providing safe environments and reducing the risks of virus exposure play pivotal roles in our daily lives. Contact tracing is a well-established and widely-used approach to track and suppress the spread of viruses. Most digital contact tracing systems can detect direct face-to-face contact based on estimated proximity, without quantifying the exposed virus concentration. In particular, they rarely allow for quantitative analysis of indirect environmental exposure due to virus survival time in the air and constant airborne transmission. In this work, we propose an indoor spatiotemporal contact awareness framework (iSTCA), which explicitly considers the self-containing quantitative contact analytics approach with spatiotemporal information to provide accurate awareness of the virus quanta concentration in different origins at various times. Smartphone-based pedestrian dead reckoning (PDR) is employed to precisely detect the locations and trajectories for distance estimation and time assessment without the need to deploy extra infrastructure. The PDR technique we employ calibrates the accumulative error by identifying spatial landmarks automatically. We utilized a custom deep learning model composed of bidirectional long short-term memory (Bi-LSTM) and multi-head convolutional neural networks (CNNs) for extracting the local correlation and long-term dependency to recognize landmarks. By considering the spatial distance and time difference in an integrated manner, we can quantify the virus quanta concentration of the entire indoor environment at any time with all contributed virus particles. We conducted an extensive experiment based on practical scenarios to evaluate the performance of the proposed system, showing that the average positioning error is reduced to less than 0.7 m with high confidence and demonstrating the validity of our system for the virus quanta concentration quantification involving virus movement in a complex indoor environment. The worldwide COVID-19 pandemic has brought about many changes in our daily lives and struck a devastating blow to the global economy. It is widely recognized that airborne transmission serves as the primary pathway for the spread of COVID-19 via expiratory droplets, especially in indoor environments ,3,4. SomContact tracing in indoor environments can complement the ones used in outdoor environments to enable comprehensive digital contact tracing. However, indoor contact tracing imposes unique technical challenges due to virus concentrations and unreliable GNSS signals in indoor environments . The virAlthough the virus concentration will gradually decrease due to inactivation, deposition, and air purification after the virus-laden droplets are exhaled, the poor air exchange rate, superspreaders, and more virulent variants will keep it at a relatively high concentration for a long time in an indoor environment ,12. The To be precisely aware of the amount of the virus one is exposed to and to detect both direct and indirect contacts, an indoor spatiotemporal contact awareness (iSTCA) framework is proposed. Since the virus concentration (at different times in the same area) is not the same because of the dispersion and diffusion of the virus and human movements, we employed a self-contained PDR technique to calculate the human trajectory with accuracy and further achieve the location and time of the expelled virus droplets for the quantitative measurement of the concentration at any time in different spots. Moreover, based on the acquired changing virus concentration and reliable trajectories, the exposure time and distance of both direct and indirect contacts can be derived via cross-examination to realize quantitative spatiotemporal contact awareness.To accurately present the virus concentrations at different times, we established quantitative virus concentration changes in various areas of indoor environments at different times by infected individuals. The viral-laden droplets were continuously released during the expiratory activities, moving forward. During the movements of viral-loaded droplets exhaled by infectious individuals at different locations and times, the virus instances met in certain spots at certain times and contributed to the calculation of the concentration. Finally, the concentration of each virus instance was integrated.We properly selected PDR for the acquisition of the trajectory to conduct contact awareness without requiring extra infrastructure or being affected by coverage limitations compared with other indoor positioning techniques.We considered various landmarks to calibrate the accumulative error for trajectory achievement by using PDR. A custom deep neural network using bidirectional long short-term memory (Bi-LSTM) and multi-head convolutional neural networks (CNNs) with residual concatenations were designed and implemented to extract temporal information in forward and backward directions and spatial features at various resolutions from built-in sensor readings for landmark identification.Additionally, we demonstrate the effectiveness of the proposed Bi-LSTM-CNN classification model for landmark identification through empirical experiments, as well as the performance of our proposed iSTCA system for quantitative spatiotemporal contact analytics.Our main contributions are as follows:The remainder of this paper is organized as follows. The related work about contact awareness and indoor localization techniques, including PDR, is reviewed in Contact tracing is used to identify and track people who may have been exposed to a virus due to the prevalence of many infectious diseases in our society. To conduct contact tracing, it is necessary for the infected individuals to provide their visited locations and people whom they encountered based on the specific definitions of meetups for different diseases. Instead of interviews and questionnaires via traditional manual tracing, technology-aided contact tracing can track people at risk conveniently and intelligently. To reduce the spread of COVID-19 effectively, digital contact tracing, which generally depends on applications installed on smartphones, has been developed in both academia and industry, using various technologies, such as GNSS, Bluetooth, and Wi-Fi.There are typically two approaches for encounter determinations, peer-to-peer proximity detection-based and geolocation-based. Peer-to-peer proximity can be estimated by the received signal strength (RSS) of wireless signals, such as Bluetooth and ultra-wideband (UWB), and the distance between two devices in geolocation-based approaches can be precisely derived from the cross-examination after obtaining the accurate location and trajectory with the help of localization techniques using various technologies, such as global positioning system (GPS), Wi-Fi, and PDR.Some systems based on peer-to-peer proximity using Bluetooth or BLE have been implemented, and part of them are deployed by the governments of various countries, such as Australia (COVIDSafe), Singapore (Trace together), and the United Kingdom (NHS COVID-19 App) due to their ubiquitous embedding in mobile phones . Among tTo achieve accurate geolocation in contact tracing, plenty of localization systems have been researched with the joint efforts of researchers and engineers in the past based on GNSS, cellular technology, radio frequency identification (RFID), and quick response (QR) code . GNSS caOne of the ultimate goals of contact awareness systems is to estimate the risk based on the recorded encounter data . MoreoveThe aforementioned contact tracing research usually only considers the static virus concentration without considering the exposure to the environmental virus and dynamism in the virus concentration. Moreover, in contrast to the qualitative estimation of exposure risks that can be achieved in previous works, there is a lack of sufficient quantitative awareness about the concentrations of contracted viruses. Such awareness would be useful in our daily lives to protect ourselves from virus infections.Virus-encapsulating secretions are continuously exhaled and aerosolized into airborne virus-laden particles with infectivity from daily expository activities. There is a great difference between the size and number of droplets expelled, depending on their origin locations in the respiratory tract . The timThe viral loads of virus-containing droplets change after leaving the human expiratory tract with airborne transmission and a combination of environmental factors. In particular, the viral load emitted is expressed in terms of the quanta emission rate  [t 50 Hz) ,36. It it 50 Hz) ,37.For the quantitative evaluation of the virus quanta concentration, the precise spatial distance and temporal distance between two individuals should be efficiently estimated. To reach this objective, a variety of indoor positioning techniques have been proposed for various scenarios. The widely studied fingerprinting-based method relies on the latest fingerprint database that needs to be precisely updated in time. In addition to the time-consuming and labor-intensive collection and re-establishment, the instability of RSS due to environmental uncertainties poses another challenge to the accuracy . MoreoveSince PDR does not need additional equipment or a pre-survey, it has a wide range of potential applications for the indoor positioning of pedestrians. It relies on the inertial sensors extensively existing in mobile devices, e.g., smartphones, to acquire information about the user’s movements, which are then combined with the user’s previous location to estimate the present position and further achieve complete trajectory. The equation utilized for location estimation is as follows:destrian .As mobile technology continues to evolve, a growing number of physical sensors are being installed in smartphones and, thus, various combinations of sensors can provide increasingly rich information, which makes PDR more feasible and accessible. A typical PDR consists of three main components: step detection, step-length estimation, and heading estimation .Step detection. As the most popular method for accurate step detection, peak detection is employed in this paper, which relies on the repeating fluctuation patterns during human movement. Using the smartphone’s accelerometer to determine whether the pedestrian is stationary, or walking is straightforward as it directly reflects the moving acceleration. The magnitude of acceleration on three dimensions artphone . A peak Stride length estimation. Various linear and nonlinear methods are proposed to estimate the step length, which varies from person to person because of different walking postures determined by various factors, including height, weight, and step frequency. Therefore, it is not easy to precisely construct the same step-length estimation model. Some researchers assume that the step length is a static value affected by the individual characteristics of different users. On the contrary, the empirical Weinberg model estimates the stride length according to the dynamic movement state, which is closer to reality [ reality . The modach step .Heading estimation. Heading information is a critical component for the entire PDR implementation, which seriously affects localization accuracy. To avoid the accumulative error in the direction estimation based on the gyroscope, and short-term direction disturbances based on the magnetometer, the combination of the gyroscope and magnetometer is typically adopted for heading estimation [timation . The curtimation . As theytimation . The rotAlthough PDR methods can estimate the location and trajectory of pedestrians, low-cost inertial sensors built into smartphones provide poor-quality measurements, resulting in accuracy degradation. Moreover, the cumulative error, including the heading estimation caused by the gyroscope and step-length estimation error caused by an accelerometer, could be produced in the long-term positioning using PDR, increasing the challenge of precise localization collection. Therefore, it is necessary to prepare the reference points with the correct positions known during the movement to reduce the accumulated errors when the user passes. Spatial contexts, such as landmarks, can be properly chosen to calibrate the localization error based on the inherent spatial information without additional deployment costs. Landmark is defined as a spatial point with salient features and semantic characteristics from its near environment in indoor positioning systems, such as corners, stairs, and elevators . These fLandmark identification involves classifying the sequences of various sensor data recorded at regular intervals by sensing devices, usually smartphones, into a well-defined landmark, which has been extensively regarded as a problem of multivariate time series classification. To address this issue, it is critical to extract and learn the features comprehensively to determine the relationship between sensing information and movement patterns. In recent years, numerous features have been attained in many studies on certain raw signal statistical aspects, such as variance, mean, entropy, kurtosis, correlation coefficients, or frequency domains via the integration of cross-formal codings, such as signals with Fourier transform and wavelet transform . MoreoveTo facilitate feature engineering and improve performance, artificial neural networks based on deep learning techniques have been employed to conduct activity identification without hand-crafted extraction. Deep learning techniques have been applied in many fields to solve practical problems with remarkable performance, such as image processing, speech recognition, and natural language processing, to solve practical problems ,51. Manyeptively . In our More importantly, the convolution operation in CNN can efficiently capture the local spatial correlation features by limiting the hidden unit’s receptive field to be local . CNN conrections .Therefore, both Bi-LSTM and CNN are involved in capturing the spatial and temporal features of signals for landmark identification. The architecture of the proposed landmark identification is shown in Exhalation and inhalation respiratory activities are constantly alternating , and droplets are continuously being released from the respiratory tract with a horizontal velocity during the process of exhalation with the same direction as the movement of the human. The particles exhaled at each moment will continue to move forward, starting from the user positions when they are expelled. The viral droplets exhaled from the infectious host are transported and dispersed into the ambient airflow before finally being inhaled by a susceptible person. Each exhalation lasts several seconds , in which a long distance can be traveled for those who are in motion, and the initial position of droplets expelled cannot be accurately estimated in an indoor environment. Therefore, once complete, the exhalation period is divided into many short-term  particle ejections. Because the interval is short, the continuous virus exhalation process can be converted into an instantaneous process, i.e., the virus is released instantly at the beginning of each interval. The virus-laden droplets expelled at different intervals maintain independent and identical motion patterns and the initial positions of the particles released in each interval can be regarded as the locations of the people at the initial moments. The virus-containing particles maintain a uniform motion of initially horizontal velocity  and an Android application installed. The application can periodically read and store the readings of 11 channels  as the user walks along the prescribed routes at a normal speed in the experimental area. Moreover, participants are required to hold their smartphones at chest level, which is a reasonable position where participants can record extra information to facilitate data processing. Indeed, it is recommended that they record the timestamp and the identification of the passing landmark to construct the dataset for the landmark identification model training.The proposed landmark recognition model was extensively evaluated by a series of experiments and implemented using the Keras framework with the TensorFlow backend to minimize the cross-entropy loss. The model was performed using the collected data with 3863 samples. The dataset was divided into training (70%) and testing (30%) sets, randomly, without overlapping. There were a total of 11 landmarks, including 7 corners, 2 stairs, and 2 elevators.Moreover, the model configuration  was selected as the optimizer during the gradient descent. Other training hyperparameters were also evaluated and their recognition accuracies are presented in Following the considered model configuration and optimal training hyperparameters, the accuracy curve and loss curve of the training and testing processes are illustrated in Moreover, to evaluate the proposed network more comprehensively, further comparisons were conducted on other deep neural networks  with the same depth and training hyperparameters as shown in The path shown as the red line in As mentioned above, we regard all virus particles exhaled every 0.1 s during exhalation as virus instances. There will be many virus instances expelled during the entire movement of an infector. During the transmission of each instance, a uniform motion with a velocity of 3.9  and the device’s status variation . In addition, a large amount of power of the smartphone is consumed during the indoor positioning process, resulting in the smartphone being overheated.Technology-assisted virus exposure tracking approaches are increasingly being adopted to mitigate and tame the epidemic. In view of the complexity of quantifying virus exposure due to human movement and airborne dispersion of virus particles, we propose iSTCA, a self-containing contact awareness approach that exploits PDR-based techniques. Quantitative information support directly concerned with risk assessment is provided for self-protection and epidemic control. More precisely, to reduce and calibrate the accumulative errors of trajectories based on landmarks, we apply Bi-LSTM and multi-head CNN with residual concatenation to long-term dependency in forward and backward directions and extract local correlations at various resolutions for landmark identification. The proposed method exploits the trajectories of people with viral-laden droplets exhaled and the transmission and attenuation of viruses in the air to quantify the virus quanta concentration in an indoor environment via spatiotemporal analytics for prevention and sanitization. In future work, we will continue studying the landmark identification model with different devices and various attitudes of the device and conduct further research on the exploration of other advanced deep neural networks and fusion algorithms. We will consider employing wearable devices, such as smartwatches and smart bands, to replace mobile phones for power saving in indoor positioning. Moreover, we plan to apply the proposed techniques for the development of services in developing communities without reliable digital infrastructure.


Lassa fever (LF) is endemic to broad regions of West Africa. Infection with Lassa virus (LASV), the etiologic agent of LF, results in a spectrum of clinical signs in humans, including severe and lethal hemorrhagic disease. Person-to-person transmission occurs through direct contact with body fluids or contaminated bedding and clothing. To investigate transmission risk in acute LASV infection, we evaluated viral RNA and infectious virus obtained from conjunctival, nasal, oral, genital, and rectal swab specimens from guinea pigs modelling lethal and non-lethal LF. Viral RNA and infectious virus were detected in all specimen types beginning 8 days post infection, prior to onset of fever. In the pre-clinical and clinical period, virus was isolated from a subset of nasal, oral, genital, and rectal swabs, and from all conjunctival swabs. Overall, conjunctival and nasal specimens most frequently yielded infectious virus. These findings indicate mucosal transmission risk based on virus isolation from various sites early in infection and support potential utility of minimally invasive specimen evaluation by RT-qPCR for LASV diagnostics. Lassa virus (LASV), the etiologic agent of Lassa fever (LF), causes over 100,000 human cases annually in endemic regions of West Africa ,3. Using4 FFU of LASV strain Josiah or Sauerwald . Sauerwald was isolated from a fatal human case  and grown on Vero-E6 cells  [Groups of 25 strain 13/N guinea pigs  housed in a biosafety level 4 (BSL-4) laboratory were infected subcutaneously (SC) in the interscapular region with target dose 1 × 10Guinea pigs that develop clinical disease after LASV infection demonstrate early non-specific signs  and elevated temperatures peaking ∼12 dpi . Fever rAll animals were monitored daily. At the predetermined timepoints, weights, temperatures and clinical score were recorded (Table S1 and S2). Terminal intracardiac blood was collected in EDTA from animals under deep isoflurane anesthesia followed by euthanasia . Mucosal specimens were collected immediately post mortem from conjunctiva, nares, oropharynx, genitalia , and rectum. Sterile 6″ mini-tip (0.078″ diameter) polyester swab applicators  were used for nasal specimens; sterile 6″ polyester-tipped applicators  were used for all other sites. Two specimens were collected per site: one in 500 µL MagMAX lysis solution (1:1 with isopropanol) for RNA isolation, and one in 1 mL sterile DMEM  for virus isolation and quantification.p = 0.0036). In general, RNA levels were highest in genital followed by conjunctival and nasal specimens . Relative RNA values and positivity coincided with peak viremia and body temperature elevation (12 dpi); levels remained stable 16 dpi with Josiah infection but declined with Sauerwald.RNA from collected tissues  and swabs was isolated using MagMAX Pathogen RNA/DNA Kit . Genomic DNA was removed using BaseLine Zero DNase (Epicentre), and RT-qPCR was performed using SuperScript III Platinum One-Step RT-qPCR Kit (Invitrogen) with strain-specific primers and probe targeting LASV N gene. Standard curves generated by S-segment in vitro transcripts were used to quantify viral RNA , followed by nasal , conjunctival , rectal , and oral  samples , and staining for LASV proteins using an in-house 5 monoclonal antibody mix as the primary antibody (SPR628). Overall, 80 of 128 (63%) RT-qPCR-positive specimens yielded infectious virus. Infectious virus was most frequently isolated from RT-qPCR-positive conjunctival  and nasal  specimens . Beginning 8 dpi, we detected viral RNA and isolated virus from all specimen types evaluated, including conjunctival, oral, nasal, genital, and rectal swabs. Viral RNA and virus isolation were more frequently detected and respective titres were higher in animals infected with the Josiah strain ; however, infectious virus was also detected in many samples from animals infected with non-lethal Sauerwald strain, indicating that shedding and transmission risk early in infection are likely independent of disease outcome.In humans, LASV RNA has been detected in lacrimal fluid , saliva et al. detected viral RNA in 80% of seminal fluid specimens at discharge [Most recently, mucosal shedding and viral persistence were investigated in urine, saliva, lacrimal fluid, vaginal fluid, and seminal fluid from a large cohort of human LF survivors at discharge and serially for up to 2 years post discharge . A low pischarge . The disMastomys natalensis, the natural host of LASV [To our knowledge, shedding and transmission have not been reported in animal models of disease. Recently, oral, rectal and urinary shedding were characterized in  of LASV . RNA wasOverall, our studies indicate a high frequency of mucosal shedding and infectious virus presence early in infection in all sample types we assessed, and provide key data to consider in infection control and diagnostic assay development for LASV and other pathogenic arenaviruses.Click here for additional data file.


Human monkeypox, caused by monkeypox virus, has spread unprecedentedly to more than 100 countries since May 2022. Here we summarized the epidemiology of monkeypox through a literature review and elucidated the risks and elimination strategies of this outbreak mainly based on the summarized epidemiology. We demonstrated that monkeypox virus became more contagious and less virulent in 2022, which could result from the fact that the virus entered a special transmission network favoring close contacts  and the possibility that the virus accumulated a few adaptive mutations. We gave the reasons to investigate whether cattle, goats, sheep, and pigs are susceptible to monkeypox virus and whether infection with monkeypox virus could be latent in some primates. We listed six potential scenarios for the future of the outbreak . We also listed multiple factors aiding or impeding the elimination of the outbreak. We showed that the control measures strengthened worldwide after the World Health Organization declared the outbreak a public health emergency of international concern (PHEIC) could eliminate the outbreak in 2022. We clarified eight strategies, i.e., publicity and education, case isolation, vaccine stockpiling, risk-based vaccination or ring vaccination, importation quarantine, international collaboration, and laboratory management, for the elimination of the outbreak. Human monkeypox (MPX) is akin to smallpox and is caused by monkeypox virus (MPXV). MPXV, which mainly circulated in some African countries before 2022, has spread unprecedentedly to more than 100 countries or territories. As of November 8, 2022, 78,379 human cases with 43 deaths have been confirmed  1–6). T. T6). ThMPX virus and share the same genus Orthopoxvirus, subfamily Chordopoxvirinae, and family Poxviridae with Abatino macacapox virus, Akhmeta virus, Camelpox virus, Cowpox virus (CPXV), Ectromelia virus, Raccoonpox virus, Skunkpox virus, Vaccinia virus (VACV), Variola virus , Taterapox virus, and Volepox virus  were first identified in 1958 . MPXV inIn host distribution, MPXV can infect various mammals, including rodents , primates , carnivores , lagomorphs, and insectivores  , 12–14. In clinical epidemiology, the incubation period in humans is 5–21 days, and symptoms last 2–4 weeks , 8. EarlIn phylogenetic distribution, as shown in In transmission routes, MPXVs spread among animals and humans through three routes , 8, 26: In infection courses, usually, human MPXV infection is assumed to be transient, but we suspect that the infection in primates could sometimes be latent, as suggested by two studies. One was in 1958 in Denmark, where MPX occurred in two batches of cynomolgus monkeys only when they had been transported to Copenhagen for 51 and 62 days .via the animal-to-human route, whereas in recent years, more than 70% of human cases were infected via the human-to-human route (0) value of MPXV before 2022 was <1.0  outside Africa , we assumed that the outcome of a human MPX case would be known on the 21reported , 4. Thenreported . The sigIn risk factors, as per the epidemiology of MPX elucidated above, endemic countries, international travel, importation of mammals, humans unvaccinated with smallpox vaccines, MSM, crowded parties, close contact with infected humans, family members of infected humans, the trash of infected humans, healthcare workers in affected regions, vaccine unavailability, squirrels, rodents, non-human primates, pet animal markets or traders, and zoos are all risky for the transmission of MPX –6, 8–20.In future prediction, If we looked at the counts of new human cases shown in Multiple factors aid the world in eliminating the 2022 outbreak . The WHOThis is also supported by the fact that MPXV and VACV are close to each other in phylogenetics , 24. UnlOn the other hand, multiple factors hinder the world from eliminating the 2022 outbreak  32)..32).First, MPXV has infected too many people in many countries in 2022 , and theSecond, MPXV can infect various species of domestic and wild animals –3, 6. ThThird, the contagiousness of MPXV in humans could increase through genomic mutation. If MPXV accumulates some adaptive mutations and replicates more efficiently in humans, it could be more contagious and virulent in humans simultaneously, as per the trade-off theory regarding the long-term evolution of virulence of pathogens . This poFourth, the 2022 outbreak could not have aroused the vigilance of many threatened human populations worldwide because of its low CFRs (approximately 0.049% as mentioned above). This could lead to multiple waves of human MPX infections in the coming years.Fifth, the 2022 outbreak can provide live MPXV strains for laboratory engineering and bioterrorism attacks, which can accelerate the virus′ spread worldwide . This isSixth, some countries, particularly those in war or severe poverty, cannot eliminate human MPX infections promptly. They cannot identify and isolate human MPX cases promptly, and they have not stocked enough smallpox or MPX vaccines and antivirals. The global vaccine inequity and antiviral inequity that facilitated the spread of SARS-CoV-2 could be repeated with MPX .As per the epidemiology and risks given above, the following strategies should be considered to eliminate the 2022 outbreak outside Africa earlier .First, all countries should strengthen publicity and education regarding the risks, prevention, treatment, and control of the disease, preferably through new media, which are more efficient in communication and more capable of avoiding stigmatization than traditional media. All people should know how to report suspected human or animal cases of MPX to the government, public health agencies, hospitals, etc. Likely due to strengthened publicity and education, MSM in the USA have taken steps to protect themselves and their partners from MPX  .Second, infected people should be isolated until they are no longer infectious, and their rubbish should be properly treated, to cut off the transmission of MPXV in humans and animals.Third, MPX vaccines should be well stockpiled worldwide. The USA decided to stockpile 13 million doses of MPX vaccines in May 2022. The vaccines stockpiled worldwide can be employed in endemic African countries if the 2022 outbreak is eliminated in the near future.Fourth, epidemiological investigation of humans and animals living in threatened areas should be strengthened in order to identify new human or animal cases promptly. As explained above, it is desirable to investigate whether cattle, goats, sheep, and pigs are susceptible to MPXV, which is important for the elimination of the 2022 outbreak as they are populous and frequently in close contact with humans. It is also desirable to investigate whether MPXV infection could be latent or persistent in some primates and humans infected with HIV.Fifth, if possible, family members, sexual partners, healthcare workers, and other close contacts of human MPX cases should be vaccinated imminently (ring vaccination) to block the transmission in affected regions . If possSixth, the quarantine of imported animals should be strengthened. This is supported by the fact that pet rodents imported from Ghana spread the virus to pet prairie dogs in the USA in 2003, which further infected more than 70 people .Seventh, robust international collaboration in sharing epidemiological information and vaccines is needed to aid low- and middle-income countries to eliminate the 2022 outbreak . Mass vaEighth, all countries should consider strictly restricting laboratory research with live MPXV to avoid laboratory spill-over events , 40.Here we summarized the epidemiology of MPX through a literature review and elucidated the risks and the elimination strategies of this outbreak mainly based on the summarized epidemiology. We demonstrated that MPXV became more contagious and less virulent in 2022, which could result from the fact that the virus entered a special transmission network favoring close contacts outside Africa and the possibility that the virus accumulated a few adaptive mutations. We gave the reasons investigating whether cattle, goats, sheep, and pigs are susceptible to MPXV and whether infection with MPXV could be latent in some primates. We listed six potential scenarios for the future of the outbreak and multiple factors aiding or impeding its elimination. We showed that the control measures strengthened worldwide after the WHO declared the outbreak a PHEIC could eliminate the outbreak in 2022. We listed eight strategies for the elimination of the 2022 outbreak. Collectively, this analysis provides novel insights into the risks and elimination of the 2022 outbreak.J-MC conceived, designed and supported this study, analyzed the data, and drafted the manuscript. G-HZ and J-WC made the core conclusion, analyzed the data, and revised the manuscript. R-XC, H-YG, M-MZ, M-HS, Y-FJ, G-HL, and S-MT collected and analyzed relevant data and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Open Fund of Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control (No. ZMM2021) and the Open Competition Program of Top-Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG02).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pathogens initiated this Special Issue. Epidemiology is a field that combines specialist knowledge of many disciplines, considered a facilitator of communication and various diverse skills.One Health is a popular headline for an all-inclusive concept of our scientific work. It is not a new concept, but one along that we have been researching for many decades, if not centuries. Nevertheless, the term has become a keyword used worldwide for an inclusive approach to investigating issues such as pathogens in our various environments and host species. It was with this intention that We are very pleased with to present ten original publications covering the molecular properties, distribution and surveillance of pathogens and vectors. A few examples:A sampling methodology for the surveillance of mosquitos and arboviruses in Florida was more efficient when stratified by a vertical gradient;Genotyping parvoviruses of dogs and cats in Sri Lanka allowed identification of cats as possible source of the virus for dogs;A hitherto unknown zoonotic risk emerged from a study of illegally traded companion animals in Italy and Austria;A study in Barbados resulted in the identification of the seasonal and occupational distribution of human orthohantavirus infections among febrile patients;We now know that the majority of genotypes of equine herpes virus 5 from Poland clustered within two main groups and similar viruses circulated in geographically distinct locations without any apparent age predisposition;An investigation of re-emerging rabies virus in Southeast Asia demonstrated a need for collecting data in remote locations for detecting drivers of emergence.Leptospira spp., about virulence factors of the avian-pathogenic Escherichia coli (APEC) to inform antibiotic therapy, vaccine development and virulence inhibitors, and about the economic loss of the zoonotic Ross River virus (RRV) transmitted from horses to humans. A more confined investigation described an outbreak of canine distemper in a rescue centre.In addition, three reviews detail the merits and limitations of diagnostic assays for describing the diversity of This Special Issue intended to provide ‘insights into the ecological behaviour of pathogens and their impact on human and animal populations and environments’. We believe the ten publications have significantly contributed to achieving this goal. We would like to thank our colleagues and authors who collaborated and crafted articles describing their scientific thinking and outcomes.