Hybrid gel beads based on combining a low‐molecular‐weight gelator (LMWG) with a polymer gelator (PG) demonstrate an enhanced ability to self‐propel in water, with the LMWG playing an active role. Hybrid gel beads were loaded with ethanol and shown to move in water owing to the Marangoni effect changes in surface tension caused by the expulsion of ethanol – smaller beads move farther and faster than larger beads. Flat shapes of the hybrid gel were cut using a “stamp” – circles moved the furthest, whereas stars showed more rotation on their own axes. Comparing hybrid LMWG/PG gel beads with PG‐only beads demonstrated that the LMWG speeds up the beads, enhancing the rate of self‐propulsion. Self‐assembly of the LMWG into a “solid‐like” network prevents its leaching from the gel. The LMWG also retains its own unique function – specifically, remediating methylene blue pollutant dye from basic water as a result of noncovalent interactions. The mobile hybrid beads accumulate this dye more effectively than PG‐only beads. Self‐propelling gel beads have potential applications in removal/delivery of active agents in environmental or biological settings. The ability of self‐assembling LMWGs to enhance mobility and control removal/delivery suggests that adding them to self‐propelling systems can add significant value. Motion capture: Gel beads that incorporate a self‐assembled low‐molecular‐weight gelator (LMWG) into an agarose polymer gel particle are reported; when loaded with ethanol and placed in water, they self‐propel as a result of the Marangoni effect. The presence of the LMWG significantly increases their mobility and endows them with unique functions, such as enhanced ability to adsorb a pollutant dye. Self‐assembled hydrogels are fascinating, responsive soft materials with potential applications ranging from environmental regeneration to tissue engineering.2O2 giving rise to an inhomogeneous concentration around the surface of the particle.One innovative way to spatially and temporally control LMWGs is to develop gels that can physically move in programmable ways. Such actuating systems can be considered as hydrogel machines and are an emergent area of intense interest.Gels are ideal for creating self‐propelled machines as a result of their solvent compatibility, porous structures and ability to encapsulate and release molecules in a controlled manner. Motile gel beads with their own source of propulsion have potential uses in bio‐nanotechnology and medicine, where they can explore their surroundings, and deliver bioactive systems.2 with the PG agarose  to give spherical gel beads on cooling . This experiment indicated that each gel bead incorporated 99 % of the expected DBS‐CONHNH2. If, instead, the intact gel beads were studied by 1H NMR in D2O, no signal was observed for either agarose or DBS‐CONHNH2, demonstrating that both components are fully assembled into a solid‐like form. This is important because unlike blending a simple additive with the PG, the self‐assembly of the LMWG into a “solid‐like” nanoscale network limits leaching from the beads, and helps fix this network within the hybrid system.To quantify the amount of LMWG loaded into each gel bead, we performed a simple −1. These shifts are consistent with the presence of supramolecular interactions between the LMWG and the PG networks and are similar to those previously observed for bulk samples of this hybrid gel.The supramolecular interactions between the two gel networks within the beads were studied by IR spectroscopy . The O−H and N−H stretches of the LMWG appeared broadened in the presence of agarose, and the C=O band shifted from 1642 to 1665 cm2 gels made in sample vials in order to better understand the dual network nature of these hybrid hydrogel systems . In summary, the LMWG has a G′ value of 800 Pa, the agarose PG has a G′ value of 5960 Pa, and the combination of the two has a G′ value of 29400 Pa. This clearly demonstrates that both gel networks are forming and interpenetrating to form a stiffer, more robust gel.We also performed rheological characterisation of control agarose/DBS‐CONHNHWe then loaded the gel beads with EtOH to induce spontaneous motion in water by exploiting the Marangoni effect. In principle, as ethanol diffuses out of the beads, it changes the local surface tension close to the bead, leading to self‐propulsion. Such effects are understood in polymer gel systems, but in this study, we were interested in determining the potential benefits of incorporating an LMWG into PG beads.EtOH‐loaded beads were obtained by immersing them in EtOH for 24 h. When transferred into a small petri dish (8 cm diameter) filled with water, they displayed spontaneous motion for a maximum of 10–15 mins . In general terms, the beads followed a random trajectory from the centre of the petri dish, where they were loaded, often ultimately ending up moving along the circumference  were prepared by using 20 μL droplet volumes. To obtain larger gel beads (4.0–4.5 mm diameter), we increased the droplet volume to 30 μL, whereas smaller beads (1.5–2.5 mm diameter) were prepared using 10 μL droplets. We refer to hybrid gel beads with diameters of 1.5–2.5 mm, 3.0–3.6 mm and 4.0–4.5 mm as “small”, “standard” and “large” respectively .First, we studied the effect of gel bead diameter on the total distance travelled after 5 and 30 s. Beads of different diameters were prepared by varying the drop volumes of the DBS‐CONHNHThe data indicate that the gel bead diameter significantly influences the total distance travelled and hence their average speed in the first 5 and 30 s . Initially, the standard and smaller gel beads showed similar behaviour, travelling 25–26 cm after 5 s at an average speed of about 5 cm/s (Table S4). The larger gel beads were significantly slower  and after 5 s travelled a shorter distance . After 30 s, the smaller gel beads had travelled a longer total distance . This study was performed by immersing the different gel beads loaded with EtOH in water 5 mL) and subsequently analysing the EtOH content of the water after 30 s, 60 s and 24 h. After 30 seconds, the larger gel beads released a greater amount of EtOH  that were immersed in EtOH for 24 h, with the behaviour of standard gel beads that were half‐immersed in EtOH for only 15 min . This lowered ethanol loading, the fully loaded gel beads released 8.62 μL/bead over 60 s, whereas the half‐loaded beads only released 6.15 μL/bead. In the first 30 s (Table S4), the half‐loaded beads travelled a significantly shorter distance of 31.4 cm (compared to 70.1 cm for the fully loaded beads) at a lower average speed of 1.0 cm/s (compared with 2.3 cm/s). This confirms that the EtOH loading does influence the self‐propulsion of beads, when beads with equivalent diameters and weights are compared (Section S10.2 in the Supporting Information).We had wondered whether half‐immersing the beads in EtOH for loading may influence the directionality of motion by preferentially loading one half of the gel bead with ethanol. It is well‐known that “Janus” particles can exhibit a degree of controlled motion.2/agarose gels (0.3 % w/v of DBS‐CONHNH2 and 1.0 % w/v of agarose) using small “icing cutters”  to a hybrid gel prepared in a 5×5 cm square tray . The total distance travelled by the shaped gels after 5 s was similar for the different shapes and also for the gel beads . The circle and the crescent‐shaped gels were the fastest, with an average speed of 5.8 and 5.6 cm/s, followed by the gel beads (5.2 cm/s) and the stars . After 60 s, however, a remarkable difference had opened up in the total distance travelled by the different gels. The circular gels travelled the furthest . This allowed us to determine the impact of the LMWG on self‐propulsion . Under basic conditions (pH 11.8), the hybrid gel beads took up approximately 70 mg/g of their maximum loading in the first hour, the next 70 mg/g over the following 4 h, and the remaining 35 mg/g over a 24 h period. To demonstrate the gel beads could be re‐used, after 24 h exposure to methylene blue, we washed them with basic NaOH solution until the colour was fully removed. On re‐using the gel beads, the performance in terms of total methylene blue uptake was actually even better than on the first run , with the total uptake rising to approximately 280 mg/g by the fourth use. We suggest this is likely to be a result of the base‐washing process pre‐activating the DBS‐CONHNHFinally, and importantly, we tested whether the moving beads could be used for dye recovery by performing a dye uptake experiment using the ethanol‐loaded mobile beads. The mobility of the gel particles in basic solution pH 11.8–12.0) was similar to that in pure water . However, the movement was somewhat less consistent, with gel objects showing different rates of mobility over a 60 s timescale making it more difficult to draw conclusions about the effects of shapes. Pleasingly, the mobile ethanol‐loaded gel beads were able to remediate methylene blue from solution and importantly, in each case, the mobile beads with an embedded LMWG network very significantly outperformed the agarose‐only beads  were compared with static gels . Although the mobile gels and static gels eventually bind similar amounts of methylene blue, the mobile gels show slightly slower initial uptake rates than static gels, especially over the first 15 min while the beads are in most motion. We reasoned that this may be because initially, the moving gels release the loaded EtOH, an efflux process that may somewhat limit initial dye absorption. After that phase, they then start to absorb the dye more strongly. As such, the moving gel beads have potential to access difficult to reach locations through their motion where they could then absorb pollutant dyes. This approach could, in the future, be combined with magnetic bead technologies to facilitate bead recovery.2 and agarose. The beads were prepared by emulsion methods and have been fully characterised. Once the beads are loaded with ethanol, they are able to self‐propel in water as a result of the Marangoni effect. Smaller beads move farther and faster than larger beads. Furthermore, cutting the gels into shapes has a direct impact on the mobility, with motion being switched between translational modes and internal rotation. The presence of the self‐assembled, low‐molecular‐weight gelator network amplifies the Marangoni effect and significantly enhances the mobility of the shaped gels. This is a result of more rapid efflux of ethanol from the hybrid gel beads than the agarose‐only systems, which means that, even though they have similar total ethanol loadings, the system incorporating the LMWG is better able to harness it for rapid propulsion. This is a clear benefit of the hybrid gel approach.In summary, we have reported a new family of supramolecular gel beads based on interwoven networks of DBS‐CONHNHThe self‐assembled LMWG network retains its functionality in these gels, enhancing the remediation of the pollutant dye methylene blue under basic conditions, with the hybrid gel beads significantly outperforming agarose‐only objects. Dye uptake is reversible, and the gel beads can be reused multiple times. Importantly, the mobile ethanol‐loaded beads can remediate methylene blue from solution, and there is a general correlation between the ability of the beads to move and the amount of methylene blue removed, thus suggesting the LMWG controls both processes.In terms of future perspective, we note that, in addition to binding dyes, this LMWG can bind precious heavy metals, remediating valuable waste streams and generating catalytic gel beads.5a, 242. Different LMWGs have a wide range of different functions, and the ability to assemble these functional LMWGs into agarose gel beads and use the Marangoni Effect for self‐propulsion opens up a wide range of possibilities for the combination of LMWGs with mobile gel technology.Thinking more broadly, this approach should not be limited to DBS‐CONHNHThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


African swine fever (ASF) caused by the African swine fever virus (ASFV) is ranked by OIE as the most important source of mortality in domestic pigs globally and is indigenous to African wild suids and soft ticks. Despite two ASFV genotypes causing economically devastating epidemics outside the continent since 1961, there have been no genome-level analyses of virus evolution in Africa. The virus was recently transported from south-eastern Africa to Georgia in 2007 and has subsequently spread to Russia, eastern Europe, China, and south-east Asia with devastating socioeconomic consequences. To date, two of the 24 currently described ASFV genotypes defined by sequencing of the p72 gene, namely genotype I and II, have been reported outside Africa, with genotype II being responsible for the ongoing pig pandemic. Multiple complete genotype II genome sequences have been reported from European, Russian and Chinese virus isolates but no complete genome sequences have yet been reported from Africa. We report herein the complete genome of a Tanzanian genotype II isolate, Tanzania/Rukwa/2017/1, collected in 2017 and determined using an Illumina short read strategy. The Tanzania/Rukwa/2017/1 sequence is 183,186 bp in length (in a single contig) and contains 188 open reading frames. Considering only un-gapped sites in the pairwise alignments, the new sequence has 99.961% identity with the updated Georgia 2007/1 reference isolate (FR682468.2), 99.960% identity with Polish isolate Pol16_29413_o23 (MG939586) and 99.957% identity with Chinese isolate ASFV-wbBS01 (MK645909.1). This represents 73 single nucleotide polymorphisms (SNPs) relative to the Polish isolate and 78 SNPs with the Chinese genome. Phylogenetic analysis indicated that Tanzania/Rukwa/2017/1 clusters most closely with Georgia 2007/1. The majority of the differences between Tanzania/Rukwa/2017/1 and Georgia 2007/1 genotype II genomes are insertions/deletions (indels) as is typical for ASFV. The indels included differences in the length and copy number of the terminal multicopy gene families, MGF 360 and 110. The Rukwa2017/1 sequence is the first complete genotype II genome from a precisely mapped locality in Africa, since the exact origin of Georgia2007/1 is unknown. It therefore provides baseline information for future analyses of the diversity and phylogeography of this globally important genetic sub-group of ASF viruses. The high mortality induced in naïve pig populations is devastating to pig farmers, especially given that there is no chemotherapy or vaccine currently available for disease control. Prevention of the disease relies on strict biosecurity measures which are frequently not effectively applied, particularly in the endemic areas. As a result, the virus and associated disease have spread into many new areas that have not previously been reported (reviewed by4). Historically, the disease was first described in East Africa in the 1920s5. The first occurrence outside Africa, of a virus classified in p72 genotype I, was reported in 1958 and again in 1961 from Lisbon Portugal, with subsequent spread to other regions of Europe and Latin America (reviewed by7). In 2007, an ASF virus (ASFV) within p72 genotype II8 was again exported through a human agency outside sub-Saharan Africa into Georgia. However, the precise geographical origin has not been identified, since genotype II is present in Mozambique, Madagascar, Malawi, Zambia, Zimbabwe and Southern Tanzania16 as represented in Fig. According to the World Organization for Animal Health (OIE), African swine fever virus (ASF) is the most important disease-causing pathogen affecting the domestic swine population globally17.Using a risk assessment methodology, shipment of a pork product originating from southeastern Africa, or Madagascar was highlighted as a likely source of viruses that could be disseminated into other continents18 and South-east Asia. Despite significant efforts by FAO and others to control its spread, numbers of ASFV infected animals are increasing rapidly in domestic pigs and wild boar populations, with movements of the latter facilitating the rapid geographical spread of the virus (reviewed in2).Between mid-2007 to date, ASF genotype II viruses, derived from the Georgia introduction, have been reported throughout the Caucasus, Russia, the Baltic republics, Czech Republic, Romania, Hungary, Bulgaria, Poland, Belgium, and most recently ChinaAsfivirus within the family Asfiviridae, ASFV is the only known DNA virus causing hemorrhagic fever transmitted by an arthropod, namely Argasid soft ticks in the genus Ornithodoros. The genome of the virus is composed of a linear double-stranded DNA molecule between 175 and 195 kbp in length and containing up to 190 open reading frames depending on the virus isolate19. Multiple approaches have been used to characterize the virus20, with genotyping of the virus by full length or partial sequencing of the genes encoding the p72, p54, p30 proteins and the Central variable region now widely applied.Constituting the sole member of the genus 22. Most of these genotypes can be associated with particular geographical regions, especially in sub-Saharan Africa, where the virus almost certainly originated, and is endemic in wild suids23. Among the 24 published genotypes, genotype II is currently of the highest global importance, since it is the cause of an ongoing pig pandemic encompassing a region from Eastern Europe to China and Southeast Asia. The economic losses involved are enormous, amounting to as much as 25% of global pig production. The socioeconomic impact is particularly severe in China, which produces 450 million of the world’s pigs, many of which are in small or medium scale, backyard or village systems18.Twenty-four genotypes of ASFV have been identified by sequencing the p72/B646L gene of the virus25, most of the complete genomes are derived from isolates sampled outside Africa in novel environments into which ASFV has recently been introduced by human agency. The newly infected agroecosystems in Eurasia lack both the ancestral tick vector (Ornithidorus moubata/porcinus) and also warthogs (Phacocherus africanus) and bush pigs (Potamochoerus larvatus), the mammalian hosts with which the viruses have co-evolved over millions of years in the African continent. The currently available complete genomes derived from ASFV genotypes I and II are therefore of limited relevance for reconstructing the evolutionary history of ASF viruses. The complete genome sequence of an African ASFV genotype II virus, has not yet been reported, despite the devastating impact of this genotype on domestic pig production across Eurasia32. To date, the analyses of genotype II within Africa have been confined to genotyping of the viruses at selected polymorphic loci, particularly the 3ʹ end of the gene encoding the p72 surface protein (locus B646L) where most mutations sampled appear synonymous at the protein level33. The p72 gene analysis is typically combined with sequencing the region encoding the tetrapeptide central variable region (CVL) within locus p602L, which provides high-resolution data for differentiating lineages that have emerged on short timescales. Such data, as seen in Supplementary Figure Multiple complete genome sequences of ASFV have been determined with a strong bias towards p72 genotypes 1 and II. However, with a few exceptions, for example, Benin and South African isolates in p72 genotype 1We generated the whole genome sequence of a p72 genotype II ASFV that was isolated from a clinically ill domestic pig in the Rukwa region of the southern highlands of Tanzania following infection of PBMCs using a virus suspension prepared from spleen tissue. The location within Tanzania of the district from which the virus was isolated and its geographical separation from the presumptive origin of Georgia 2007/1 in Southeastern Africa is shown in Fig. For comparative genomics analysis, reference genotype II whole genome sequences published in previous studies were retrieved from the European Nucleotide Archive. While all publicly available genotype II genomes were closely related to the Tanzania/Rukwa/2017/1 sequence, we concentrated on three that were representative of different geographical regions spanning the extent of the current pandemic of ASFV across Eurasia. These were the original genotype II isolate sampled outside of Africa, considered to represent the index case of the current pandemic affecting Asia and Europe , the Polish virus Pol16_29413_o23  and a Chinese isolate ASFV-wbBS01 sampled in 2018 (MK645909.1). Initial comparison of the new Tanzanian viral genome determined in this study was performed by mapping with the minimap2 program to the Georgia/2007/1 isolate (FR682468.2). A Circos plot representing the mapping of reads to the recently corrected Georgia 2007/1 (FR682468.2) genome sequence was drawn and is presented in Fig. The overall sequence similarity between the two genomes based on an un-gapped alignment was 99.671% and the relationship between Tanzania/Rukwa/2017/1 and other ASFV genotype II complete genomes are shown as a matrix of pairwise identity values in Fig. Since indels represent the major sources of variation between ASFV genomes, we focus primarily on this aspect in detail here. Major differences included the apparent absence of the single-copy locus ASFV_G_ACD_00120 from the Tanzania/Rukwa/2017/1 genome and a series of differences in 5ʹ and 3ʹ multicopy gene families (MGFs). Regarding the MGFs, specific differences include: (1) at the 5ʹ end of the Tanzania/Rukwa/2017/1 virus, the MGF360-1L gene is absent; (2) there is almost complete loss of MGF360_21R near to the 3ʹend of the Tanzania/Rukwa/2017/1 genome; (3) the copy of MGF110-7L in Tanzania/Rukwa/2017/1 is truncated  relative to Georgia 2007/1 (FR682468.2) and encodes only 39 amino acids, due to the insertion of a G resulting in a frameshift and introduction of a stop codon; (4) a deletion of one nucleotide in MGF360-14L results in a major truncation of this gene compared to Georgia 2007/1. (5) Two SNPs affect MGF505-4R in Tanzania/Rukwa/2017/1, the second one introducing a premature stop codon, so the length of this ORF is 369 aa versus 506 aa in Georgia/2007/1.One indel not associated with the MGFs is an insertion of 16 bases that yields a tandem repeat (aaaaaaataaacaaca) located just 5ʹ of the ATG codon of locus KP177R as presented in the Supplementary Figure 34 and will be efficiently addressed only by long-read sequencing including the full terminal hairpin loops.It is noteworthy that the final inverted terminal repeat of Georgia 2007/1  has abnormally high coverage of more than 1000×. One possible explanation is that sequential repeats have collapsed during assembly of both the ‘original’ reference Georgia 2007/1 genome (FR682468.1) and the re-sequenced Georgia 2007/1 (FR682468.2), but to a lesser degree. This problem of lack of resolution of the copy number in the terminal repeat region of ASFV has been noted previously31) exhibited 99.960% similarity to Tanzania/Rukwa/2017/1 and the Chinese virus ASFV-wbBS01 (MK645909.128) exhibited 99.957% similarity. The Tanzania/Rukwa/2017/1 sequence is noticeably shorter than the Polish sequence . This is because the 5ʹ (3090 bp) and 3ʹ (2356 bp) overhang ends that are present in the Polish genome, could not be sequenced using our protocol. A number of indels also differentiated the Tanzanian and Polish virus genomes, including a long deletion of 688 bp close to the 5ʹ end of the Tanzania/Rukwa/2017/1 virus. Additionally, nucleotides 7423 to 8110 of the Polish virus  are apparently absent in Tanzania/Rukwa/2017/1. There is also an insertion of 16 bp (taaacaacaaaaaaaa) at alignment positions 3250 to 3265 between nucleotides 3249 and 3250 of the Polish virus. Other deletions observed included 11 bp corresponding to nucleotides 25,319 to 25,329 of the Polish virus , 66 bp corresponding to nucleotides 175,453 to 175,518 of the Polish virus . The remaining shorter insertion and deletions range from one to six nucleotides in length and are located primarily in homopolymer runs throughout the assembly.Searches using BLAST indicated that genome sequence of Tanzania/Rukwa/2017/1 was very closely related to both the Chinese and Polish viruses. Pol16_29413_o23  at position 9711 of the reference. Other features include an insertion of one base (G) between positions 9660 and 9661 of the Polish genome Among the 189,420 nucleotide sites present in the pairwise alignment of Tanzania/Rukwa/2017/1 with Pol16_29413_o23, there are 183,159 complete sites (with no gaps and no undetermined nucleotides), among which there are only 73 variable sites. In total, we counted 126 SNPs from the mapping of our short reads to the Polish virus sequence MG939586.1.The alignment of Tanzania/Rukwa/2017/1 with the Chinese ASFV-wbBS01 (accession: MK645909) comprises 189,427 sites, with 183,153 complete sites (without gaps or undetermined nucleotides), among which only 78 are variable. Unlike the Polish viral genome, the Chinese strain is annotated, with 185 predicted CDS and corresponding conceptual translations. At the 5ʹ end of the genome, reads mapping to the inverted tandem repeat region (nucleotides 1 to 422 of MK645909) were present. This was followed by a gap without reads in Tanzania/Rukwa/2017/1 corresponding to base pairs 400 to 2175 of MK645909. As already mentioned, MGF360-1L is missing in the Tanzanian isolate as compared to Georgia 2007/1 and viruses derived from the index isolate. However, the next representative of the 360-multigene family, encoded at the 5ʹ end of ASFV, MGF360-2L, is present downstream in Tanzania/Rukwa/2017/1. Two intergenic inserts of one base each were observed in Tanzania/Rukwa/2017/1 between MGF360-3L and MGF110-1L. There is also insertion of a T in Tanzania/Rukwa/2017/1 between positions 5818 and 5819 in MK645909 and insertion of a G in Tanzania/Rukwa/2017/1 between sites 6050 and 6051. There is a T/C SNP towards the beginning of MGF110-1L (position 6102 of the MK645909) corresponding to the second position with the TAG stop codon which becomes a tryptophan codon TGG, hence extending MGF110-1L from 196 amino acids in ASFV-wbBS01, to 269 a.a. in Tanzania/Rukwa/2017/1. The MGF110-1L open reading frame is also affected by two SNPs: C/T at position 6365 and A/T at position 6367 of the MK645909. These two SNPs are respectively in the third and first positions of a codon. The codon TGG (tryptophan) in wbBS01 is mutated to encode arginine (AGA) in Tanzania/Rukwa/2017/1. Additionally, MGF110-3L is truncated at the 5ʹ end. Within MGF110-4L, there are also SNPs, specifically T/C at position 8160 and C/A at position 8204. In the fused MGF 110 protein MGF110-5L-6L, a deletion of three nucleotides (ATG) across a codon boundary in Tanzania/Rukwa/2017/1, corresponding to positions 8670 to 8672 in wbBS01 was observed. In respect of the multicopy gene families located at the 3ʹ end of the genome, as already mentioned above, MGF360-21R is almost entirely deleted from the Tanzania/Rukwa/2017/1, but it is present in Georgia 2007/1 and the Polish and Chinese viruses derived from it over the last 10 years.35 a non-synonymous SNP (T/C), is present at position 72,854 of MK645909, resulting in the substitution of Phe by Ser in Tanzania/Rukwa/2017/1. At the 3ʹ end of the virus, gene DP60R is altered by two single-nucleotide deletions and one SNP. These are the deletion of one A (from an A10 homopolymer in wbBS01) at position 189,148, followed by deletion of another A (from an A9 homopolymer in wbBS01) at position 189,172 and a SNP (A to G) at position 189,218. The comparative analysis of Belgium 2018/1 and Georgia 2007/1 also detected one such deletion/insertion of an A in the same homopolymer region of DP60R34.In EP152R, an essential protein that has been shown to interact with the host protein BAG6A total of 54 additional full-length ASFV genome sequences obtained from GenBank was used to generate a maximum-likelihood tree (using FastTree with a GTR evolutionary model). The tree is depicted in Fig. The tree was inferred from full-genome sequences: gappy and hypervariable (saturated) sites were trimmed out using BMGE, and FastTree was used to generate a phylogenetic tree with a GTR evolutionary model. The p72 genotypes are indicated with roman numerals between parentheses.36 for a partial analysis of this).This phylogeny, generated using whole genome sequence data, clearly places Tanzania/Rukwa/2017/1 as a member of the genotype II ASFV taxonomic unit, a monophyletic clade is highly conserved, except for Estonia 2014 (accession LS478113.1), which displays some divergence. The differences in the Estonia sequence may be attributable to several indels not shared with other ASFV genotype II viruses . We then aligned, trimmed the alignment (BMGE with options -t DNA -g 0.0 -h 0.4 -b 10) and inferred a phylogenetic tree with FastTree as above. The resulting cladogram is presented in Fig. 38. Although this virus group is of major concern as the cause of a serious on-going pig pandemic, our study provides the first full genome sequence of a genotype II ASFV from Africa. Our analyses demonstrate close similarity of the Tanzanian sequence to viral sequences from Georgia (2007), Poland (2017) and China (2018), consistent with widespread dissemination of genotype II ASFV following its introduction into Georgia from southeastern Africa in 2007. Based on genotyping and whole genome sequencing, the Georgia 2007/1 virus is believed to have originated in South East Africa, but the exact location within the range of this genotype which includes Mozambique, Malawi, Zambia, southern Tanzania and Madagascar, is not known9. The divergent sequence of the K177R gene in Rukwa/2017/1 suggests that Tanzania is not the source of the introduction.As a result of the recent introduction to Georgia and rapid subsequent dissemination North to the Russian Federation and subsequently East to China and Southeast Asia and West as far as East Germany, genotype II p72 group ASFV represents by far the most geographically widespread of the twenty-four viral genotypes that are currently known36. The genotype II ASFV from Tanzania exhibits a high level of similarity over the entire length of the genome with the recently re-assembled and annotated Georgia 2007 isolate genome34 a Polish genotype from 201731 and a 2018 Chinese isolate28. The analysis provides strong evidence that among the set of available full-genome sequences of genotype II, Tanzania/Rukwa/2017/1 is the closest to the phylogenetic divergence point (representing the most likely recent common ancestor) between genotypes I and II. The Tanzania/Rukwa/2017/1 virus was isolated > 1000 km from the southeast African locations thought to have been the likely source of ASFV introduced to Georgia in 2007 through infected pork products 8. We, therefore, presume that as more ASFV genotype II genome sequences are determined from other parts of the extensive geographical range of this ASFV subtype, even more closely related genomes to Georgia/2007/1 will likely be discovered. Detailed analysis indicates that, although genotype II viruses are highly related to each other, Tanzania/Rukwa/2017/1 shows molecular divergence from Georgia 2007/1, and other group II genomes in the form of indels and a limited number of SNPs. This presumably relates to the fact that mutations differentiating Tanzania/Rukwa/2017/1 from Georgia 2007, are likely to have accumulated over a longer timescale, than the 13 years that have elapsed since the introduction of ASFV to Georgia in 2007.The Tanzanian genome determined in this study is relatively closely related to other genotype II viruses that are ultimately derived from Georgia/2007/1, as indicated by the clustering of the Tanzanian genome together with these viruses in a maximum likelihood tree constructed from all publicly available completed ASFV genomes Fig. . and a p39. The reason for this mode of genome evolution being more prevalent is not fully understood. However, the virus encodes a proof-reading enzyme 3ʹ to 5ʹ exonuclease, which possibly represents an adaptation to survival in the oxidizing environment of the macrophage, and may contribute to the relative paucity of SNPs (reviewed by19). At the same time, the virus also encodes a promiscuous DNA ligase, which may facilitate the generation of indels through non-homologous end joining19, although there are some conflicting reports in the literature regarding the fidelity of this enzyme which may be explained by conditions used for in vitro assays.The pattern in which the majority of the mutations separating Tanzania/Rukwa/2017/I from other genotype II viruses are inserts/deletions (indels) rather than SNPs has been noted previously when all available ASFV genomes were compared40. Such rearrangements appear to be frequent in genotype II isolates originating from wild boar41. However, despite the fact there are multiple copy number and potential coding differences in the MGFs (mainly reductions) that differentiate Tanzania/Rukwa/2017/1 from Georgia 2007 and recently modified Eurasian ASF genomes that have re-arranged subsequent to the introduction of this virus to Georgia, the Tanzanian genotype II ASFV that we have sequenced, nonetheless remains sufficiently virulent to cause disease, and was isolated from a clinically reacting domestic pig.The organization of the terminal multicopy gene families may also contribute to the high observed frequency of evolution through genomic deletions in ASFV. Loss of certain open reading frames in the multicopy families has been associated with reduction of virulence in domestic pigs, including in recent genotype II isolates from wild boar and domestic pigs42. The p72 major surface protein gene (B646L) is also 100% conserved between Tanzania/Rukwa/2017/I and the Georgia reference MK645909. This is an important finding given that the primers used for diagnosis (PPA1 and PPA2) and C-terminal sequencing by genotyping (e.g. P72-U and P72-D primers) are designed from this gene that encodes a major viral surface protein. However, by contrast, certain unique ORFs appear to be absent from Tanzania/Rukwa/2017/1, including ASFV_Ch_ACD_00120, which is present in Georgia 2007/1 progeny viruses. This ORF is currently of unknown function, but is presumably not essential for virus survival. The insertion of 16 bases that creates a tandem repeat (aaaaaaataaacaaca) to the p177R locus encoding the K177R structural protein, does not affect the coding sequence of the protein, but might play a role altering expression by modulating transcription or translation.Some genes of known function are entirely conserved throughout the genotype II group of viruses, for example, p30 (CP204L) is 100% conserved, with no SNPs. This gene has been shown to be key to viral replicationThe annotation of Tanzania/Rukwa/2017/1 revealed 188 putative open reading frames compared to 185 coding sequences (CDS) identified in the annotated Chinese virus genome. However, three of the Tanzania/Rukwa/2017/1 CDS lacked in-frame ATG codons near the N terminal end of the ORF and were therefore annotated as pseudogenes. It is difficult to specify a precise ‘core number’ of coding sequences that are common to all genotype II ASFV viruses, since many of the genomes in the public databases are not yet fully annotated.43. The Tanzania/Rukwa/2017/1 virus was passaged in culture only once to minimize the risk of mutations, and sequencing was performed on total DNA extracted from ASFV infected porcine PBMCs. There were no additional procedures used to enrich for viral sequences or reduce the host DNA contamination. This process provided sufficient viral DNA to assemble an almost complete genome, excluding the terminal repeats, as a single contig, using only the medium-throughput Illumina MiSeq platform. In the future, ‘deep sequencing’ of specific loci, using a platform with higher throughput, such as Illumina Hiseq, would provide information on micro-variation that may be present at low levels within the genomes of ASFV field isolates.Virus isolation in tissue culture remains a gold standard for confirmation of viruses in samples. Given that the virus used in this study is hemadsorbing, a combination of virus isolation and hemadsorption was used as a confirmatory test for the sample and increased the virus titreOrnithodoros ticks at the wildlife domestic pig interface in Mozambique9, relatively close to the presumed origin of the genotype II ASFV that was anthropogenically transported to Georgia in 2007. By contrast genotyping using the polymorphic p72 and CVL genes, to type ASFV circulating in Zambia was consistent with one of the two major distinct genotypes representing a recent introduction of genotype II viruses from Tanzania44. A comparative analysis including all the public sequences for East-African isolates (including Madagascar and Mauritius) as well as the resequenced Georgia 2007/1 isolate (FR682468.2) and based on the commonly-used genotyping markers p72/B646L, CVR/B604L, p30/CP204L and p54/E183L seen in Supplentary Figures 45. The Tanzanian Rukwa/2017/1 virus is thus confirmed as being relatively close to the African virus that was the precursor of Georgia/2007/1, with the 16 bp insertion immediately upstream the p22/KP177R gene representing a notable point of difference. In addition, some MGFs differed in length and copy number, and Rukwa/2017/1 was further differentiated from Georgia/2007/1 by a synonymous SNP in the p30/CP204L coding sequence and SNPs in other single-copy loci .The tissue slice was placed on a sterile sieve positioned on top of a sterile dish prior to being crushed using a pestle allowing the debris to remain on the sieve and the supernatant to pass onto the dish. The sieve was rinsed using 500 µl sterile PBS (pH7.0) containing 5 µg/ml gentamycin sulfate (Bio Whittaker) allowing the lysate to drain onto the dish. The tissue lysate containing PBS was then pipetted out of the dish and transferred into a sterile 1.5 ml Eppendorf tube. The lysate was centrifuged at 14,000 rpm for 10 min to remove tissue debris and the resultant supernatant was pipetted out and transferred into a sterile 1.5 ml Eppendorf tube and stored at − 20 °C. The lysate was used for both extraction of genomic DNA for diagnostic confirmation, for genotyping and as an inoculum for virus isolation.21. The purified PCR amplicon was commercially sequenced using the Sanger method by Macrogen (South Korea).To determine the p72 genotype, we performed partial sequencing of the C-terminal region of B646L gene that encodes the major capsid protein p72 using primers p72-U (5ʹGGCACAAGTTCGGACATGT3ʹ) and p72-D (5ʹGTACTGTAACGCAGCACAG3ʹ) which amplify a 478 bp region routinely used for initial genotyping of ASFV isolates47. Briefly, 0.5 × 106 cells in a 2 ml volume of the complete RPMI were seeded into each well of a 6-well Corning Costar TC-Treated culture plate manufactured by Sigma-Aldrich. The cells were incubated in a humidified chamber at 37 °C and 5% CO2 for 3 days to differentiate mononuclear cells into macrophages. They were then inoculated with the thawed tissue suspension (obtained from the tissue processing step above) at a multiplicity of infection (MOI) of 1:10 and incubated for an additional 24 h. 50 µl of 1% autologous red blood cells diluted in sterile PBS (pH 7.0) was added to each well and the plate was examined for evidence of hemadsorption over a 6-day period. The ASFV isolates were blind passaged once.Peripheral blood mononuclear cells (PBMCs) were prepared from fresh porcine blood using standard procedures and ASFV was isolated as described byDNA from single passaged porcine PBMC was isolated using the DNeasy Blood and Tissue Kit  following the manufacturer's instructions. Quality and quantity of DNA was assessed using Nanodrop and Qubit spectrophotometry and verified by agarose gel electrophoresis. The total DNA extracted from ASFV infected porcine PBMCs was used to prepare libraries for sequencing without further ASFV DNA enrichment procedure or steps to eliminate DNA host contamination.Library preparation for sequencing was performed using a Nextera XT DNA Library Prep Kit  according to the manufacturer’s preparation manual.An Illumina MiSeq machine was used for the sequencing. Briefly, the genomic library was normalized following the manufacturer’s instructions outlined in the Nextera XT protocol. The flow cell used for the sequencing run was thawed at room temperature together with the sequencing reagents. Thawed reagents were pipetted to mix and 5 μl of the library were transferred into a new tube. The library was diluted to the loading concentration of the sequencing system following the manufacturer’s instructions.Sus scrofa data in GenBank. This exercise resulted in overall alignment rates of 94.82% for paired reads and 91.44% for unpaired survivors. Virus genome assembly proceeded with the reads that failed to align to the Sus scrofa reference using a total of 473,890 paired reads plus 72,928 unpaired reads. We used Unicycler version 0.4.7, which on short-read data acts as a wrapper and enhancer for SPAdes (version 3.13.0). Results with or without the "–linear-seqs 1" option did not differ. We combined all unmapped reads as the input to Unicycler . The best k-mer size for the assembly was 127, which yielded 505 contigs. The longest assembled segment we obtained was of length 183,186 bp. As part of the Unicycler pipeline, all segments were further polished by Pilon (version 1.23). The final assembly file contained 160 sequences. The resulting longest contig of length 183,186 bp is presented herein as the Tanzania/Rukwa/2017/1 genome.The paired reads that were analysed were first trimmed using Trimmomatic (version 0.38) to remove adapter content and low-quality read ends. The exact trimming steps were as follows: ILLUMINACLIP:NexteraPE-PE.fa:2:30:12.LEADING:10.TRAILING:10 SLIDINGWINDOW:4:15 MINLEN:20. Porcine reads were removed by alignment to Whole-genome ASFV sequences from Europe, Africa and Asia were downloaded from the European Nucleotide Archive (ENA) for the purpose of phylogenetic analysis of the Tanzania/Rukwa/2017/1 virus genome. After adding the Tanzania/Rukwa/2017/1 sequence, we generated a multiple sequence alignment using mafft version 7.453 . In order to trim the alignment from its gappy (> 10% gaps) and saturated (entropy h > 0.5) sites, we then used BMGE v.1.12 (options -t DNA -g 0.1 -h 0.5 -b 10), which enabled reduction from 212,659 sites to 169,170 sites suitable for phylogenetic inference. We proceeded using FastTree version 2.1.11 (multi-threaded executable with OpenMP) to infer a phylogenetic tree using the GTR model of nucleotide evolution and a discrete gamma model with 20 rate categories (options -gtr -gamma -spr 4 -mlacc 2 -slownni for more exhaustive NNI steps during maximum-likelihood topology optimization).This study was conducted at the Biosciences East and central Africa (BecA) Hub : a regionally approved institution to work with ASFV. All experiments were performed in a biosafety level two laboratory in a biosafety cabinet dedicated for this work.African Swine Fever (ASF) is listed by OIE as a notifiable disease of swine at national, regional and international levels hence no ethical approvals were required for the investigation reported herein. Relevant permits to engage with farmers and collect samples were obtained from regional offices.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


Marnaviridae comprises small non-enveloped viruses with positive-sense RNA genomes of 8.6–9.6 kb. Isolates infect marine single-celled eukaryotes (protists) that come from diverse lineages. Some members are known from metagenomic studies of ocean virioplankton, with additional unclassified viruses described from metagenomic datasets derived from marine and freshwater environments. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Marnaviridae, which is available at ictv.global/report/marnaviridae.The family  Chaetenuissarnavirus II) [Structurally characterized members have polyhedral virions, 22–35 nm in diameter, without envelopes or discernible projections , Fig. 1.Locarnavirus, Marnavirus) or two ORFs ; members of the genus Labyrnavirus have a third small overlapping open reading frame (ORF) at the 3′-end of the genome  tail terminates the genome of those viruses that have been isolated.Members possess monopartite positive-sense RNA genomes of 8.6–9.6 kb  containie genome . Virus gViral proteins are synthesized as part of one or two polyproteins. Regardless of whether the genomes are mono- or di-cistronic, the non-structural proteins are encoded in the 5′-region and the structural proteins are encoded in the 3′-region. Replication is cytolytic and various cytopathic effects have been observed . Membranwww.ictv.global/taxonomy. The genus Marnavirus includes the species Heterosigma akashiwo RNA virus, whose only member has a mono-cistronic genome and infects a harmful algal bloom-forming raphidophyte. Labyrnavirus includes the species Aurantiochytrium single-stranded RNA virus 01, whose only member has a di-cistronic genome and a third small overlapping ORF, which is transcribed as a sub-genomic RNA during infection but may not be functional [Aurantiochytrium sp., a marine thraustochytrid. Locarnavirus includes the species Jericarnavirus B, Sanfarnavirus 1, Sanfarnavirus 2 and Sanfarnavirus 3, members of which were discovered through metagenomic analysis of marine virioplankton. Both mono- and di-cistronic genome organizations are found. Kusarnavirus includes the species Astarnavirus, whose only member infects the diatom Asterionellopsis glacialis and has a di-cistronic genome. Bacillarnavirus includes the species Rhizosolenia setigera RNA virus 01, Chaetoceros tenuissimus RNA virus 01 and Chaetoceros socialis forma radians RNA virus 1, members of which infect diatoms and have di-cistronic genomes. Salisharnavirus includes the species Britarnavirus 1, Britarnavirus 4, Palmarnavirus 128 and Palmarnavirus 473, members of which were discovered by metagenomics and have di-cistronic genomes. Sogarnavirus includes the species Chaetenuissarnavirus II, Chaetarnavirus 2, Britarnavirus 2, Britarnavirus 3, Palmarnavirus 156 and Jericarnavirus A. Members have di-cistronic genomes and have been isolated from diatoms or discovered by metagenomics.Current taxonomy: nctional . The hosMarnaviridae: https://www.ictv.global/report/marnaviridaeFull ICTV Report on the family


Cryptococcus comprises more than 80 species, including C. neoformans and C. gattii, which are pathogenic to humans, mainly affecting the central nervous system. The two species differ in geographic distribution and environmental niche. C. neoformans has a worldwide distribution and is often isolated from bird droppings. On the contrary, C. gattii is reported in tropical and subtropical regions and is associated with Eucalyptus species. This review aims to describe the distribution of environmental isolates of the Cryptococcus neoformans species complex and the Cryptococcus gattii species complex in Colombia. A systematic investigation was carried out using different databases, excluding studies of clinical isolates reported in the country. The complex of the species of C. gattii is recovered mainly from trees of the genus Eucalyptus spp., while the complex of the species of C. neoformans is recovered mainly from avian excrement, primarily Columba livia (pigeons) excrement. In addition, greater positivity was found at high levels of relative humidity. Likewise, an association was observed between the presence of the fungus in places with little insolation and cold or temperate temperatures compared to regions with high temperatures.The genus  Cryptococcus includes yeast fungi that develop a specific biotrophic association with different host plants and comprises more than 80 species, with two being pathogenic to humans. Previously known as a single species, Cryptococcus neoformans was divided into two varieties, C. neoformans variety gattii and C. neoformans variety neoformans [C. neoformans species complex and C. gattii species complex [C. neoformans complex has several varieties , while the complex of the species of C. gattii is divided into four genotypes  [C. neoformans species , C. deneoformans (referred to as C. neoformans var. neoformans with serotype D y genotype VNIV), and a hybrid classification composed of C. neoformans and C. deneoformans (with serotype AD and genotype VNIII) be recognized. The complex of the species of C. gattii has been reorganized as five species: C. gattii with genotype VGI, C. deuterogattii with genotype VGII, C. bacillisporus with genotype VGIII, C. tetragattii with genotype VGIV, and finally C. decagattii with genotypes VGIV and VGIIIc, in addition to hybrids such as C. deneoformans with C. gattii, the hybrid C. neoformans with C. gattii, and the hybrid C. neoformans with C. deuterogattii [C. neoformans the worldwide cause of cryptococcal meningoencephalitis [C. neoformans complex affects immunocompromised individuals, mainly those affected by the human immunodeficiency virus (HIV) [C. gattii complex is associated with infections in apparently immunocompetent patients, indicating that even people with a healthy immune system can be affected by this pathogen [Cryptococcus spp. is capable of remaining latent without affecting the host, which makes it highly adaptive [The genus oformans ,2; nowad complex ,4,5. C. nd VGIV) ,7,8,9. Vnd VGIV) ,11. Neverogattii ,7. Theseus (HIV) ,13,14,15pathogen ,17. It sC. neoformans complex has a wide distribution in different departments of Colombia, such as Cauca [C. livia) [C. neoformans complex and the soil contaminated with it [C. neoformans complex [C. neoformans complex has been reported in tree species, with higher prevalence compared to the C. gattii complex, for example in Eucalyptus spp. [Terminalia catappa [Olea europea [Ceratonia (carob tree) [The as Cauca , Córdobaas Cauca , Cundinaas Cauca ,21, Huilas Cauca , Nariño as Cauca  Norte deas Cauca ,25,26,27as Cauca ,29. In tas Cauca , Nigeriaas Cauca ,32, Chinas Cauca , South Aas Cauca , Brazil as Cauca ,36, Unitas Cauca , Italy [as Cauca , Guinea as Cauca , Argentias Cauca ,41, and as Cauca , among oas Cauca ,45,46,47. livia) , saproph with it ,50,51,52 with it ,54. The  complex . Additiotus spp. ,56, Term catappa , Olea eu europea , and Cerob tree) .C. gattii complex has been described in temperate regions such as Canada, California, and Oregon [Conversely, the d Oregon . Thus, id Oregon ,26, Cundd Oregon ,60, and d Oregon , and acrd Oregon , Africa d Oregon , India [d Oregon ,62, Itald Oregon , the Unid Oregon  and soutd Oregon , Canada d Oregon , Spain [d Oregon , and Chid Oregon , among od Oregon ,71,72,73C. gattii complex are associated with tree species, with reported isolation from flowers, bark, and leaves [C. neoformans complex, especially when associated with the genus Eucalyptus [Acacia, Ficus, and Terminalia catappa, in several regions of the world [C. gattii complex has also been isolated from other environments, such as soil, air, and water [C. gattii complex; however, different studies on the environmental conditions have reported that the favorable characteristics for the development of this yeast depend on the climatic conditions of the area [The habitats of d leaves ,74,75. Dcalyptus . Howeverhe world ,59,63,67nd water ,76,77. Rthe area ,73,78,79Studies published from 2006 to 2016 in different regions of Colombia reported cases of cryptococcosis. The prevalent risk factor reported was HIV infection in 83.5% sufferers and an increase in cases in the female population compared to the previous period of 1997–2005 . Thus, tC. neoformans species complex and C. gattii species complex in Colombia and their associations with environmental conditions.The objective of this systematic review is to describe the distribution of environmental isolates of the Cryptococcus spp., environment, meteorological conditions, and ecology. The articles were selected considering the following inclusion criteria: studies of environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in Colombia, mainly articles in Spanish and English. Studies of clinical isolates reported in Colombia were excluded. The titles and abstracts of 103 articles were evaluated for relevance; after removing duplicate articles and study reviews, 53 articles underwent full-text review, taking into account environmental variables, microbiological identification methods, and molecular characterization, which were included in the total synthesis of the document.A systematic search was carried out in eight online databases: BioMed, DialNet, DOAJ, Medline, PubMed, ScienceDirect, Redalyc, and SciELO, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, which include identification, selection, and inclusion of the reviewed literature. The years of publication were 1990 to 2020. The descriptors used were: C. neoformans species complex and C. gattii species complex is associated with different environments, from cold and temperate climates to tropical and subtropical climates. In this way, the studies carried out in Colombia aimed to identify the distribution of the infectious agent of cryptococcosis, and thus identify the ecological niche of this fungus.Colombia has different thermal floors, producing multiple climatic conditions throughout the national territory. The natural habitat of the Cryptococcus spp. and environmental variables. Indeed, Granados et al. and Anacona et al. reported a correlation between high and mean temperature values and recovered isolates of C. neoformans [C. gattii serotype C had fewer isolates due to extreme minimum and maximum temperatures according to Granados et al. [C. neoformans serotype A and C. gattii serotype C [C. neoformans, as reported by Anacon 2018 et al. [C. gattii serotype B exhibited a positive correlation with relative humidity, associated with the high recovery under low evaporation conditions [Of the included studies, five reports showed associations between isolates of oformans ,81. Convrotype C ,81 in coC. neoformans serotype A evidenced a strong correlation [C. neoformans serotype A and C. gattii serotype C exhibited a positive correlation with high solar radiation values, in contrast to C. gattii serotype B, which showed a positive correlation with low values of solar radiation [For the climatic variable precipitation, the relation . For soladiation ,81.C. neoformans fungus [Regarding the sample source, Caicedo and Vallejo reported that the accumulation of excreta and the high presence of pigeons result in increases in the recovery of the s fungus ,28.C. neoformans [Finally, Caicedo reported that wooden boxes and flat ceilings were the nesting areas where there were more significant isolates of oformans .Cryptococcus in the region, with the C. gattii species complex being reported in tropical to temperate climates, demonstrating that this species is highly adaptable to different ecological conditions in the country [C. neoformans have been reported in temperate and tropical climates, associated mainly with avian excreta [C. neoformans and C. gattii in different parts of Colombia are shown.The studies carried out in Colombia confirm the presence of pathogenic species of the genus  country ,60. Othe excreta ,23,56,82 excreta , Pasto [ excreta ,27, Mont excreta , Bogotá  excreta ,19, and  excreta ,29,61, w excreta ,81. ThesC. neoformans molecular type VNI in almond trees and roses. The researchers concluded that C. neoformans, a molecular type VNI, had a higher prevalence than C. gattii and was associated with human exposure in this geographic region [The city of Barranquilla is the capital of the department of Atlántico, in which a warm climate predominates, with an annual temperature of 28 °C; this region has bimodal rainy seasons that go from May to June and from September to November, with average annual rainfall ranging between 500 and 1500 mm, with dry seasons from December to March and from June to July. Between 2012 and 2014, Noguera et al. conducted a clinical and environmental study, whereby 32 sampling points were recorded, consisting of local parks and areas with a high density of trees. From 1068 environmental samples, 0.4% were positive for c region .Cryptococcus species with the eucalyptus trees E. camaldulensis and E. terericornis, distributed in 13 areas located in the northeast, east, west, and center of Bogotá. This city has an altitude of 2630 m above sea level (masl), while at the sampling time the city had a humidity of 65.5% and average temperature of 16.4 °C; the location is shown in Cryptococcus genus, distributed as follows; 8 were C. laurentii; 5 were C. macerans; 4 were C. ater and 4 were C. uniguttulatus; 2 were C. neoformans; and 1 each was C. hungaricus, C. albidus, C. kuetzingii, and C. heveanesis. For the remaining 500 samples, the authors did not report any identification. An important finding reported in this study was the establishment of the leaves as the main sources of isolates for Cryptococcus [In Bogotá in 1994, Duarte et al. evaluated the association of tococcus .Cryptococcus species and Eucalyptus trees in a park located in the northeast of Bogotá. No environmental data were recorded during the investigation; however, according to the climatic parameters reported by IDEAM in this period, Bogotá had an average temperature of 13.4 °C and an annual rainfall of 453.91 mm [C. laurentii was the mainly isolated species, with 17 positive samples, followed by C. neoformans var. neoformans with two positive isolates, and finally one C. albidus isolate. Contrary to what is reported in the literature, where a close association between C. gattii and E. camaldulensis is described, in this study no isolates of this species were obtained [Another environmental report by Castañeda and Castañeda in 2001 showed the association between isolates of 53.91 mm ,85. The C. neoformans.The study published by Granados and Castañeda showed the results of sampling carried out in 2003 during February to May in 3 areas of Bogotá: The José Celestino Mutis Botanical Garden (JB) , El Lago metropolitan park (PEL) , and the campus of Universidad Nacional de Colombia (UN) . Climatological data for this period showed an average long-term temperature of 19.1 °C, with precipitation being between 500 and 1000 mm ,86. In tC. neoformans var. gattii and 1% (5) for C. neoformans var. grubii, which were significantly higher in the dry excrement than the humid excreta. Based on the results, exposure to sunlight does not represent a decisive factor influencing the recovery of yeast. In this study, the fungus showed more frequent colonization in the debris found around trees, supporting the concept that the main niche appears to be the result of the biodegradation of wood. In contrast, the rainy months (April and May), characterized by high rainfall, humidity, few hours of sunlight, and less extreme temperatures (an average temperature of 5 °C higher), favored the appearance and spread of C. neoformans in the environment more than the dry months. Finally, this study shows how C. neoformans can colonize several zones in a city, and how its population densities can vary within the zones; this distribution could be explained by the capacity of birds to disperse propagules in the wind [It should be noted that in those trees that were inhabited by birds, the frequency of isolation was lower 21.87%) compared to samples isolated from the trees or in decomposing wood (48.75%), which were not frequented by birds. For the statistical analysis, ANOVA, MANOVA, canonical discriminant analysis, Pearson’s correlation, and Chi squared were performed, using the Statistical Analysis System (SAS) for Windows v. 8.02. The frequency of isolation in the excreta samples was 6.7% [% comparethe wind .5URA gene, while PCR was used for the determination of mating types. Of the 128 samples collected, 15 (11.7%) isolates were identified as C. gattii molecular type VGIII, mating type a; of these isolates, three were from the extract of the inflorescence of red flowers, while the rest were from the detritus of Corymbia ficifolia. Of the 113 remaining samples, the researchers did not specify any identification. The present study reports the isolation of C. gattii in C. ficifolia trees in Colombia, as represented in In 2010, Escandón et al. collected samples over six months, between February and August 2007. The authors did not report environmental data during the investigation; however, according to the climatic parameters reported by IDEAM in this period, Bogotá had an average temperature of 19.124 °C and precipitation range of 500–1000 mm ,88. In tC. livia) from ceilings, roofs, and wooden boxes were recovered. All samples collected on roofs were negative, while all those obtained from ceilings were positive for C. neoformans. Molecular characterization was not performed. The statistical analysis consisted of descriptive statistics and the Chi square test, however the program used was not indicated. In total, 47 % of the samples were identified as C. neoformans. Researchers associated high exposure to ultraviolet rays with the absence of C. neoformans in roof samples because the sunlight exposure limited the growth of yeast. They also found that where there was a higher number of pigeons, the recovery rate of the fungus was higher  from excreta of caracara (Polyborus plancus) and the other from a petri dish exposed in the area where the same species of falconiform bird lived , and 13 air samples were also collected. The authors did not report on the molecular characterization. The researchers were able to isolate four positive samples for C. gattii serotype C (2.54%), corresponding to plant debris; they did not report on the identification for the remaining 153 samples. The researchers concluded that there may be an association between plant dendrites and the positive isolates of C. gattii, this being a type of habitat in which yeast is found [In 1998, Callejas et al. published a study that aimed to search the is found , as showC. gattii (at that time classified as C. neoformans var. gattii) with the species T. catappa . The authors reported on a total of 370 samples, of which 160 were from almond debris and 210 were from 9 new almond trees, selected from January 1998 to July 1999. The study reported that 31 samples (19.3%) were positive for C. gattii serotype C (formerly C. neoformans var. gattii) of the 160 samples initially collected in the study, while of the 210 samples collected from the nine additional almond trees, only 1 (0.48%) was positive for C. gattii. The authors established that it was not possible to isolate the C. neoformans species complex from any of the almond trees, thus suggesting that there is a specific association of C. gattii with this tree species [T. catappa , Ficus sp. (Higuerón), Licania tomentosa (Oiti), Pithecellobium dulce (Chiminango), Fagara rhoifolia (Tachuelo), Melicoccus bijugatus (mamón), Enterolobium cyclocarpum (piñon), and Mangifera indica (mango)  for C. gattii and three (0.07%) for C. neoformans. From C. gattii-positive samples, seven isolates were C serotype, VGIII molecular type; and one was serotype B, VGI molecular type. Of the positive samples for C. neoformans, 13 isolates were classified as a C. neoformans var. grubii, serotype A, VNI molecular type. All recovered isolates were from the soil samples. Contrary to expectations for the study area, which has a high rate of cryptococcosis due to C. gattii, there was a low rate of environmental recovery for this species, suggesting the importance of establishing environmental variables that directly affect the survival of microorganisms in the environment, meaning samplings should be taken at specific times of the year [In 2001, Castañeda et al. published a study that sought to determine the association of the presence of  species . Another (mango) . Moleculthe year .Cryptococcus environmental isolates in ten public areas. The study did not report environmental data for the months of sample collection; however, as reported by IDEAM for this period, the average temperature was 24.95 °C, with annual rainfall ranging between 50 and 74 mm [5URA gene were used to determine the molecular pattern. The results obtained in this study established the presence of C. neoformans in 19 trees (4.3%) and C. gattii in one tree (0.2%), for a total of 21 isolates and 20 positive trees. Santander Park, with 47.6% (10 out of 21) of the isolates, was the area that presented the most significant number of positive samples, followed by La Victoria Park with 23.8% (5 of 21 isolates), all of which were identified as C. neoformans. Using a different method, C. gattii was isolated from the Ficus benjamina tree located in the Mercedes Ábrego park  of the municipalities studied, indicating that 104 (14%) samples were positive, whereby the pigeon droppings (21%) were the principal substrate. A total of 92 isolates (88%) were recovered from the cold thermal floor. In the Páramo area, the yeast was not isolated due to the environmental conditions, such as the precipitation and high relative humidity, few hours of sunlight, and temperate thermal floor between 18 and 24 °C, which favor the propagation of this yeast in the environment, as well as the high altitudes of between 1000 and 2000 masl. In conclusion, the authors confirmed the association between C. neoformans var. grubii serotype A and pigeon feces, in addition to confirming eucalyptus as an environmental niche for C. gattii serotypes B and C, while almonds were also established as possible hosts for serotype C , white snakeroot tree , Laurel (Laurus nobilis), Raque , and Guaba (Phytolacca bogotensis). Of the 167 samples taken with a swab, C. gattii was found in February in 3 out of 40 samples (7.5%), on March 6 32 out of 40 samples (80%), and on March 12 in 11 out of 87 samples . Additionally, C. gattii serotype B was recovered from 46 of the 167 samples of Eucalyptus (27.5%). The presence of yeasts with small capsules was confirmed in 30 (65.2%) of these, while the remaining 16 were strains without capsules. This study was the first report on C. gattii serotype B. It should be noted that the fungus was found on different trees species, as observed in However, a study carried out by Escandón P. in the municipality of La Calera (Cundinamarca) from February to March of 2003 evaluated the presence and distribution of species of the T. catappa), and 86 from pigeon droppings (C. livia). The samples were processed using conventional techniques. Species were determined by seeding on Canavanine-Glycine-Bromothymol Blue (CGB) agar, and molecular typing was performed using PCR fingerprinting and RFLP of the 5URA gene. Of the total samples processed, 40 colonies obtained from eight (6.8%) samples were identified as C. neoformans var. grubii and isolated from bird droppings, which can be observed in C. gattii was not isolated from the collected samples [In 2018, Virviescas B. et al. performed a study in the city of Neiva to determine the distribution of the etiological agents of cryptococcosis in the region. The municipality has an average annual temperature of 27.7 °C, is located at a latitude of 2°59′55” north and a longitude of 75°18′16” west, and has an altitude of 442 masl, with an approximate population of 345,911 inhabitants. In total, 118 samples were collected—32 from the fruits, leaves, and debris of almond trees  present in the urban area of the city. Monteria has an altitude of 20 masl, a warm climate with temperatures ranging between 28 and 35 °C, a relative humidity of 85%, and pluvial precipitation of 1200 to 2500 mm annually. The researchers collected 2445 samples from 163 T. catappa trees. The samples were collected, taking into account the parts of the bark with grooves and hollows. The study did not include molecular characterization. The statistical analyses included non-parametric analysis, Tukey’s hypothesis test, and Chi-squared tests using Statgraphics Centurion version 15.2.Between 2008 and 2009, an investigation was carried out by Contreras et al. in Monteria, the capital of the department of Córdoba D, locateC. gattii was isolated from 217 samples, which corresponded to 8.9%. The authors highlighted that the sample collection corresponded to a rainy period involving more humidity and a higher accumulation of debris. Given the obtained results, the researchers consider almond trees an ecological niche for C. gattii , since in the region the ripe fruit is edible and could indicate a point of spread for the fungus. These findings were the first report of the isolation of C. gattii from environmental sources in the department of Córdoba [Of the total samples processed,  Córdoba .For the municipality of Pasto, Vallejo et al. in 2016 reported a study that carried out an analysis of the residential and populated areas with a high density of pigeons. The study did not report environmental data; however, according to the climatic parameters reported by the IDEAM, Pasto has an altitude of 2527 masl, an average temperature of 19.2 °C, and a maximum average annual rainfall of 106,267 mm . The citC. neoformans. The number of isolates was lower in fresh samples, while wet samples (not necessarily fresh) provided a higher number of positive isolates, similar to the results reported by Granados and Castañeda in 2006 [Cryptococcus isolates  and herons (Bubulcus ibis) between September 2012 and June 2013. These associations can be observed in Popayán is the capital of the Colombian department of Cauca, with an elevation of 1735 masl, a subtropical highland climate with an annual average temperature of 18–19 °C, with maximum temperatures occurring from July to September (high temperature of 29 °C and minimum temperature of 10 °C) and a precipitation of 1941 mm. The city is located on the west of Colombia, as shown in C. neoformans species complex was also carried out by means of conventional procedures, along with the molecular characterization of the isolates. One-way ANOVA, post hoc analysis, Tukey’s test, and the Chi-squared test were performed using SPSS version 15 for statistical analysis. A total of 118 (38.94%) samples were positive for Cryptococcus neoformans var. grubii, with 99.2% corresponding to the VNI molecular pattern and the remaining 0.8% to VNII. Contrary to the report for the pigeon excreta samples, no species of the C. neoformans species complex was isolated from B. ibis. In this way, the study allows us to confirm that bird droppings, especially of C. livia, are a fundamental environmental niche for C. neoformans var. grubii molecular type VNI, since they allow growth and reproduction of the fungus, representing a potential source of infection for the susceptible population in Popayán [The authors collected and processed the samples using conventional techniques. The identification of presumptive isolates of the Cryptococcus genus comprises a group of fungal pathogens that frequently affects humans; without timely and adequate treatment, it can be fatal. In order to establish control measures for infectious diseases, it is highly relevant to provide a report on the fungal environmental distribution throughout a country.The According to the environmental studies reported in Colombia until 2019, climatic conditions are essential when analyzing the requirements that favor the appearance of the fungus . The stuCryptococcus spp., it can be said that the most significant number of samples collected was in the city of Cúcuta at 4338 samples, of which only 0.14% were positive, which may be due to the temperatures at this site (27.8 °C to 28.8 °C) [C. neoformans species [Cryptococcus from the 765 samples collected under temperature conditions ranging between 12 and 18 °C [Cryptococcus isolates is higher in environments with low temperatures, since such conditions promote their conservation [Regarding the numbers of samples collected for the isolation of 28.8 °C) . This lo28.8 °C) . In the  species . Similarnd 18 °C . The freervation .Cryptococcus species complex have mainly involved extraction with saline phosphate buffer and identification using G. abyssinica seed agar. In some studies, Indian ink staining was used to identify the fungus capsule, being a simple and inexpensive method. The molecular methods generally used in the studies were restriction fragment length polymorphism (RFLP) of the 5URA gene and PCR fingerprinting with primer (GTG)5.The methods used for the identification and characterization of the Data analysis was not performed in most of the studies, except when reporting the percentages of positive samples found for each species according to the nomenclature used by each study. However, some studies have reported statistical data analysis, including Granados et al. in Bogotá , CaicedoC. neoformans complex with excreta of C. livia pigeons and of some C. neoformans species with P. plancus, thus affirming the association of the fungus with different avian species. Bird droppings contain xanthine, urea, uric acid, and creatinine, substrates that the fungus can assimilate within [The studies carried out in Colombia, as with the reports worldwide, demonstrate the association of the e within ,31, givee within . This pre within .Cryptococcus is higher if the samples are wet, as shown by Quintero et al. In Cundinamarca region, 88% of the isolates were recovered from the cold thermal floor, with high precipitation and relatively humid environmental conditions, which favored the occurrence and spread of the fungus in the environment, as reported by Cogliati et al., who found a higher occurrence of the fungus in rainy seasons or wet periods [Cryptococcus species.In addition, the probability of finding  periods ,93. Simi periods ,94. Cont periods . This waCryptococcus species complex in places exposed to constant sunlight, thus relating the amount of brightness to the presence of the fungus [Vallejo et al. in 2016 obtained positive samples for the e fungus , since te fungus ,95. Thise fungus .C. neoformans complex is generally associated with debris from trees of the genus Eucalyptus and Ficus sp., as stated by Lazéra et al. [C. livia) [C. neoformans as being pigeon droppings, which was contrary to the findings reported by Cogliati et al., who that suggested that poultry feces is a secondary and transitory niche [According to the samples registered for the different studies, the presence of the a et al.  and Cogla et al. ,93. Simi. livia) , which i. livia) , who estry niche ,93.C. gattii has been found in different regions, such as Cúcuta and Monteria, which have average temperatures of around 27 °C, as well as Cundinamarca, which has an average temperature of 13 °C. In agreement with the study by Cogliati et al., where identified that warm temperatures favors the distribution of C. gattii complex [T. catappa and Ficus sp., as reported by Castañeda et al. [C. gattii complex was shown for up to 12 months in these trees species [Ficus sp. [C. gattii in soil samples from Ficus spp. [C. gattii complex and rubber and almond species has been reported in different parts of the world. For example, the study by Randhawa et al. in 2003 reported decomposed wood in Ficus religiosa trees as an environmental source of C. gattii [C. gattii complex [C. gattii complex is difficult to isolate from the environment [C. neoformans complex, the number of positive isolates reported in each study was low, however the number of studies is much higher [ complex ,93. The a et al.  and corr species . Additioicus sp. . In the cus spp. . This as. gattii . These f complex , describ complex ,97,98. Tironment ,100. Howh higher ,26,27, aC. neoformans complex and the C. gattii complex have been reported in clinical isolates; however, the C. neoformans complex is mainly isolated from the environment, indicating the presence of the two species in the environment [C. gattii complex in the environment, the number of studies has increased, with the largest numbers of isolated samples being obtained for the species of C. gattii, especially serotype C in Cúcuta, which has a high incidence of C. gattii serotype B in immunocompetent patients [C. gattii with serotype B have been isolated in low proportions [Both the ironment . Thus, bpatients ,25,26,27portions . The twoportions .C. gattii VGII is the most common in clinical isolates, while the VGIII genotype is less frequent [C. neoformans is VNI [Regarding genotypes, frequent . Howeverfrequent ,57. Addis is VNI ,103, whis is VNI ,26,27.C. neoformans species, while the excreta of birds was considered an important reservoir for this species [Finally, it should be mentioned that as in other regions, in Colombia the soil samples represented a poor environment for the recovery of the  species ,99,100.C. neoformans species complex and C. gattii species complex share some niches in terms of where they are established, being distributed in regions ranging from 20 to 3000 masl. Similarly, positive isolates were found in cold and temperate climates, with low sunlight and high relative humidity, thus showing that in wet samples, the probability of finding species of the genus Cryptococcus is higher. In contrast, although to a lesser extent, it has been established that in some cases these yeast species can grow in areas with a significant influence of sunlight, which may be related to the production of melanin as a protective factor against UV rays, giving this species adaptability to these environments [The studies carried out in Colombia have shown that the ronments  In this Cryptococcus complex species, such as G. abyssinica agar, urease test, Indian ink staining, CGB agar, RFLP of the 5URA gene, and PCR fingerprinting with primer (GTG)5, correspond to the methods mostly used in Colombian studies. It is worth highlighting their effectiveness for the characterization of these two species.Furthermore, the methods used for isolation and identification of the Cryptococcus spp. species were isolated in urban areas, such as squares, parks, churches, and zoos, which could be related to human activity, therefore increasing the possibility of infection through these sources.Finally, the reviewed studies allowed us to establish that the most significant numbers of