Taxonomic and functional characterization of biofilms from a photovoltaic panel reveals high genetic and metabolic complexity of the communities.

AIMS: Biofilms are complex microbial cell aggregates that attach to different surfaces in nature, industrial environments, or hospital settings. In photovoltaic panels (PVs), biofilms are related to significant energy conversion losses. In this study, our aim was to characterize the communities of microorganisms and the genes involved in biofilm formation. METHODS AND RESULTS: In this study, biofilm samples collected from a PV system installed in southeastern Brazil were analyzed through shotgun metagenomics, and the microbial communities and genes involved in biofilm formation were investigated. A total of 2030 different genera were identified in the samples, many of which were classified as extremophiles or producers of exopolysaccharides. Bacteria prevailed in the samples (89%), mainly the genera Mucilaginibacter, Microbacterium, Pedobacter, Massilia, and Hymenobacter. The functional annotation revealed >12 000 genes related to biofilm formation and stress response. Genes involved in the iron transport and synthesis of c-di-GMP and c-AMP second messengers were abundant in the samples. The pathways related to these components play a crucial role in biofilm formation and could be promising targets for preventing biofilm formation in the PV. In addition, Raman spectroscopy analysis indicated the presence of hematite, goethite, and ferrite, consistent with the mineralogical composition of the regional soil and metal-resistant bacteria. CONCLUSIONS: Taken together, our findings reveal that PV biofilms are a promising source of microorganisms of industrial interest and genes of central importance in regulating biofilm formation and persistence.

A clinical metagenomic study of biopsies from Mexican endophthalmitis patients reveals the presence of complex bacterial communities and a diversity of resistance genes.

Infectious endophthalmitis is a severe ophthalmic emergency. This infection can be caused by bacteria and fungi. For efficient treatment, the administration of antimicrobial drugs to which the microbes are susceptible is essential. The aim of this study was to identify micro-organisms in biopsies of Mexican endophthalmitis patients using metagenomic next-generation sequencing and determine which antibiotic resistance genes were present in the biopsy samples. In this prospective case study, 19 endophthalmitis patients were recruited. Samples of vitreous or aqueous humour were extracted for DNA extraction for metagenomic next-generation sequencing. Analysis of the sequencing results revealed the presence of a wide variety of bacteria in the biopsies. Resistome analysis showed that homologues of antibiotic resistance genes were present in several biopsy samples. Genes possibly conferring resistance to ceftazidime and vancomycin were detected in addition to various genes encoding efflux pumps. Our findings contrast with the widespread opinion that only one or a few bacterial strains are present in the infected tissues of endophthalmitis patients. These diverse communities might host many of the resistance genes that were detected, which can further complicate the infections.

Diverse bacterial consortia: key drivers of rhizosoil fertility modulating microbiome functions, plant physiology, nutrition, and soybean grain yield.

Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability.

One versus two-stage codigestion of sugarcane vinasse and glycerol: Assessing combinations at mesophilic and (hyper) thermophilic conditions.

Sugarcane vinasse exits the distillation process at high temperatures, which may differ from the optimal temperatures for dark fermentation and anaerobic digestion. A 15 °C temperature increase, for example, stops sugarcane vinasse methane generation, making distillery vinasse digestion complicated. Conversely, in other aspects, co-digesting vinasse and glycerol has been proven to stabilize methane production from vinasse because of sulfate dilution. However, glycerol has not been tested to stabilize vinasse digestion under temperature changes. Thus, this study compared the effects of different temperature settings on the co-digestion of 10 g COD L-1 of vinasse and glycerol (50 %:50 % on a COD basis) in anaerobic fluidized bed reactors (AFBR), i.e., an acidogenic and a methanogenic one-stage AFBRs operated at 55, 60, and 65 °C, and two methanogenic AFBRs fed both with acidogenic effluent (one operated at room temperature (25 °C) and the other at 55, 60, and 65 °C). The co-digestion provided steady methane generation at all AFBRs, with methane production rates ranging from 2.27 to 2.93 L CH4 d-1 L-1, whether in one or two stages. A feature of this research was to unravel the black box of the role of sulfate in the digestion of sugarcane vinasse, which was rarely studied. Desulfovibrio was the primary genus degrading 1,3-propanediol into 3-hydroxypropanoate after genome sequencing. Phosphate acetyltransferase (EC: 2.3.1.8, K00625) and acetate kinase (EC: 2.7.2.1, K00925) genes were also found, suggesting propionate was metabolized. In practical aspects, regarding the two-stage systems, the thermophilic-mesophilic (acidogenic-methanogenic) configuration is best for extracting additional value-added products because 1,3-propanediol may be recovered at high yields with steady methane production at reduced energy expenditure in a reactor operated at room temperature. However, the one-stage design is best for methane generation per system volume since it remained stable with rising temperatures, and all systems presented similar methane production rates.

A First Insight into the Microbial and Viral Communities of Comau Fjord-A Unique Human-Impacted Ecosystem in Patagonia (42∘ S).

While progress has been made in surveying the oceans to understand microbial and viral communities, the coastal ocean and, specifically, estuarine waters, where the effects of anthropogenic activity are greatest, remain partially understudied. The coastal waters of Northern Patagonia are of interest since this region experiences high-density salmon farming as well as other disturbances such as maritime transport of humans and cargo. Here, we hypothesized that viral and microbial communities from the Comau Fjord would be distinct from those collected in global surveys yet would have the distinctive features of microbes from coastal and temperate regions. We further hypothesized that microbial communities will be functionally enriched in antibiotic resistance genes (ARGs) in general and in those related to salmon farming in particular. Here, the analysis of metagenomes and viromes obtained for three surface water sites showed that the structure of the microbial communities was distinct in comparison to global surveys such as the Tara Ocean, though their composition converges with that of cosmopolitan marine microbes belonging to Proteobacteria, Bacteroidetes, and Actinobacteria. Similarly, viral communities were also divergent in structure and composition but matched known viral members from North America and the southern oceans. Microbial communities were functionally enriched in ARGs dominated by beta-lactams and tetracyclines, bacitracin, and the group macrolide-lincosamide-streptogramin (MLS) but were not different from other communities from the South Atlantic, South Pacific, and Southern Oceans. Similarly, viral communities were characterized by exhibiting protein clusters similar to those described globally (Tara Oceans Virome); however, Comau Fjord viromes displayed up to 50% uniqueness in their protein content. Altogether, our results indicate that microbial and viral communities from the Comau Fjord are a reservoir of untapped diversity and that, given the increasing anthropogenic impacts in the region, they warrant further study, specifically regarding resilience and resistance against antimicrobials and hydrocarbons.

Microbial and functional characterization of granulated sludge from full-scale UASB thermophilic reactor applied to sugarcane vinasse treatment.

Considering the scarcity of data in the literature regarding phylogenetic and metabolic composition of different inocula, especially those from thermophilic conditions, this research aimed at characterizing the microbial community and preferable metabolic pathways of an UASB reactor sludge applied to the thermophilic treatment (55°C) of sugarcane vinasse, by means of shotgun metagenomics. After its metabolic potential was depicted, it was possible to observe several genes encoding enzymes that are of great importance to anaerobic digestion processes with different wastes as substrate, especially regarding the biodegradation of carbohydrates and ligninolytic compounds, glycerolypids, volatile fatty acids and alcohols metabolism and biogas (H2 and CH4) production. The genera identified in higher relative abundances for Bacteria domain were Sulfirimonas (37.52 ± 1.8%), possibly related to the sludge endogenic activity due to its strong relation with a peptidoglycan lyase enzymes family, followed by Fluviicola (5.01 ± 1.0%), Defluviitoga (4.36 ± 0.2%), Coprothermobacter (4.32 ± 0.5%), Fervidobacterium (2.93 ± 0.3%), Marinospirillum (2.75 ± 0.2%), Pseudomonas (2.14 ± 0.2%) and Flavobacterium (1.78 ± 0.1%), mostly related with carbohydrates fermentations and/or H2 production. For Archaea domain, Methanosarcina (0.61 ± 0.1%), Methanothermobacter (0.38 ± 0.0%), Methanoculleus (0.30 ± 0.1%), Thermococcus (0.03 ± 0.0%), Methanolobus (0.02 ± 1.8%), Methanobacterium (0.013 ± 0.0%), Aciduliprofundum and Pyrococcus (0.01 ± 0.0%) were the most dominant ones, being Methanosarcina the most related with methanogenesis. It was concluded that the robust inoculum description performed in this study may subside future biotechnological researches by using similar inocula (UASB sludges), focusing on the obtainment of value-added by-products by means of anaerobic digestion, such as volatile fatty acids, alcohols and biogas (H2 and CH4), by using several types of waste as substrate.

Microbiomes Associated With the Surfaces of Northern Argentinian Fruits Show a Wide Species Diversity.

The fiber, vitamin, and antioxidant contents of fruits contribute to a balanced human diet. In countries such as Argentina, several tropical fruits are witnessing a high yield in the harvest season, with a resulting surplus. Fruit fermentation using autochthonous starter cultures can provide a solution for food waste. However, limited knowledge exists about the microbiota present on the surfaces of fruits and the preceding flowers. In the present exploratory study, the microbiomes associated with the surfaces of tropical fruits from Northern Argentina, such as white guava, passion fruit and papaya were investigated using a shotgun metagenomic sequencing approach. Hereto, one sample composed of 14 white guava fruits, two samples of passion fruits with each two to three fruits representing the almost ripe and ripe stage of maturity, four samples of papaya with each two to three fruits representing the unripe, almost ripe, and ripe stage of maturity were processed, as well as a sample of closed and a sample of open Japanese medlar flowers. A considerable heterogeneity was found in the composition of the fruits' surface microbiota at the genus and species level. While bacteria dominated the microbiota of the fruits and flowers, a small number of the metagenomic sequence reads corresponded with yeasts and filamentous fungi. A minimal abundance of bacterial species critical in lactic acid and acetic acid fermentations was found. A considerable fraction of the metagenomic sequence reads from the fruits' surface microbiomes remained unidentified, which suggested that intrinsic species are to be sequenced or discovered.

Core of the saliva microbiome: an analysis of the MG-RAST data.

BACKGROUND: Oral microbiota is considered as the second most complex in the human body and its dysbiosis can be responsible for oral diseases. Interactions between the microorganism communities and the host allow establishing the microbiological proles. Identifying the core microbiome is essential to predicting diseases and changes in environmental behavior from microorganisms. METHODS: Projects containing the term "SALIVA", deposited between 2014 and 2019 were recovered on the MG-RAST portal. Quality (Failed), taxonomic prediction (Unknown and Predicted), species richness (Rarefaction), and species diversity (Alpha) were analyzed according to sequencing approaches (Amplicon sequencing and Shotgun metagenomics). All data were checked for normality and homoscedasticity. Metagenomic projects were compared using the Mann-Whitney U test and Spearman's correlation. Microbiome cores were inferred by Principal Component Analysis. For all statistical tests, p < 0.05 was used. RESULTS: The study was performed with 3 projects, involving 245 Amplicon and 164 Shotgun metagenome datasets. All comparisons of variables, according to the type of sequencing, showed significant differences, except for the Predicted. In Shotgun metagenomics datasets the highest correlation was between Rarefaction and Failed (r = - 0.78) and the lowest between Alpha and Unknown (r = - 0.12). In Amplicon sequencing datasets, the variables Rarefaction and Unknown (r = 0.63) had the highest correlation and the lowest was between Alpha and Predicted (r = - 0.03). Shotgun metagenomics datasets showed a greater number of genera than Amplicon. Propionibacterium, Lactobacillus, and Prevotella were the most representative genera in Amplicon sequencing. In Shotgun metagenomics, the most representative genera were Escherichia, Chitinophaga, and Acinetobacter. CONCLUSIONS: Core of the salivary microbiome and genera diversity are dependent on the sequencing approaches. Available data suggest that Shotgun metagenomics and Amplicon sequencing have similar sensitivities to detect the taxonomic level investigated, although Shotgun metagenomics allows a deeper analysis of the microorganism diversity. Microbiome studies must consider characteristics and limitations of the sequencing approaches. Were identified 20 genera in the core of saliva microbiome, regardless of the health condition of the host. Some bacteria of the core need further study to better understand their role in the oral cavity.

Microbial and functional characterization of an allochthonous consortium applied to hydrogen production from Citrus Peel Waste in batch reactor in optimized conditions.

Energy recovery from lignocellulosic waste has been studied as an alternative to the problem of inappropriate waste disposal. The present study aimed at characterizing the microbial community and the functional activity of reactors applied to H2 production through lignocellulosic waste fermentation in optimized conditions. The latter were identified by means of Rotational Central Composite Design (RCCD), applied to optimize allochthonous inoculum concentration (2.32-5.68 gTVS/L of granular anaerobic sludge), pH (4.32-7.68) and Citrus Peel Waste (CPW) concentration (1.55-28.45 g/L). After validation, the conditions identified for optimal H2 production were 4 gSTV/L of allochthonous inoculum, 29.8 g/L of CPW (substrate) and initial pH of 8.98. In these conditions, 48.47 mmol/L of H2 was obtained, which is 3.64 times higher than the concentration in unoptimized conditions (13.31 mmol H2/L using 15 g/L of CPW, 2 gTVS/L of allochthonous inoculum, pH 7.0). Acetogenesis was the predominant pathway, and maximal concentrations of 3,731 mg/L of butyric acid and 3,516 mg/L of acetic acid were observed. Regarding the metataxonomic profile, Clostridium genus was dramatically favored in the optimized condition (79.78%) when compared to the allochthonous inoculum (0.43%). It was possible to identify several genes related to H2 (i.e dehydrogenases) and volatile fatty acids (VFA) production and with cellulose degradation, especially some CAZymes from the classes Auxiliary Activities, Glycoside Hydrolases and Glycosyl Transferase. By means of differential gene expression it was observed that cellulose degradation and acetic acid production pathways were overabundant in samples from the optimized reactors, highlighting endo-ß-1,4-glucanase/cellulose, endo-ß-1,4-xylanase, ß-glucosidase, ß-mannosidase, cellulose ß-1,4-cellobiosidase, cellobiohydrolase, and others, as main the functions.

Detection of Trypanosoma cruzi in the saliva of diverse neotropical bats.

Trypanosoma cruzi is widely reported in bats, yet transmission routes remain unclear. We present evidence from metagenomic sequence data that T. cruzi occurs in the saliva of diverse Neotropical bats. Phylogenetic analyses demonstrated that the bat-associated T. cruzi sequences described here formed part of a bat-specific clade, suggesting an independent transmission cycle. Our results highlight the value in repurposing metagenomic data generated for viral discovery to reveal insights into the biology of other parasites. Evaluating whether the presence of T. cruzi in the saliva of two hematophagous bat species represents an ecological route for zoonotic transmission of Chagas disease is an interesting avenue for future research.

Shotgun metagenomics and microscopy indicate diverse cyanophytes, other bacteria, and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria).

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free-living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Demographic and environmental drivers of metagenomic viral diversity in vampire bats.

Viruses infect all forms of life and play critical roles as agents of disease, drivers of biochemical cycles and sources of genetic diversity for their hosts. Our understanding of viral diversity derives primarily from comparisons among host species, precluding insight into how intraspecific variation in host ecology affects viral communities or how predictable viral communities are across populations. Here we test spatial, demographic and environmental hypotheses explaining viral richness and community composition across populations of common vampire bats, which occur in diverse habitats of North, Central and South America. We demonstrate marked variation in viral communities that was not consistently predicted by a null model of declining community similarity with increasing spatial or genetic distances separating populations. We also find no evidence that larger bat colonies host greater viral diversity. Instead, viral diversity follows an elevational gradient, is enriched by juvenile-biased age structure, and declines with local anthropogenic food resources as measured by livestock density. Our results establish the value of linking the modern influx of metagenomic sequence data with comparative ecology, reveal that snapshot views of viral diversity are unlikely to be representative at the species level, and affirm existing ecological theories that link host ecology not only to single pathogen dynamics but also to viral communities.

Investigation of tylosin in feed of feedlot cattle and effects on liver abscess prevalence, and fecal and soil microbiomes and resistomes1.

Liver abscesses in feedlot cattle are detrimental to animal performance and economic return. Tylosin, a macrolide antibiotic, is used to reduce prevalence of liver abscesses, though there is variable efficacy among different groups of cattle. There is an increased importance in better understanding the etiology and pathogenesis of this condition because of growing concern over antibiotic resistance and increased scrutiny regarding use of antibiotics in food animal production. The objective of this study was to compare the microbiomes and antimicrobial resistance genes (resistomes) of feces of feedlot cattle administered or not administered tylosin and in their pen soil in 3 geographical regions with differing liver abscess prevalences. Cattle (total of 2,256) from 3 geographical regions were selected for inclusion based on dietary supplementation with tylosin (yes/no). Feces and pen soil samples were collected before harvest, and liver abscesses were identified at harvest. Shotgun and 16S rRNA amplicon sequencing were used to evaluate the soil and feces. Microbiome and resistome composition of feces (as compared by UniFrac distances and Euclidian distances, respectively) did not differ (P > 0.05) among tylosin or no tylosin-administered cattle. However, feedlot location was associated with differences (P ≤ 0.05) of resistomes and microbiomes. Using LASSO, a statistical model identified both fecal and soil microbial communities as predictive of liver abscess prevalence in pens. This model explained 75% of the variation in liver abscess prevalence, though a larger sample size would be needed to increase robustness of the model. These data suggest that tylosin exposure does not have a large impact on cattle resistomes or microbiomes, but instead, location of cattle production may be a stronger driver of both the resistome and microbiome composition of feces.

Using noninvasive metagenomics to characterize viral communities from wildlife.

Microbial communities play an important role in organismal and ecosystem health. While high-throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low-input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time.

The Geographic Structure of Viruses in the Cuatro Ciénegas Basin, a Unique Oasis in Northern Mexico, Reveals a Highly Diverse Population on a Small Geographic Scale.

The Cuatro Ciénegas Basin (CCB) is located in the Chihuahuan desert in the Mexican state of Coahuila; it has been characterized as a site with high biological diversity despite its extreme oligotrophic conditions. It has the greatest number of endemic species in North America, containing abundant living microbialites (including stromatolites and microbial mats) and diverse microbial communities. With the hypothesis that this high biodiversity and the geographic structure should be reflected in the virome, the viral communities in 11 different locations of three drainage systems, Churince, La Becerra, and Pozas Rojas, and in the intestinal contents of 3 different fish species, were analyzed for both eukaryotic and prokaryotic RNA and DNA viruses using next-generation sequencing methods. Double-stranded DNA (dsDNA) virus families were the most abundant (72.5% of reads), followed by single-stranded DNA (ssDNA) viruses (2.9%) and ssRNA and dsRNA virus families (0.5%). Thirteen families had dsDNA genomes, five had ssDNA, three had dsRNA, and 16 had ssRNA. A highly diverse viral community was found, with an ample range of hosts and a strong geographical structure, with very even distributions and signals of endemicity in the phylogenetic trees from several different virus families. The majority of viruses found were bacteriophages but eukaryotic viruses were also frequent, and the large diversity of viruses related to algae were a surprise, since algae are not evident in the previously analyzed aquatic systems of this ecosystem. Animal viruses were also frequently found, showing the large diversity of aquatic animals in this oasis, where plants, protozoa, and archaea are rare.IMPORTANCE In this study, we tested whether the high biodiversity and geographic structure of CCB is reflected in its virome. CCB is an extraordinarily biodiverse oasis in the Chihuahuan desert, where a previous virome study suggested that viruses had followed the marine ancestry of the marine bacteria and, as a result of their long isolation, became endemic to the site. In this study, which includes a larger sequencing coverage and water samples from other sites within the valley, we confirmed the high virus biodiversity and uniqueness as well as the strong biogeographical diversification of the CCB. In addition, we also analyzed fish intestinal contents, finding that each fish species eats different prey and, as a result, presents different viral compositions even if they coexist in the same pond. These facts highlight the high and novel virus diversity of CCB and its "lost world" status.