A direct examination of microbial specialized metabolites associated with ocean sediments from Baffin Bay and the Gulf of Maine.

Specialized metabolites produced by microorganisms found in ocean sediments display a wide range of clinically relevant bioactivities, including antimicrobial, anticancer, antiviral, and anti-inflammatory. Due to limitations in our ability to culture many benthic microorganisms under laboratory conditions, their potential to produce bioactive compounds remains underexplored. However, the advent of modern mass spectrometry technologies and data analysis methods for chemical structure prediction has aided in the discovery of such metabolites from complex mixtures. In this study, ocean sediments were collected from Baffin Bay (Canadian Arctic) and the Gulf of Maine for untargeted metabolomics using mass spectrometry. A direct examination of prepared organic extracts identified 1468 spectra, of which ∼45% could be annotated using in silico analysis methods. A comparable number of spectral features were detected in sediments collected from both locations, but 16S rRNA gene sequencing revealed a significantly more diverse bacterial community in samples from Baffin Bay. Based on spectral abundance, 12 specialized metabolites known to be associated with bacteria were selected for discussion. The application of metabolomics directly on marine sediments provides an avenue for culture-independent detection of metabolites produced under natural settings. The strategy can help prioritize samples for novel bioactive metabolite discovery using traditional workflows.

Distinct epidemiological profiles of porcine circovirus 3 and fox circovirus in Canadian foxes (Vulpes spp.).

Circoviruses (genus Circovirus, family Circoviridae) are ssDNA viruses that infect mammals, and they sometimes can transmit among different species. We investigated the distribution and diversity of porcine circovirus 3 (PCV-3, species Porcine circovirus 3) and fox circovirus (species Canine circovirus 1) in different populations of foxes (Vulpes spp.) inhabiting the Canadian province of Newfoundland and Labrador to compare their epidemiological profiles. Of the 210 samples tested in this study 9 were positive for PCV-3 and 99 were positive for fox circovirus. Eight foxes were PCV-3-positive (8/128, 6.3%) and the virus was only found in the most human-populated areas. The PCV-3 positivity rate was significantly higher in stool (7/180, 8.8%) than in spleen (2/120, 1.7%: p < 0.05). Phylogenetic analyses showed that sequences from different animals were unrelated to each other. Fox circovirus was identified in 66 animals (51.6%) and positivity rates were the highest in the least human-populated areas. There were no significant differences between positivity rates in stool (32/80, 40.0%), spleen (59/120, 49.2%), or lymph nodes (8/10, 80.0%). Among 54 positive animals for which both spleen and stool samples were available, 25 (46.3%) had detectable virus in both samples. All fox circovirus sequences recovered in this study formed a monophyletic clade, and no geographic segregation of study strains was observed. The high prevalence and high genetic diversity observed for fox circovirus implies that the virus has been circulating in this population for a long time. PCV-3 cases were consistent with sporadic infections from multiple sources, possibly related to scavenging behavior and consumption of meat by-products and human waste, while fox circovirus was endemic, indicating that foxes are likely the maintenance host for this virus. To the best of our knowledge this is the first study demonstrating the presence of fox circovirus in North America and to show that PCV-3 can be detected in foxes. Future studies should evaluate the pathogenic potential of these viruses for wildlife.

A novel calicivirus discovered in trumpeter swans (Cygnus buccinator) expands the richness of known avian caliciviruses.

Caliciviruses are ssRNA viruses that can infect a wide range of hosts, including birds. While several avian caliciviruses have been discovered, their taxonomy and host distribution are largely unknown. We molecularly characterized a novel calicivirus (trumpeter swan calicivirus: TruSCV) in trumpeter swans over-wintering in south-west British Columbia, Canada. The positivity rate was 20.3% (14/69) and there were no significant differences in infection rates between males (5/34, 14.7%) and females (9/35, 25.7%) or among considered age groups (juveniles: 4/14, 28.6%; sub-adults: 1/9, 11.1%; adults: 9/46, 19.6%). Twelve infected swans died of lead poisoning, one because of starvation, and one from physical injuries. TruSCV complete genome possessed the typical organization and protein motifs of caliciviruses and a type 2 IRES and its closest relative was a virus circulating in Australian ducks. Phylogenetic analyses showed the existence of 34 different but monophyletic avian caliciviruses. These viruses, while having conserved genomic organization and protein motifs, possess different IRES types and group in several divergent clades, with only two of them corresponding to currently defined genera, highlighting the need for epidemiological investigations and systematic analyses to better define their taxonomy. Follow-up studies are needed to elucidate the diversity, distribution, and pathogenic potential of TruSCV.

A Novel Iridovirus Discovered in Deep-Sea Carnivorous Sponges.

Carnivorous sponges (family Cladorhizidae) use small invertebrates as their main source of nutrients. We discovered a novel iridovirus (carnivorous sponge-associated iridovirus, CaSpA-IV) in Chondrocladia grandis and Cladorhiza oxeata specimens collected in the Arctic and Atlantic oceans at depths of 537-852 m. The sequenced viral genome (~190,000 bp) comprised 185 predicted ORFs, including those encoding 26 iridoviral core proteins, and phylogenetic analyses showed that CaSpA-IV is a close relative to members of the genus Decapodiridovirus and highly identical to a partially sequenced virus pathogenic to decapod shrimps. CaSpA-IV was found in various anatomical regions of six C. grandis (sphere, stem, root) from the Gulf of Maine and Baffin Bay and of two C. oxeata (sphere, secondary axis) from Baffin Bay. Partial MCP sequencing revealed a divergent virus (CaSpA-IV-2) in one C. oxeata. The analysis of a 10 nt long tandem repeat showed a number of repeats consistent across sub-sections of the same sponges but different between animals, suggesting the presence of different strains. As the genetic material of crustaceans, particularly from the zooplanktonic copepod order Calanoida, was identified in the investigated samples, further studies are required to elucidate whether CaSpA-IV infects the carnivorous sponges, their crustacean prey, or both.

Novel Divergent Members of the Kitrinoviricota Discovered through Metagenomics in the Intestinal Contents of Red-Backed Voles (Clethrionomys gapperi).

Metagenomic methods are powerful tools to investigate viral diversity in biological or environmental samples and to identify previously unknown viruses. We used RNA metagenomics to identify, in the gut of red-backed voles, the nearly complete genomes of two novel members of the Kitrinoviricota, a phylum including viruses with positive-sense ssRNA genomes encoding an RNA-directed RNA polymerase. The genome of a novel member of the Tombusviridae presented four open reading frames (ORFs); a -1 frameshift is potentially involved in generating the viral replicase. This sequence was part of a phylogenetic clade that did not include any officially classified species. The second genome presented a large ORF coding for a viral polyprotein containing the typical protein domains common to flexiviruses. The sequence clustered with currently known members of the Deltaflexiviridae. Both viruses appear to represent the first members of novel species in yet undefined genera. The identified viruses likely originated from the vole diet as members of the two viral families are known to infect plants and fungi, respectively. Investigating public databases demonstrated that a much higher richness than currently recognized exists for these two viral families, highlighting the need to update taxonomy systems and possibly also include genomes identified through metagenomics.

Newlavirus, a Novel, Highly Prevalent, and Highly Diverse Protoparvovirus of Foxes (Vulpes spp.).

The genus Protoparvovirus (family Parvoviridae) includes several viruses of carnivores. We describe a novel fox protoparvovirus, which we named Newlavirus as it was discovered in samples from Newfoundland and Labrador, Canada. Analysis of the full non-structural protein (NS1) sequence indicates that this virus is a previously uncharacterized species. Newlavirus showed high prevalence in foxes from both the mainland (Labrador, 54/137, 39.4%) and the island of Newfoundland (22/50, 44%) but was not detected in samples from other carnivores, including coyotes (n = 92), lynx (n = 58), martens (n = 146), mink (n = 47), ermines (n = 17), dogs (n = 48), and ringed (n = 4), harp (n = 6), bearded (n = 6), and harbor (n = 2) seals. Newlavirus was found at similar rates in stool and spleen (24/80, 30% vs. 59/152, 38.8%, p = 0.2) but at lower rates in lymph nodes (2/37, 5.4%, p < 0.01). Sequencing a fragment of approximately 750 nt of the capsid protein gene from 53 samples showed a high frequency of co-infection by more than one strain (33.9%), high genetic diversity with 13 genotypes with low sequence identities (70.5-87.8%), and no geographic segregation of strains. Given the high prevalence, high diversity, and the lack of identification in other species, foxes are likely the natural reservoir of Newlavirus, and further studies should investigate its distribution.

Investigating the Diversity and Host Range of Novel Parvoviruses from North American Ducks Using Epidemiology, Phylogenetics, Genome Structure, and Codon Usage Analysis.

Parvoviruses are small single-stranded DNA viruses that can infect both vertebrates and invertebrates. We report here the full characterization of novel viruses we identified in ducks, including two viral species within the subfamily Hamaparvovirinae (duck-associated chapparvovirus, DAC) and a novel species within the subfamily Densovirinae (duck-associated ambidensovirus, DAAD). Overall, 5.7% and 21.1% of the 123 screened ducks (American black ducks, mallards, northern pintail) were positive for DAC and DAAD, respectively, and both viruses were more frequently detected in autumn than in winter. Genome organization and predicted transcription profiles of DAC and DAAD were similar to viruses of the genera Chaphamaparvovirus and Protoambidensovirus, respectively. Their association to these genera was also demonstrated by subfamily-wide phylogenetic and distance analyses of non-structural protein NS1 sequences. While DACs were included in a highly supported clade of avian viruses, no definitive conclusions could be drawn about the host type of DAAD because it was phylogenetically close to viruses found in vertebrates and invertebrates and analyses of codon usage bias and nucleotide frequencies of viruses within the family Parvoviridae showed no clear host-based viral segregation. This study highlights the high parvoviral diversity in the avian reservoir with many avian-associated parvoviruses likely yet to be discovered.

Virus discovery reveals frequent infection by diverse novel members of the Flaviviridae in wild lemurs.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

ViDiT-CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications.

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (less than US$5 per sample), and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing, and microbiome profiling. ViDiT-CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal-cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT-CACTUS demonstrated its validity in a wide range of microbiology applications, and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

Temporal Bacterial Surveillance of Salmon Aquaculture Sites Indicates a Long Lasting Benthic Impact With Minimal Recovery.

Coastal aquaculture has experienced substantial growth in the last few decades and associated impacts on natural environments are of increasing importance. Understanding both the effects of aquaculture on marine ecosystems and the processes of recovery during fallowing periods is crucial for the development of a more environmentally sustainable industry. Because bacteria are sensitive to environmental change, surveying fluctuations in bacterial communities is a promising tool for monitoring the status of benthic environments. Here, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in flocculent matter samples collected over a period of 3 years and at various distances from cages (0-200 meters) at production and fallow (3-35 months) salmon aquaculture sites in southern Newfoundland to evaluate the environmental impact of aquaculture on predominantly hard-bottom substrates. Bacterial composition analysis revealed four clusters, three of which (defined as "recently disturbed," "intermediate impact," and "high impact") differed markedly from a fourth "low impact" cluster that contained far-field samples collected >500 m from cages. Samples within the high impact group were most often collected directly under cages, whereas those in the intermediate impact group were mainly sampled from 20 to 40 m from cages. Large scale phylum shifts (increases of Bacteroidetes, Firmicutes, Spirochaetes, and decreases in Proteobacteria and Epsilonbacteraeota) and a decline in bacterial diversity were observed in the high impact cluster, indicating significant ecological change. Samples from sites of different fallow duration were found in the high impact cluster, indicating a lack of recovery, even after 35 months of fallowing. Finally, we identified 28 genera as bacterial biomarkers, specific to one or more clusters, including genera associated with organically enriched environments and previously reported in the context of aquaculture impacts. Tracking the relative abundance of biomarkers in relation to different lengths of fallowing in the three more impacted clusters showed that these markers remained significantly above low impact cluster levels at all times, further pointing toward incomplete recovery. Our results suggest that coastal aquaculture on hard-bottom substrates is prone to long lasting impacts on bacterial communities, especially below cages, and that effects can be accurately tracked using bacterial community profiles or specific biomarkers.

Coronafacoyl Phytotoxin Biosynthesis and Evolution in the Common Scab Pathogen Streptomyces scabiei.

Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei Structural elucidation of accumulated biosynthetic intermediates in the ΔCYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria.IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.

Microbiome analysis shows enrichment for specific bacteria in separate anatomical regions of the deep-sea carnivorous sponge Chondrocladia grandis.

The Cladorhizidae is a unique family of carnivorous marine sponges characterised by either the absence or reduction of the aquiferous system and by the presence of specialised structures to trap and digest mesoplanktonic prey. Previous studies have postulated a key role of host-associated bacteria in enabling carnivory in this family of sponges. In this study, we employed high-throughput Illumina-based sequencing to identify the bacterial community associated with four individuals of the deep-sea sponge Chondrocladia grandis sampled in the Gulf of Maine. By characterising the V6 through V8 region of the 16S rRNA gene, we compared the bacterial community composition and diversity in three distinct anatomical regions with predicted involvement in prey capture (sphere), support (axis) and benthic substrate attachment (root). A high abundance of Tenacibaculum, a known siderophore producing bacterial genus, was present in all anatomical regions and specimens. The abundance of Colwellia and Roseobacter was greater in sphere and axis samples, and bacteria from the hydrocarbon-degrading Robiginitomaculum genus were most abundant in the root. This first description of the bacterial community associated with C. grandis provides novel insights into the contribution of bacteria to the carnivorous lifestyle while laying foundations for future cladorhizid symbiosis studies.

Autologous antibody capture to enrich immunogenic viruses for viral discovery.

Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.

Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.