Scavengers as Prospective Sentinels of Viral Diversity: the Snowy Sheathbill Virome as a Potential Tool for Monitoring Virus Circulation, Lessons from Two Antarctic Expeditions.

Antarctica is a unique environment due to its extreme meteorological and geological conditions. In addition to this, its relative isolation from human influences has kept it undisturbed. This renders our limited understanding of its fauna and its associated microbial and viral communities a relevant knowledge gap to fill. This includes members of the order Charadriiformes such as snowy sheathbills. They are opportunistic predator/scavenger birds distributed on Antarctic and sub-Antarctic islands that are in frequent contact with other bird and mammal species. This makes them an interesting species for surveillance studies due to their high potential for the acquisition and transport of viruses. In this study, we performed whole-virome and targeted viral surveillance for coronaviruses, paramyxoviruses, and influenza viruses in snowy sheathbills from two locations, the Antarctic Peninsula and South Shetland. Our results suggest the potential role of this species as a sentinel for this region. We highlight the discovery of two human viruses, a member of the genus Sapovirus GII and a gammaherpesvirus, and a virus previously described in marine mammals. Here, we provide insight into a complex ecological picture. These data highlight the surveillance opportunities provided by Antarctic scavenger birds. IMPORTANCE This article describes whole-virome and targeted viral surveillance for coronaviruses, paramyxoviruses, and influenza viruses in snowy sheathbills from the Antarctic Peninsula and South Shetland. Our results suggest an important role of this species as a sentinel for this region. This species' RNA virome showcased a diversity of viruses likely tied to its interactions with assorted Antarctic fauna. We highlight the discovery of two viruses of likely human origin, one with an intestinal impact and another with oncogenic potential. Analysis of this data set detected a variety of viruses tied to various sources (from crustaceans to nonhuman mammals), depicting a complex viral landscape for this scavenger species.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

Characterisation of the Upper Respiratory Tract Virome of Feedlot Cattle and Its Association with Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a major health problem within the global cattle industry. This disease has a complex aetiology, with viruses playing an integral role. In this study, metagenomics was used to sequence viral nucleic acids in the nasal swabs of BRD-affected cattle. The viruses detected included those that are well known for their association with BRD in Australia (bovine viral diarrhoea virus 1), as well as viruses known to be present but not fully characterised (bovine coronavirus) and viruses that have not been reported in BRD-affected cattle in Australia (bovine rhinitis, bovine influenza D, and bovine nidovirus). The nasal swabs from a case-control study were subsequently tested for 10 viruses, and the presence of at least one virus was found to be significantly associated with BRD. Some of the more recently detected viruses had inconsistent associations with BRD. Full genome sequences for bovine coronavirus, a virus increasingly associated with BRD, and bovine nidovirus were completed. Both viruses belong to the Coronaviridae family, which are frequently associated with disease in mammals. This study has provided greater insights into the viral pathogens associated with BRD and highlighted the need for further studies to more precisely elucidate the roles viruses play in BRD.

Metagenomic Analysis of Respiratory RNA Virome of Children with and without Severe Acute Respiratory Infection from the Free State, South Africa during COVID-19 Pandemic Reveals Higher Diversity and Abundance in Summer Compared with Winter Period.

Viral respiratory infections contribute to significant morbidity and mortality in children. Currently, there are limited reports on the composition and abundance of the normal commensal respiratory virome in comparison to those in severe acute respiratory infections (SARIs) state. This study characterised the respiratory RNA virome in children ≤ 5 years with (n = 149) and without (n = 139) SARI during the summer and winter of 2020/2021 seasons in South Africa. Nasopharyngeal swabs were, collected, pooled, enriched for viral RNA detection, sequenced using Illumina MiSeq, and analysed using the Genome Detective bioinformatic tool. Overall, Picornaviridae, Paramoxyviridae, Pneumoviridae, Picobirnaviridae, Totiviridae, and Retroviridae families were the most abundant viral population in both groups across both seasons. Human rhinovirus and endogenous retrovirus K113 were detected in most pools, with exclusive detection of Pneumoviridae in SARI pools. Generally, higher viral diversity/abundance was seen in children with SARI and in the summer pools. Several plant/animal viruses, eukaryotic viruses with unclear pathogenicity including a distinct rhinovirus A type, were detected. This study provides remarkable data on the respiratory RNA virome in children with and without SARI with a degree of heterogeneity of known viruses colonizing their respiratory tract. The implication of the detected viruses in the dynamics/progression of SARI requires further investigations.

The gut virome in health and disease: new insights and associations.

PURPOSE OF REVIEW: Recent years have seen great strides made in the field of viral metagenomics. Many studies have reported alterations in the virome in different disease states. The vast majority of the human intestinal virome consists of bacteriophages, viruses that infect bacteria. The dynamic relationship between gut bacterial populations and bacteriophages is influenced by environmental factors that also impact host health and disease. In this review, we focus on studies highlighting the dynamics of the gut virome and fluctuations associated with disease states. RECENT FINDINGS: Novel correlations have been identified between the human gut virome and diseases such as obesity, necrotizing enterocolitis and severe acute respiratory syndrome coronavirus 2 infection. Further associations between the virome and cognition, diet and geography highlight the complexity of factors that can influence the dynamic relationship between gut bacteria, bacteriophages and health. SUMMARY: Here, we highlight some novel associations between the virome and health that will be the foundation for future studies in this field. The future development of microbiome-based interventions, identification of biomarkers, and novel therapeutics will require a thorough understanding of the gut virome and its dynamics.

Serological responses to human virome define clinical outcomes of Italian patients infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.

Faecal virome of the Australian grey-headed flying fox from urban/suburban environments contains novel coronaviruses, retroviruses and sapoviruses.

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalian-infecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

Zoonotic disease and virome diversity in bats.

The emergence of zoonotic viral diseases in humans commonly reflects exposure to mammalian wildlife. Bats (order Chiroptera) are arguably the most important mammalian reservoir for zoonotic viruses, with notable examples including Severe Acute Respiratory Syndrome coronaviruses 1 and 2, Middle East Respiratory Syndrome coronavirus, henipaviruses and lyssaviruses. Herein, we outline our current knowledge on the diversity of bat viromes, particularly through the lens of metagenomic next-generation sequencing and in the context of disease emergence. A key conclusion is that although bats harbour abundant virus diversity, the vast majority of bat viruses have not emerged to cause disease in new hosts such that bats are better regarded as critical but endangered components of global ecosystems.

Myotis fimbriatus Virome, a Window to Virus Diversity and Evolution in the Genus Myotis.

Significant efforts have been made to characterize viral diversity in bats from China. Many of these studies were prospective and focused mainly on Rhinolophus bats that could be related to zoonotic events. However, other species of bats that are part of ecosystems identified as virus diversity hotspots have not been studied in-depth. We analyzed the virome of a group of Myotis fimbriatus bats collected from the Yunnan Province during 2020. The virome of M. fimbriatus revealed the presence of families of pathogenic viruses such as Coronavirus, Astrovirus, Mastadenovirus, and Picornavirus, among others. The viral sequences identified in M. fimbriatus were characterized by significant divergence from other known viral sequences of bat origin. Complex phylogenetic landscapes implying a tendency of co-specificity and relationships with viruses from other mammals characterize these groups. The most prevalent and abundant virus in M. fimbriatus individuals was an alphacoronavirus. The genome of this virus shows evidence of recombination and is likely the product of ancestral host-switch. The close phylogenetic and ecological relationship of some species of the Myotis genus in China may have played an important role in the emergence of this alphacoronavirus.

The gut virome: A new microbiome component in health and disease.

The human gastrointestinal tract harbours an abundance of viruses, collectively known as the gut virome. The gut virome is highly heterogeneous across populations and is linked to geography, ethnicity, diet, lifestyle, and urbanisation. The currently known function of the gut virome varies greatly across human populations, and much remains unknown. We review current literature on the human gut virome, and the intricate trans-kingdom interplay among gut viruses, bacteria, and the mammalian host underlying health and diseases. We summarise evidence on the use of the gut virome as diagnostic markers and a therapeutic target. We shed light on novel avenues of microbiome-inspired diagnosis and therapies. We also review pre-clinical and clinical studies on gut virome-rectification-based therapies, including faecal microbiota transplantation, faecal virome transplantation, and refined phage therapy. Our review suggests that future research effort should focus on unravelling the mechanisms exerted by gut viruses/phages in human pathophysiology, and on developing phage-prompted precision therapies.

Characterization of Pipistrellus pygmaeus Bat Virome from Sweden.

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78-99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88-94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

Dynamics of nasopharyngeal tract phageome and association with disease severity and age of patients during three waves of COVID-19.

In December 2019, several patients were hospitalized and diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which subsequently led to a global pandemic. To date, there are no studies evaluating the relationship between the respiratory phageome and the SARS-CoV-2 infection. The current study investigated the phageome profiles in the nasopharyngeal swabs collected from 55 patients during the three different waves of coronavirus disease 2019 (COVID-19) in the Campania Region (Southern Italy). Data obtained from the taxonomic profiling show that phage families belonging to the order Caudovirales have a high abundance in the patient samples. Moreover, the severity of the COVID-19 infection seems to be correlated with the phage abundance.

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses-The Example of SARS-CoV-2.

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known-up to 2015-vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits' design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits' panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats.

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline panleukopenia virus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used meta-transcriptomic and viral particle enrichment metagenomic approaches to characterize the enteric viromes of 23 cats naturally infected with FPV (FPV-cases) and 36 age-matched healthy shelter cats (healthy controls). Sequencing reads from mammalian infecting viral families largely belonged to the Coronaviridae, Parvoviridae and Astroviridae. The most abundant viruses among the healthy control cats were feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (feline bocavirus), with frequent coinfections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6 out of 36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2, feline coronavirus and C. bocaparvovirus 4 (feline bocaparvovirus 2). The latter and feline bocaparvovirus 3 were detected significantly more frequently in FPV-cases than in healthy controls. Feline calicivirus was present in a higher proportion of FPV-cases (11 out of 23, 47.8%) compared to healthy controls (5 out of 36, 13.9%, p = 0.0067). Feline kobuvirus infections were also common among FPV-cases (9 out of 23, 39.1%) and were not detected in any healthy controls (p < .0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19 out of 23, 82.6%) than in healthy controls (18 out of 36, p = .0142). The differences in eukaryotic virome composition revealed here indicate that further investigations are warranted to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL.

Assessment of porcine Rotavirus-associated virome variations in pigs with enteric disease.

Enteric disease is the predominant cause of morbidity and mortality in young mammals including pigs. Viral species involved in porcine enteric disease complex (PEDC) include rotaviruses, coronaviruses, picornaviruses, astroviruses and pestiviruses among others. The virome of three groups of swine samples submitted to the Kansas State University Veterinary Diagnostic Laboratory for routine testing were assessed, namely, a Rotavirus A positive (RVA) group, a Rotavirus co-infection (RV) group and a Rotavirus Negative (RV Neg) group. All groups were designated by qRT-PCR test results for Porcine Rotavirus A, B, C and H such that samples positive for RVA only went in the RVA group, samples positive for > 1 rotavirus went in the RV group and samples negative for all were grouped in the RVNeg group. All of the animals had clinical enteric disease resulting in scours and swollen joints/lameness, enlarged heart and/or a cough. All samples were metagenomic sequenced and analyzed for viral species composition that identified 14 viral species and eight bacterial viruses/phages. Sapovirus and Escherichia coli phages were found at a high prevalence in RVA and RV samples but were found at low or no prevalence in the RVNeg samples. Picobirnavirus was identified at a high proportion and prevalence in RVNeg and RV samples but at a low prevalence in the RVA group. Non-rotaviral diversity was highest in RVA samples followed by RV then RV Neg samples. A sequence analysis of the possible host of Picobirnaviruses revealed fungi as the most likely host. Various sequences were extracted from the sample reads and a phylogenetic update was provided showing a high prevalence of G9 and P[23] RVA genotypes. These data are important for pathogen surveillance and control measures.

Alterations in the Composition of Intestinal DNA Virome in Patients With COVID-19.

Patients with Coronavirus Disease 2019 (COVID-19), due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection mainly present with respiratory issues and related symptoms, in addition to significantly affected digestive system, especially the intestinal tract. While several studies have shown changes in the intestinal flora of patients with COVID-19, not much information is available on the gut virome of such patients. In this study, we used the viromescan software on the latest gut virome database to analyze the intestinal DNA virome composition of 15 patients with COVID-19 and investigated the characteristic alternations, particularly of the intestinal DNA virome to further explore the influence of COVID-19 on the human gut. The DNA viruses in the gut of patients with COVID-19 were mainly crAss-like phages (35.48%), Myoviridae (20.91%), and Siphoviridae (20.43%) family of viruses. Compared with healthy controls, the gut virome composition of patients with COVID-19 changed significantly, especially the crAss-like phages family, from the first time of hospital admission. A potential correlation is also indicated between the change in virome and bacteriome (like Tectiviridae and Bacteroidaceae). The abundance of the viral and bacterial population was also analyzed through continuous sample collection from the gut of patients hospitalized due to COVID-19. The gut virome is indeed affected by the SARS-CoV-2 infection, and along with gut bacteriome, it may play an important role in the disease progression of COVID-19. These conclusions would be helpful in understanding the gut-related response and contribute to the treatment and prevention strategies of COVID-19.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

Virome characterization of game animals in China reveals a spectrum of emerging pathogens.

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high-risk viruses. We inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

The Human Virome: Viral Metagenomics, Relations with Human Diseases, and Therapeutic Applications.

The human body is colonized by a wide range of microorganisms. The field of viromics has expanded since the first reports on the detection of viruses via metagenomic sequencing in 2002. With the continued development of reference materials and databases, viral metagenomic approaches have been used to explore known components of the virome and discover new viruses from various types of samples. The virome has attracted substantial interest since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic. Increasing numbers of studies and review articles have documented the diverse virome in various sites in the human body, as well as interactions between the human host and the virome with regard to health and disease. However, there have been few studies of direct causal relationships. Viral metagenomic analyses often lack standard references and are potentially subject to bias. Moreover, most virome-related review articles have focused on the gut virome and did not investigate the roles of the virome in other sites of the body in human disease. This review presents an overview of viral metagenomics, with updates regarding the relations between alterations in the human virome and the pathogenesis of human diseases, recent findings related to COVID-19, and therapeutic applications related to the human virome.

Diversity and evolution of the animal virome.

The COVID-19 pandemic has given the study of virus evolution and ecology new relevance. Although viruses were first identified more than a century ago, we likely know less about their diversity than that of any other biological entity. Most documented animal viruses have been sampled from just two phyla - the Chordata and the Arthropoda - with a strong bias towards viruses that infect humans or animals of economic and social importance, often in association with strong disease phenotypes. Fortunately, the recent development of unbiased metagenomic next-generation sequencing is providing a richer view of the animal virome and shedding new light on virus evolution. In this Review, we explore our changing understanding of the diversity, composition and evolution of the animal virome. We outline the factors that determine the phylogenetic diversity and genomic structure of animal viruses on evolutionary timescales and show how this impacts assessment of the risk of disease emergence in the short term. We also describe the ongoing challenges in metagenomic analysis and outline key themes for future research. A central question is how major events in the evolutionary history of animals, such as the origin of the vertebrates and periodic mass extinction events, have shaped the diversity and evolution of the viruses they carry.