During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

UNASSIGNED: A high prevalence of onchocerciasis-associated epilepsy (OAE) has been observed in onchocerciasis-endemic areas with high ongoing Onchocerca volvulus transmission. However, the pathogenesis of OAE remains to be elucidated. We hypothesise that the O. volvulus virome could be involved in inducing epilepsy. With this study, we aim to describe the O. volvulus virome and identify potential neurotropic viruses linked to OAE.
UNASSIGNED: In Maridi County, an onchocerciasis endemic area in South Sudan with a high prevalence of OAE, we will conduct an exploratory case-control study enrolling 40 persons aged 12 years and above with palpable onchocerciasis nodules. Cases will be participants with OAE (n=20), who will be age- and village-matched with controls without epilepsy (n=20). For each study participant, two skin snips at the iliac crest will be obtained to collect O. volvulus microfilariae, and one nodulectomy will be performed to obtain adult worms. A viral metagenomic study will be conducted on microfilariae and adult worms, and the O. volvulus virome of persons with and without OAE will be compared. The number, size, and localisation of onchocerciasis nodules in persons with and without OAE will be described. Moreover, the pre- and post-nodulectomy frequency of seizures in persons with OAE will be compared.
UNASSIGNED: The protocol has been approved by the Ethics Committee of the University of Antwerp and the Ministry of Health of South Sudan. Findings will be disseminated nationally and internationally via meetings and peer-reviewed publications.
UNASSIGNED: ClinicalTrials.gov registration NCT05868551 ( https://clinicaltrials.gov/study/NCT05868551).
UNASSIGNED: 1.1, dated 09/05/2023.

Stool samples for fecal immunochemical tests (FIT) are collected in large numbers worldwide as part of colorectal cancer screening programs. Employing FIT samples from 1034 CRCbiome participants, recruited from a Norwegian colorectal cancer screening study, we identify, annotate and characterize more than 18000 DNA viruses, using shotgun metagenome sequencing. Only six percent of them are assigned to a known taxonomic family, with Microviridae being the most prevalent viral family. Linking individual profiles to comprehensive lifestyle and demographic data shows 17/25 of the variables to be associated with the gut virome. Physical activity, smoking, and dietary fiber consumption exhibit strong and consistent associations with both diversity and relative abundance of individual viruses, as well as with enrichment for auxiliary metabolic genes. We demonstrate the suitability of FIT samples for virome analysis, opening an opportunity for large-scale studies of this enigmatic part of the gut microbiome. The diverse viral populations and their connections to the individual lifestyle uncovered herein paves the way for further exploration of the role of the gut virome in health and disease.

Studies on the human virome based on the application of metagenomic approaches involve overcoming a series of challenges and limitations inherent not only to the biological features of viruses, but also to methodological pitfalls which different approaches have tried to minimize. These approaches fall into two main categories: bulk-metagenomes and virus-like particle (VLP) enrichment. In order to address issues associated with commonly used experimental procedures to assess the degree of reliability, representativeness, and reproducibility, we designed a comparative analysis applied to three experimental protocols, one based on bulk-metagenomes and two based on VLP enrichment. These protocols were applied to stool samples from 10 adult participants, including two replicas per protocol and subject. We evaluated the performances of the three methods, not only through the analysis of the resulting composition, abundance, and diversity of the virome via taxonomical classification and type of molecule (DNA versus RNA, single stranded vs. double stranded), but also according to how the a priori identical replicas differed from each other according to the extraction methods used. Our results highlight the strengths and weaknesses of each approach, offering valuable insights and tailored recommendations for drawing reliable conclusions based on specific research goals.

Viruses play a crucial role in microbial mortality, diversity and biogeochemical cycles. Groundwater is the largest global freshwater and one of the most oligotrophic aquatic systems on Earth, but how microbial and viral communities are shaped in this special habitat is largely unexplored. In this study, we collected groundwater samples from 23 to 60 m aquifers at Yinchuan Plain, China. In total, 1920 non-reductant viral contigs were retrieved from metagenomes and viromes constructed by Illumina and Nanopore hybrid sequencing. Only 3% of them could be clustered with known viruses, most of which were Caudoviricetes. Coupling 1.2 Tb Hi-C sequencing with CRISPR matching and homology search, we connected 469 viruses with their hosts while some viral clusters presented a broad-host-range trait. Meanwhile, a large proportion of biosynthesis related auxiliary metabolism genes were identified. Those characteristics might benefit viruses for a better survival in this special oligotrophic environment. Additionally, the groundwater virome showed genomic features distinct from those of the open ocean and wastewater treatment facilities in GC distribution and unannotated gene compositions. This paper expands the current knowledge of the global viromic records and serves as a foundation for a more thorough understanding of viruses in groundwater.

The lungs have their own microbiota which seems to be altered in disease processes such as asthma. Viral infection accounts for many asthma exacerbations. Little is known about the lung virome, and the role that viruses play in non-exacerbating asthmatics. We aimed to assess if detection of virus in bronchoscopy samples of asthmatic patients in a non-exacerbating state influences their asthma control and modulates airway cytokine composition. Patients were recruited from a specialist asthma clinic and underwent bronchoscopy with standardised bronchoalveolar lavage (BAL). Viral analysis was performed; cell differential and cytokine levels were measured. Forty-six samples were obtained of which 10.8% demonstrated evidence of airway virus, and 91.3% of patients in the cohort were classed as severe asthmatics. Oral steroid use was significantly higher in severe asthmatic patients with virus detected, and the forced expiratory volume in one second tended to be lower in the virus-detected group. It was also found that BAL interleukin-13 and tumor necrosis factor-α levels were significantly higher in severe asthmatic patients with virus detected. Our results suggest that in severe asthmatics in a non-exacerbating state, the presence of virus resulted in overall poorer asthma control. The pattern of cytokine elevation seen in asthmatic patients with virus detected may provide insight to the pathophysiology involved.

Several previous studies have identified a potential role that the gut microbiome can play in autism spectrum disorder (ASD) in children, but little is known about how variations in the virome may be involved in ASD. We aimed to understand the changes in the gut DNA virome of children with ASD.
A case-control study was presented, in which 13 two-children families were observed while considering the age, mode of birth, history of antibiotic use, and vaccination history to minimize the influence of confounding factors. DNA viral metagenomic sequencing was successfully performed on stool samples from 11 children with ASD and 12 healthy non-ASD children. The basic composition and gene function of the participants\' fecal DNA virome were detected and analyzed. Finally, the abundance and diversity of the DNA virome of children with ASD and their healthy siblings were compared.
The gut DNA virome in children aged 3-11 years was found to be dominated by the Siphoviridae family of Caudovirales. The proteins encoded by the DNA genes mainly carry out the functions of genetic information transmission and metabolism. Compared the gut DNA virome of ASD and healthy non-ASD children, their abundance of Caudovirales and Petitvirales both showed a significant negative correlation (r = -0.902, P < 0.01), there was no statistically significant difference in the relative abundance of viruses at the order and family levels, and a difference in the relative abundance at the genus level for Skunavirus (Ζ = -2.157, P = 0.031). Viral α diversity was reduced in children with ASD, but α diversity and β diversity did not differ statistically between groups.
This study indicates that elevated Skunavirus abundance and decreased α diversity in the gut DNA virulence group of children with ASD, but no statistically significant difference in the change in alpha and beta diversity. This provides preliminary cumulative information on virological aspects of the relationship between the microbiome and ASD, and should benefit future multi-omics and large sample studies on the gut microbes in children with ASD.

In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles of viruses in agroecosystems. Such unbiased exploration is available through viromics, which could generate biological and ecological insights from immense high-throughput sequencing (HTS) data.
Here, we implemented HTS-based viromics to explore viral diversity in tomatoes and weeds in farming areas at a nation-wide scale. We detected 125 viruses, including 79 novel species, wherein 65 were found exclusively in weeds. This spanned 21 higher-level plant virus taxa dominated by Potyviridae, Rhabdoviridae, and Tombusviridae, and four non-plant virus families. We detected viruses of non-plant hosts and viroid-like sequences and demonstrated infectivity of a novel tobamovirus in plants of Solanaceae family. Diversities of predominant tomato viruses were variable, in some cases, comparable to that of global isolates of the same species. We phylogenetically classified novel viruses and showed links between a subgroup of phylogenetically related rhabdoviruses to their taxonomically related host plants. Ten classified viruses detected in tomatoes were also detected in weeds, which might indicate possible role of weeds as their reservoirs and that these viruses could be exchanged between the two compartments.
We showed that even in relatively well studied agroecosystems, such as tomato farms, a large part of very diverse plant viromes can still be unknown and is mostly present in understudied non-crop plants. The overlapping presence of viruses in tomatoes and weeds implicate possible presence of virus reservoir and possible exchange between the weed and crop compartments, which may influence weed management decisions. The observed variability and widespread presence of predominant tomato viruses and the infectivity of a novel tobamovirus in solanaceous plants, provided foundation for further investigation of virus disease dynamics and their effect on tomato health. The extensive insights we generated from such in-depth agroecosystem virome exploration will be valuable in anticipating possible emergences of plant virus diseases and would serve as baseline for further post-discovery characterization studies. Video Abstract.

Mosquitoes are a formidable reservoir of viruses and important vectors of zoonotic pathogens. Blood-fed mosquitoes have been utilized to determine host infection status, overcoming the difficulties associated with sampling from human and animal populations. Comprehensive surveillance of potential pathogens at the interface of humans, animals, and the environment is currently an accredited method to provide an early warning of emerging or re-emerging infectious diseases and to proactively respond to them. Herein we performed comprehensive sampling of mosquitoes from seven habitats (residential areas, hospital, airplane, harbor, zoo, domestic sheds, and forest park) across five cities in Guangdong Province, China. Our aim was to characterize the viral communities and blood feeding patterns at the human-animal-environment interface and analyze the potential risk of cross-species transmission using meta-transcriptomic sequencing. 1898 female adult mosquitoes were collected, including 1062 Aedes and 836 Culex mosquitoes, of which approximately 12% (n = 226) were satiated with blood. Consequently, 101 putative viruses were identified, which included DNA and RNA viruses, and positive-stranded RNA viruses (+ssRNA) were the most abundant. According to viral diversity analysis, the composition of the viral structure was highly dependent on host species, and Culex mosquitoes showed richer viral diversity than Aedes mosquitoes. Although the virome of mosquitoes from different sampling habitats showed an overlap of 39.6%, multiple viruses were specific to certain habitats, particularly at the human-animal interface. Blood meal analysis found four mammals and one bird bloodmeal source, including humans, dogs, cats, poultry, and rats. Further, the blood feeding patterns of mosquitoes were found to be habitat dependent, and mosquitoes at the human-animal interface and from forests had a wider choice of hosts, including humans, domesticated animals, and wildlife, which in turn considerably increases the risk of spillover of potential zoonotic pathogens. To summarize, we are the first to investigate the virome of mosquitoes from multiple interfaces based on the One Health concept. The characteristics of viral community and blood feeding patterns of mosquitoes at the human-animal-environment interface were determined. Our findings should support surveillance activities to identify known and potential pathogens that are pathogenic to vertebrates.

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.