Vultures, as nature’s most successful obligate scavenging birds, play crucial ecological, cultural, and public health roles by consuming rotting carcasses in natural and anthropogenic environments (García-Jiménez et al., 2022). With 23 vulture species worldwide, evolved from separate phylogenetic lineages including New World vultures (7 species in the family Cathartidae) and Old World vultures (16 species in the family Accipitridae) (Jarvis et al., 2014), these avian groups face significant threats. Specifically, approximately 73% of vulture species are at a high risk of extinction, with 77% of global populations experiencing dramatic declines in the late 20th century (Buechley and Sekercioglu, 2016). The decline is attributed to various factors such as dietary toxins [residual veterinary drugs in livestock carcasses (Swan et al., 2006; Adawaren et al., 2018) and poisonous baits (Margalida et al., 2019; van den Heever et al., 2019)], habitat loss, lack of food resources, nest abandonment, low reproductive rate, hunting for traditional medicine or belief-based use (Mckean et al., 2013), climate change (Marneweck et al., 2021), heavy metal bioaccumulation (Yamac et al., 2019; Bassi et al., 2021), and collisions with wind turbines (de Lucas et al., 2012). Consequently, political measures, such as banning the veterinary use of diclofenac in India, Pakistan, Nepal, and Bangladesh, as well as establishing supplementary feeding stations, are being implemented for vulture protection and conservation efforts (Cortes-Avizanda et al., 2010; Moleon et al., 2014; Morales-Reyes et al., 2018; Blanco et al., 2019).

Eight Old World vulture species inhabit China, with three species (Gypaetus barbatus, Aegypius monachus, and Gyps himalayensis) widely distributed in the Qinghai-Tibetan Plateau and performing the crucial ecological function of removing the rotting carcasses of domestic animals (e.g., yaks and Tibetan sheep) and wildlife, which could be disease sources, from the plateau. Among these three vulture species, the Himalayan vulture G. himalayensis has larger populations compared with the other species (Lu et al., 2009) and is more accessible for scientific research. Currently listed as “Near Threatened” on the International Union for Conservation of Nature’s Red List, Himalayan vultures may reach “Vulnerable” status owing to ongoing population decline (Paudel et al., 2016). However, limited research on this species hampers effective conservation efforts.

The gastrointestinal tracts of animals harbor complex microbial ecosystems (viruses, bacteria, archaea, fungi, and protists), forming coevolutionary relationships with hosts and playing vital roles in digestion, nutrient acquisition, immune regulation, development, disease resistance, and overall health (Colston and Jackson, 2016; Kumar et al., 2022). Although research on gut bacterial microbiomes in wild birds (representing 30% of known tetrapod diversity) is progressing rapidly (Bodawatta et al., 2021), studies on gut virome diversity (viral metagenomes) in birds have progressed comparatively slowly. However, the importance of studying viromes in birds and other wild animals has increased owing to the need for better prevention and control of emerging viral infectious diseases originating from wildlife (Albery et al., 2021; Chaffringeon et al., 2021; Li et al., 2021). The emergence of next-generation sequencing and improved bioinformatics tools has facilitated the identification of eukaryotic and prokaryotic viral sequences, revealing new pathogenic viruses in various bird species as well as their role in viral disease transmission (Shan et al., 2022; Zhu et al., 2022). However, our understanding of bird viromes is still in its early stages (François and Pybus, 2020).

Previous studies have shown that scavenging vultures could be potential reservoirs of bacterial pathogens due to their feeding habits (Roggenbuck et al., 2014; Zepeda Mendoza et al., 2018; Wang et al., 2021). However, the gut viromes of vultures, as vital components of the microbiome, remain poorly understood. Therefore, understanding the viral composition, and function associated with vultures is essential for conservation efforts. The host–gut virome alliance is influenced by various factors, including host genetics, diet, geographical environment, age, gender, lifestyle, and physiological status (Perlman et al., 2022). Additionally, captivity has been shown to alter the gut microbial composition and diversity in various vertebrates, including mammals, birds, fish, reptiles, and amphibians, potentially due to differences in diets and living environments in captivity compared with the wild (Hird, 2017; McKenzie et al., 2017). Therefore, it is important to investigate the changes that occur in gut viromes between wild Himalayan vultures and those in captivity, where artificially provided food is relatively clean and fresh compared with the complex scavenging diets of wild populations. In this study, we aimed to analyze the composition, diversity, and function of the gut virome in Himalayan vultures in different living environments, comparing captive and wild populations. Using viral metagenomic sequencing, given its capacity for detecting known and unknown viruses in the animal host, we aimed to (1) investigate gut viral compositional and functional variations between captive and wild Himalayan vultures, and (2) analyze the phylogenetic diversity of the main virus groups. To the best of our knowledge, this is the first study to analyze and compare the gut virome of Himalayan vultures in different living environments; therefore, our findings will contribute to our understanding of the coevolution and transmission of the gut virome in scavenging vultures and improve management knowledge regarding gut viromes in enteric diseases of captive individuals.

In the past decade, advancements in next-generation sequencing technologies and bioinformatic analysis of viral metagenomes have greatly facilitated the study of viromes in diverse wildlife species (Mokili et al., 2012; Li et al., 2021). These methods have allowed for the characterization of both known and unknown viruses in various wildlife species, including mosquitoes, ticks, bats, and rodents (Zhou et al., 2022). Among these species, wild birds exhibit high biodiversity and serve as significant reservoirs of different viruses, such as avian influenza virus, Newcastle disease virus, and West Nile virus which have the potential for cross-species transmission, posing a serious threat to human health (Barroso et al., 2021) especially considering their migration, and flocking behavior. Despite the growing body of research on the bacteriome of birds, our understanding of bird viromes remains limited (François and Pybus, 2020; Tyson-Pello and Olsen, 2020). Only a few previous studies have explored bird viromes, providing valuable insights into the composition, function, and evolution of avian viruses. For instance, Shan et al. (2022) conducted an extensive investigation of viral communities in cloacal swabs collected from 3,182 wild and breeding birds belonging to 10 different orders within the Aves class across eight provinces in China, resulting in the identification of 707 viral genomes. This large-scale study significantly expanded our knowledge of avian viral diversity. Although most avian virus studies have focused on birds from the orders Anseriformes and Galliformes, which are closely related to common domestic poultry (Ramírez-Martínez et al., 2018; Vibin et al., 2020), as well as resident birds in the order Passeriformes (Truchado et al., 2020), there is a lack of information regarding viral diversity, evolution, and ecology in scavenging birds from the Qinghai-Tibetan Plateau, particularly vulture species (Zhu et al., 2022). Therefore, investigating the viromes of understudied Himalayan vulture populations can contribute to the prevention of potential viral outbreaks in the future, given their feeding habits that involve consuming the carcasses of various wild and domestic animals on the plateau. In this study, we conducted the first comprehensive analysis of the gut DNA viromes in Himalayan vultures using viral metagenomics, aiming to explore their composition, function, and the differences between wild and captive individuals.

Keeping birds in captivity in zoos represents a drastic departure from their natural living environment, which can have profound effects on their gut bacterial diversity. Previous studies have demonstrated a significant reduction in gut bacterial diversity in captive birds compared with their wild counterparts (Oliveira et al., 2020; Wu et al., 2021). Consistent with these findings, our study revealed a noteworthy decrease in the richness and diversity of viruses in the captive groups, consistent with the previous investigations of the gut bacteriome. Captive birds undergo various changes that likely impact their gut microbiome, including alterations or limitations in diet, antibiotic treatments, reduced exposure to diverse microbial communities from different habitats, and heightened contact with human-associated microbes (McKenzie et al., 2017). The diet provided to the Himalayan vultures in the zoo was consistent and hygienic, and the relatively confined living area restricted their exposure to microbes, potentially explaining the lower diversity observed in their gut viromes. Due to small sample sizes, the PERMANOVA p-value for beta-diversity was not significant. The resulting NMDS plot appeared to show that the high degree of separation between the two groups across the first axis. Changes in beta-diversity further supported the notion of stable gut viral communities in captive individuals, distinct from those observed in the wild group. Unsurprisingly, the shared diet, and living environment contribute to the higher similarity in gut viromes within the zoo group. Conversely, free-living individuals from the wild group, with their expansive territories, and diverse diets, exhibited a certain degree of variation in their gut viromes.

In line with the prevalence of bacteria in the gut microbiome, gut viral communities appear to be primarily composed of prokaryotic viruses (Dion et al., 2020). In this study, 41 viral families and 140 viral genera were identified, primarily consisting of bacteriophages. Within the wild group, 64.94% of gut DNA virome sequences exhibited homology to phages from the Siphoviridae, Microviridae, Myoviridae, and Inoviridae families. In the zoo group, 79.55% of gut DNA virome sequences showed homology to phages from the Siphoviridae and Herelleviridae families. Bacteriophages, the apex predators of the bacterial realm, were found to be the most abundant group of viruses in the gut virome of humans and other animals (Liang and Bushman, 2021; Harvey and Holmes, 2022). Bacteriophages can influence the structure of the gut bacteriome and maintain high levels of microbial community diversity through kill-the-winner dynamics (Maslov and Sneppen, 2017). We hypothesized that bacteriophages likely play a vital role in modulating the bacterial community in the gut of Himalayan vultures, particularly the dominant microbial members of the gut microbiota. However, in our study, we solely predicted the putative hosts of the viral contigs and did not sequence the bacteriome. The interactions between bacteriophages and bacteria are intricate and challenging to predict owing to the considerable genetic diversity present in bacterial and bacteriophage populations. Therefore, future investigations should incorporate experimental evidence to elucidate these interactions and enhance our understanding of the impact of phages within the gastrointestinal environment.

In the wild group, a certain proportion of viral sequences exhibited homology with animal viruses from the Mimiviridae and Herpesviridae families, plant viruses from the Phycodnaviridae and Geminiviridae families, and insect viruses from the Baculoviridae and Iridoviridae families. In the zoo group, a certain proportion of viral sequences displayed homology with animal viruses from the Poxviridae, Herpesviridae, and Anelloviridae families, plant viruses from the Phycodnaviridae and Caulimoviridae families, insect viruses from the Baculoviridae family, and archaea viruses from the Bicaudaviridae and Lipothrixviridae families. These findings highlight the high diversity of viral communities in Himalayan vultures, and the presence of animal, plant, and insect viruses often reflects their acquisition from the host’s diet and living environment. As scavenger birds, wild Himalayan vultures consume numerous bacteria and viruses present in decaying food. Although this could explain the origin of these gut viruses, it remains unclear whether the viruses selected and retained by the host during the long evolutionary process can fulfill beneficial physiological functions or protective roles similar to those played by gut commensal bacteria, and this matter requires further research for confirmation. In captive Himalayan vultures, the diversity and abundance of the gut microbiota undergo changes due to dietary restrictions and the captive living environment. Thus, changes in the composition and abundance of the gut virome may result from direct or indirect effects associated with changes in the gut microbiota between wild and captive states.

The functional capacity of the gut virome was characterized by the identification of 93 KEGG pathways, which exhibited a high enrichment in metabolism and genetic information processing. This finding aligns with the predicted function of the gut bacteriome in Himalayan vultures, as previously reported by our research group (Wang et al., 2021), in which metabolism (47.30%) and genetic information processing (18.93%) ranked in the first two categories. Metabolism, as the most prevalent functional category, likely signifies a high metabolic activity within the viral communities. This could be attributed to the collection of fecal samples after the Himalayan vultures had completed their feeding. Another notable functional category is genetic information processing, particularly the replication and repair pathway, which may be induced by increased exposure to harmful substances from carrion-borne pathogens. Furthermore, a diverse array of AMGs, including CAZymes and ARGs, were identified in the gut virome of Himalayan vultures. The presence of these AMGs in viruses is presumed to enhance host metabolism and facilitate the generation of new viruses. In our study, we observed a significant presence of viral CAZymes, including core hydrolysis enzymes essential for the degradation of complex polysaccharides. These results were in line with our previously published prediction of gut microbiota function in Himalayan vultures, where carbohydrate metabolism (22.97%) was the highest subtype under the metabolic category (Wang et al., 2021). We speculate that these viral CAZymes might participate in the decomposition and utilization of complex carbohydrates, thereby promoting viral replication within the gut ecosystem of Himalayan vultures. Notably, both the bacteriome and virome in bird guts are considered reservoirs of ARGs. The presence of ARGs encoded by gut viruses in Himalayan vultures suggests their potential involvement in the carriage and transmission of functional ARGs, although further investigation is required to confirm this hypothesis.

This study includes several limitations that provide a direction for future research. First, the small sample size is an obvious limitation, primarily because collecting Himalayan vultures feces in both wild and captive settings is challenging. Fresh feces are difficult to collect given the irregularity of excretion from the birds. Virome sequencing is also expensive, which is another objective reason for the low sample size of this study. In future works, a larger sample size of Himalayan vultures and broader geographic area coverage, including more wild environments, are needed. Second, although many software and approaches have been developed for viromes analyzes, the process from sample collection, sequencing preparation to bioinformatics analysis is challenging, and can lead to biases. For example, many viruses occur as integrated prophages in the genomes of bacteria. In our study, the enrichment of virus-like particles before high-throughput sequencing can reduce the background noise of cellular organisms and enrich extracellular viruses to a certain extent, but unfortunately cannot capture intracellular viruses such as phages. Identifying the host range of viral sequences is also essential for understanding gut viral communities as a whole. Computational methods used to predict bacteriophage hosts fall into two categories: sequence alignment-dependent or-independent. Alignment-dependent methods are usually performed by screening for CRISPR spacers matching to phages. However, only about 40% of bacteria carry a CRISPR-Cas system (Horvath and Barrangou, 2010). Alignment-independent tools tend to exhibit relatively low accuracy (Ahlgren et al., 2017). In this study, VPF-Class, as the alignment-dependent method, was used to predict the hosts of viral contigs based on the assignment of their proteins to a set of classified viral protein families. In the future, we anticipate that alignment-independent and alignment-based approaches will be integrated to improve the overall sensitivity of host assignments without compromising accuracy. Lastly, the lack of sufficient virome databases limits our ability to identify additional viruses that may be present in our viral sequencing data, which can only be addressed by significantly expanding the virome sequences and annotations in existing databases. The limitations above do not affect the robustness of the current results, but follow-up studies in a larger sample sizes will still complement some of the shortcomings of the current study and provide additional new findings.

In conclusion, our study provides initial insights into the DNA viral composition of the gut virome in Himalayan vultures. The comparative analysis of viromes enhances our understanding of viral communities in wild and captive Himalayan vultures. However, future studies should validate these findings using cultivation-based whole-genome sequencing. Additionally, broader sampling from different locations is necessary to enhance our understanding of the viral diversity present in the gastrointestinal tracts of this scavenger bird species.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was reviewed and approved by this animal study was reviewed and approved by the Ethical Committee of Qinghai University.

CL and WW designed the experiments. YW, BT, SZ, SH, and WZ completed the field sampling. JZ, HC, JL, FL, and YB performed the data analysis and prepared the figures. JZ and WW wrote the manuscript. ZL, KS, CL, and WW contributed to the revision of manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by the National Natural Science Foundation of China (grant No. 31960277); the National Natural Science Foundation of China and Russian Foundation for Basic Research Cooperative Exchange Project (grant No. 32111530018, 21-54-53031); the Program of Science and Technology International Cooperation Project of Qinghai Province (grant No. 2022-HZ-812).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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