Viral Diversity Research Articles

Full-genome sequencing of dozens of new DNA viruses found in Spanish bat feces.

Metagenomics has become a fundamental tool to characterize the global virosphere, allowing us not only to understand the existing viral diversity and its ecological implications but also to identify new and emerging viruses. RNA viruses have a higher zoonotic potential, but this risk is also present for some DNA virus families. In our study, we analyzed the DNA fraction of fecal samples from 22 Spanish bat species, identifying 52 complete or near-complete genomes of different viral families with zoonotic potential. This doubles the number of genomes currently described in Europe. Metagenomic data often produce partial genomes that can be difficult to analyze. Our work, however, has characterized a large number of complete genomes, thus facilitating their taxonomic classification and enabling different analyses to be carried out to evaluate their zoonotic potential. For example, recombination studies are relevant since this phenomenon could play a major role in cross-species transmission.

Diverse Circular DNA Viral Communities in Blood, Oral, and Fecal Samples of Captive Lemurs.

Few studies have addressed viral diversity in lemurs despite their unique evolutionary history on the island of Madagascar and high risk of extinction. Further, while a large number of studies on animal viromes focus on fecal samples, understanding viral diversity across multiple sample types and seasons can reveal complex viral community structures within and across species. Groups of captive lemurs at the Duke Lemur Center (Durham, NC, USA), a conservation and research center, provide an opportunity to build foundational knowledge on lemur-associated viromes. We sampled individuals from seven lemur species, i.e., collared lemur (Eulemur collaris), crowned lemur (Eulemur coronatus), blue-eyed black lemur (Eulemur flavifrons), ring-tailed lemur (Lemur catta), Coquerel's sifaka (Propithecus coquereli), black-and-white ruffed lemur (Varecia variegata variegata), and red ruffed lemur (Varecia rubra), across two lemur families (Lemuridae, Indriidae). Fecal, blood, and saliva samples were collected from Coquerel's sifaka and black-and-white ruffed lemur individuals across two sampling seasons to diversify virome biogeography and temporal sampling. Using viral metagenomic workflows, the complete genomes of anelloviruses (n = 4), cressdnaviruses (n = 47), caudoviruses (n = 15), inoviruses (n = 34), and microviruses (n = 537) were determined from lemur blood, feces, and saliva. Many virus genomes, especially bacteriophages, identified in this study were present across multiple lemur species. Overall, the work presented here uses a viral metagenomics approach to investigate viral communities inhabiting the blood, oral cavity, and feces of healthy captive lemurs.

Analysis of viral diversity in dogs with acute gastroenteritis from Brazilian Amazon

Viral gastroenteritis is commonly reported in dogs and involves a great diversity of enteric viruses. In this research, viral diversity was investigated in dogs with diarrhea in Northern Brazil using shotgun metagenomics. Furthermore, the presence of norovirus (NoV) was investigated in 282 stool/rectal swabs of young/adult dogs with or without diarrhea from two public kennels, based on one-step reverse transcription polymerase chain reaction (RT-PCR) for genogroup VI and VII (GVI and GVII) and real-time RT-PCR for GI, GII, and GIV. Thirty-one viral families were identified, including bacteriophages. Phylogenetic analyses showed twelve complete or nearly complete genomes belonging to the species of Protoparvovirus carnivoran1, Mamastrovirus 5, Aichivirus A2, Alphacoronavirus 1, and Chipapillomavirus 1. This is the first description of the intestinal virome of dogs in Northern Brazil and the first detection of canine norovirus GVII in the country. These results are important for helping to understand the viral groups that circulate in the canine population.

The evolution, complexity, and diversity of swine influenza viruses in China: A hidden public health threat

Swine influenza viruses (SIVs), including H1N1, H1N2, and H3N2, have spread throughout the global pig population. Potential pandemics are a concern with the recent sporadic cross-species transmission of SIVs to humans. We collected 1421 samples from Guangdong, Fujian, Henan, Yunnan and Jiangxi provinces during 2017–2018 and isolated 29 viruses. These included 21H1N1, 5H1N2, and 3H3N2 strains. Genome analysis showed that the domestic epidemic genotypes of H1N1 were mainly G4 and G5 reassortant EA swine H1N1. These genotypes have a clear epidemic advantage. Two strains were Clade 6B.1 pdm/09H1N1, suggesting a possible pig-to-human transmission route. Notably, three new H1N2 genotypes were identified using the genomic backbones of G4 or G5 viruses for recombination. The identification of various subtypes and genotypes highlight the complexity and diversity of SIVs in China and need for continuous monitoring of SIV evolution to assess the risks and prepare for potential influenza pandemics.

Tropical Fruit Virus Resistance in the Era of Next-Generation Plant Breeding

Plant viral diseases constitute a major contributor to agricultural production losses, significantly impacting the economies of exporting countries by more than USD 30 billion annually. Understanding and researching the biology and genomics of viruses is crucial for developing virus-resistant genetically edited or genetically modified plants. Genetic modifications can be targeted to specific regions within genes of target plants which are important or essential for the virus to establish a systemic infection, thus fostering resistance or enabling plants to effectively respond to invading agents while preserving their yield. This review provides an overview of viral incidence and diversity in tropical fruit crops and aims to examine the current state of the knowledge on recent research efforts aimed at reducing or eliminating the damage caused by viral diseases, with emphasis on genetically edited products that have reached the market in recent years.

Refining SARS-CoV-2 intra-host variation by leveraging large-scale sequencing data.

Understanding viral genome evolution during host infection is crucial for grasping viral diversity and evolution. Analyzing intra-host single nucleotide variants (iSNVs) offers insights into new lineage emergence, which is important for predicting and mitigating future viral threats. Despite next-generation sequencing's potential, challenges persist, notably sequencing artifacts leading to false iSNVs. We developed a workflow to enhance iSNV detection in large NGS libraries, using over 130 000 SARS-CoV-2 libraries to distinguish mutations from errors. Our approach integrates bioinformatics protocols, stringent quality control, and dimensionality reduction to tackle batch effects and improve mutation detection reliability. Additionally, we pioneer the application of the PHATE visualization approach to genomic data and introduce a methodology that quantifies how related groups of data points are represented within a two-dimensional space, enhancing clustering structure explanation based on genetic similarities. This workflow advances accurate intra-host mutation detection, facilitating a deeper understanding of viral diversity and evolution.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

Reconstruction of the historic time course of blood-borne virus contamination of clotting factor concentrates, 1974-1992.

Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.

Metagenomic analysis of the gut virome in patients with irritable bowel syndrome.

Irritable bowel syndrome (IBS), a chronic functional gastrointestinal disorder, is recognized for its association with alterations in the gut microbiome and metabolome. This study delves into the largely unexplored domain of the gut virome in IBS patients. We conducted a comprehensive analysis of the fecal metagenomic data set from 277 IBS patients and 84 healthy controls to characterize the gut viral community. Our findings revealed a distinct gut virome in IBS patients compared to healthy individuals, marked by significant variances in between-sample diversity and altered abundances of 127 viral operational taxonomic units (vOTUs). Specifically, 111 vOTUs, predominantly belonging to crAss-like, Siphoviridae, Myoviridae, and Quimbyviridae families, were more abundant in IBS patients, whereas the healthy control group exhibited enrichment of 16 vOTUs from multiple families. We also investigated the interplay between the gut virome and bacteriome, identifying a correlation between IBS-enriched bacteria like Klebsiella pneumoniae, Fusobacterium varium, and Ruminococcus gnavus, and the IBS-associated vOTUs. Furthermore, we assessed the potential of gut viral signatures in predicting IBS, achieving a notable area under the receiver operator characteristic curve (AUC) of 0.834. These findings highlight significant shifts in the viral diversity, taxonomic distribution, and functional composition of the gut virome in IBS patients, suggesting the potential role of the gut virome in IBS pathogenesis and opening new avenues for diagnostic and therapeutic strategies targeting the gut virome in IBS management.

Characterization of CRESS-DNA viruses in human vaginal secretions: An exploratory metagenomic investigation.

The Phylum Cressdnaviricota consists of a large number of circular Rep-encoding single-stranded (CRESS)-DNA viruses. Recently, metagenomic analyzes revealed their ubiquitous distribution in a diverse range of eukaryotes. Data relating to CRESS-DNA viruses in humans remains scarce. Our study investigated the presence and genetic diversity of CRESS-DNA viruses in human vaginal secretions. Vaginal swabs were collected from 28 women between 29 and 43 years old attending a fertility clinic in New York City. An exploratory metagenomic analysis was performed and detection of CRESS-DNA viruses was confirmed through analysis of near full-length sequences of the viral isolates. A phylogenetic tree was based on the REP open reading frame sequences of the CRESS-DNA virus genome. Eleven nearly complete CRESS-DNA viral genomes were identified in 16 (57.1%) women. There were no associations between the presence of these viruses and any demographic or clinical parameters. Phylogenetic analysis indicated that one of the sequences belonged to the genus Gemycircularvirus within the Genomoviridae family, while ten sequences represented previously unclassified species of CRESS-DNA viruses. Novel species of CRESS-DNA viruses are present in the vaginal tract of adult women. Although they be transient commensal agents, the potential clinical implications for their presence at this site cannot be dismissed.

Virus ecology and 7-year temporal dynamics across a permafrost thaw gradient.

Soil microorganisms are pivotal in the global carbon cycle, but the viruses that affect them and their impact on ecosystems are less understood. In this study, we explored the diversity, dynamics, and ecology of soil viruses through 379 metagenomes collected annually from 2010 to 2017. These samples spanned the seasonally thawed active layer of a permafrost thaw gradient, which included palsa, bog, and fen habitats. We identified 5051 virus operational taxonomic units (vOTUs), doubling the known viruses for this site. These vOTUs were largely ephemeral within habitats, suggesting a turnover at the vOTU level from year to year. While the diversity varied by thaw stage and depth-related patterns were specific to each habitat, the virus communities did not significantly change over time. The abundance ratios of virus to host at the phylum level did not show consistent trends across the thaw gradient, depth, or time. To assess potential ecosystem impacts, we predicted hosts in silico and found viruses linked to microbial lineages involved in the carbon cycle, such as methanotrophy and methanogenesis. This included the identification of viruses of Candidatus Methanoflorens, a significant global methane contributor. We also detected a variety of potential auxiliary metabolic genes, including 24 carbon-degrading glycoside hydrolases, six of which are uniquely terrestrial. In conclusion, these long-term observations enhance our understanding of soil viruses in the context of climate-relevant processes and provide opportunities to explore their role in terrestrial carbon cycling.

Molecular Diagnosis and Genetic Diversity of Tomato Yellow Leaf Curl Virus in Basra Governorate

Molecular Diagnosis and Genetic Diversity of Tomato Yellow Leaf Curl Virus in Basra Governorate

Eutrophication impacts the distribution and functional traits of viral communities in lakes

Viruses play a crucial role in aquatic ecosystems by regulating microbial composition and impacting biogeochemical cycling. While the response of viral diversity to the trophic status has been preliminarily explored in lake ecosystems, there is limited integrated exploration of the biogeography of viruses, host associations, and the auxiliary metabolic genes (AMGs), particularly for plateau lakes. Therefore, this research investigated the viral biogeography, virus-host association, and AMGs in the surface waters of 11 lakes varying in trophic levels (eutrophic and oligo-mesotrophic) in the Yunnan-Guizhou plateau region of China. A total of 73,105 viral operational taxonomic units were obtained from 11 samples, with 84.8 % remaining unannotated at the family level, indicating a predominance of novel viruses within these lakes. The most abundant viral family was Kyanoviridae (24.4 %), recognized as a common cyanophage. The vast majority of cyanobacteria and several eukaryotic algae were predicted as hosts for the viruses, with a lytic lifestyle predominating the life strategy of these cyanophages, implying the potential influence of the virus on algae. The viral community structure significantly correlated with both trophic status and the bacterial community. The structure equation model analysis revealed chlorophyll a was the primary factor affecting viral communities. Moreover, numerous AMGs linked to carbon metabolism, phosphorus metabolism, sulfur metabolism, and photosynthesis were found in these lakes, some of which showed virus preference for the trophic statuses, suggesting a vital role of the virus in driving biogeochemical cycling in the lake crossing different nutrient levels. In addition, a restricted presence of viruses was found to infect humans or harbor antibiotic resistance genes in the lakes, suggesting a subtle yet potential link to human health. Overall, these findings offer insights into the response of viral communities to eutrophication and their potential role in biogeochemical cycling and controlling algal propagation.

High‐throughput sequencing‐based detection and characterization of sugarcane viruses in Ethiopia

AbstractA virus discovery investigation using high‐throughput sequencing in sugarcane samples from Ethiopia revealed the presence of sugarcane mild mosaic virus (SCMMV, genus Ampelovirus) and sugarcane umbra‐like virus (SULV, genus Umbravirus). The genome sequences of two isolates of SCMMV and one SULV were determined. The sequences of the two SCMMV isolates were 13,005 nucleotides in length and showed ~73.5% nucleotide identity along the genome and ~90.2, 96.8, and 90.4% amino acid sequence identity among each other in the RdRp, CP, and HSP70h, respectively. Isolate SCMMV ET2 showed a close relationship to group A isolates from Colombia, the USA, and the Philippines, with amino acid sequence identity of the predicted virus proteins in the range of 94–98.9%. Conversely, SCMMV ET1 shared a closer relationship with group B isolates from Colombia, Ivory Coast, and Argentina, exhibiting a 93–99% amino acid sequence identity. The complete genome sequence of SULV, comprising 3041 nucleotides, exhibited the highest identity with its counterpart from South Africa (MN868593). These findings contribute to expanding our understanding of the viral diversity within the sugarcane crop in Ethiopia.

Host-virus coevolution drives soil microbial function succession along a millennium land reclamation chronosequence

IntroductionGene exchange between viruses and hosts plays an important role in driving virus-host coevolution, enabling adaptation of both viruses and hosts to environmental changes. However, the mechanisms and functional significance of virus-host gene exchanges over long-term scales remain largely unexplored. ObjectiveThe present study aimed to gain insights into the role of viruses in virus-host interactions and coevolution by monitoring virome dynamics along a millennium-long land reclamation chronosequence. MethodsWe collected 24 soil samples from 8 stages of a millennium-long land reclamation chronosequence, including non-reclamation, and reclamation periods of 10, 50, 100, 300, 500, 700, and 1000 years. We characterized their metagenomes, and identified DNA viruses within these metagenomes. ResultsOur findings reveal a significant shift in viral community composition after 50 years of land reclamation, but soil viral diversity reached a stable phase approximately 300 years after the initial reclamation. Analysis of the virus-host network showed a scale-free degree distribution and a reduction in complexity over time, with generalist viruses emerging as key facilitators of horizontal gene transfer. ConclusionThese findings highlight the integral role of viruses, especially generalist types, in mediating gene exchanges between viruses and hosts, thereby influencing the coevolutionary dynamics in soil ecosystems over significant timescales. This study offers novel insights into long-term virus-host interactions, showing how the virome responds to environmental changes, driving shifts in various microbial functions in reclaimed land.

Viromic and Metagenomic Analyses of Commercial Spirulina Fermentations Reveal Remarkable Microbial Diversity.

Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.

Viral Envelope Evolution in Simian-HIV-Infected Neonate and Adult-Dam Pairs of Rhesus Macaques.

We recently demonstrated that Simian-HIV (SHIV)-infected neonate rhesus macaques (RMs) generated heterologous HIV-1 neutralizing antibodies (NAbs) with broadly-NAb (bNAb) characteristics at a higher frequency compared with their corresponding dam. Here, we characterized genetic diversity in Env sequences from four neonate or adult/dam RM pairs: in two pairs, neonate and dam RMs made heterologous HIV-1 NAbs; in one pair, neither the neonate nor the dam made heterologous HIV-1 NAbs; and in another pair, only the neonate made heterologous HIV-1 NAbs. Phylogenetic and sequence diversity analyses of longitudinal Envs revealed that a higher genetic diversity, within the host and away from the infecting SHIV strain, was correlated with heterologous HIV-1 NAb development. We identified 22 Env variable sites, of which 9 were associated with heterologous HIV-1 NAb development; 3/9 sites had mutations previously linked to HIV-1 Env bNAb development. These data suggested that viral diversity drives heterologous HIV-1 NAb development, and the faster accumulation of viral diversity in neonate RMs may be a potential mechanism underlying bNAb induction in pediatric populations. Moreover, these data may inform candidate Env immunogens to guide precursor B cells to bNAb status via vaccination by the Env-based selection of bNAb lineage members with the appropriate mutations associated with neutralization breadth.

Diversity of Picorna-Like Viruses in the Teltow Canal, Berlin, Germany.

The viromes of freshwater bodies are underexplored. The Picornavirales order, with 371 acknowledged species, is one of the most expansive and diverse groups of eukaryotic RNA viruses. In this study, we add 513 picorna-like viruses to the assemblage of more than 2000 unassigned picorna-like viruses. Our set of the aquatic Picornavirales virome of the Teltow Canal in Berlin, Germany, consists of 239 complete and 274 partial genomes. This urban freshwater body is characterized by the predominance of marna-like viruses (30.8%) and dicistro-like viruses (19.1%), whereas picornaviruses, iflaviruses, solinvi-like viruses, polycipi-like viruses, and nora-like viruses are considerably less prevalent. Caliciviruses and secoviruses were absent in our sample. Although presenting characteristic domains of Picornavirales, more than 100 viruses (20.8%) could not be assigned to any of the 9 Picornavirales families. Thirty-three viruses of the Marnaviridae-mostly locarna-like viruses-exhibit a monocistronic genome layout. Besides a wealth of novel virus sequences, viruses with peculiar features are reported. Among these is a clade of untypeable marna-like viruses with dicistronic genomes, but with the capsid protein-encoding open reading frame located at the 5' part of their RNA. A virus with a similar genome layout but clustering with dicistroviruses was also observed. We further detected monocistronic viruses with a polymerase gene related to aparaviruses. The detection of Aichi virus and five novel posa-like viruses indicates a slight burden in municipal wastewater.

A metagenomic investigation of the faecal RNA virome structure of asymptomatic chickens obtained from a commercial farm in Durban, KwaZulu-Natal province, South Africa

BackgroundVirome studies on birds, including chickens are relatively scarce, particularly from the African continent. Despite the continuous evolution of RNA viruses and severe losses recorded in poultry from seasonal viral outbreaks, the information on RNA virome composition is even scantier as a result of their highly unstable nature, genetic diversity, and difficulties associated with characterization. Also, information on factors that may modulate the occurrence of some viruses in birds is limited, particularly for domesticated birds. Viral metagenomics through advancements in sequencing technologies, has enabled the characterization of the entire virome of diverse host species using various samples.MethodsThe complex RNA viral constituents present in 27 faecal samples of asymptomatic chickens from a South African farm collected at 3-time points from two independent seasons were determined, and the impact of the chicken’s age and collection season on viral abundance and diversity was further investigated. The study utilized the non-invasive faecal sampling method, mRNA viral targeted enrichment steps, a whole transcriptome amplification strategy, Illumina sequencing, and bioinformatics tools.ResultsThe results obtained revealed a total of 48 viral species spanning across 11 orders, 15 families and 21 genera. Viral RNA families such as Coronaviridae, Picornaviridae, Reoviridae, Astroviridae, Caliciviridae, Picorbirnaviridae and Retroviridae were abundant, among which picornaviruses, demonstrated a 100% prevalence across the three age groups (2, 4 and 7 weeks) and two seasons (summer and winter) of the 27 faecal samples investigated. A further probe into the extent of variation between the different chicken groups investigated indicated that viral diversity and abundance were significantly influenced by age (P = 0.01099) and season (P = 0.00099) between chicken groups, while there was no effect on viral shedding within samples in a group (alpha diversity) for age (P = 0.146) and season (P = 0.242).ConclusionThe presence of an exceedingly varied chicken RNA virome, encompassing avian, mammalian, fungal, and dietary-associated viruses, underscores the complexities inherent in comprehending the causation, dynamics, and interspecies transmission of RNA viruses within the investigated chicken population. Hence, chickens, even in the absence of discernible symptoms, can harbour viruses that may exhibit opportunistic, commensal, or pathogenic characteristics.

Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022–2023

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022–April 2023. The number of outbreaks during the winter of 2022–2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022–2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021–2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021–2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022–2023.