Metagenomics analysis of viruse populations from feces of diarrheal geese in Sichuan Province

doi:10.21203/rs.3.rs-5330999/v1

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Metagenomics analysis of viruse populations from feces of diarrheal geese in Sichuan Province

https://doi.org/10.21203/rs.3.rs-5330999/v1

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In recent years, novel viruses have brought serious economic losses to the goose industry. In this study, we performed viral metagenomics analysis on fecal samples from sick geese with clinical symptoms of diarrhea in a goose farm in Sichuan, China. The results showed that six viruses belonging to four virus families were identified in the fecal samples, including goose calicivirus, a member of the proposed genus sanovirus in the family Caliciviridae; two novel species in Picornaviridae, goose megrivirus and goose picornavirus; goose astrovirus type 1, a lesser-reported member of goose astrovirus; and partial segments of Rotavirus F and Rotavirus G. All of these viruses are associated with avian diarrhea and may be the pathogens causing diarrhea in geese on this farm. This research enhances the diversity of viruses that infect geese and serves as a valuable reference for the prevention and control of goose diseases.

goose

viral metagenome

phylogenetic analysis

Avian is an important host for zoonotic viruses, for example, waterfowl (geese and ducks) are natural hosts of influenza A (H5N1) viruses and play an important role in the transmission of the viruses (Ringenberg et al., 2024); enteroviruses such as Rotaviruses and Astroviruses(AstV) cause human and avian diarrhea (Dhama et al., 2015), and these viruses pose serious hazards to human health and husbandry. China is one of the most developed countries in the world for goose farming. In recent years, novel viruses have caused serious economic losses to the goose industry, such as goose astrovirus (GoAstV) (Xiang et al., 2024), goose reovirus (GRV) (Zhang et al. 2024), novel goose parvovirus (NGPV) (Zhu et al., 2024), and so on. Therefore, the detection, host range and evolutionary relationship assessment of virus carried by geese is crucial.

Viral metagenomics is used to sequence and identify viruses by high-throughput sequencing technology to reveal information on virus diversity, genome structure, gene function, and more. It has been widely used in the differential diagnosis of infectious diseases. For instance, Sun utilized viral metagenomics to detect a variety of novel picornaviruses in fecal samples of bar-headed goose (Sun et al., 2024). In this study, we performed viral metagenomics on the fecal samples of diarrheal geese from a goose farm in Sichuan, and analyzed the genetic evolution of the detected viruses in order to determine the species of viruses in the fecal samples of diarrheal geese and their genomic characteristics, which can provide data support for prevention and control of goose diseases.

Clinical symptoms and sample collection

The disease was observed in approximately 2000 geese aged of 1–3 weeks raised in a goose farm in Sichuan Province, and the mortality peak was 50–70 geese/day. The main clinical symptoms of the sick geese included diarrhea, mental depression, and emaciation. Enteritis was found in the postmortem examination of the dead geese. Thirty fecal samples were collected from geese with diarrhea.

Viral metagenomics analysis of viruses in fecal samples

The fecal samples of the diseased geese were added to sterile PBS in the ratio of 1:5, vortexed and mixed. After centrifugation at 12000 rpm and 4℃ for 15 min, the supernatant was collected and filtered through a 0.22 µm filter to remove bacteria. After treatment, 30 samples were mixed into a 1.5 mL mixture with 50 µL of each sample. FastPure Viral DNA/RNA Mini Kit (Vazyme, Nanjing, China) was used to extract total RNA from the sample. The qualified total RNA was sequenced by Illumina NovaSeq 6000 by Sangon Bioengineering (Shanghai). The raw data were quality-controlled to remove raw reads, and the clean reads were subjected to downstream analysis. Host-contaminated sequences were removed using minimap2 software. Species annotation of reads was performed using Kraken2. Sequences were obtained by assembling the optimized clean reads for each sample using metaSPAdes (http://spades.bioinf.spbau.ru/release3.11.0/) software to screen for Contigs ≥ 1kb length and cyclization. Virus-derived Contigs were identified using VirSorter2 and VirFinder software.

Phylogenetic analysis of the complete genome of the virus

The complete genome sequence of the virus was downloaded from the GenBank database, and the multiple sequence alignment was performed by Clustal W in the software MEGA-X. The phylogenetic tree was constructed based on the Neighbor-joining method, and the p-distance method was used to calculate the genetic distance. The bootstrap was set to 1000. Identity analyses were performed using the Clustal W in MegAlign software.

Illumina sequencing results

A total of 47,067,100 raw reads were obtained from goose feces samples using the Illumina Novaseq 6000 platform, and a total of 200,218 clean reads related to avian viruses were obtained after removing host, fungal, and bacterial genes. The viruses annotated in the feces samples included four virus families, in the following order of reads number: Astroviridae, Picornaviridae, Caliciviridae, Sedoreoviridae; six viruses were annotated at the species level, in the following order of reads number: GoAstV, goose calicivirus, Rotavirus F, Rotavirus G, goose picornavirus, goose megrivirus (Fig. 1).

Genome and phylogenetic analysis of GoAstV

A total of 41,424 reads were annotated to GoAstV in the feces samples, accounting for 20.69% of the total number of reads, and a complete GoAstV genome was assembled, named AAstV/Goose/CHN/2023/C102. The length of the assembled GoAstV-C102 was 7268 nt, and the base distribution was A (32.08%), T (26.28%), C (18.05%), and G (23.59%). Identity analyses showed that GoAstV-C102 had the highest identity with GoAstV TZ03 (97.38%), followed by GoAstV-1 representative strain FLX (96.68%). Based on the phylogenetic tree of the complete genome sequence of AstV, the strain was clustered into goose astrovirus type 1 (GoAstV-1) (Fig. 2).

Genome and phylogenetic analysis of goose calicivirus

A total of 34,670 reads were annotated to goose calicivirus, accounting for 17.32% of the total reads. A complete genome of goose calicivirus was constructed and named goose calicivirus SC01/SWUN/2023. The length of the assembled SC01 was 8,116 nt, and the base distribution was A(25.91%), T(25.10%), C(26.56%), and G(22.42%). Goose calicivirus SC01 had the highest identity (91.99%) with goose calicivirus MA/CHN/2018, followed by goose calicivirus representative strain H146 (91.30%). The phylogenetic tree based on the complete genome sequence of goose calicivirus showed that the strain was clustered into a single branch with two other Caliciviruses, which belonged to unclassified Caliciviridae (Fig. 3).

Genome and phylogenetic analysis of Rotavirus

A total of 33,343 reads were annotated to Rotavirus F and G. 11,666 reads were annotated to Rotavirus F, accounting for 5.83% of the total reads; 21,677 reads were annotated to Rotavirus G, accounted for 10.83% of the total reads. Partial segments of Rotavirus F and G were assembled. The VP1 and NSP3 proteins were annotated in Rotavirus F, and the VP1, VP2, VP3, VP6, VP7, NSP2, NSP3, NSP4, NSP5 and NSP6 were annotated in Rotavirus G .The phylogenetic tree based on VP1 nucleotide sequences showed that Rotavirus strains were mainly clustered into two main branches, including Rotavirus B/G and Rotavirus A/D/F (Fig. 4).

Genome and phylogenetic analysis of goose picornavirus

A total of 19,288 reads were annotated to goose picornavirus in gossamer stool samples, accounting for 9.63% of the total reads, and a complete goose picornavirus genome was assembled. It was named goose picornavirus SC01/SWUN/2023. The length of the assembled SC01 was 8,015 nt, and the base distribution was A(25.02%), T(27.81%), C(24.73%), and G(22.45%). Goose picornavirus SC01 and goose picornavirus Abbotsbury/A/2016 had the highest identity of 90.87%. Phylogenetic tree based on the complete genome sequence of goose picornavirus showed that the strain was clustered into Ludopivirus with three other goose picornaviruses (Fig. 5).

Genome and phylogenetic analysis of goose picornavirus

A total of 15,251 reads were annotated to goose megrivirus, accounting for 7.61% of the total reads, and two complete Megrivirus genomes were assembled, named goose megrivirus A3/SC01/China/2023 and goose megrivirus A3/SC02/China/2023. The length of SC01 strain was 9,945 nt, and the base distribution was A(25.39%), T(28.38%), C(24.60%) and G(21.64%). Megrivirus SC01 and Megrivirus A3 isolate W18 had the highest identity of 91.06%. The length of SC02 strain was 9,984 nt, and the base distribution was A(25.30%), T(28.21%), C(24.80%) and G(21.69%). SC02 strain also had the highest identity with W18 strain (90.68%). The identity between SC01 and SC02 was 95.6%. The phylogenetic tree based on the complete genome sequence of Megrivirus showed that the two strains identified in this study were clustered into a small branch with Megrivirus A3 isolate W18 and a large branch with the rest of Megrivirus A strains, suggesting that the two strains were classified as Megrivirus A genotype, Megrivirus A3 subgenotype (Fig. 6).

Virus metagenomics based on high-throughput sequencing is of great value for the rapid identification of novel viruses, and the comprehensive characterization of viruses carried by animals is of critical importance for disease prevention and control, animal health and public health. In this study, we performed viral metagenomics analysis on fecal samples of diarrheal geese collected from a goose farm in Sichuan Province, China, in July 2023, and the results showed that the fecal samples contained six viruses from four virus families, including goose calicivirus, goose megrivirus, Rotavirus F, Rotavirus G, goose picornavirus, and goose astrovirus, and phylogenetic analysis of the six viruses were further analyzed.

In this study, we identified two novel viruses of Picornaviridae, goose megrivirus and goose picornavirus. Megrivirus is a new genus of the Picornaviridae, which contains five species, Megrivirus A, Megrivirus B, Megrivirus C, Megrivirus D, and Megrivirus E (Zhang et al., 2023), and the hosts include ducks (Liao et al., 2014), geese (Wang et al., 2017), chickens (Haji et al., 2023), turkeys ( Boros et al., 2013), penguin (Yinda et al., 2019) and harrier (Boros et al., 2017) among other avian species. In this study, the goose megrivirus SC01 and SC02 exhibited the highest nucleotide identity with the goose megrivirus W18, followed by another goose megrivirus HN56 and duck-origin Megrivirus LY. Based on the results of phylogenetic analysis, the identified of the two goose megrivirus strains were classified as Megrivirus A. Goose Picornavirus SC01 had the highest identity with goose picornavirus Abbotsbury/A/2016 at 90.87%, and according to the phylogenetic analysis results, the goose picornavirus SC01 was classified as Ludopivirus, the epidemiological and pathological characteristics of Ludopivirus are currently unknown and need to be further studied. Avian species infected with Picornaviruses can be reservoir hosts for novel Picornaviruses viruses in humans (Woo et al., 2006). Comprehensive detection of Picornaviruses carried by avian species is of great significance for disease prevention and control.

Caliciviridae are non-encapsulated, single-stranded, positive-strand RNA viruses characterized by cup-shaped depressions. ICTV classifies Caliciviridae into 11 genera: Bavovirus, Lagovirus, Minovirus, Nacovirus, Nebovirus, Norovirus, Recovirus, Salovirus, Sapovirus, Valovirus, and Vesivirus, many Caliciviruses remain unclassified. Some members of the Caliciviridae are significant pathogens associated with gastroenteritis in humans and respiratory diseases in felines. For example, Norovirus can cause acute gastroenteritis in humans (de Graaf et al. 2016). In recent years, the virus has begun to be detected in avian feces. Wolf S et al. detected a calicivirus in the intestinal contents of a chicken suffering from developmental delay (Wolf et al., 2011). Wang detected goose calicivirus H146 in the intestinal contents of geese with enteritis, which was highly consistent with goose calicivirus SC01 found in this study, and both were unclassified caliciviruses. According to Wang's study, goose calicivirus SC01 can be classified as a member of the proposed genus: Sanovirus (Wang et al., 2017).

Rotavirus is a segmented double-stranded RNA virus, classified into groups A to H. Rotavirus is one of the significant pathogens causing viral diarrhea in humans and animals (Dhama et al., 2015). In this study, most of the segments of Rotavirus G and a few segments of Rotavirus F were obtained, and the incomplete genomic information obtained may be due to the low abundance of Rotaviruses in the sample. According to the results of phylogenetic analysis, Rotavirus F is most closely related to Rotavirus A/D, and Rotavirus G is most closely related to Rotavirus B. This result is consistent with the findings of Kindler E et al. (Kindler et al., 2013), which revealed the Rotavirus evolved in two separate evolutionary branches (Johne et al., 2011).

AstV are non-enveloped, single-stranded, sense RNA viruses, which are divided into Mamastrovirus and Avastrovirus genera. AstV are one of the important pathogens causing diarrhea in humans and animals (Li et al., 2022). GoAstV is a novel astrovirus, which was first identified in 2017. It can be divided into GoAstV-1 represented by FLX strain (Zhang et al., 2017), and GoAstV-2 represented by SD01 strain (Zhang et al., 2018). At present, GoAstV is an unclassified Avastrovirus. Infection with GoAstV leads to gout and growth retardation in goslings, with a mortality rate of up to 70% (Xu et al., 2023), causing huge losses to the goose industry. In this study, a near full-length GoAstV-1 genome sequence was obtained, and GoAstV-C102 showed the highest concordance with GoAstV-1 TZ03 (Wang et al., 2021) at 97.38%, followed by GoAstV-1 representative strain FLX at 96.68%. Notably, geese in this study did not exhibit typical gout symptoms; instead, they primarily showed pathological signs of enteritis, similar to the FLX strain isolated from geese with enteritis (Zhang et al., 2017). The pathogenic characteristics of GoAstV-C102 require further investigation.

In conclusion, six viruses from four virus families were identified from the feces of diarrhea-affected geese in a goose farm in Sichuan using viral metagenomics. The genetic evolution of these viruses was analyzed to clarify their genetic characteristics, which enriched the diversity of viruses that infect geese and provided a reference for the prevention and control of goose disease.

Funding

This work was supported by Central University Basic Scientific Research Business Expenses Special Funds (ZYN2024087).

Conflict of interest

The authors have no competing interests to declare.

Ethics approval and consent to participate

This study was conducted in accordance with the ethical guidelines of the College of Animal and Veterinary Sciences, Southwest Minzu University. All animal experiments were approved by the Ethical Committee for Animal Experiments of Southwest Minzu University. The research involved the use of deceased animals, and the animals used in this study were found dead prior to the research due to natural causes. The origin of the deceased animals was a goose farm in Sichuan, China, where the geese had exhibited symptoms of diarrhea and enteritis. No animals were sacrificed specifically for this study.

Data availability

The data supporting this study’s findings are in the manuscript and are also available from the corresponding author upon reasonable request.

Author Contributions

Huanrong Zhang was responsible for the design of the experiments and made significant revisions to the manuscript. Guo Chen was responsible for the execution of the experiments. Rongzhi Ma was responsible for the analysis of the data and drafted the initial manuscript. Both authors read and approved the final manuscript.

Data Availability Statement

The sequence data generated in this study have been deposited in approved databases with the following accession numbers:

Organism	Strain	Accession NO.
rotavirus G	SC01/SWUN/2023/G01 VP1	PQ465873
rotavirus G	SC01/SWUN/2023/G02 VP2	PQ465874
rotavirus G	SC01/SWUN/2023/G03 VP3	PQ465875
rotavirus G	SC01/SWUN/2023/G06 VP6	PQ465876
rotavirus G	SC01/SWUN/2023/G07 VP7	PQ465877
rotavirus G	SC01/SWUN/2023/G12 NSP2	PQ465878
rotavirus G	SC01/SWUN/2023/G14 NSP4	PQ465879
rotavirus G	SC01/SWUN/2023/G15 NSP5	PQ465880
rotavirus F	SC01/SWUN/2023/F01 VP1	PQ465881
rotavirus F	SC01/SWUN/2023/F13 NSP3	PQ465882
goose calicivirus	SC01/SWUN/2023	PQ368549
goose megrivirus	SC01/China/2023	PQ368550
goose megrivirus	SC02/China/2023	PQ368551
goose picornavirus	SC01/SWUN/2023	PQ368552
goose astrovirus	AAstV/Goose/CHN/2023/C102	PP763758

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No competing interests reported.

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You are reading this latest preprint version

Metagenomics analysis of viruse populations from feces of diarrheal geese in Sichuan Province

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Clinical symptoms and sample collection

Viral metagenomics analysis of viruses in fecal samples

Phylogenetic analysis of the complete genome of the virus

RESULTS

Illumina sequencing results

Genome and phylogenetic analysis of GoAstV

Genome and phylogenetic analysis of goose calicivirus

Genome and phylogenetic analysis of goose picornavirus

Genome and phylogenetic analysis of goose picornavirus

DISCUSSION

Declarations

References

Additional Declarations

Status:

Version 1