In a poultry viral agent discovery project, 40 duck liver samples were collected from live poultry market located in Hengyang city, Hunan province, China, between August and September 2023 (Figure 1). Approximately 50 mg of liver tissue was homogenized with 500 μL sterile phosphate-buffered saline (PBS). Total RNA was extracted from 200 μL homogenates by using RNAiso Plus reagent (TaKaRa, Dalian, China) and subsequently purified using the RNeasy Plus Mini Kit (Qiagen, Germany). The quantity and quality of the extracted RNA were assessed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, United States). Subsequently, the individual RNA solutions were pooled in equal quantities, and the quality of the pooled RNA was evaluated using an Agilent 2,100 Bioanalyzer (Agilent Technologies) before library construction and sequencing.

The RNA library preparation followed the methodology as previously described (12, 13). Briefly, ribosomal RNA (rRNA) was depleted using a Ribo-Zero-Gold (Epidemiology) kit (Illumina Inc., United States) following the manufacturer’s instructions. The remaining RNA was fragmented, reverse-transcribed, adapted, and purified using the TruSeq total RNA library preparation kit (Illumina Inc., United States). The quality of the library quality was assessed using the Qubit high-sensitivity RNA/DNA assays (Thermo Fisher Scientific) and the Agilent 2,100 Bioanalyzer (Agilent Technologies). Paired-end sequencing with 150-bp read length was conducted on the Illumina Hiseq2500 platform. All library preparation and sequencing procedures were carried out by Novogene (Tianjin, China).

The sequencing reads were demultiplexed, trimmed for the adaptor, and subjected to quality control using the fastp program (14). Subsequently, de novo assembly was performed using the Megahit program (15) with default parameters. The resulting contigs were compared against the entire non-redundant protein (nr) database downloaded from GenBank using the diamond blastx program (16) with an e-value threshold of 1e–3. Viral contigs with unassembled overlaps or originating from the same scaffold were merged using the SeqMan program implemented in the Lasergene software package (version 7.1, DNAstar).

To confirm the assembly results, reads were mapped back to the target contigs with Bowtie2 (17), and assembly errors were inspected using Integrated Genomics Viewer (IGV). Any gaps between these contigs were filled by reverse transcription PCR (RT-PCR) and Sanger sequencing (Table 1). The virus genome termini were determined using 5′/3′ RACE kits (TaKaRa, Dalian, China) following the producer’s instructions. The final virus genome sequence was obtained by consensus mapping assembly and confirmed by Sanger sequencing using overlapping primers that covered the entire sequence.

The identification of potential open reading frames (ORFs) was conducted using ORFfinder¹ and annotated by comparing against the non-redundant protein database. The nucleotide sequence similarity between this newly identified Hepacivirus and other hepaciviruses was calculated using the MegAlign program, available in the Lasergene software package (version 7.1, DNAstar).

To determine potential recombination events that occurred in the evolutionary history of this newly identified Hepacivirus, seven methods (RDP, GENECONV, bootscan, maximum chi square, Chimera, SISCAN, and distance plot) within RDP4 program (18) were employed. Analyzes were conducted using complete genome sequences with default settings for the different test methods and a Bonferroni corrected p-value cutoff of 0.05. Only sequences with significant evidence (p < 0.05) of recombination detected by at least two methods and confirmed by phylogenetic analysis were considered to have strong evidence for recombination. Furthermore, similarity plot analyzes were performed to further characterize potential recombination events, including the location of possible breakpoints, using Simplot version 3.5.1 (19).

To determine the phylogenetic relationship between the newly identified Hepacivirus and other known hepaciviruses, amino acid sequences of the complete polyprotein, NS3 (peptidase and helicase), and NS5 (RNA-dependent RNA polymerase) proteins were aligned using the E-INS-i algorithm implemented in MAFFT program (20). Phylogenetic trees were reconstructed using the maximum-likelihood method (ML) implemented in PhyML version 3.0 (21). The LG amino acid substitution model with a gamma (Γ)-distribution model (i.e., LG + Γ) determined using Prot-Test 3 (22), was employed. Bootstrap support values were calculated from 1,000 replicate trees using a Subtree Pruning and Regrafting (SPR) branch-swapping algorithm. For clarity purposes, all phylogenetic trees were rooted at the mid-point.

To gain insight into the prevalence of the newly identified Hepacivirus circulating in domestic ducks, liver tissues were collected from live poultry markets located in five cities in Hunan (Figure 1). All individual samples, including those that subjected to meta-transcriptome sequencing, were screened for the presence of the virus using PCR with primers targeting the NS5B region (Table 1). The PCR product with the expected size was confirmed by Sanger sequencing.

Statistical analyzes were conducted using Statistical Package for Social Sciences (SPSS) Version 21.0 software. Descriptive statistics were used to calculate frequency and percentage, while Fisher exact test was utilized to determine the p-value and assess the differences in positive rates between sampling sites. A p value <0.05 was considered as statistically significant.

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The procedures for sampling and sample processing were approved by the ethics committee of Foshan University. All animals were treated in strict accordance with the Rules for the Implementation of Laboratory Animal Medicine (1998) from the Ministry of Health, China.

In a viral agent discovery project involving domestic ducks, RNA solutions extracted from 40 individual liver tissues were pooled as one sample. This sample was screened for both known and putative novel viruses through meta-transcriptome sequencing. After de novo assembly and comparison against the nr database, 20 contigs ranging from 257 to 995 nt in length were annotated as Hepacivirus Q, with 95.4 to 100% amino acid identities. After filling the gaps between these contigs through RT-PCR, and determining the terminal sequences using 5′/3′ RACE, the complete genome sequence of this virus was obtained. Using this sequence as the reference sequence, 393 reads were remapped, providing a genome coverage of 92.8% (9,194 nt/9,904 nt) and a pairwise identity of 96.2% at a mean depth of 9.9× (Figure 2).

This newly identified Hepacivirus has a complete genome sequence comprising 9,893 nucleotides and contains a single large ORF that encodes a polyprotein of 2,999 amino acids. Comparative analysis of the sequence revealed that this novel virus shares a genome-wide nucleotide identity of 94.95 to 96.94% with the available sequences of Hepacivirus Q in GenBank. Additionally, the putative complete polyprotein of this novel Hepacivirus exhibits an amino acid identity of 98.13 to 98.93% with the polyprotein of Hepacivirus Q. Consequently, this newly identified Hepacivirus can be classified as a new subspecies of Hepacivirus Q.

No statistically supported recombination event was detected within this novel Hepacivirus after systematic analyzes. Phylogenetic trees reconstructed using the amino acid sequence of the complete polyprotein, NS3, and NS5B proteins consistently revealed that this newly identified Hepacivirus grouped together with the known Hepacivirus Q strains and formed a sister lineage to a Hepacivirus that was identified from Bald eale in the USA (Figure 3). Moreover, the Hepacivirus Q group, which includes this newly identified hepacivirus, exhibits significant genetic divergence from other duck Hepacivirus identified in China, indicating a high level of genetic diversity within hepaciviruses. Notably, the NS3 tree shows a closer phylogenetic relationship among the Hepacivirus Q group and both Bald eagle Hepacivirus and Jogalong virus, while the NS5B tree reveals a closer relationship with Jogalong virus (Figure 3). This phylogenetic incongruence suggests that recombination events may have occurred in the evolutionary history of Hepacivirus Q, although no statistically supported recombination event was detected within Hepacivirus Q strains or other hepaciviruses during systematic analysis.

A total of 480 domestic duck liver tissues, including those previously subjected to meta-transcriptomic sequencing, were screened for the presence of Hepacivirus Q using nested RT-PCR. Following PCR screening, sequencing, and BLAST analysis, 64 individual RNA samples tested positive for Hepacivirus Q, resulting in an overall prevalence rate of 13.3% (95% CI: 10.3–16.3%) (Table 2). All NS5B sequences determined herein shared 98.9 to 99.8%. Additionally, the positive rates of Hepacivirus Q in Hengyang, Chenzhou, Yongzhou, Shaoyang, and Huaihua were 19.2, 11.4, 10.0, 14.1, and 10.0%, respectively. No significant difference was observed among the positive rates across the different sampling sites (χ² = 5.610, p = 0.230).