Hepatitis A virus (HAV), a member of the genus Hepatovirus (Picornaviridae HepV), remains a significant viral pathogen, frequently causing enterically transmitted hepatitis worldwide. In this study, we conducted an epidemiological survey of HepVs carried by small terrestrial mammals in the wild in Yunnan Province, China. Utilizing HepV-specific broad-spectrum RT-PCR, next-generation sequencing (NGS), and QNome nanopore sequencing (QNS) techniques, we identified and characterized two novel HepVs provisionally named EpMa-HAV and EpLe-HAV, discovered in the long-tailed mountain shrew (Episoriculus macrurus) and long-tailed brown-toothed shrew (Episoriculus leucops), respectively. Our sequence and phylogenetic analyses of EpMa-HAV and EpLe-HAV indicated that they belong to the species Hepatovirus I (HepV-I) clade II, also known as the Chinese shrew HepV clade. Notably, the codon usage bias pattern of novel shrew HepVs is consistent with that of previously identified Chinese shrew HepV. Furthermore, our structural analysis demonstrated that shrew HepVs differ from other mammalian HepVs in RNA secondary structure and exhibit variances in key protein sites. Overall, the discovery of two novel HepVs in shrews expands the host range of HepV and underscores the existence of genetically diverse animal homologs of human HAV within the genus HepV.

Black-headed gulls have been confirmed as the natural hosts of Deltacoronavirus (δ-CoV) and Gammacoronavirus (γ-CoV). A total of 59 CoV-PCR-positive fecal samples were identified among 509 fecal samples collected from overwintering black-headed gulls in Yunnan Province, China. The prevalence of black-headed gull deltacoronavirus (BHG-DCoV) was 3.54% (18/509), while that of black-headed gull gammacoronavirus (BHG-GCoV) was 8.06% (41/509). The prevalence of BHG-GCoV was significantly higher than that of BHG-DCoV (χ² = 9.518, p < 0.01). Two complete genome sequences of BHG-GCoVs were obtained, with lengths of 27,358 bp and 27,355 bp, respectively, from the fecal samples of black-headed gulls. The nucleotide similarity between the two complete genomes is 98.75%. Phylogenetic analysis based on the whole genome has confirmed that the two strains of BHG-GCoVs clustered into the species Gammacoronavirus anatis. Although BHG-GCoVs belong to the species Gammacoronavirus anatis, they are distantly related to the representative strain Duck_CoV 2714 and exhibit a closer genetic relationship with GCoVs from Xenus cinereus (AvXc-GCoV) and Numenius phaeopus (AvNp-GCoV). Similarity analysis of the five conserved domains revealed a high amino acid similarity not only with AvXc-GCoV and AvNp-GCoV but also with GCoVs from common gulls detected in Poland and those from ruddy turnstones identified in Australia. Additionally, we found that, except for the common gull, the amino acid sequences of the S protein of BHG-GCoVs showed a 88.69% to 96.44% similarity with those of GCoVs carried by Charadriiformes, while the similarity with GCoVs carried by Anseriformes ranged from 31.15% to 54.81%. Furthermore, recombination events were detected in BHG-GCoVs, suggesting that these strains are likely recombinant strains of common gull GCoV and the GCoV of Arenaria interpres (AvAi-GCoV), indicating that recombination events may occur frequently among GCoVs.

Black-headed gulls have been confirmed to be the natural hosts of Deltacoronavirus (δ-CoV) and Gammacoronavirus (γ-CoV). A total of 59 coronaviruses (CoVs) were detected in 509 fecal samples collected from overwintering black-headed gulls in Yunnan Province, China. Among them, the prevalence of black-headed gull Deltacoronavirus (BHG-DCoV) was 3.54% (18/509), and that of black-headed gull Gammacoronaviru (BHG-GCoV) was 8.06% (41/509). The prevalence of BHG-GCoV was higher than that of BHG-DCoV (χ² = 9.518, P < 0.01). It was obtained that two complete genome sequences of BHG-GCoVs with lengths of 27,358 nt and 37,355 nt respectively from the fecal samples of black-headed gulls. The nu-cleotide similarity between the two complete genomes is 98.75%. Phylogenetic analysis based on the whole genome has confirmed that the two stains of BHG-GCoVs clustered into the species Gammacoronavirus anatis. Although BHG-GCoVs belong to the species Gammacoronavirus anatis, they are distantly related to the representative strain Duck_CoV 2714, and have a relatively close genetic relationship with the GCoV from Xenus cinereus (AvXc-GCoV) and the GCoV from Numenius phaeopus (AvNp-GCoV). Based on the similar-ity analysis of the five conserved domains, there is a high amino acid similarity not only with AvXc-GCoV and AvNp-GCoV, but also with the GCoV of the common gull detected in Poland and the GCoV from ruddy turnstone detected in Australia. We also found that the amino acids of the S protein of BHG-GCoVs had a relatively high similarity with GCoV from most of the Charadriiformes except for the common gull, while having a rela-tively low amino acid similarity with that from the Anseriformes. Meanwhile, we also de-tected recombination events in BHG-GCoVs, indicating that recombination events might occur frequently during the viral evolution process.