Hepacivirus is a genus of small enveloped spherical viruses with positive-sense RNA genomes of approximately 8.9–10.5 kb [1], which contain a single long open reading frame (ORF) flanked by 5′- and 3′-terminal non-coding regions. The ORF encodes a polyprotein of approximately 3,000 amino acids that are proteolytically cut into Core, E1, E2, p7/p13, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins [2]. Hepacivirus comprises 14 species (Hepacivirus A to N) according to the International Committee on Taxonomy of Viruses (ICTV) classification [3]. Members of different hepacivirus species show characteristically restricted host ranges including human (Hepacivirus C), primates (Hepacivirus B&N), monkeys (Hepacivirus D), horses (Hepacivirus A), rodents (Hepacivirus E to J), bats (Hepacivirus K to M), and cows (Hepacivirus N) [2, 4, 5]. Small mammals in Rodentia and bovine are important reservoirs of hepaciviruses, while few investigations have addressed hepacivirus in wild mammals in China [6-12]. The present study aimed to close this knowledge gap, and a hepacivirus screening was conducted in small mammals captured in a wide geographical area of the Yunnan province, in China, during 2016 to 2017.
From 2016 to 2017, small mammals were collected at 12 sites in the Yunnan province (Fig. 1). All animals were euthanized, dissected and their intestine, spleen, lung, kidney, and liver were collected in the liquid nitrogen tank and preserved at −80°C after transporting the laboratory. This study was approved by and conducted according to the guidelines of the Animal Ethics Committee of the Yunnan Institute of Endemic Diseases Control and Prevention (approval no. 2017007).
Viral RNA was extracted from liver tissue using the Viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Hepacivirus RNA was detected by a 400bp segment of NS3 analysis via reverse transcription-nested polymerase chain reaction (RT-PCR) with degenerate primers (HCV F1: GCICCIACIGGIAGYGGIAA; HCV R1: CCIGTCATIAGRGCRTCIGT; HCV R2: CARTCIRTIACIGARTCRAARTYICC; HCV F2: TAYGAYGTIATIATITGYGAYGARTG; HCV F3: TAYGAYGTIATIATITGYGAYGA;HCVF4:GCIACIGCIACICCICCIGG;HCVR3:CCIGTCATIAGRGCRTCIGT; HCV R4: CARTCIRTIACIGARTCRAARTYICC) [13]. PCR was performed using Super Script Ⅲ One-Step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The obtained PCR final product was sequenced by Tsingke Biotechnology (Wuhan, China).
Next, the full genome sequence of hepacivirus was evaluated by next generation sequencing using an Illumina HiSeq 3000 system (Illumina, San Diego, CA, USA) and QNome-3841 (Qitan Technology Ltd., Chengdu, China). The sequencing reads were assembled and analyzed using MEGAHIT (v1.2.9) and Geneious (v10.0.9), respectively. The remaining genome gaps were filled by specific primers through traditional PCR. The genome end sequences were obtained by rapid amplification of cDNA ends using Clontech-SMARTer RACE 5' and 3' Kit (Takara, Kusatsu, Japan) and by poly (A) tailing.
Sequence alignments contained genomic nucleotide sequences of the virus identified in this study together with 27 sequences obtained from representative members of the Hepacivirus genus by ClustalW. Phylogenetic trees were inferred for coding sequences of the polyprotein genes using the maximum likelihood (ML) method and Jukes-Cantor model implemented in the MEGA X software. To assess support for individual nodes, 1000 bootstrap replicates were obtained under the same procedure. The phylogenetic trees were visualized using Adobe Illustrator CC 2018.
Overall, 1,602 small mammals—comprising 21 species, 11 genera, 4 families, and 3 orders—were trapped in 12 counties (Fig. 1, Table 1). Two liver tissue samples (0.12%, 2/1602) of Crocidura dracula (commonly known as Southeast Asian shrew) were positive for hepacivirus nucleic acids. One Crocidura dracula specimen was collected from Nanjian County (Hepacivirus Cf/NJ12) and another was collected from Xiangyun County (Hepacivirus Cf/XY174) in 2017. The positive rates of hepacivirus infection among Crocidura draculas was 12.5% (2/16). Especially, Hepacivirus Cf/NJ12 had 73.7% nucleotide identity with Hepacivirus Cf/XY174 based on partial NS3 sequence. In particular, Cf/NJ12 shared a high similarity (69.25%) with the Suncus murinus hepacivirus SZCDC19 (GenBank No. MF775363) based on partial NS3 sequence, whereas Cf/XY174 was most similar (68.90%) with Suncus sp. hepacivirus SoSm-HepaV/Cs2009 (GenBank No. MT085224) based on partial NS3 sequence.
Because of poor assembly quality of Cf/XY174, only the complete genome sequence of Cf/NJ12 was determined, which was 10,383 nucleotides in length and with a G +C content of 52.3% (genome warehouse number: GWHBHEC01000000). Similar with other hepaciviruses, the genome of Cf/NJ12 contained one ORF encoding a polyprotein of 2,842 amino acids flanked by the 5′- and 3′-untranslated regions (822 and 57 nucleotides respectively) and the polyprotein was putatively cleaved into ten proteins (Fig. 2). The Cf/NJ12 ORF shared 30.3–37.7% nucleotide and 20.6–31.1% amino acid identity with other known hepacivirus species (Table 2), and the predicted polyprotein shared a high amino acid similarity (72.49%) with that of a hepacivirus detected in an Asian house shrew (S. murinus) (GenBank No. QUS47367) from Wenzhou, China.
Phylogenetic analysis based on the ORF region nucleotide sequence showed that the hepacivirus strain detected in C. dracula formed an independent branch (Fig. 3). And we did not find any evidence of recombination in the Cf/NJ12 genome. The amino acid p-distances of the conserved regions 996–1,437 and 2,693–3,115 (relevant to positions 1,123–1,566 and 2,536–2,959 of Hepacivirus C1a [AF011751]) of Cf/NJ12 ranged within 0.518–0.590 and 0.464–0.546 with known species of the Hepacivirus genus, respectively (Table 2). The ICTV species demarcation criteria for this genus defines that a novel hepacivirus must exhibit a p-distance greater than 0.25 between amino acids 1,123–1,566 of NS3 and greater than 0.3 between amino acids 2,536–2,959 NS5, as numbered in the HCV genotype 1a of H77 reference strain (AF011751) [3]. Therefore, we propose that Cf/NJ12 should constitute a new hepacivirus species.
In addition to hepaciviruses, which have been reported by ICTV, novel hepaciviruses have been gradually discovered around the world. For example, novel hepaciviruses were found in Suncus. So far, Hepacivirus genus includes 14 species, and their reservoirs are extensive, including primates, dogs, horses, cows, bats and rodents. Rodents are the main natural reservoirs. Among the 14 species of hepaciviruses from ICTV, the reservoirs of 6 hepaciviruses (Hepacivirus E to J) were Peromysus maniculateus [14], Myodes glareolus [13], Rattus norvegicus [15] and Rhabdomys pumilio [13]. However, hepacivirus was only detected in Soricomorpha animals in this survey, but not in rodents. It is speculated that the possible reasons are the small number of sampling sites and samples or the unenough sensitive detection method due to various reasons. In addition, hepacivirus was only found in Xiangyun county and Nanjian County, and no hepacivirus was found in other locations, indicating that the distribution of these viruses may be limited to these two regions. However, considering the movement of Soricomorpha animals due to ecological changes, we cannot exclude the possibility of hepacivirus spreading in the future. Therefore, it is necessary to long-term surveillance for hepacivirus in their natural reservoirs and its potential zoonotic transmission risk should be evaluated.
In addition to hepaciviruses, which have been reported by ICTV, novel hepaciviruses have been gradually discovered around the world. For example, novel hepaciviruses were found in Suncus murinus in Shenzhen city, China [7], Akodon montensis, Calomys tener, Oligoryzomys nigripes, Necromys lasiurus and Mus musculus from São Paulo State, Brazil [4], Spermophilus undulatus from Xinjiang Uygur Autonomous Region, China [8], cows from Yunnan, China [11], Trichosurus vulpecula from Sydney, Australia [16], and Bradypus variegatus from the Sloth Sanctuary, Penshurt, Limón, Costa Rica [17]. Phylogenetic analysis showed that Cf/NJ12 was independent of the other 14 hepaciviruses in this study. However, Cf/NJ12 formed a large clade with 4 hepaciviruses (Hepacivirus E, F, H, G), indicating that Cf/NJ12 may had a common ancestor with them. Based on the species demarcation criteria of ICTV, the Cf/NJ12 obtained in this study is a novel hepacivirus after p-distance analysis. These were the first hepaciviruses detected in C. dracula and the first whole genome sequences of the novel hepacivirus ever reported. The expanded host range of hepacivirus suggests that hepacivirus can cross-species transmission and thus contribute to their genetic diversification. It is worth noting that rodent hepaciviruses contain six types of hepaciviruses and bat hepaciviruses contain 3 types of hepaciviruses, indicating that hepaciviruses are highly genetically diverse, rodents and bats may be the initial hosts of hepaciviruses [2, 13, 18]. In addition, different hepacivirus can infect the same host. For example, Hepaciviruses G and H infect Rattus norvegicus [19]. Hepacivirus K and L infect Hipposideros vittatus [20] and this genetic diversity of hepaciviruses may have evolved genetically through interspecies transmission.
We found that in addition to the liver, hepacivirus RNA was also detected in the spleen, kidney, and intestine (Data was not shown). Similar to other reported hepaciviruses, the highest viral load was detected in the liver [7, 8, 13]. Wide tissue tropism of C. dracula hepacivirus may represent more transmitted pathways rather than major blood transfusion of Hepatitis C virus (HCV). However, the herein described data was obtained from only one positive animal, which is not enough for a comprehensive understanding of the epidemiologic characteristics of C. dracula hepacivirus.
The discovery of Cf/NJ12 extends our understanding of the genetic diversity and host range of hepaciviruses, and helps to elucidate the origin and evolution of hepaciviruses. Chimpanzees (Pan troglodytes) and tree Shrews (Tupaia telangeri) are so far known to have clinical symptoms similar to humans after inoculating HCV [21, 22]. And the course and pathological signs of Bank Vole (Clethrionomys glareolus) inoculated with Hepacivirus F and Hepacivirus J were similar to those of human infected with HCV [23]. But chimpanzees are relatively rare and expensive subjects and studying the pathogenesis of HCV replication in chimpanzees may take 10 to 20 years. In fact, the pathogenesis of Hepacivirus infection in tree shrews, Bank Vole and humans is quite different. Therefore, there is still a lack of suitable animal models to study the pathogenesis and immunity of hepacivirus. The first discovery of novel hepacivirus in C. dracula may provide a new option for future development of hepacivirus models.