Molecular questioning of potential efficacy of epsilon targeted antiviral treatment option for Domestic Cat Hepadnavirus

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Taylan KOÇ, Ece Adiguzel, T. Cigdem Oguzoglu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4249164/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We aimed to elucidate the molecular and secondary structure of DCH to predict the development of antiviral drugs. We performed a series of polymerase chain reactions to obtain complete sequences of DCH. The complete sequences were processed using computational tools. The phylogenetic analysis showed that our sequences belong to one clade, but four are not part of this monophyletic clade. A recombination detection program identified four cases as potential recombination events. The secondary structure of the cis-acting RNA region (ε) was evaluated and revealed motifs similar to those found in HBV. This similarity highlights the potential for new-generation therapeutics in this region. Figures Figure 1 Figure 2 Figure 3 Full Text Domestic Cat Hepadnavirus (DCH) is a novel feline virus that is believed to cause chronic hepatic failure in cats, according to authorities [1-4]. It is known that the Hepatitis B virus (HBV), which is the homolog of DCH in humans, induces chronic hepatic failure and cirrhosis in particular immune-suppressed individuals [4-6]. It is presumed that DCH causes hepatic failure in immune-suppressed cats, although this has not yet been revealed. Researchers have highlighted that DCH may frequently occur in cases of dual or mixed infections by feline retroviruses [7,8]. This presumption has naturally led to the investigation of the pathogenicity of DCH. DCH has gained priority over other infectious agents in cats due to its frequent detection, despite the presence of prominent feline viruses such as feline immunodeficiency virus, feline leukemia virus, feline panleukopenia virus, and feline coronavirus [2-4,7]. This has led to a surge in research on DCH since 2018, with many papers focusing on its detection and molecular characterization [2-4,7-17]. DCH belongs to the family Hepadnaviridae , genus Orthohepadnavirus . DCH is a partially double-stranded DNA molecule and approximately 3200 nucleotides in length [5]. It possesses a unique enzyme that can convert RNA to DNA, which has led some researchers to refer to it as a 'DNA retrovirus' [18,19]. Hepatitis B virus, a homolog of DCH, replicates using closed circular DNA (cccDNA) as an RNA intermediate, also known as pre-genomic RNA (pgRNA) [20]. pgRNA is the critical intermediate form in the replication. It is employed like an mRNA for the formation of viral proteins. In the formation of pgRNA, cis-acting RNA or so-called epsilon (ε) binds to its counterpart region on the P protein, and the P-ε packaging complex occurs [20]. This mechanism is probably also present in the replication of DCH. However, there is not enough information specific to DCH. In this study, we investigate the last phylogenetic situation of DCH and the RNA secondary structure of the ε region, which is crucial for DCH replication. Comparing the stem-loop ε RNA secondary structures of DCH and HBV may provide insight into the potential development of a generic antiviral against the P-ε packaging complex. All examinations were conducted on cats according to international and national ethical guidelines. Ankara University Local Ethics Committee for Animal Experimentation approved this study (permission license no. 2020/13/111). The cats were selected according to their clinical manifestations and biochemical findings, especially those with suspected hepatic failure or disease. We conducted viral genome extractions using the slightly modified Phenol:Chloroform:Isoamyl Alcohol (25:24:1) method [21]. We used new primer sets and performed a series of polymerase chain reactions [4]. Gel electrophoresis was performed after PCR, and the relevant amplified bands were visualized under a blue-light transilluminator (Blook, Genedirex, Taiwan). The amplicons were purified using a commercial PCR purification kit (Qiagen, Hilden, Germany). Sequence analysis was then implemented using the Sanger sequencing method for each amplicon. The quality of the sequences was checked using a bioinformatic tool (DNA Baser, Heracle BioSoft SRL, Romania), and any noisy or ambiguous sequences were cleaned using the same tool. We downloaded 40 complete sequences of DCH from GenBank (as of February 4th, 2024) to conduct molecular analysis. Multiple sequence alignment (MSA) was performed using ClustalW [22]. The resulting alignment was used to construct a phylogenetic tree and evaluate the genetic situation of DCH. For the phylogenetic analysis, we used the MSA to construct a maximum likelihood tree (ML) in MEGA software. To construct the tree, we analyzed the best model for machine learning and then calculated the machine learning tree using the indicated parameters, Kimura-2, and 1000 bootstrap replicates [23]. To investigate potential recombination, we used various algorithms, including LARD, SiSCAN, 3SEQ, BOOTSCAN/RECSCAN, RDP, MAXCHI, CHIMAERA, and GENECOV, through the recombination detection program 5 (RDP5) software [24]. Representative sequences, excluding TR-sequences for other DCH complete sequences available in GenBank, were determined using MMseq2 [25]. Before conducting cis-acting RNA (ε) prediction analysis and secondary structure of pgRNA and ε, we imported the determined representative sequences into alignment. Cis- acting RNA (ε) prediction was conducted using the Jpred4 [26] and RNAalifold [27] tools implicated in JalView [28]. The output stem-loop was visualized using Varna [29]. Human HBV stem-loop formation, which originated from the consensus sequence of predetermined HBVs, was also visualized in Varna. We used JalView to obtain the 3D structure after protein alignment. The results of the phylogenetic analysis show that Turkish (TR) sequences are mainly included in one clade, but four of them are not part of this monophyletic clade. At first glance, three main clades were observed in Genotype-A. According to the ML phylogenetic tree, many Turkish sequences form a separate monophyletic clade. However, TR-03-PEY, TR-275, SV-8, and SV-15 are in other branches within the same clade. The DCH sequences from Thailand formed a distinct clade was located far from the putative Clade 1. Due to a sharp bifurcation for Clade 1 that existed on the phylogenetic tree, we labeled this clade as Clade A.1.2 (Fig. 1). The recombination detection program RDP5 was used to investigate potential recombination events. A full exploratory recombination scan was conducted using all available methods. RDP5 identified four cases as potential recombination events. Three of these cases indicated the minor parents as 'Unknown'. The analysis identified TR-SV8, TR-SV15, and TR-296 as potential recombination sequences. Additionally, the analysis predicted a higher likelihood of recombination involving PK83-B/THA/2022, TR-404, and TR-382, which were identified as the major parent, minor parent, and potential recombinant, respectively (Fig. 2). The secondary structure of the cis-acting RNA (ε region) was evaluated using Jpred, revealing motifs similar to those found in HBV (Fig. 3). Conserved putative (ε) regions were found in all TR sequences, with only one amino acid substitution at K572M, which was a synonymous mutation. This substitution had no observed functional effect on the secondary structure. The apical stem-loop of the ε region of DCH showed similar motifs to the apical stem-loop of the ε region of HBV, as contextualized in the 3D visualization of the encapsidation signal region of HBV (PBD no: 2ixy) (Fig. 3). Domestic cat hepadnavirus is a recently discovered virus that become as important as other prominent feline viruses, FIV, FeLV, FPV, and FIP [4]. It has been shown to have a close genetic relationship with HBV in several studies, and this case usually recalls similar dynamics that might be efficient during the replication of both viruses. From its clinical point of view, antiviral treatment options have raised curiosity about whether they could be applied similarly to HBV treatment [30]. Unveiling of molecular dynamics of specific regions of DCH therefore likely to pave the way for experimental and antiviral development studies. The phylogenetic tree shows that Thailand's DCH sequences, previously placed in Clade A1 on the tree constructed by Capozza et al. [3], have recently formed a separate group after the initial bifurcation. Additionally, Turkish sequences are grouped in a clade that Capozza et al. [3] referred to as Clade A2. There are currently no official genotypes or genotyping methods for DCH. However, we propose the following putative clades or subgroups in Genotype A: Clade A1.1, Clade A1.2, and Clade A2, for consideration by authorities and other researchers. TR sequences have been classified as Clade A1.2 according to this recommended grouping. The detection of pre-existing or new mutations and/or recombination is crucial in predicting virus-host or virus-virus interactions due to the dynamic nature of viruses and their genetic components. In this study, we utilized RDP5 to investigate potential recombination events. The results indicate that four potential recombination cases may have occurred, with one being more strongly supported than the other three. TR-sequences were predicted and assigned as potential recombinant sequences in all recombination cases. These preliminary results may indicate genetic variations of DCHs and their incidence. The study conducted recently did not find any evidence of recombination. However, we have identified four potential recombination events using the same tool [31]. In two of the predicted events, the recombination signals were detected using five models (3SEQ, BOOTSCAN, MAXCHI, CHIMAERA, and GENECOV). In drug design studies, comparative studies would be important to consider the conservation of DCH and HBV in both ε regions. Neglecting the ε region belonging to DCH in the comparison could be considered a deficiency in a study that emphasized the existence of a common ancestor of hepadnavirus and nackednavirus [32]. A recent study has revealed a similarity in the cell entry pathway between HBV and DCH. The researchers have pointed out that DCH infection in cats could be an animal model for HBV research. The molecular and structural analysis of the ε region in this study also supports the idea of DCH being a suitable animal model for HBV [30]. Recent literature suggests that N6 -methyladenosine (m6A) modification may play an important role in the regulation of the hepadnaviral life cycle [33-35]. As the most abundant epitranscriptomic internal modification factor, m6A can be expressed by cellular or viral RNAs. This has been studied to a limited extent in HBV and HCV pathogenesis [36]. Based on these studies, m6A expression levels can cause bilateral effects. However, m6A is known to play a regulatory role in many biological activities such as immune response, tumourigenesis, and viral replication [33-35]. When we compared the stem-loop structure of DCH and HBV epsilon by secondary structure analysis, we found that they have almost the same conformation on the X protein, although the nucleotide spacing is different. In a previous study, researchers who revealed the importance of m6a for HBV replication emphasized that nucleotides 1815 and 1950 have m6a peaks found in all HBV transcripts [33]. Therefore, they claimed that this modification could be a target for a new-generation antiviral drug. The high molecular similarity of DCH to HBV may help to rapidly develop an antiviral targeting m6a. In conclusion, this study presents the phylogenetic, recombinant, and partial structural analysis of DCH. The complete genomes of the strains obtained from our country were examined in terms of this hypothesis. The data obtained exhibit similar molecular dynamics to the preliminary data for DCH. The study reveals a close resemblance between the RNA secondary structure of ε and that of HBV, highlighting the potential for new-generation therapeutics in this region. Additionally, this study emphasizes the significance of the comparative evaluation of orthohepadnaviruses, particularly DCH and HBV. Declarations Funding Ece Adiguzel was supported financially by Ankara University-Coordinatorship of scientific research projects (PhD thesis grant: 21L0239016). Data Availability Turkish DCH sequences obtained in this study were deposited in GenBank under accession numbers ON325584, ON293153, and OQ130240-OQ130250. References Beatty JA, Tu T, Pesavento PA et al (2023) Domestic Cat Hepadnavirus and Lymphoma. Viruses 15:. https://doi.org/10.3390/v15122294 Capozza P, Pellegrini F, Camero M et al (2023) Hepadnavirus Infection in a Cat with Chronic Liver Disease: A Multi-Disciplinary Diagnostic Approach. Vet Sci 10. https://doi.org/10.3390/vetsci10120668 Capozza P, Carrai M, Choi YR et al (2023) Domestic Cat Hepadnavirus: Molecular Epidemiology and Phylogeny in Cats in Hong Kong. Viruses 15. https://doi.org/10.3390/v15010150 Adıgüzel E, Erdem-Şahinkesen E, Koç BT et al (2023) The detection and full genomic characterization of domestic cat Orthohepadnaviruses from Türkiye. Vet Med Sci 9:1965–1972. https://doi.org/10.1002/vms3.1217 Aghazadeh M, Shi M, Barrs VR et al (2018) A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia. https://doi.org/10.3390/v10050269 . Viruses 10: Takahashi K, Kaneko Y, Shibanai A et al (2022) Identification of domestic cat hepadnavirus from a cat blood sample in Japan. J Vet Med Sci 84:648–652. https://doi.org/10.1292/jvms.22-0010 Beatty JA, Choi YR, Nekouei O et al (2024) Epidemiology of Pathogenic Retroviruses and Domestic Cat Hepadnavirus in Community and Client-Owned Cats in Hong Kong. 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Exp Mol Med 53:339–345. https://doi.org/10.1038/s12276-021-00581-3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4249164","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":302943073,"identity":"522b8a08-9069-4b80-b5e5-abdd7bd7a6ec","order_by":0,"name":"B. Taylan KOÇ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACAwY2IFnBwNgApCSI08IG0nIGrsWASC2MbaRoMZdvS3zwc56d7IYDzAdv8zD8ySeoxbKN7bBh77Zk4w0H2JKteRgMLBsIOuwYe5s04zbmxA0HeMykgVoIuwyiZU49UAv/N2K1sB2TZmw4DLKFjTgtlm1pyYY9x44bzzzMZmw5x8CYsBZz5mOGD37UVMv2HW9+eONNhRwRoQwHzGB3kqBhFIyCUTAKRgFuAAD0njT+VPd+SwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4279-6233","institution":"Adnan Menderes Universitesi","correspondingAuthor":true,"prefix":"","firstName":"B.","middleName":"Taylan","lastName":"KOÇ","suffix":""},{"id":302943074,"identity":"dfdbe103-90b6-43ba-a3db-7d5aed284c99","order_by":1,"name":"Ece Adiguzel","email":"","orcid":"","institution":"Republic of Turkey Ministry of Agriculture and Forestry: Turkiye Cumhuriyeti Tarim ve Orman Bakanligi","correspondingAuthor":false,"prefix":"","firstName":"Ece","middleName":"","lastName":"Adiguzel","suffix":""},{"id":302943075,"identity":"f0bebe87-c162-4d28-8272-636f50e1846c","order_by":2,"name":"T. Cigdem Oguzoglu","email":"","orcid":"","institution":"Ankara University: Ankara Universitesi","correspondingAuthor":false,"prefix":"","firstName":"T.","middleName":"Cigdem","lastName":"Oguzoglu","suffix":""}],"badges":[],"createdAt":"2024-04-10 20:34:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4249164/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4249164/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57443002,"identity":"810a0088-d801-4aa9-955c-d1bcc4be37e5","added_by":"auto","created_at":"2024-05-30 18:49:25","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340184,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4249164/v1/d3094ba00ed1f906fe0ff0af.jpeg"},{"id":57443001,"identity":"d11b4591-8ed1-492e-8b67-a0138a626f31","added_by":"auto","created_at":"2024-05-30 18:49:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRDP5 screenshot after accomplishing the analyses.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eThe potential recombination analysis between TR- and representative sequences. The regions in the pink-colored frames represent potential breakpoints in the recombination cases. In (A) and (D), potential major parent, minor parent, and recombinant sequences were determined in TR DCH sequences. In (B) and (C), potential parents and recombinant sequences were determined in both TR, and the representative DCH sequences, which were selected by MMseq2.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4249164/v1/1b464c88d7482f4edec6867c.png"},{"id":57443003,"identity":"626010f4-758a-4898-83e1-982bc3aa0026","added_by":"auto","created_at":"2024-05-30 18:49:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRNA secondary structure prediction of \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eε\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e region. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA.\u003c/strong\u003e ε region multiple alignment of TR DCH sequences for RNA secondary structure prediction and conserved region. \u003cstrong\u003eB.\u003c/strong\u003e Stem-loop of ε region of alignment based DCH and HBV. \u003cstrong\u003eC.\u003c/strong\u003e 3D visualization of stem-loop. 3D visualization was provided from Jpred, an RNA secondary prediction tool implicated in JalView software.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4249164/v1/5ea8e3104c55c0adf1b9ad41.png"},{"id":58292257,"identity":"52ef162a-bc6e-4f0d-a4cd-80421dedcdb7","added_by":"auto","created_at":"2024-06-13 13:55:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1083299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4249164/v1/3de8b5a6-31c9-4f06-a566-9ba93f3a671d.pdf"}],"financialInterests":"","formattedTitle":"Molecular questioning of potential efficacy of epsilon targeted antiviral treatment option for Domestic Cat Hepadnavirus","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDomestic Cat Hepadnavirus (DCH) is a novel feline virus that is believed to cause chronic hepatic failure in cats, according to authorities\u0026nbsp;[1-4]. It is known that the Hepatitis B virus (HBV), which is the homolog of DCH in humans, induces chronic hepatic failure and cirrhosis in particular immune-suppressed individuals [4-6]. It is presumed that DCH causes hepatic failure in immune-suppressed cats, although this has not yet been revealed. Researchers have highlighted that DCH may frequently occur in cases of dual or mixed infections by feline retroviruses [7,8]. This presumption has naturally led to the investigation of the pathogenicity of DCH. DCH has gained priority over other infectious agents in cats due to its frequent detection, despite the presence of prominent feline viruses such as feline immunodeficiency virus, feline leukemia virus, feline panleukopenia virus, and feline coronavirus [2-4,7]. This has led to a surge in research on DCH since 2018, with many papers focusing on its detection and molecular characterization [2-4,7-17].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCH belongs to the family \u003cem\u003eHepadnaviridae\u003c/em\u003e, genus \u003cem\u003eOrthohepadnavirus\u003c/em\u003e. DCH is a partially double-stranded DNA molecule and approximately 3200 nucleotides in length [5]. It possesses a unique enzyme that can convert RNA to DNA, which has led some researchers to refer to it as a 'DNA retrovirus' [18,19]. Hepatitis B virus, a homolog of DCH, replicates using closed circular DNA (cccDNA) as an RNA intermediate, also known as pre-genomic RNA (pgRNA) [20]. pgRNA is the critical intermediate form in the replication. It is employed like an mRNA for the formation of viral proteins. In the formation of pgRNA, \u003cem\u003ecis-acting\u003c/em\u003e RNA or so-called epsilon (ε) binds to its counterpart region on the P protein, and the P-ε packaging complex occurs [20]. This mechanism is probably also present in the replication of DCH. However, there is not enough information specific to DCH.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigate the last phylogenetic situation of DCH and the RNA secondary structure of the\u0026nbsp;ε\u0026nbsp;region, which is crucial for DCH replication. Comparing the stem-loop\u0026nbsp;ε\u0026nbsp;RNA secondary structures of DCH and HBV may provide insight into the potential development of a generic antiviral against the P-ε\u0026nbsp;packaging complex.\u003c/p\u003e\n\u003cp\u003eAll examinations were conducted on cats according to international and national ethical guidelines. Ankara University Local Ethics Committee for Animal Experimentation approved this study (permission license no. 2020/13/111). The cats were selected according to their clinical manifestations and biochemical findings, especially those with suspected hepatic failure or disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted viral genome extractions using the slightly modified Phenol:Chloroform:Isoamyl Alcohol (25:24:1) method [21]. We used new primer sets and performed a series of polymerase chain reactions [4]. Gel electrophoresis was performed after PCR, and the relevant amplified bands were visualized under a blue-light transilluminator (Blook, Genedirex, Taiwan).\u003c/p\u003e\n\u003cp\u003eThe amplicons were purified using a commercial PCR purification kit (Qiagen, Hilden, Germany). Sequence analysis was then implemented using the Sanger sequencing method for each amplicon. The quality of the sequences was checked using a bioinformatic tool (DNA Baser, Heracle BioSoft SRL, Romania), and any noisy or ambiguous sequences were cleaned using the same tool.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe downloaded 40 complete sequences of DCH from GenBank (as of February 4th, 2024) to conduct molecular analysis. Multiple sequence alignment (MSA) was performed using ClustalW [22]. The resulting alignment was used to construct a phylogenetic tree and evaluate the genetic situation of DCH. For the phylogenetic analysis, we used the MSA to construct a maximum likelihood tree (ML) in MEGA software. To construct the tree, we analyzed the best model for machine learning and then calculated the machine learning tree using the indicated parameters, Kimura-2, and 1000 bootstrap replicates [23].\u003c/p\u003e\n\u003cp\u003eTo investigate potential recombination, we used various algorithms, including LARD, SiSCAN, 3SEQ, BOOTSCAN/RECSCAN, RDP, MAXCHI, CHIMAERA, and GENECOV, through the recombination detection program 5 (RDP5) software [24]. Representative sequences, excluding TR-sequences for other DCH complete sequences available in GenBank, were determined using MMseq2 [25].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBefore conducting cis-acting RNA (ε) prediction analysis and secondary structure of pgRNA and ε, we imported the determined representative sequences into alignment. \u003cem\u003eCis-\u003c/em\u003eacting RNA (ε) prediction was conducted using the Jpred4 [26]\u0026nbsp;and RNAalifold [27]\u0026nbsp;tools implicated in JalView [28]. The output stem-loop was visualized using Varna [29]. Human HBV stem-loop formation, which originated from the consensus sequence of predetermined HBVs, was also visualized in Varna. We used JalView to obtain the 3D structure after protein alignment.\u003c/p\u003e\n\u003cp\u003eThe results of the phylogenetic analysis show that Turkish (TR) sequences are mainly included in one clade, but four of them are not part of this monophyletic clade. At first glance, three main clades were observed in Genotype-A. According to the ML phylogenetic tree, many Turkish sequences form a separate monophyletic clade. However, TR-03-PEY, TR-275, SV-8, and SV-15 are in other branches within the same clade. The DCH sequences from Thailand formed a distinct clade was located far from the putative Clade 1. Due to a sharp bifurcation for Clade 1 that existed on the phylogenetic tree, we labeled this clade as Clade A.1.2 (Fig. 1).\u003c/p\u003e\n\u003cp\u003eThe recombination detection program RDP5 was used to investigate potential recombination events. A full exploratory recombination scan was conducted using all available methods. RDP5 identified four cases as potential recombination events. Three of these cases indicated the minor parents as 'Unknown'. The analysis identified TR-SV8, TR-SV15, and TR-296 as potential recombination sequences. Additionally, the analysis predicted a higher likelihood of recombination involving PK83-B/THA/2022, TR-404, and TR-382, which were identified as the major parent, minor parent, and potential recombinant, respectively (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe secondary structure of the \u003cem\u003ecis-acting RNA\u003c/em\u003e (ε region) was evaluated using Jpred, revealing motifs similar to those found in HBV (Fig. 3). Conserved putative (ε) regions were found in all TR sequences, with only one amino acid substitution at K572M, which was a synonymous mutation. This substitution had no observed functional effect on the secondary structure. The apical stem-loop of the ε region of DCH showed similar motifs to the apical stem-loop of the ε region of HBV, as contextualized in the 3D visualization of the encapsidation signal region of HBV (PBD no: 2ixy) (Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDomestic cat hepadnavirus is a recently discovered virus that become as important as other prominent feline viruses, FIV, FeLV, FPV, and FIP [4]. It has been shown to have a close genetic relationship with HBV in several studies, and this case usually recalls similar dynamics that might be efficient during the replication of both viruses. From its clinical point of view, antiviral treatment options have raised curiosity about whether they could be applied similarly to HBV treatment [30]. Unveiling of molecular dynamics of specific regions of DCH therefore likely to pave the way for experimental and antiviral development studies. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree shows that Thailand's DCH sequences, previously placed in Clade A1 on the tree constructed by Capozza et al. [3], have recently formed a separate group after the initial bifurcation. Additionally, Turkish sequences are grouped in a clade that Capozza et al. [3]\u0026nbsp;referred to as Clade A2. There are currently no official genotypes or genotyping methods for DCH. However, we propose the following putative clades or subgroups in Genotype A: Clade A1.1, Clade A1.2, and Clade A2, for consideration by authorities and other researchers. TR sequences have been classified as Clade A1.2 according to this recommended grouping.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe detection of pre-existing or new mutations and/or recombination is crucial in predicting virus-host or virus-virus interactions due to the dynamic nature of viruses and their genetic components. In this study, we utilized RDP5 to investigate potential recombination events. The results indicate that four potential recombination cases may have occurred, with one being more strongly supported than the other three. TR-sequences were predicted and assigned as potential recombinant sequences in all recombination cases. These preliminary results may indicate genetic variations of DCHs and their incidence. The study conducted recently did not find any evidence of recombination. However, we have identified four potential recombination events using the same tool [31].\u0026nbsp;In two of the predicted events, the recombination signals were detected using five models (3SEQ, BOOTSCAN, MAXCHI, CHIMAERA, and GENECOV).\u003c/p\u003e\n\u003cp\u003eIn drug design studies, comparative studies would be important to consider the conservation of DCH and HBV in both ε regions. Neglecting the ε region belonging to DCH in the comparison could be considered a deficiency in a study that emphasized the existence of a common ancestor of hepadnavirus and nackednavirus [32]. A recent study has revealed a similarity in the cell entry pathway between HBV and DCH. The researchers have pointed out that DCH infection in cats could be an animal model for HBV research. The molecular and structural analysis of the ε region in this study also supports the idea of DCH being a suitable animal model for HBV [30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecent literature suggests that \u003cem\u003eN6\u003c/em\u003e-methyladenosine (m6A) modification may play an important role in the regulation of the hepadnaviral life cycle [33-35]. As the most abundant epitranscriptomic internal modification factor, m6A can be expressed by cellular or viral RNAs. This has been studied to a limited extent in HBV and HCV pathogenesis [36]. Based on these studies, m6A expression levels can cause bilateral effects. However, m6A is known to play a regulatory role in many biological activities such as immune response, tumourigenesis, and viral replication [33-35]. When we compared the stem-loop structure of DCH and HBV epsilon by secondary structure analysis, we found that they have almost the same conformation on the X protein, although the nucleotide spacing is different. In a previous study, researchers who revealed the importance of m6a for HBV replication emphasized that nucleotides 1815 and 1950 have m6a peaks found in all HBV transcripts [33]. Therefore, they claimed that this modification could be a target for a new-generation antiviral drug. The high molecular similarity of DCH to HBV may help to rapidly develop an antiviral targeting m6a.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study presents the phylogenetic, recombinant, and partial structural analysis of DCH. The complete genomes of the strains obtained from our country were examined in terms of this hypothesis. The data obtained exhibit similar molecular dynamics to the preliminary data for DCH. The study reveals a close resemblance between the RNA secondary structure of ε and that of HBV, highlighting the potential for new-generation therapeutics in this region. Additionally, this study emphasizes the significance of the comparative evaluation of orthohepadnaviruses, particularly DCH and HBV.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eEce Adiguzel was supported financially by Ankara University-Coordinatorship of scientific research projects (PhD thesis grant: 21L0239016).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eTurkish DCH sequences obtained in this study were deposited in GenBank under accession numbers ON325584, ON293153, and OQ130240-OQ130250.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeatty JA, Tu T, Pesavento PA et al (2023) Domestic Cat Hepadnavirus and Lymphoma. 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Exp Mol Med 53:339\u0026ndash;345. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s12276-021-00581-3\u003c/span\u003e\u003cspan address=\"10.1038/s12276-021-00581-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4249164/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4249164/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe aimed to elucidate the molecular and secondary structure of DCH to predict the development of antiviral drugs. We performed a series of polymerase chain reactions to obtain complete sequences of DCH. The complete sequences were processed using computational tools. The phylogenetic analysis showed that our sequences belong to one clade, but four are not part of this monophyletic clade. A recombination detection program identified four cases as potential recombination events. The secondary structure of the cis-acting RNA region (ε) was evaluated and revealed motifs similar to those found in HBV. This similarity highlights the potential for new-generation therapeutics in this region.\u003c/p\u003e","manuscriptTitle":"Molecular questioning of potential efficacy of epsilon targeted antiviral treatment option for Domestic Cat Hepadnavirus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 18:49:20","doi":"10.21203/rs.3.rs-4249164/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"18d0a30e-5eba-4f61-a86c-dc6618441e23","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T13:47:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-30 18:49:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4249164","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4249164","identity":"rs-4249164","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}