Conserved Functions of Orthohepadnavirus X Proteins to Inhibit Type-I Interferon Signaling.

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-ß (TRIF)-mediated IFN-ß signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNß signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins.

A comparative study of clinicopathological and imaging features of HBV-negative and HBV-positive intrahepatic cholangiocarcinoma patients with different pathologic differentiation degrees.

Hepatitis B is a risk factor for the development of intrahepatic cholangiocarcinoma. The prognosis of HBV-related ICC remains to be further investigated. To investigate the clinical, pathological and imaging features of intrahepatic cholangiocarcinoma of hepatitis B virus-positive and -negative patients. Data from January 31, 2012 to December 31, 2019 of 138 patients were retrospectively analyzed. The patients were divided into hepatitis B virus-positive group (group A[n = 66]) and virus-negative group (group B[n = 72]), and the patients were divided into groups according to pathological differentiation degree and tumor size. The differences in clinical, imaging characteristics and the progression-free survival between groups were analyzed. There were significant differences in gender, age, HBc antibody, CA125 and AFP, tumor distribution site, maximum diameter, plain scan density, inferior hepatic angle, peritumoral bile duct dilatation, vascular encasement invasion, intrahepatic bile duct dilatation and lymphadenopathy between the two groups (P < 0.05); There were statistical differences in signs of vascular encasement invasion between the two groups with well-to-moderately differentiated tumors (P < 0.05); there were statistical differences in tumor density uniformity, signs of vascular encasement invasion and lymphadenopathy between the two groups with poorly differentiated tumors (P < 0.05). Large groups A and B showed differences in tumor density uniformity, vascular encasement invasion, arterial phase, overall reinforcement pattern, peritumoral bile duct stones and biliary dilatation (P < 0.05). There was no statistical difference in postoperative PFS between the two groups (P > 0.05). The clinical and imaging features of ICC of hepatitis B virus-positive and -negative patients are different, and there is little difference in postoperative disease-free survival time.

The detection and full genomic characterization of domestic cat Orthohepadnaviruses from Türkiye.

BACKGROUND: Domestic cat hepadnaviruses (DCHs) have been described as a novel virus that can infect cats. OBJECTIVE: The aim of our study is the first identification and molecular characterizations of DCH infection in Turkish domestic cats. METHODS: The blood, organ and ascites fluid samples from 550 cats were randomly sampled. The presence of DCH nucleic acid was investigated by using both in the literature and newly designed primers. RESULTS: It was found that the hepadnavirus positivity rate is 4% (22/550) in Türkiye. The full genomic characterization was performed on 13 of 22 samples, and others were characterized as nearly full genome. In this study, we highlight that whole blood samples should be also screened for DCH, not only serum samples as has frequently been done in other studies. DCH-infected cats were also found positive (54.54%, 12/22) for Feline leukaemia virus infection. BLAST results revealed that Turkish DCHs have 86.32%-99.08% homology with strains in the GenBank database, enabling us to construct phylogenetic trees. CONCLUSIONS: According to this study's results, it is suggested that this infection should be added to veterinary diagnostic panels worldwide. Additionally, we suggest that our new synthesized primers for the amplification of X gene can also be used for diagnosis.

Liver fibrosis is closely related to metabolic factors in metabolic associated fatty liver disease with hepatitis B virus infection.

This case-control study aimed to identify the clinical characteristics and explore the risk factors for liver fibrosis in metabolic associated fatty liver disease (MAFLD) patients with hepatitis B virus (HBV) infection. The patients were grouped into MAFLD + HBV and MAFLD (without HBV infection). Propensity score matching (PSM) was used to match baseline features between the groups. We included 401 patients with biopsy-proven MAFLD, 179 of whom had HBV infection. A total of 83 pairs were successfully matched via PSM, and steatosis scores and ballooning in the MAFLD + HBV group were lower than those in the MAFLD group, while the inflammation scores and liver fibrosis stages were higher. After adjusted for confounding factors, HBV infection was associated with a higher risk of significant liver fibrosis in patients with MAFLD [odds ratio (OR): 3.140, P = 0.003]. Overall, 43.58% (78/179) of patients in the MAFLD + HBV group had significant liver fibrosis. Further multivariate regression analysis, hypertension (OR: 2.640; P = 0.031), type 2 diabetes (OR: 4.939; P = 0.035), and elevated glutamyl-transferase levels (OR: 3.980; P = 0.001) were risk factors for liver fibrosis in the MAFLD + HBV group. This suggests metabolic rather than viral factors are more closely associated with liver fibrosis in MAFLD patients with HBV infection.

Genetic characterization of hepatitis B virus genotypes among patients with chronic infection in Sulaimaniyah city, Iraq.

Background: Hepatitis B virus (HBV) genotypes are distributed unevenly throughout the world's regions. The researchers' goal in this study was to find out which HBV genotypes are now prevalent in the blood of chronic HBV patients in Iraq's Kurdistan Region's Sulaimaniyah governorate. Methods: Genotyping was carried out utilizing Polymerase Chain Reaction (PCR) type-specified primers. Thirty-three chronic HBV patients were included in the HBV genotyping assay. Phylogenic trees of Pre-S1/Pre S2/S genes' nucleotide sequences were constructed using 36 HBV isolates. Results: All the patients had HBV genotype D. Additionally, two samples were further analyzed by sequencing and deposited in GenBank as HBV/Sul-1/2021 accession numbers MZ077051 and HBV/Sul-2/2021 accession numbers MZ077052. Phylogenic analysis indicated that the HBV isolates belong to sub-genotype D1/serotype ayw2. The HBV/Sul-2/2021 had two sequence deletion mutations from G61del-T87del, which accounted for 27 amino acid deletions, and ten other mutations were identified in the carboxylic terminus of the pre-S1 from Q104del-R113del. Accordingly, 37 amino acids were deleted in the S promoter region. Several other substitution mutations were recorded in both HBV isolates. Conclusion: Patients with chronic HBV were found to have the HBV sub-genotype D1/subtype ayw2 with no mixed genotypes. HBV/Sul-1/2022, a new strain with a 37-amino acid mutation, was found to be distinct from any previously known HBV isolates.

[Modern views on the role of X gene of the hepatitis B virus (Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus) in the pathogenesis of the infection it causes].

The review presents information on the role of hepatitis B virus (Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus) (HBV) X gene and the protein it encodes (X protein) in the pathogenesis of viral hepatitis B. The evolution of HBV from primordial to the modern version of hepadnaviruses (Hepadnaviridae), is outlined as a process that began about 407 million years ago and continues to the present. The results of scientific works of foreign researchers on the variety of the influence of X protein on the infectious process and its role in the mechanisms of carcinogenesis are summarized. The differences in the effect of the X protein on the course of the disease in patients of different ethnic groups with regard to HBV genotypes are described. The significance of determining the genetic variability of X gene as a fundamental characteristic of the virus that has significance for the assessment of risks of hepatocellular carcinoma (HCC) spread among the population of the Russian Federation is discussed.

[Identification by enzyme immunoassay of escape mutants S143L and G145R of hepatitis B virus (Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus)].

INTRODUCTION: The achievement of the goal of the World Health Organization to eliminate viral hepatitis B by 2030 seems to be problematic partly due to the presence of escape mutants of its etiological agent, hepatitis B virus (HBV) (<i>Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus</i>), that are spreading mainly in the risk groups. Specific routine diagnostic assays aimed at identification of HBV escape mutants do not exist.The study aimed the evaluation of the serological fingerprinting method adapted for routine detection of escape mutations in 143 and 145 aa positions of HBV surface antigen (HBsAg). MATERIAL AND METHODS: HBV DNA from 56 samples of HBsAg-positive blood sera obtained from donors, chronic HBsAg carriers and oncohematology patients has been sequenced. After the identification of mutations in HBsAg, the samples were tested in the enzyme-linked immunosorbent assay (ELISA) kit «Hepastrip-mutant-3K¼. RESULTS AND DISCUSSION: Escape mutations were detected mainly in patients with hematologic malignancies. Substitutions in 143 and 145 aa were found in 10.81% and in 8.11% of such patients, respectively. The G145R mutation was recognized using ELISA kit in almost all cases. The kit specifically recognized the S143L substitution in contrast to the S143T variant. The presence of neighbor mutation D144E can be assumed due to it special serological fingerprint. CONCLUSION: ELISA-based detection of escape mutations S143L, D144E and G145R can be used for routine diagnostics, especially in the risk groups. The diagnostic parameters of the kit can be refined in additional studies. This immunoassay and methodology are applicable for the development and quality control of vaccines against escape mutants.

Orthohepadnavirus infection in a neotropical bat (Platyrrhinus lineatus).

Hepatitis B virus (HBV) is the prototype of the Orthohepadnavirus genus and represents an important cause of chronic hepatitis, liver cirrhosis, and hepatic cancer in humans worldwide. To verify the occurrence and genetic variability of orthohepadnavirus among neotropical bats, we tested 81 liver samples of New World bats from São Paulo State, Southeastern Brazil, collected during 2012. PCR, sequencing, and phylogenetic analysis of Surface/Polymerase and Core viral genes confirmed the occurrence of the first isolate of bat orthohepadnavirus detected in South America. These results may contribute to subsequent studies of the origin, variability, host species, and evolution of bat orthohepadnaviruses in South America.

Identification of Novel Rodent-Borne Orthohantaviruses in an Endemic Area of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka.

We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.

Exosomal miR-145-5p derived from orthohantavirus-infected endothelial cells inhibits HTNV infection.

Human infection of orthohantavirus can cause potentially fatal diseases, such as hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) in Eurasia. Exosomes are new carriers for information exchange between cells. Cumulative findings suggest that exosomes released from parental infected cells can block or promote viral infection in recipient cells, but the role of exosomes in hantavirus infection is poorly understood. In our study, we identified the exosomes derived from HTNV-infected human vascular endothelial cells (HUVECs) (Exo-HV) and found the antiviral properties of Exo-HV in the uninfected recipient cells. High-throughput sequencing revealed the distinctly expressed miRNAs transcriptomes in Exo-HV. MiR-145-5p, one of the abundant miRNAs packaged into Exo-HV, was found to be able to transferred to recipient cells and functioned by directly targeting M RNA of HTNV 76-118 and inducing type I interferon (IFN-I) response, thus, blocking the viral replication. Concluding, this study indicated that exosomes released by HTNV-infected HUVECs were able to transfer active molecules, miR-145-5p as a proving sample, to mediate novel anti-HTNV activity in the neighboring uninfected cells, which will help us to explore new strategies for the treatment of infectious disease utilizing exosomes with miRNA.

A Retrospective Survey of Rodent-borne Viruses in Rural Populations of Brazilian Amazon.

INTRODUCTION: The Amazon tropical rainforest has the most dense and diverse ecosystem worldwide. A few studies have addressed rodent-borne diseases as potential hazards to humans in this region. METHODS: A retrospective survey was conducted using enzyme-linked immunosorbent assay for detecting mammarenavirus and orthohantavirus antibodies in 206 samples collected from rural settlers of the Brazilian Western Amazonian region. RESULTS: Six (2.91%) individuals in the age group of 16 to 36 years were found to possess antibodies against mammarenavirus. CONCLUSION: Evidence of previous exposure to mammarenavirus in the rural population points to its silent circulation in this region.

Isolation and characterization of new Puumala orthohantavirus strains from Germany.

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies.

Orthohantavirus Isolated in Reservoir Host Cells Displays Minimal Genetic Changes and Retains Wild-Type Infection Properties.

: Orthohantaviruses are globally emerging zoonotic pathogens. While the reservoir host role of several rodent species is well-established, detailed research on the mechanisms of host-othohantavirus interactions has been constrained by the lack of an experimental system that is able to effectively replicate natural infections in controlled settings. Here we report the isolation, and genetic and phenotypic characterization of a novel Puumala orthohantavirus (PUUV) in cells derived from its reservoir host, the bank vole. The isolation process resulted in cell culture infection that evaded antiviral responses, persisted cell passaging, and had minor viral genome alterations. Critically, experimental infections of bank voles with the new isolate resembled natural infections in terms of viral load and host cell distribution. When compared to an attenuated Vero E6 cell-adapted PUUV Kazan strain, the novel isolate demonstrated delayed virus-specific humoral responses. A lack of virus-specific antibodies was also observed during experimental infections with wild-type PUUV, suggesting that delayed seroconversion could be a general phenomenon during orthohantavirus infection in reservoir hosts. Our results demonstrate that orthohantavirus isolation on cells derived from a vole reservoir host retains wild-type infection properties and should be considered the method of choice for experimental infection models to replicate natural processes.

Orthohantavirus pulmonary syndrome in Santa Cruz and Tarija, Bolivia, 2018.

INTRODUCTION: Orthohantaviruses are still a significant public health threat in endemic countries, with high case fatality rates (CFR). In Bolivia, the reporting of small outbreaks occurred until 2012. The findings of 40 laboratory-confirmed cases diagnosed in two departments are reported herein. METHODS: This was an observational, retrospective and cross-sectional study. Data on laboratory-confirmed cases in 2018 were collected from the hospitals and departmental health services (SEDES) of Santa Cruz and Tarija. An ELISA was used for the detection of IgM antibody to hantavirus in the patient blood samples. RESULTS: Forty patients were IgM-positive. The median age of the patients was 24 years (interquartile range 19-41 years) and 72.5% were male. All patients were hospitalized; 57.5% were admitted to the intensive care unit and had cardiopulmonary compromise, with 83% of these presenting acute respiratory distress syndrome and 89.5% of these requiring mechanical ventilation. Six patients died (CFR 15%). Patients <15 or >60 years old were more prone to die (odds ratio 10.33, 95% confidence interval 1.411-75.694), as were those with comorbidities (odds ratio 16.5, 95% confidence interval 1.207-225.540). CONCLUSIONS: Orthohantavirus infections were associated with a high CFR. These cases occurred in areas with eco-epidemiological conditions facilitating viral transmission, including the presence of rodents, as well as the risk of spillover to humans due to social, environmental, and occupational factors.

Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules.

Virus infection frequently triggers host cell stress signaling resulting in translational arrest; as a consequence, many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study, we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs) but does so in only a limited proportion of infected cells. Our data indicate some cell type-specific and hantavirus species-specific variability in SG prevalence and show SG formation to be dependent on the activation of protein kinase R (PKR). Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like endoplasmic reticulum (ER) kinase (PERK).IMPORTANCE Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

A Retrospective Survey of Rodent-borne Viruses in Rural Populations of Brazilian Amazon

Abstract INTRODUCTION: The Amazon tropical rainforest has the most dense and diverse ecosystem worldwide. A few studies have addressed rodent-borne diseases as potential hazards to humans in this region. METHODS: A retrospective survey was conducted using enzyme-linked immunosorbent assay for detecting mammarenavirus and orthohantavirus antibodies in 206 samples collected from rural settlers of the Brazilian Western Amazonian region. RESULTS: Six (2.91%) individuals in the age group of 16 to 36 years were found to possess antibodies against mammarenavirus. CONCLUSION: Evidence of previous exposure to mammarenavirus in the rural population points to its silent circulation in this region.

Novel Genetic Rearrangements Termed "Structural Variation Polymorphisms" Contribute to the Genetic Diversity of Orthohepadnaviruses.

The genetic diversity of orthohepadnaviruses is not yet fully understood. This study was conducted to investigate the role of structural variations (SVs) in their diversity. Genetic sequences of orthohepadnaviruses were retrieved from databases. The positions of sequence gaps were investigated, since they were found to be related to SVs, and they were further used to search for SVs. Then, a combination of pair-wise and multiple alignment analyses was performed to analyze the genomic structure. Unique patterns of SVs were observed; genetic sequences at certain genomic positions could be separated into multiple patterns, such as no SV, SV pattern 1, SV pattern 2, and SV pattern 3, which were observed as polymorphic changes. We provisionally referred to these genetic changes as SV polymorphisms. Our data showed that higher frequency of sequence gaps and lower genetic identity were observed in the pre-S1-S2 region of various types of HBVs. Detailed examination of the genetic structure in the pre-S region by a combination of pair-wise and multiple alignment analyses showed that the genetic diversity of orthohepadnaviruses in the pre-S1 region could have been also induced by SV polymorphisms. Our data showed that novel genetic rearrangements provisionally termed SV polymorphisms were observed in various orthohepadnaviruses.

A Novel Orthohepadnavirus Identified in a Dead Maxwell's Duiker (Philantomba maxwellii) in Taï National Park, Côte d'Ivoire.

New technologies enable viral discovery in a diversity of hosts, providing insights into viral evolution. We used one such approach, the virome capture sequencing for vertebrate viruses (VirCapSeq-VERT) platform, on 21 samples originating from six dead Maxwell's duikers (Philantomba maxwellii) from Taï National Park, Côte d'Ivoire. We detected the presence of an orthohepadnavirus in one animal and characterized its 3128 bp genome. The highest viral copy numbers were detected in the spleen, followed by the lung, blood, and liver, with the lowest copy numbers in the kidney and heart; the virus was not detected in the jejunum. Viral copy numbers in the blood were in the range known from humans with active chronic infections leading to liver histolytic damage, suggesting this virus could be pathogenic in duikers, though many orthohepadnaviruses appear to be apathogenic in other hosts, precluding a formal test of this hypothesis. The virus was not detected in 29 other dead duiker samples from the Côte d'Ivoire and Central African Republic, suggesting either a spillover event or a low prevalence in these populations. Phylogenetic analysis placed the virus as a divergent member of the mammalian clade of orthohepadnaviruses, though its relationship to other orthohepadnaviruses remains uncertain. This represents the first orthohepadnavirus described in an artiodactyl. We have tentatively named this new member of the genus Orthohepadnavirus (family Hepadnaviridae), Taï Forest hepadnavirus. Further studies are needed to determine whether it, or some close relatives, are present in a broader range of artiodactyls, including livestock.

Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells.

Orthohantaviruses, previously known as hantaviruses, are zoonotic viruses that can cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) in humans. The HPS-causing Andes virus (ANDV) and the HFRS-causing Hantaan virus (HTNV) have anti-apoptotic effects. To investigate if this represents a general feature of orthohantaviruses, we analysed the capacity of six different orthohantaviruses - belonging to three distinct phylogroups and representing both pathogenic and non-pathogenic viruses - to inhibit apoptosis in infected cells. Primary human endothelial cells were infected with ANDV, HTNV, the HFRS-causing Puumala virus (PUUV) and Seoul virus, as well as the putative non-pathogenic Prospect Hill virus and Tula virus. Infected cells were then exposed to the apoptosis-inducing chemical staurosporine or to activated human NK cells exhibiting a high cytotoxic potential. Strikingly, all orthohantaviruses inhibited apoptosis in both settings. Moreover, we show that the nucleocapsid (N) protein from all examined orthohantaviruses are potential targets for caspase-3 and granzyme B. Recombinant N protein from ANDV, PUUV and the HFRS-causing Dobrava virus strongly inhibited granzyme B activity and also, to certain extent, caspase-3 activity. Taken together, this study demonstrates that six different orthohantaviruses inhibit apoptosis, suggesting this to be a general feature of orthohantaviruses likely serving as a mechanism of viral immune evasion.