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The risk of emerging infectious diseases (EID) is increasing globally. More than 60% of EIDs worldwide are caused by animal-borne pathogens, and most viral pathogens are rodent-borne. This study aimed to characterise the virome and analyse the phylogenetic evolution and diversity of rodent-borne viruses in Hainan Province, China. We collected 588 anal and throat samples from rodents, combined them into 28 pools according to their species and location, and processed them for next-generation sequencing and bioinformatics analysis. The diverse viral reads closely related to mammals were assigned to 15 viral families. Molecular clues of the important rodent-borne viruses were further identified by polymerase chain reaction for phylogenetic analysis and annotation of genetic characteristics such as coronavirus, arenavirus, picornavirus. We identified a pestivirus in Leopoldoms edwardsi and two bocaviruses in Rattus andamanensis and Leopoldoms edwardsi from the national nature reserves of Jianfengling and Bangxi with low amino acid identity to known pathogens are proposed as the novel species, and their rodent hosts have not been previously reported to carry these viruses. These results expand our knowledge of viral classification and host range and suggest that there are highly diverse, undiscovered viruses that have evolved independently in their unique wildlife hosts in inaccessible areas, which may cause zoonosis if they cross their host barrier. Our virome and phylogenetic analyses of rodent-borne viruses provide basic data for the prevention and control of human infectious diseases caused by rodent-borne viruses in the subtropical area of China.

Background: Emerging infectious diseases are mainly dominated by zoonotic diseases and rats are potential reservoirs for zoonotic infectious diseases. It is essential for preventing and treating such diseases to understand the composition of gut microbial communities in rats. As a tropical island locating in the south of China, Hainan province has abundant rat species. Here, we examined the gut microbial composition in adult rats from Hainan province of China.Methods: A total of 162 fresh faecal samples were collected from adult wild rats, including three species (Rattus norvegicus, Leopoldamys edwardsi, and Rattus losea), from nine regions of Hainan province in 2018. We analyzed the composition of gut microbiota using the 16S rRNA gene amplicon sequencing.Results: We identified 4,903 bacterial operational taxonomic units (OTUs) (30 phyla, 175 families, and 498 genera), which is different among distinct geographical distributions. Totally, Firmicutes were the most abundant phyla, followed by Bacteroidetes (15.55%), Proteobacteria (6.13%) and Actinobacteria (4.02%). The genus Lactobacillus (20.08%), unidentified_Clostridiales (5.16%), Romboutsia (4.33%), unidentified_Ruminococcaceae (3.83%), Bacteroides (3.66%), Helicobacter (2.40%) and Streptococcus (2.37%) were dominant in the samples. Among locations for rat species, the gut microbial communities were different in composition and abundance.Conclusion: This work provides information to identify microbial communities using for disease control in Hainan province.

Enolase is a crucial enzyme involved in the glycolytic pathway and gluconeogenesis in parasites. It also has been reported to function as a plasminogen receptor and may be involved in tissue invasion. In this study, the biochemical properties of the enolase of Spirometra mansoni (Smenolase) were investigated. The Smenolase gene was found to cluster closely with the enolase genes of Clonorchis sinensis and Echinococcus granulosus, and some functional motifs were identified as conserved. Smenolase was confirmed to be a component of the secretory/excretory products (ESPs) and a circulating antigen of spargana. Recombinant Smenolase expressed in vitro was able to bind to human plasminogen. Smenolase was detected in the eggs, testicles, and vitellaria of adult worms and the tegument of spargana. The transcription level of Smenolase was highest at the gravid proglottid stage. When spargana were cultured with glucose of different concentration in vitro, it was observed that the expression levels of Smenolase in the low-glucose groups were consistent with that of Smenolase in vivo. These results indicate that Smenolase is a critical enzyme involved in supplying energy to support the development and reproduction of the parasite, and it may also play a role in sparganum invasion.

Dengue virus is an arthropod-borne pathogen that is transmitted to humans primarily by Aedes spp. mosquitos, causing the acute infectious disease, dengue fever (DF). Until 2019, no dengue outbreak had been reported in Hainan Province for over 20 years. However, in early September of 2019, an increasing number of infected cases appeared and the DF outbreak lasted for over one month in Haikou City, Hainan Province. In our study, we collected 97 plasma samples from DF patients at three hospitals, as well as 1585 mosquito larvae samples from puddles in different areas of Haikou. There were 49 (50.5%) plasma samples found to be strongly positive and 9 (9.3%) plasma samples were weakly positive against the NS1 antigen. We discovered DENV both in the patient’s plasma samples and mosquito larvae samples, and isolated the virus from C6/36 cells inoculated with the acute phase serum of patients. Phylogenetic analysis revealed that the new strains were the most closely related to the epidemic strain in the southern regions of China, belonging to lineage IV, genotype I, DENV-1. Compared to the seven closest strains from neighboring countries and provinces, a total of 18 amino acid mutations occurred in the coding sequences (CDS) of the new isolated strain, DENV1 HMU-HKU-2. Our data shows that dengue virus is re-emerged in Hainan, and pose new threats for public health. Thus regular molecular epidemiological surveillance is necessary for control and prevention of DENV transmission. Electronic supplementary material The online version of this article (10.1007/s12250-020-00335-x) contains supplementary material, which is available to authorized users.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Recent studies showed that the distribution of hepatitis B virus (HBV) genotype exhibited geographical and ethnic characteristics. Haikou city is the largest city on Hainan Island that geographically isolated from mainland of China, and is the home of multiple ethnic groups. The aim of the study was to investigate the characteristics of the HBV genotype/subgenotype distribution in Haikou city. HBV DNA was isolated from180 serum samples derived from the Han and Li groups. The HBV genotype was detected by polymerase chain reaction using genotype-specific primers and was further determined by full-length genome sequences. The results revealed that the genotype B (37.2%) and C (62.8%) were the predominant HBV genotypes in Haikou, regardless of ethnic background., Additionally, the genotype distribution was not significantly different regarding ethnicity, sex or level of serum HBV DNA. Moreover, there were multiple subgenotypes circulating in the region. In conclusion, our study revealed the diverse HBV genotypes/subgenotypes in Haikou. These findings provide a preliminary study of the distribution of HBV genotypes circulating on Hainan Island.

Currently, multiplex-PCR with genotype-specific primers is widely used for preliminary screening of hepatitis B virus (HBV) genotyping, despite its relatively lower accuracy compared with whole genome sequencing. Here, we present the discrepant results of HBV genotyping by PCR and full-length sequencing. HBV DNA was isolated from chronic hepatitis B serum and the HBV genotype was detected by PCR using genotype-specific primers and full-length genome sequencing. As a result, the determination of genotype B by the PCR method was consistent with the DNA sequencing results; however, PCR revealed that genotype C exhibited a mixed genotype of B and C in the current study. In conclusion, the PCR-based genotyping method may not provide accurate information of the HBV genotype and whole genome sequencing remains the “gold standard” method for HBV genotyping.

The discovery of new viruses is important for predicting their potential threats to the health of humans and other animals. A novel picornavirus was identified from oral, throat, and anal swab samples collected from belugas (Delphinapterus leucas), from Dalian Sun Asia Tourism Holding Co., China, between January and December 2018, using a metagenomics approach. The genome of this novel PicoV-HMU-1 strain was 8197 nucleotides (nt) in length, with a open reading frame (from 1091 to 8074 nt) that encoded a polyprotein precursor of 2328 amino acids. Moreover, the genomic length and GC content of PicoV-HMU-1 were within the ranges found in other picornaviruses, and the genome organization was also similar. Nevertheless, PicoV-HMU-1 had a lower amino acid identity and distinct host species compared with other members of the Picornaviridae family. Phylogenetic trees were constructed based on the P1 and 3D amino acid sequences of PicoV-HMU-1 along with representative members of the Picornaviridae family, which showed that PicoV-HMU-1 was related to unclassified bat picornaviruses groups. These findings suggest that the PicoV-HMU-1 strain represents a potentially novel genus of picornavirus. These data can enhance our understanding of the picornavirus genetic diversity and evolution.

Enolase is a crucial enzyme involved in the glycolytic pathway and gluconeogenesis in parasites. It also has been reported to function as a plasminogen receptor and may be involved in tissue invasion. In this study, the biochemical properties of the enolase of Spirometra mansoni (Smenolase) were investigated. The Smenolase gene was found to cluster closely with the enolase genes of Clonorchis sinensis and Echinococcus granulosus, and some functional motifs were identified as conserved. Smenolase was confirmed to be a component of the secretory/excretory products (ESPs) and a circulating antigen of spargana. Recombinant Smenolase expressed in vitro was able to bind to human plasminogen. Smenolase was detected in the eggs, testicles, and vitellaria of adult worms and the tegument of spargana. The transcription level of Smenolase was highest at the gravid proglottid stage. When spargana were cultured with glucose of different concentration in vitro, it was observed that the expression levels of Smenolase in the low-glucose groups were consistent with that of Smenolase in vivo. These results indicate that Smenolase is a critical enzyme involved in supplying energy to support the development and reproduction of the parasite, and it may also play a role in sparganum invasion.

Two strains (NLN63T and NLN82) of Gram-stain-negative, oxidase- and catalase-positive, bacilli-shaped organisms were isolated from the faecal samples of two separate Rattus norvegicus in Baisha county of Hainan Province, Southern PR China. Phylogenetic analysis based on the near full-length 16S rRNA sequences revealed that strain NLN63T belongs to the genus Pelistega , having maximum similarity to Pelistega suis CCUG 64465T (97.1 %), Pelistega europaea CCUG 39967T (96.2 %) and Pelistega indica DSM 27484T (96.2 %), respectively. The phylogenomic tree built on 553 core genes from genomes of 20 species in the genus Pelistega and other adjacent genera further confirmed that strains NLN63T and NLN82 form a distinct subline and exhibit specific phylogenetic affinity with P. europaea CCUG 39967T. In digital DNA–DNA hybridization analyses, strain NLN63T showed low estimated DNA reassociation values (21.4–22.6 %) with the type strains of the species in the genus Pelistega . The DNA G+C contents of strains NLN63T and NLN82 were 37.3 and 37.1 mol%, respectively. Strain NLN63T had a unique MALDI-TOF MS profile, contained Q-8 as the major quinone and C16 : 0, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c or both) and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c or both) as the dominant fatty acids. Based upon these polyphasic characterization data obtained from the present study, a novel species of the genus Pelistega , Pelistega ratti sp. nov., is proposed with NLN63T (=GDMCC 1.1697T=JCM 33788T) as the type strain.

Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication. IMPORTANCE Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence.

Enterocytozoon bieneusi is a potentially important zoonotic pathogen. However, there is no information on E. bieneusi infection of captive long-tailed macaques (Macaca fascicularis) in Hainan Province, China. Here 193 fecal specimens of M. fascicularis were collected from a breeding base in Hainan Province, China, housing non-human primates for experimental use. E. bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene. A total of 59 (30.6%) specimens were PCR-positive for E. bieneusi and 16 ITS genotypes were identified including nine known genotypes: Type IV (n = 19), D (n = 11), CM1 (n = 8), PigEBITS7 (n = 4), Pongo2 (n = 4), Peru8 (n = 3), Peru11 (n = 1), WL21 (n = 1) and CM2 (n = 1) and seven novel genotypes HNM-I to HNM-VII (one each). Importantly, genotypes D, Type IV, Peru8, PigEBITS7, and Peru11, which were the predominant (38/59, 64.4%) genotypes identified among captive M. fascicularis in this study, are also well-known human-pathogenic genotypes. All the genotypes of E. bieneusi identified here, including the seven novel ones, belonged to zoonotic Group 1. This is the first report of the identification of E. bieneusi in M. fascicularis in Hainan Province, China. The finding that the numerous known human-pathogenic types and seven novel genotypes of E. bieneusi all belong to zoonotic Group 1 indicates the possibility of transmission of this important pathogenic parasite between M. fascicularis and humans.

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.

Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV. IMPORTANCE Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill defined, but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCC DNA. Our studies have thus revealed a novel mechanism of controlling viral persistence via regulating nucleocapsid disassembly.

Mutations in the basal core promoter (BCP) and precore (PC) regions of the hepatitis B virus (HBV) are more common in genotypes B and C than in genotype A, suggesting that these mutations might affect replication competency depending on genotype. The purpose of the study was to investigate the influence of these mutations on the capacity of HBV for replication and antiviral drug susceptibility according to genotype. Genotypes A, B, and C of HBV strains with a BCP mutation, PC mutation, or BCP + PC mutation were made by site-directed mutagenesis. Replication competency of each construct and susceptibility to nucleos(t) ide analogues were tested in an Huh7 cell line. In genotype A, the BCP and BCP + PC mutations increased the viral replication around 6.5 times compared with the wild type, and the PC mutation alone similarly increased the viral replication around three times. In genotypes B and C, all three mutant types increased viral replication to a similar extent, regardless of mutation pattern. Interestingly, the BCP mutation appeared to have a greater effect on viral replication in genotype A than in genotypes B and C. This finding was unexpected because the BCP mutation is more common in HBV genotypes B and C. Moreover, the BCP, PC, and BCP + PC mutations decreased the sensitivity of HBV to antiviral agents to various degrees (2- to 10-fold) regardless of genotype. In conclusion, BCP and PC mutations increased viral replication regardless of HBV genotype and decreased in vitro antiviral susceptibility to the nucleos(t) ide analogues. J. Med. Virol. 87:601–608, 2015. © 2015 Wiley Periodicals, Inc.

Background: Adefovir (ADV) resistance is more frequent in lamivudine (LMV)-resistant chronic hepatitis B (CHB) patients than in nucleos(t)ide analogue-naive patients. The majority of LMV-resistant mutants harbor the rtM204V/I mutation, while a minor fraction harbor the rtA181V/T mutation. We aimed to elucidate the mechanism of the high rate of ADV resistance in LMV-resistant patients during ADV therapy. Methods: We performed a clonal analysis of HBV reverse transcriptase in treatment-naive (n = 3) and LMV-resistant patients before ADV therapy (n = 14). Dynamic changes in the viral population (n = 9) during ADV therapy were also analyzed. Results: Before ADV therapy, rtA181V/T was observed in 30 of 680 clones (4.4%) from 7 patients with LMV resistance under dominant rt204V/I mutation and in one of 150 clones in treatment-naive patients. The rtA181V/T mutation was more frequently found in clones from LMV-resistant patients than in treatment-naive patients (p = 0.029). The rtN236T mutation was not observed in any clone. During ADV therapy, most rtM204V/I mutants were replaced by wild type in all 3 patients without the rtA181V/T mutation and in one patient with the rtA181V/T mutation. Subsequently, wild type was replaced by the rtN236T and/or rtA181V/T mutant. In patients with the rtA181V/T mutation (n = 6), the rtA181V/T mutant overtook the rtM204V/I mutant in 3 of 4 patients with ADV resistance. In 2 patients without ADV resistance, most of the viral population was replaced by wild type by the last follow-up. Conclusion: The high rate of ADV resistance in patients with LMV-resistance might be attributable to preexisting rtA181V/T mutant virus.

Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3′ exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3′ end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5′ end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC DNA to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved.

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion.

Angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are expressed in both vascular and adipose tissues. Angiotensin II (AG II) has an adipogenic effect and increases PAI-1 expression. To evaluate the chronic effects of AG II type 1 receptor (AT(1)R) antagonism on adipose mass and PAI-1 expression in vascular and adipose tissues, losartan (30mg/kg/day) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes, for 20 weeks. Adipose mass and regional fat distribution in the abdomen did not change after chronic AT(1)R antagonism in OLETF rats. AGT and PAI-1 mRNA expressions in adipose tissue of OLETF rats were significantly increased compared with Long-Evans Tokushima Otsuka (LETO) rats, the normal control. Chronic losartan therapy further increased the level of adipose AGT in OLETF rats, but did not affect the level of adipose PAI-1 mRNA. In contrast, aortic PAI-1 expression in OLETF rats was attenuated by chronic losartan therapy. Our results have two implications. First, adipose tissue may be an important source of AG II in metabolic syndrome even after chronic losartan therapy. Second, chronic AT(1)R antagonism with losartan causes differential effects on vascular and adipose PAI-1 expression.

Objectives: It has been reported that distribution of hepatitis B virus (HBV) genotypes shows geographic difference and are associated with clinical outcomes of HBV infection, including response to antiviral therapy and progression of chronic liver diseases. In this study, we analyzed the distribution of HBV genotypes according to the various clinical outcomes of chronic HBV infection in Korea, which is one of the most endemic areas of HBV infection. Methods: A total of 200 patients with chronic HBV infection were enrolled. Clinical diagnoses of the 200 patients with chronic liver diseases were as follows: hepatitis B e antigen (HBeAg)-positive healthy carrier (defined as HBeAg+, anti-HBe–, HBV DNA+ by hybridization, normal transaminase; n = 40); inactive HBsAg carrier (n = 40); chronic hepatitis B (n = 40); liver cirrhosis (n = 40); hepatocellular carcinoma (n = 40). HBV genotypes were determined by nested polymerase chain reaction using genotype-specific primers. Results: All patients except 2 (inactive HBsAg carriers) were positive for nested PCR and they have genotype C regardless of clinical outcomes. Conclusions: HBV genotype was genotype C regardless of various clinical outcomes of chronic HBV infection in Korea. Considering that HBV genotypes have clinical relevance, distribution of HBV genotype in each area should be monitored when management for chronic HBV infection is planned.

It has been hypothesized that naturally occurring mutations in HBV may play a role in the pathogenesis of HBV-related disease. We determined the molecular characteristics of naturally occurring HBV isolates and performed a functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B. The 10 HBV clones isolated were identified as HBV genotype B4 and subtype adw. In most clones, amino acid substitutions and nucleotide changes occurred in a specific region of the core protein, X protein and in the core promoter. In the core protein, cI3L, cL60M, and cI97L were detected in 8 of 10 clones and cP130T was detected in all 10 clones. In the X protein, xI127M was detected in 5 clones. In the basal core promoter, the A1762T/G1764A mutation was found only in 1 clone. An 11-bp nucleotide insertion between nucleotides 1772 and 1773, was found in 2 clones. Six clones that were replication-competent exhibited variation in the level of replicating capacity in vitro even though the average genetic distance between the HBV clones was only 0.5% (range: 0.3–0.7%). Among the replication-competent 6 clones, 5 clones showed higher replication competency compared with clone B9 (reference clone used in this study) in Huh7 cells. However, 4 clones showed lower replication competency compared with clone B9 in HepG2 cells. In conclusion, the HBV virus exhibits genetic variation in the form of quasispecies with different mutation patterns, and these quasispecies may be recognized by distinct viral replication patterns even in patients with subtle genetic mutation.

Objective: In vitro studies showed that mutations in the basal core promoter (BCP) or precore (PC) region restore the replication inefficiency of the lamivudine-resistant mutant. The aim of this study was to clarify the effect of molecular characteristics on the antiviral response to adefovir in patients with lamivudine-resistant chronic hepatitis B (CHB). Methods: Sixty-six lamivudine-resistant patients who were treated with adefovir monotherapy were studied. Sequences of BCP, PC region and reverse transcriptase were determined before adefovir therapy. In patients with virologic breakthrough, reverse transcriptase sequencing was performed. Results: The cumulative probabilities of virologic response were 23.3, 46, 52.7 and 59.5% at years 1, 2, 3 and 4, respectively. PC mutation, the absence of compensatory mutations (rtL80I/V or rtV173L), and a decrease in serum hepatitis B virus (HBV) DNA by 3 log or greater at 6 months were independent predictors of virologic response. The cumulative probabilities of virologic breakthrough were 0, 12.9, 30.7 and 44.5% at years 1, 2, 3 and 4, respectively. BCP mutation and a less than 3 log decrease in serum HBV DNA at 6 months were 2 independent risk factors for virologic breakthrough. Conclusion: Response to adefovir depends on mutation patterns in the BCP, PC region and reverse transcriptase, and on-treatment decreases in serum HBV DNA in lamivudine-resistant CHB patients.

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo., Funding for this work came from the National Institutes of Health, http://www.nih.gov, R01 AI074982 to JH, R21 AI101558 to CS, and T32 CA060395 to RM.

Objectives: Despite the pathogenic role of the basal core promoter (BCP) and the precore mutations in chronic hepatitis B virus (HBV) infection, their role in the progression of liver disease is still controversial. We analyzed whether the accumulation of these mutations might enhance the progression of HBV-related chronic liver disease. Methods: Forty consecutive patients at each clinical status were analyzed. Clinical statuses were as follows: HBeAg-positive asymptomatic carrier (HBeAg+ ASC) (defined as HBeAg+, anti-HBe–, HBV-DNA+ by hybridization, normal ALT); inactive HBsAg carrier; chronic hepatitis B; liver cirrhosis. The genotype and the BCP/precore regions were determined by PCR using genotype specific primers and direct sequencing, respectively. Results: All patients except one were infected with genotype C. The A to T mutation at nucleotide 1762 and/or G to A mutation at nucleotide 1764 were found in 30% in HBeAg+ ASC, 65.7% in inactive HBsAg carrier, 95% in chronic hepatitis B, and 90% in liver cirrhosis (p < 0.001). The prevalence of the G to A mutation at nucleotide 1896 was 5% in HBeAg+ ASC, 22.5% in inactive HBsAg carrier, 32.5% in chronic hepatitis B, and 50% in liver cirrhosis, respectively (p < 0.001). The T to C/A mutation at nucleotide 1753 in the BCP and G to A mutation at nucleotide 1899 in the precore were more frequent in liver cirrhosis than in the other clinical statuses (p < 0.05). Conclusion: Sequential accumulation of mutations in the BCP/precore has an important role in the progression of HBV-related liver disease.