Virome survey of the bat, Rhinolophus affinis, in Hainan Province, China.

Bats are important mammal reservoirs of zoonotic pathogens. However, due to research limitations involving species, locations, pathogens, or sample types, the full diversity of viruses in bats remains to be discovered. We used next-generation sequencing technology to characterize the mammalian virome and analyze the phylogenetic evolution and diversity of mammalian viruses carried by bats from Haikou City and Tunchang County in Hainan Province, China. We collected 200 pharyngeal swab and anal swab samples from Rhinolophus affinis, combining them into nine pools based on the sample type and collection location. We subjected the samples to next-generation sequencing and conducted bioinformatics analysis. All samples were screened via specific PCR and phylogenetic analysis. The diverse viral reads, closely related to mammals, were assigned into 17 viral families. We discovered many novel bat viruses and identified some closely related to known human/animal pathogens. In the current study, 6 complete genomes and 2 partial genomic sequences of 6 viral families and 8 viral genera have been amplified, among which 5 strains are suggested to be new virus species. These included coronavirus, pestivirus, bastrovirus, bocavirus, papillomavirus, parvovirus, and paramyxovirus. The primary finding is that a SADS-related CoV and a HoBi-like pestivirus identified in R. affinis in Hainan Province could be pathogenic to livestock. This study expands our understanding of bats as a virus reservoir, providing a basis for further research on the transmission of viruses from bats to humans.

Genomic and phylogenetic profiling of RNA of tick-borne arboviruses in Hainan Island, China.

Ticks act as vectors and hosts of numerous arboviruses. Examples of medically important arboviruses include the tick-borne encephalitis virus, Crimean Congo hemorrhagic fever, and severe fever with thrombocytopenia syndrome. Recently, some novel arboviruses have been identified in blood specimens of patients with unexplained fever and a history of tick bites in Inner Mongolia. Consequently, tick-borne viruses are a major focus of infectious disease research. However, the spectrum of tick-borne viruses in subtropical areas of China has yet to be sufficiently characterized. In this study, we collected 855 ticks from canine and bovine hosts in four locations in Hainan Province. The ticks were combined into 18 pools according to genus and location. Viral RNA-sequence libraries were subjected to transcriptome sequencing analysis. Molecular clues from metagenomic analyses were used to classify sequence reads into virus species, genera, or families. The diverse viral reads closely associated with mammals were assigned to 12 viral families and important tick-borne viruses, such as Jingmen, Beiji nairovirus, and Colorado tick fever. Our virome and phylogenetic analyses of the arbovirus strains provide basic data for preventing and controlling human infectious diseases caused by tick-borne viruses in the subtropical areas of China.

Diversity and independent evolutionary profiling of rodent-borne viruses in Hainan, a tropical island of China.

The risk of emerging infectious diseases (EID) is increasing globally. More than 60% of EIDs worldwide are caused by animal-borne pathogens. This study aimed to characterize the virome, analyze the phylogenetic evolution, and determine the diversity of rodent-borne viruses in Hainan Province, China. We collected 682 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 contigs closely related to mammals were assigned to 22 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 arenavirus, coronavirus, astrovirus, pestivirus, parvovirus, and papillomavirus. We identified pestivirus and bocavirus in Leopoldoms edwardsi from Huangjinjiaoling, and bocavirus in Rattus andamanensis from the national nature reserves of 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.

Comparative analysis of the gut microbiota of wild adult rats from nine district areas in Hainan, China.

BACKGROUND: Wild rats have the potential to hold zoonotic infectious agents that can spread to humans and cause disease. AIM: To better understand the composition of gut bacterial communities in rats is essential for preventing and treating such diseases. As a tropical island located in the south of China, Hainan province has abundant rat species. Here, we examined the gut bacterial composition in wild adult rats from Hainan province. METHODS: Fresh fecal samples were collected from 162 wild adult rats, including three species (Rattus norvegicus, Leopoldamys edwardsi, and Rattus losea), from nine regions of Hainan province between 2017-2018. RESULTS: We analyzed the composition of gut microbiota using the 16S rRNA gene amplicon sequencing. We identified 4903 bacterial operational taxonomic units (30 phyla, 175 families, and 498 genera), which vary between samples of different rat species in various habitats at various times of the year. In general, 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. CONCLUSION: The composition and abundance of the gut microbial communities varied between rat species and locations. This work provides fundamental information to identify microbial communities useful for disease control in Hainan province.

Lack of geographical and ethnic distribution of Hepatitis B virus genotypes in Hainan Island, China.

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.

Cyclocytidine hydrochloride inhibits the synthesis of relaxed circular DNA of hepatitis B virus.

Background: Cyclocytidine hydrochloride (HCl) has been reported to inhibit DNA synthesis by affecting DNA polymerase. Here, we tested the antiviral effect of cyclocytidine on hepatitis B virus (HBV) DNA synthesis, which is reliant on DNA polymerase activity. Materials and Methods: Cyclocytidine HCl was treated to HBV-producing HepAD38 cells or added to an endogenous polymerase reaction, and HBV DNA was detected by Southern blot. Results: Treatment of 20 µM cyclocytidine HCl significantly decreased the production of relaxed circular (rc) DNA in HepAD38 cells and block rcDNA synthesis in endogenous polymerase reaction (EPR), a cell free assay, possibly by inhibiting the HBV DNA polymerase activity. Conclusion: Cyclocytidine HCl could inhibit the synthesis of HBV rcDNA, the precursor of covalently closed circular DNA, and this result provides a case for the usage of "old" drugs for "new" applications.

Identification of a Novel Astrovirus in Pinnipeds.

Astroviruses infect human and animals and cause diarrhea, fever, and vomiting. In severe cases, these infections may be fatal in infants and juvenile animals. Previous evidence showed that humans in contact with infected animals can develop serological responses to astroviruses. Mamastrovirus 11 is a species of Mamastrovirus and was first reported in 2018. It was detected in the fecal samples of a California sea lion. The genome sequence of its capsid protein (CP) was submitted to GenBank. However, the genome sequence of its non-structural protein region was not elucidated. In the present study, we characterized the genome sequences of the novel astroviruses AstroV-HMU-1 and AstroV-like-HMU-2. These were obtained from California sea lions (Zalophus californianus) and walruses (Odobenus rosmarus) presenting with loose stools. A phylogenetic analysis revealed that the CP of AstroV-HMU-1 closely clustered with Mamastrovirus 11 while its RNA-dependent RNA polymerase (RdRp) and serine protease (SP) were closely related to the mink astrovirus in the genus Mamastrovirus. The genome of AstroV-HMU-1 provided basic information regarding the NS protein regions of Mamastrovirus 11. Recombination analyses showed that the genomes of Z. californianus AstroV-HMU-1, VA2/human and the mink astrovirus may have recombined long ago. The NS of AstroV-like-HMU-2 segregated from the Astroviridae in the deep root of the phylogenetic tree and exhibited 36% amino acid identity with other mamastroviruses. Thus, AstroV-like-HMU-2 was proposed as a member of a new genus in the unclassified Astroviridae. The present study suggested that that the loose stools of pinnipeds may be the result of occasional infection by this novel astrovirus. This discovery provides a scientific basis for future investigations into other animal-borne infectious diseases.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

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.

Discrepant results of hepatitis B virus genotype determination by PCR and DNA sequencing.

Currently, multiplex-PCR with genotype-specific primers is widely used for preliminary screening of hepatitis B virus (HBV) genotypes, 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 showed a mix of B and C genotypes in the current study. In conclusion, the PCR-based genotyping may not provide accurate information of the HBV genotype and whole genome sequencing remains the "gold standard" for HBV genotyping.

Identification and genome analysis of a novel picornavirus from captive belugas (Delphinapterus leucas) in China.

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.

Identification of an enolase gene and its physiological role in Spirometra mansoni.

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.

Phylogenetic Analysis of the Dengue Virus Strains Causing the 2019 Dengue Fever Outbreak in Hainan, China.

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.

Genotyping of Enterocytozoon bieneusi among captive long-tailed macaques (Macaca fascicularis) in Hainan Province: High genetic diversity and zoonotic potential.

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.

Identification of a Novel Ichthyic Parvovirus in Marine Species in Hainan Island, China.

Parvoviruses are a diverse group of viruses that are capable of infecting a wide range of animals. In this study, we report the discovery of a novel parvovirus, tilapia parvovirus HMU-HKU, in the fecal samples of crocodiles and intestines of tilapia in Hainan Province, China. The novel parvovirus was firstly identified from crocodiles fed with tilapia using next-generation sequencing (NGS). Screening studies revealed that the prevalence of the novel parvovirus in crocodile feces samples fed on tilapia (75-86%) was apparently higher than that in crocodiles fed with chicken (4%). Further studies revealed that the prevalence of the novel parvovirus in tilapia feces samples collected at four areas in Hainan Province was between 40 and 90%. Four stains of the novel parvovirus were identified in this study based on sequence analyses of NS1 and all the four strains were found in tilapia in contrast only two of them were detected in crocodile feces. The nearly full-length genome sequence of the tilapia parvovirus HMU-HKU-1 was determined and showed less than 45.50 and 40.38% amino acid identity with other members of Parvoviridae in NS1 and VP1 genes, respectively. Phylogenetic analysis based on the complete helicase domain amino acid sequences showed that the tilapia parvovirus HMU-HKU-1 formed a relatively independent branch in the newly proposed genus Chaphamaparvovirus in the subfamily Hamaparvovirinae according to the ICTV's most recent taxonomic criteria for Parvoviridae classification. Tilapia parvovirus HMU-HKU-1 likely represented a new species within the new genus Chaphamaparvovirus. The identification of tilapia parvovirus HMU-HKU provides further insight into the viral and genetic diversity of parvoviruses and its infections in tilapia populations need to be evaluated in terms of pathogenicity and production losses in tilapia farming.

Pelistega ratti sp. nov. from Rattus norvegicus of Hainan island.

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.

Complete Genome Sequence of a Rodent Torque Teno Virus in Hainan Island, China.

Torque teno virus (TTV) has been reported in a wide range of mammals. In this study, we sequenced and analyzed the complete genome of a genetic variant of a rodent TTV, RoTTV3-HMU1 (Hainan Medical University 1). The virus was found in a rat (Rattus norvegicus) in a residential area of Hainan Island, China.

Complete Genome Sequence of a Mammarenavirus Harbored by Rodents on Hainan Island, China.

Wenzhou virus (WENV) is a rodent-borne mammarenavirus that was recently found to infect humans. In this study, we sequenced and analyzed the complete genome of a genetic variant of WENV, HMU (Hainan Medical University) virus. The virus was harbored by a Rattus norvegicus individual in the residential areas of Hainan Province in southern China.

Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection.

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.

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes.

UNLABELLED: 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.

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

UNLABELLED: 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.