Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.     

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

Biological characterization of acute infection with ground squirrel hepatitis virus.

Ground squirrel hepatitis virus (GSHV) is a small DNA virus, structurally and antigenically related to the human hepatitis B virus, which occurs naturally among certain wild populations of ground squirrels (P. L. Marion et al., Proc. Natl. Acad. Sci. U.S.A. 77:2941-2945, 1980). Serum from naturally infected animals was used to transmit GSHV in the laboratory by parenteral inoculation of susceptible squirrels. Sixty percent of recipient animals developed viral surface antigenemia after a latent period of 2 to 3 months; three of these animals have remained viremic for over 9 months. Like hepatitis B virus, GSHV demonstrates marked hepatotropism, with viral DNA detected in significant quantities only in the liver, where an average of 6 X 10(2) to 6 X 10(3) viral DNA molecules per cell were found by molecular hybridization. However, histological signs of liver injury after acute infection are minimal. In contrast to infection of its natural host, parenteral administration of GSHV to rats, mice, guinea pigs, and hamsters did not result in demonstrable antigenemia, suggesting that the host range of GSHV, like that of hepatitis B virus, is narrow.     

Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning.

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.     

A new hepadnavirus endemic in arctic ground squirrels in Alaska.

We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.     

Analysis of integrated ground squirrel hepatitis virus and flanking host DNA in two hepatocellular carcinomas.

We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.     

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus.

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

嗜肝病毒家族成员间进化关系的初步研究

比较分析了嗜肝病毒家族四成员间的全基因核苷酸序列,得出了它们之间的相对进化距离,DHBV 分野最早,GSHV,WHV 次之,HBV 最晚;其进化方式属趋 异性进化.进一步比较各 ORF 的保守性,发现各 ORF 间及同一 ORF 内的不同区域核苷酸发生替换的频率有明显差别;讨论了各 ORF 的特点及意义.     

Failure to detect polyalbumin-binding sites on the woodchuck hepatitis virus surface antigen: implications for the pathogenesis of hepatitis B virus in humans.

Binding sites for polymerized albumin on hepatitis B virus components were reported in human hepatitis B virus chronic carriers predominantly with active viral replication (HB e antigen positive). The presence of comparable albumin-binding sites in the woodchuck hepatitis virus (WHV) model was examined on WHV components obtained from woodchucks with active viral replication (DNA polymerase positive). Binding sites for polymerized woodchuck serum albumin were not detected on the intact WHV virion, on 22-nm woodchuck hepatitis surface antigen (WHsAg), or on WHsAg polypeptides. Woodchuck albumin was not detected in purified 22-nm WHsAg, and anti-albumin antibodies were not detected in WHV chronic-carrier woodchucks. Our results in the WHV model argue against a role for viral polyalbumin-binding sites in tissue- and host-specific virus infectivity.     

Nucleotide sequence of a cloned woodchuck hepatitis virus genome: evolutional relationship between hepadnaviruses.

We have determined the complete nucleotide sequence of a cloned DNA of woodchuck hepatitis virus (WHV), the most oncogenic virus among hepadnaviruses. The genome, designated WHV2, is 3,320 base pairs long and contains four major open reading frames (ORFs) coded on the same strand of nucleotide sequence as in the human hepatitis B virus (HBV) genome. Comparison of the nucleotide sequence and amino acid sequences deduced from it among the genomes of various hepadnaviruses demonstrates that each protein shows an intrinsic property in conserving its amino acid sequence. A parameter, the ratio of the number of triplets with one-letter change but no amino acid substitution to the total number of triplets in which one-letter change occurred, was introduced to measure the intrinsic properties quantitatively. For each ORF, the parameter gave characteristic values in all combinations. Therefore, the relative evolutional distance between these hepadnaviruses can be measured by the amino acid substitution rate of any ORF. These comparisons suggest that (i) the difference between two WHV clones, WHV1 and WHV2, corresponds to that among clones of a HBV subtype, HBVadr, and (ii) WHV and ground squirrel hepatitis virus can be categorized in a way similar to the subgroups of HBV.     

荧光定量PCR检测土拨鼠肝炎病毒核酸方法的建立

目的建立土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）核酸的荧光定量PCR（Real-time PCR）检测方法,应用于土拨鼠肝炎病毒模型的研究。方法分别根据土拨鼠肝炎病毒核心抗原（WHcAg）和表面抗原（WHsAg）的DNA序列设计13对扩增引物,从中筛选无非特异性扩增及引物二聚体且灵敏度高的引物,用于土拨鼠血清中WHV DNA的Real-time PCR检测。建立感染土拨鼠肝炎病毒的土拨鼠血清中WHV核酸的Real-timePCR检测方法。结果根据WHsAg基因的5＇端设计的一对引物WHVSF1与WHVSR1,检测灵敏度可达1×101拷贝/μL,病毒拷贝数与Real-time PCR Ct值的标准曲线的R2值为0.997,且电泳未见明显非特异性条带及引物二聚体。结论建立了土拨鼠血清中WHV DNA的Real-time PCR检测方法,该方法为进一步研究土拨鼠肝炎病毒模型奠定了基础。     

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.     

Nonoverlapping antigenic sites of woodchuck hepatitis virus surface antigen and their cross-reactivity with ground squirrel hepatitis virus and hepatitis B virus surface antigens.

Five nonoverlapping antigenic sites (sites I through V) of woodchuck hepatitis virus surface antigen were identified with competitive binding assays involving monoclonal antibodies. Site I contributed to cross-reactions among surface antigens of hepatitis B-like viruses infecting woodchucks, ground squirrels, and humans. At least three distinct sites (sites I, II, and III) are responsible for cross-reactions between woodchuck and ground squirrel hepatitis virus surface antigens. Sites IV and V of woodchuck hepatitis virus surface antigen are not major cross-reactive sites, suggesting that these elicit virus-specific antibodies. There were no cross-reactions with duck hepatitis B virus surface antigen.