Mammalian deltavirus without hepadnavirus coinfection in the neotropical rodent Proechimys semispinosus.

Hepatitis delta virus (HDV) is a human hepatitis-causing RNA virus, unrelated to any other taxonomic group of RNA viruses. Its occurrence as a satellite virus of hepatitis B virus (HBV) is a singular case in animal virology for which no consensus evolutionary explanation exists. Here we present a mammalian deltavirus that does not occur in humans, identified in the neotropical rodent species Proechimys semispinosus The rodent deltavirus is highly distinct, showing a common ancestor with a recently described deltavirus in snakes. Reverse genetics based on a tandem minus-strand complementary DNA genome copy under the control of a cytomegalovirus (CMV) promoter confirms autonomous genome replication in transfected cells, with initiation of replication from the upstream genome copy. In contrast to HDV, a large delta antigen is not expressed and the farnesylation motif critical for HBV interaction is absent from a genome region that might correspond to a hypothetical rodent large delta antigen. Correspondingly, there is no evidence for coinfection with an HBV-related hepadnavirus based on virus detection and serology in any deltavirus-positive animal. No other coinfecting viruses were detected by RNA sequencing studies of 120 wild-caught animals that could serve as a potential helper virus. The presence of virus in blood and pronounced detection in reproductively active males suggest horizontal transmission linked to competitive behavior. Our study establishes a nonhuman, mammalian deltavirus that occurs as a horizontally transmitted infection, is potentially cleared by immune response, is not focused in the liver, and possibly does not require helper virus coinfection.

Identification of a Novel Deltavirus in Boa Constrictors.

Hepatitis D virus (HDV) forms the genus Deltavirus unassigned to any virus family. HDV is a satellite virus and needs hepatitis B virus (HBV) to make infectious particles. Deltaviruses are thought to have evolved in humans, since for a long time, they had not been identified elsewhere. Herein we report, prompted by the recent discovery of an HDV-like agent in birds, the identification of a deltavirus in snakes (Boa constrictor) designated snake HDV (sHDV). The circular 1,711-nt RNA genome of sHDV resembles human HDV (hHDV) in its coding strategy and size. We discovered sHDV during a metatranscriptomic study of brain samples of a Boa constrictor breeding pair with central nervous system signs. Applying next-generation sequencing (NGS) to brain, blood, and liver samples from both snakes, we did not find reads matching hepadnaviruses. Sequence comparison showed the snake delta antigen (sHDAg) to be 55% and 37% identical to its human and avian counterparts. Antiserum raised against recombinant sHDAg was used in immunohistology and demonstrated a broad viral target cell spectrum, including neurons, epithelial cells, and leukocytes. Using RT-PCR, we also detected sHDV RNA in two juvenile offspring and in a water python (Liasis macklotisavuensis) in the same snake colony, potentially indicating vertical and horizontal transmission. Screening of 20 randomly selected boas from another breeder by RT-PCR revealed sHDV infection in three additional snakes. The observed broad tissue tropism and the failure to detect accompanying hepadnavirus suggest that sHDV could be a satellite virus of a currently unknown enveloped virus.IMPORTANCE So far, the only known example of deltaviruses is the hepatitis delta virus (HDV). HDV is speculated to have evolved in humans, since deltaviruses were until very recently found only in humans. Using a metatranscriptomic sequencing approach, we found a circular RNA, which resembles that of HDV in size and coding strategy, in a snake. The identification of similar deltaviruses in distantly related species other than humans indicates that the previously suggested hypotheses on the origins of deltaviruses need to be updated. It is still possible that the ancestor of deltaviruses emerged from cellular RNAs; however, it likely would have happened much earlier in evolution than previously thought. These findings open up completely new avenues in evolution and pathogenesis studies of deltaviruses.

A Divergent Hepatitis D-Like Agent in Birds.

Hepatitis delta virus (HDV) is currently only found in humans and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release, and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence analysis revealed secondary structures that were shared with HDV, including self-complementarity and ribozyme features. The predicted viral protein shares 32% amino acid similarity to the small delta antigen of HDV and comprises a divergent phylogenetic lineage. The discovery of an avian HDV-like agent has important implications for the understanding of the origins of HDV and sub-viral agents.

Prevalence and phylogenetic analysis of hepatitis B virus among nonhuman primates in Taiwan.

Hepatitis B virus (HBV) is a public health problem worldwide, and apart from infecting humans, HBV has been found in nonhuman primates. This study investigated the prevalence and phylogenetic analysis of hepatitis B virus (HBV) and hepatitis D virus (HDV) among nonhuman primates in Taiwan, an area where human HBV remains endemic. Serum samples from 286 captive nonhuman primates (i.e., 32 great apes [Pan troglodytes and Pongo pygmaeus], 42 gibbons [Hylobates sp. and Nomascus sp.], and 212 Cercopithecidae monkeys) were collected and tested for the presence of HBV- and HDV-specific serologic markers. None of the Cercopithecidae monkeys were reactive against serologic markers of HBV. In contrast, 21.9% (7/32) of great apes and 40.5% (17/42) of gibbons tested positive for at least one serologic marker of HBV. Of these, five gibbons were chronic HBV carriers, characterized by presence of HBV DNA and hepatitis B surface antigen in the serum. HBV DNA was also detected in the saliva of three of the chronic carries. None of these HBV carrier gibbons exhibited symptoms or significant change in serum clinical chemistry related to HBV infection. Phylogenetic analysis of the complete HBV genome revealed that gibbon viruses clustered with other HBV isolates of great apes and gibbons from Southeast Asia and separately from human-specific HBV. None of the HBV-infected animals were reactive against HDV. These findings indicate that HBV found in these animals is indigenous to their respective hosts and might have been introduced into Taiwan via the direct import of infected animals from Southeast Asia. To reduce the horizontal and vertical transmission of HBV in captive animals, the HBV carriers should be kept apart from uninfected animals.

Animal models of hepatitis delta virus infection and disease.

Hepatitis delta virus (HDV) is a defective RNA virus with similarities to unusual subviral pathogens of higher plants. It requires hepatitis B virus (HBV) for its replication/transmission, and HBV-infected humans are the only established host. HDV causes both severe acute hepatitis and rapidly progressive chronic disease in some individuals. The HDV life cycle involves remarkable features, such as ribozyme- mediated autocatalytic processes, Pol II-directed RNA synthesis from a single-stranded circular RNA template, and RNA editing. Much of our understanding of the nature of this pathogen derives from experimental studies in the chimpanzee model of HBV infection. The hepadnavirus-infected eastern woodchuck also is capable of supporting HDV replication and offers opportunities for the development of control strategies that might be applicable to human type D hepatitis.

A novel form of hepatitis delta antigen.

Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication.

Chronic infection in woodchucks infected by a cloned hepatitis delta virus.

Two woodchucks (Marmota monax) intrahepatically inoculated with hepatitis delta virus (HDV) complementary DNA clones pSVL-D3 and pSVL-Ag showed virological and pathological signs of acute and chronic HDV infection. HDV-RNA and hepatitis delta antigen (HDAg) were detected in serum by slot-blot hybridization and by western blot five weeks after inoculation. Liver biopsy specimens collected at 8th week post inoculum were positive for HDV-RNA. Anti-HDV antibodies were detected at the 11th and 9th weeks, respectively. Histological finding of hepatocarcinoma and persistence of circulating HDV-RNA and anti-HDV were observed up to the 10th month. Both woodchucks produced "small" and "large" HDAg antigen, although the inoculated cloned DNA bears the coding capability solely for the small antigen. A transient decrease of woodchuck hepatitis virus DNA (WHV-DNA) level was observed during the peak of HDV infection. Successive inoculation of acute-phase serum in three woodchucks resulted in a successful infection in one of the animals.

Tissue culture system for infection with human hepatitis delta virus.

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.