Adaptive Immune Responses, Immune Escape and Immune-Mediated Pathogenesis during HDV Infection.

The hepatitis delta virus (HDV) is the smallest known human virus, yet it causes great harm to patients co-infected with hepatitis B virus (HBV). As a satellite virus of HBV, HDV requires the surface antigen of HBV (HBsAg) for sufficient viral packaging and spread. The special circumstance of co-infection, albeit only one partner depends on the other, raises many virological, immunological, and pathophysiological questions. In the last years, breakthroughs were made in understanding the adaptive immune response, in particular, virus-specific CD4+ and CD8+ T cells, in self-limited versus persistent HBV/HDV co-infection. Indeed, the mechanisms of CD8+ T cell failure in persistent HBV/HDV co-infection include viral escape and T cell exhaustion, and mimic those in other persistent human viral infections, such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), and HBV mono-infection. However, compared to these larger viruses, the small HDV has perfectly adapted to evade recognition by CD8+ T cells restricted by common human leukocyte antigen (HLA) class I alleles. Furthermore, accelerated progression towards liver cirrhosis in persistent HBV/HDV co-infection was attributed to an increased immune-mediated pathology, either caused by innate pathways initiated by the interferon (IFN) system or triggered by misguided and dysfunctional T cells. These new insights into HDV-specific adaptive immunity will be discussed in this review and put into context with known well-described aspects in HBV, HCV, and HIV infections.

Combination of Novel Therapies for HDV.

Treatment options for HDV have been limited to interferon alfa-based therapies with its poor efficacy to side effects ratio. Several novel therapies have now advanced into the clinic. As they each have a different mechanism of action, there is the potential for combination therapy. Here we review how studying the HDV life cycle has led to the development of these novel therapies, the key developments leading to, and the details of, the first combination study of novel anti-HDV therapies, and suggest what additional combinations of novel therapies can be anticipated as we enter this exciting new area of HDV treatments.

Short '1.2× Genome' Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells.

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported "2× genome" and the "1.2× genome" infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

Constraint of Base Pairing on HDV Genome Evolution.

The hepatitis delta virus is a single-stranded circular RNA virus, which is characterized by high self-complementarity. About 70% of the genome sequences can form base-pairs with internal nucleotides. There are many studies on the evolution of the hepatitis delta virus. However, the secondary structure has not been taken into account in these studies. In this study, we developed a method to examine the effect of base pairing as a constraint on the nucleotide substitutions during the evolution of the hepatitis delta virus. The method revealed that the base pairing can reduce the evolutionary rate in the non-coding region of the virus. In addition, it is suggested that the non-coding nucleotides without base pairing may be under some constraint, and that the intensity of the constraint is weaker than that by the base pairing but stronger than that on the synonymous site.

A Rapid Point-of-Care Test for the Serodiagnosis of Hepatitis Delta Virus Infection.

Hepatitis Delta virus (HDV) is a satellite of the Hepatitis B virus (HBV) and causes severe liver disease. The estimated prevalence of 15-20 million infected people worldwide may be underestimated as international diagnostic guidelines are not routinely followed. Possible reasons for this include the limited awareness among healthcare providers, the requirement for costly equipment and specialized training, and a lack of access to reliable tests in regions with poor medical infrastructure. In this study, we developed an HDV rapid test for the detection of antibodies against the hepatitis delta antigen (anti-HDV) in serum and plasma. The test is based on a novel recombinant large hepatitis delta antigen that can detect anti-HDV in a concentration-dependent manner with pan-genotypic activity across all known HDV genotypes. We evaluated the performance of this test on a cohort of 474 patient samples and found that it has a sensitivity of 94.6% (314/332) and a specificity of 100% (142/142) when compared to a diagnostic gold-standard ELISA. It also works robustly for a broad range of anti-HDV titers. We anticipate this novel HDV rapid test to be an important tool for epidemiological studies and clinical diagnostics, especially in regions that currently lack access to reliable HDV testing.

Cell Culture Models for the Study of Hepatitis D Virus Entry and Infection.

Chronic hepatitis D is one of the most severe and aggressive forms of chronic viral hepatitis with a high risk of developing hepatocellular carcinoma (HCC). It results from the co-infection of the liver with the hepatitis B virus (HBV) and its satellite, the hepatitis D virus (HDV). Although current therapies can control HBV infection, no treatment that efficiently eliminates HDV is available and novel therapeutic strategies are needed. Although the HDV cycle is well described, the lack of simple experimental models has restricted the study of host-virus interactions, even if they represent relevant therapeutic targets. In the last few years, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key cellular entry factor for HBV and HDV has allowed the development of new cell culture models susceptible to HBV and HDV infection. In this review, we summarize the main in vitro model systems used for the study of HDV entry and infection, discuss their benefits and limitations and highlight perspectives for future developments.

Variable In Vivo Hepatitis D Virus (HDV) RNA Editing Rates According to the HDV Genotype.

Human hepatitis delta virus (HDV) is a small defective RNA satellite virus that requires hepatitis B virus (HBV) envelope proteins to form its own virions. The HDV genome possesses a single coding open reading frame (ORF), located on a replicative intermediate, the antigenome, encoding the small (s) and the large (L) isoforms of the delta antigen (s-HDAg and L-HDAg). The latter is produced following an editing process, changing the amber/stop codon on the s-HDAg-ORF into a tryptophan codon, allowing L-HDAg synthesis by the addition of 19 (or 20) C-terminal amino acids. The two delta proteins play different roles in the viral cell cycle: s-HDAg activates genome replication, while L-HDAg blocks replication and favors virion morphogenesis and propagation. L-HDAg has also been involved in HDV pathogenicity. Understanding the kinetics of viral editing rates in vivo is key to unravel the biology of the virus and understand its spread and natural history. We developed and validated a new assay based on next-generation sequencing and aimed at quantifying HDV RNA editing in plasma. We analyzed plasma samples from 219 patients infected with different HDV genotypes and showed that HDV editing capacity strongly depends on the genotype of the strain.

HDV-Like Viruses.

Hepatitis delta virus (HDV) is a defective human virus that lacks the ability to produce its own envelope proteins and is thus dependent on the presence of a helper virus, which provides its surface proteins to produce infectious particles. Hepatitis B virus (HBV) was so far thought to be the only helper virus described to be associated with HDV. However, recent studies showed that divergent HDV-like viruses could be detected in fishes, birds, amphibians, and invertebrates, without evidence of any HBV-like agent supporting infection. Another recent study demonstrated that HDV can be transmitted and propagated in experimental infections ex vivo and in vivo by different enveloped viruses unrelated to HBV, including hepatitis C virus (HCV) and flaviviruses such as Dengue and West Nile virus. All this new evidence, in addition to the identification of novel virus species within a large range of hosts in absence of HBV, suggests that deltaviruses may take advantage of a large spectrum of helper viruses and raises questions about HDV origins and evolution.

Hepatitis D virus in 2021: virology, immunology and new treatment approaches for a difficult-to-treat disease.

Approximately 5% of individuals infected with hepatitis B virus (HBV) are coinfected with hepatitis D virus (HDV). Chronic HBV/HDV coinfection is associated with an unfavourable outcome, with many patients developing liver cirrhosis, liver failure and eventually hepatocellular carcinoma within 5-10 years. The identification of the HBV/HDV receptor and the development of novel in vitro and animal infection models allowed a more detailed study of the HDV life cycle in recent years, facilitating the development of specific antiviral drugs. The characterisation of HDV-specific CD4+ and CD8+T cell epitopes in untreated and treated patients also permitted a more precise understanding of HDV immunobiology and possibly paves the way for immunotherapeutic strategies to support upcoming specific therapies targeting viral or host factors. Pegylated interferon-α has been used for treating HDV patients for the last 30 years with only limited sustained responses. Here we describe novel treatment options with regard to their mode of action and their clinical effectiveness. Of those, the entry-inhibitor bulevirtide (formerly known as myrcludex B) received conditional marketing authorisation in the European Union (EU) in 2020 (Hepcludex). One additional drug, the prenylation inhibitor lonafarnib, is currently under investigation in phase III clinical trials. Other treatment strategies aim at targeting hepatitis B surface antigen, including the nucleic acid polymer REP2139Ca. These recent advances in HDV virology, immunology and treatment are important steps to make HDV a less difficult-to-treat virus and will be discussed.

Investigating the prevalence and clinical effects of hepatitis delta viral infection in Taiwan.

PURPOSE: To clarify and investigate the prevalence and clinical impact of hepatitis D virus (HDV) infection in Taiwan's communities. METHODS: HDV infection in patients with chronic hepatitis B viral (HBV) infection was examined using an anti-HDV antibody in Yonghe Cardinal Tien Hospital (YCTH), a district hospital in Taiwan. Clinical characteristics of anti-HDV-positive and anti-HDV-negative patients were collected and compared. These characteristics were also compared with the data collected from a medical center. Continuous variables and confounding factor adjustments were compared using the analysis of covariance method, whereas categorical variables were compared using the logistic regression method. RESULTS: A total of 346 patients with chronic HBV infection were assessed from 2018 to 2019. Among them, 4 (1.15%) were positive for anti-HDV. The clinical, virological, and biochemical characteristics were similar between anti-HDV-positive and anti-HDV-negative groups. None of the four patients was positive for serum HDV RNA. Another 18 anti-HDV-positive patients were identified from National Taiwan University Hospital (NTUH). The clinical, virological, and biochemical characteristics of anti-HDV-positive patients from YCTH and NTUH were also similar. CONCLUSION: The prevalence of HDV and the serum HDV RNA-positive rate were low in district hospitals in Taiwan. Coexisting HDV infection did not influence the clinical manifestation of patients with chronic HBV infection in Taiwan. However, because the number of HDV RNA cases was very small, our findings may not be conclusive. Besides, since the sensitivity of current anti-HDV kit is not 100%, more sensitive methods are needed to achieve reliable prevalence data.

Hepatitis D Virus and Hepatocellular Carcinoma.

Hepatitis D virus (HDV) is a small, defective RNA virus that depends on hepatitis B virus (HBV) for virion assembly and transmission. It replicates within the nucleus of hepatocytes and interacts with several cellular proteins. Chronic hepatitis D is a severe and progressive disease, leading to cirrhosis in up to 80% of cases. A high proportion of patients die of liver decompensation or hepatocellular carcinoma (HCC), but the lack of large prospective studies has made it difficult to precisely define the rate of these long-term complications. In particular, the question of whether HDV is an oncogenic virus has been a matter of debate. Studies conducted over the past decade provided evidence that HDV is associated with a significantly higher risk of developing HCC compared to HBV monoinfection. However, the mechanisms whereby HDV promotes liver cancer remain elusive. Recent data have demonstrated that the molecular profile of HCC-HDV is unique and distinct from that of HBV-HCC, with an enrichment of upregulated genes involved in cell-cycle/DNA replication, and DNA damage and repair, which point to genome instability as an important mechanism of HDV hepatocarcinogenesis. These data suggest that HBV and HDV promote carcinogenesis by distinct molecular mechanisms despite the obligatory dependence of HDV on HBV.

AAV-HDV: An Attractive Platform for the In Vivo Study of HDV Biology and the Mechanism of Disease Pathogenesis.

Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, but little is known about the molecular mechanisms involved. We have recently developed an HDV mouse model based on the delivery of HDV replication-competent genomes using adeno-associated vectors (AAV), which developed a liver pathology very similar to the human disease and allowed us to perform mechanistic studies. We have generated different AAV-HDV mutants to eliminate the expression of HDV antigens (HDAgs), and we have characterized them both in vitro and in vivo. We confirmed that S-HDAg is essential for HDV replication and cannot be replaced by L-HDAg or host cellular proteins, and that L-HDAg is essential to produce the HDV infectious particle and inhibits its replication. We have also found that lack of L-HDAg resulted in the increase of S-HDAg expression levels and the exacerbation of liver damage, which was associated with an increment in liver inflammation but did not require T cells. Interestingly, early expression of L-HDAg significantly ameliorated the liver damage induced by the mutant expressing only S-HDAg. In summary, the use of AAV-HDV represents a very attractive platform to interrogate in vivo the role of viral components in the HDV life cycle and to better understand the mechanism of HDV-induced liver pathology.

Hepatitis D Virus Entry Inhibitors Based on Repurposing Intestinal Bile Acid Reabsorption Inhibitors.

Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as high-affinity hepatic entry receptor for the Hepatitis B and D viruses (HBV/HDV) opened the field for target-based development of cell-entry inhibitors. However, most of the HBV/HDV entry inhibitors identified so far also interfere with the physiological bile acid transporter function of NTCP. The present study aimed to identify more virus-selective inhibitors of NTCP by screening of 87 propanolamine derivatives from the former development of intestinal bile acid reabsorption inhibitors (BARIs), which interact with the NTCP-homologous intestinal apical sodium-dependent bile acid transporter (ASBT). In NTCP-HEK293 cells, the ability of these compounds to block the HBV/HDV-derived preS1-peptide binding to NTCP (virus receptor function) as well as the taurocholic acid transport via NTCP (bile acid transporter function) were analyzed in parallel. Hits were subsequently validated by performing in vitro HDV infection experiments in NTCP-HepG2 cells. The most potent compounds S985852, A000295231, and S973509 showed in vitro anti-HDV activities with IC50 values of 15, 40, and 70 µM, respectively, while the taurocholic acid uptake inhibition occurred at much higher IC50 values of 24, 780, and 490 µM, respectively. In conclusion, repurposing of compounds from the BARI class as novel HBV/HDV entry inhibitors seems possible and even enables certain virus selectivity based on structure-activity relationships.

Demographics and outcomes of hepatitis B and D: A 10-year retrospective analysis in a Swiss tertiary referral center.

BACKGROUND: Hepatitis B virus (HBV) is a major global health challenge with approximately 250-350 million chronically infected individuals. An improved understanding of the demographic features and outcomes of chronic HBV infection and hepatitis D virus (HDV) infection in low-endemic areas may improve prevention, early identification and management both at individual and community levels. Here, we retrospectively analyzed the demographic and clinical characteristics, treatment rates and outcomes of adult patients with chronic HBV infection with or without HDV coinfection examined at Lausanne University Hospital, Switzerland over a 10-year period. METHODS: We analyzed the medical records of all adult patients with chronic HBV and HDV infection examined in our center between 2007 and 2016. Liver-related outcome was defined as the occurrence of cirrhosis, hepatocellular carcinoma, liver transplantation or liver-related death. Analyses were performed using logistic regression and results were reported as odds ratio (OR) and 95% confidence interval (CI). RESULTS: Of 672 consecutive patients, 421 (62.6%) were male, median age was 36 years (interquartile range, 28-46 years), and 233 (34.7%) were of African origin. The prevalence of HDV coinfection was 7.1% and the proportion of anti-HDV-positive patients with detectable HDV RNA was 70.0%. In multivariate analysis, HDV coinfection was the strongest predictor for liver-related outcome (OR 6.06, 95% CI 2.93-12.54, p<0.001), followed by HBeAg positivity (OR 2.47, 95% CI 1.30-4.69, p = 0.006), age (OR per 10-year increase 2.03, 95% CI 1.63-2.52, p<0.001) and sex (OR for female 0.39, 95% CI 0.22-0.71, p = 0.002). The predictive accuracy of the multivariate model was high (receiver operator characteristic area under the curve 0.81). CONCLUSION: This retrospective study underscores the importance of migration in the epidemiology of chronic hepatitis B in low-endemic areas. HDV coinfection, HBeAg positivity and age predicted liver-related outcomes while female sex had a protective effect.

Forty-Five Years after the Discovery of the Hepatitis D Virus: Where Do We Stand?

The discovery of the Australia Antigen in the mid-1960s led, in a few years, to the identification of the virus of Hepatitis B [...].

Predominance of HBV Genotype B and HDV Genotype 1 in Vietnamese Patients with Chronic Hepatitis.

Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n = 115), liver cirrhosis (LC; n = 21), and hepatocellular carcinoma (HCC; n = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the "a" determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (n = 204) and 7% (n = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; n = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; n = 134), followed by HBV-C (31%; n = 64), HBV-D, and HBV-G (3%; n = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; n = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (n = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections.

Hepatitis D virus antibodies and liver function profile among patients with chronic hepatitis B infection in Abuja, Nigeria.

INTRODUCTION: Hepatitis D virus (HDV) is a satellite virus of hepatitis B virus (HBV). An estimated 5% of HBV infected individuals worldwide have HDV infection. There is paucity of studies in Nigeria on the burden of HDV infection. This study aimed at determining the prevalence rate of HDV antibodies among individuals with chronic hepatitis B (CHB) infection and comparing the liver function test (LFT) and disease severity among the anti-HDV positive (anti-HDV+) and anti-HDV negative (anti-HDV-) individuals. METHODOLOGY: A cross-sectional study of 180 CHB infected individuals who were clinically evaluated and tested for HDV antibodies using the Enzyme-linked Immunoassay method. Their LFT profile and Child-Turcotte-Pugh (CTP) were also assessed. Data were analyzed using the SPSS version 17. RESULTS: Their mean age was 35.2 ± 10.4 years. There were 150 (83.3%) and 30 (16.7%) individuals with uncomplicated and complicated CHB infection respectively. Thirty-four (18.9%) of the participants were anti-HDV+. The mean serum ALT, AST, albumin and INR of the anti-HDV+ subjects were 16.5 ± 13.8 IU/L, 26.3 ± 32.6 IU/L, 38.9 ± 7.6 g/L, and 1.2 ± 0.2 respectively. The mean values for the same parameters of the anti-HDV- subjects were 10.8 ± 9.5 IU/L, 13.4 ± 11.2 IU/L, 41.4 ± 6.0 g/L and 1.1 ± 0.2 respectively (p < 0.05). The mean CTP scores in the anti-HDV+ and anti-HDV- subjects were 6.1 ± 2.1 and 5.5 ± 1.2 respectively (p= 0.03). CONCLUSIONS: Anti-HDV sero-prevalence rate was 18.9% and anti-HDV+ CHB patients had worse LFT results compared to those who were anti-HDV-.

Hepatitis delta: In vitro evaluation of cytotoxicity and cytokines involved in PEG-IFN therapy.

The treatment for hepatitis Delta virus (HDV) still consists of Pegylated interferon (PEG-IFN) combined with inhibitors of Hepatitis B virus (HBV) replication. In some patients may be occur a virological response, which means a negative HDV RNA 6 months after stopping treatment. In this study it was conducted an in vitro approach with the aim to mimic possible immunological events that are observed in patients responding to PEG-IFN therapy. Jurkat cells (human T lymphocyte cell line) were employed alone or co-cultured with THP-1 (human monocytic cell line) and stimulated with controls and HBV Surface Antigen (HBsAg), Small-Delta Antigen (SHDAg), and HBsAg + SHDAg combined. Twenty-four hours stimulation with SHDAg and/or HBSAg led to a toxic profile in a co-culture condition and cell supernatants were collected for cytokines quantification. PEG-IFN was added and cells were incubated for additional 24 h. Co-cultured cells incubated with the association (SHDAg + PEG-IFN) significantly produced levels of IFN-γ, IL-2 and IL-12. On the other hand, the HBsAg alone was able to inhibit the production of IFN-γ, suggesting that this antigen may hinder the treatment exclusively with PEG-IFN.

Diversification of mammalian deltaviruses by host shifting.

Hepatitis delta virus (HDV) is an unusual RNA agent that replicates using host machinery but exploits hepatitis B virus (HBV) to mobilize its spread within and between hosts. In doing so, HDV enhances the virulence of HBV. How this seemingly improbable hyperparasitic lifestyle emerged is unknown, but it underpins the likelihood that HDV and related deltaviruses may alter other host-virus interactions. Here, we show that deltaviruses diversify by transmitting between mammalian species. Among 96,695 RNA sequence datasets, deltaviruses infected bats, rodents, and an artiodactyl from the Americas but were absent from geographically overrepresented Old World representatives of each mammalian order, suggesting a relatively recent diversification within the Americas. Consistent with diversification by host shifting, both bat and rodent-infecting deltaviruses were paraphyletic, and coevolutionary modeling rejected cospeciation with mammalian hosts. In addition, a 2-y field study showed common vampire bats in Peru were infected by two divergent deltaviruses, indicating multiple introductions to a single host species. One vampire bat-associated deltavirus was detected in the saliva of up to 35% of individuals, formed phylogeographically compartmentalized clades, and infected a sympatric bat, illustrating horizontal transmission within and between species on ecological timescales. Consistent absence of HBV-like viruses in two deltavirus-infected bat species indicated acquisitions of novel viral associations during the divergence of bat and human-infecting deltaviruses. Our analyses support an American zoonotic origin of HDV and reveal prospects for future cross-species emergence of deltaviruses. Given their peculiar life history, deltavirus host shifts will have different constraints and disease outcomes compared to ordinary animal pathogens.

First molecular epidemiological study of hepatitis B and D in individuals infected with human T-lymphotropic virus 1/2 from Argentina.

Human T-lymphotropic virus 1/2 (HTLV-1/2), hepatitis B virus (HBV), and hepatitis D virus (HDV) share transmission routes. Argentina shows low prevalence of HTLV-1/2, HBV, and HDV infections; however, this situation may vary according to the geographic region and group studied. The aim of this study was to estimate the prevalence of HBV and HDV infections and detect both viral genotypes in HTLV-1/2 individuals from Argentina. A total of 202 HTLV-1/2 confirmed samples (blood donors [BD] and individuals with risk factors for HTLV-1/2 [RF]) were tested for HBsAg and total anti-HBc by enzyme-linked immunosorbent assay. All reactive samples for some HBV markers were analyzed for HBV DNA characterization and HDV serological and molecular analysis. Total prevalence was 1.5% for HBsAg and 6.4% for anti-HBc. Prevalence was 23.1% for anti-HDV in all HBV-reactive samples. No significant difference was observed for HBV and HDV prevalence within HTLV subtypes. The population study showed that prevalence of anti-HBc was higher in the RF than in the BD population, with no significant differences between them. The HBsAg marker and anti-HDV were only found in RF, showing significant differences when compared to BD. Regarding molecular detection, one sample amplified for HBV DNA and none for HDV RNA. HBV sequence was classified as subgenotype F1b. New and updated background on serological markers of HBV and HDV infection in patients with HTLV-1/2 was provided.