Changing delta hepatitis patient profile:A single center experience in Valencia region,Spain

BACKGROUND Delta hepatitis is a rare infection with an aggressive disease course.For almost three decades,however,there have been no epidemiological studies in our traditionally endemic area.AIM To investigate the prevalence of delta hepatitis in a sample of patients with chronic hepatitis B virus(HBV)infection followed at a Hepatology Unit in Valencia,Spain.METHODS Retrospective evaluation of anti-hepatitis D virus-immunoglobulin G seroprevalence among patients with chronic HBV infection(n=605)followed at a reference Hepatology Unit in Spain.RESULTS The prevalence of anti-hepatitis D virus-immunoglobulin G among HBV-infected patients was 11.5%:Male(63%)and median age of 52 years.The majority were born in Spain(67%)and primarily infected through intravenous drug use.However,a significant percent(24.5%),particularly those diagnosed in more recent years,were migrants presumably nosocomially infected.Comorbidities such as diabetes(8.5%),obesity/overweight(55%),and alcohol consumption(34%)were frequent.A high proportion of patients developed liver complications such as cirrhosis(77%),liver decompensation(81%),hepatocellular carcinoma(HCC)(16.5%),or required liver transplantation(LT)(59.5%).Diabetes was associated with progression to cirrhosis,LT,and death.Male sex,increasing age,and alcohol were associated with LT and HCC.Compared to HBV mono-infected patients,delta individuals developed cirrhosis and liver decompensation more frequently,with no differences in HCC rates.CONCLUSION Patients infected in the 1980’s were mostly locals infected through intravenous drug use,whereas those diagnosed recently are frequently non-Spanish natives from endemic areas.Regardless of their origin,patients are predominantly male with significant comorbidities,which potentially play a major role in disease progression.We confirm a high rate of subsequent liver complications.

Management of autoimmune hepatitis induced by hepatitis delta virus

Approximately 12-72 million people worldwide are co-infected with hepatitis B virus(HBV)and hepatitis delta virus(HDV).This concurrent infection can lead to several severe outcomes with hepatic disease,such as cirrhosis,fulminant hepatitis,and hepatocellular carcinoma,being the most common.Over the past few decades,a correlation between viral hepatitis and autoimmune diseases has been reported.Furthermore,autoantibodies have been detected in the serum of patients co-infected with HBV/HDV,and autoimmune features have been reported.However,to date,very few cases of clinically significant autoimmune hepatitis(AIH)have been reported in patients with HDV infection,mainly in those who have received treatment with pegylated interferon.Interestingly,there are some patients with HBV infection and AIH in whom HDV infection is unearthed after receiving treatment with immunosuppressants.Consequently,several questions remain unanswered with the challenge to distinguish whether it is autoimmune or“autoimmune-like”hepatitis being the most crucial.Second,it remains uncertain whether autoimmunity is induced by HBV or delta virus.Finally,we investigated whether the cause of AIH lies in the previous treatment of HDV with pegylated interferon.These pressing issues should be elucidated to clarify whether new antiviral treatments for HDV,such as Bulevirtide or immu-nosuppressive drugs,are more appropriate for the management of patients with HDV and AIH.

Epidemiology of acute and chronic hepatitis B and delta over the last 5 decades in Italy

The spread of hepatitis B virus(HBV)infection has gradually decreased in Italy in the last 5 decades as shown by the steady reduction in the incidence rates of acute hepatitis B,from 10/100000 inhabitants in1984 to 0.85/100000 in 2012,and by the reduced prevalence of hepatitis B surface antigen(HBsAg)-positive cases among chronic hepatitis patients with different etiologies,from 60%in 1975 to about 10%in 2001.The prevalence of HBsAg chronic carriers in the general population also decreased from nearly 3%in the 1980s to 1%in 2010.Linked to HBV by its characteristics of defective virus,the hepatitis delta virus(HDV)has shown a similar epidemiological impact on the Italian population over time.The incidence of acute HDV infection decreased from 3.2/100000 inhabitants in 1987 to 0.8/100000 in 2010 and the prevalence of HDV infection in HBsAg chronic carriers decreased from24%in 1990 to 8.5%in 2006.Before the beneficial effects of HBV mass vaccination introduced in 1991,the decreased endemicity of HBV and HDV infection in Italy paralleled the improvement in screening blood donations,the higher standard of living and impressive reduction in the birth rate associated with a marked reduction in the family size.A further contribution to the decline in HBV and HDV infections most probably came from the media campaigns to prevent the spread of human immunodeficiency virus infection by focusing the attention of the general population on the same routes of transmission of viral infections such as unsafe sexual intercourse and parenteral exposures of different kinds.

Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

AIM： To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen （L-HDAg）. METHODS： A recombinant baculovirus expressing histidine-tagged L-HDAg （L-HDAgH） was constructed to transduce baby hamster kidney （BHK） cells by a simplified transduction protocol. RESULTS： The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION： The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

Clinical presentation and genotype of hepatitis delta in Karachi

AIM： To assess the clinical presentation and genotypes of delta hepatitis in local population. METHODS： In this prospective study, 39 consecutive patients who were positive for HBsAg and hepatitis D virus （HDV） antibody were included. The patients were divided in two groups on the basis of presence or absence of HDV RNA and a comparative study was done. Genotype of HDV was determined in PCR positive patients. RESULTS： Overall there is male dominance, in which 34 patients out of 39 （87.2%） were male. Twenty （51%） patients were from the adjacent areas of three provinces; Sindh, Punjab and Balochistan indicating the higher prevalence of delta hepatitis in this mid region of Pakistan. Patients of all age groups were affected with delta hepatitis （median 31.5 years, range 12-75）. HDV RNA was detectable in 23 patients （59%）. All the HDV strains belonged to genotype I. HBV DNA was detectable only in 3 cases who were also HBeAg and HDV RNA positive. Patients with detectable HDV RNA were younger than patients with undetectable RNA; mean age 29.7±12.8 years vs 36.8±15.2. There were no statistically significant differences in the clinical presentation and routine biochemical profile of patients with detectable or undetectable HDV RNA. Clinical cirrhosis was present in 19 （49%） patients; 12 with detectable RNA and 7 with undetectable HDV RNA （P = 0.748）. Decompensated disease was seen in eight patients; five and three respectively from each group. Four patients with undetectable RNA and two patients with detectable RNA had normal ALT and ultrasound abdomen. CONCLUSION： HDV may infect at any age, usually young adult males. Genotype I is prevalent. With time some of the patients become HDV RNA negative or asymptomatic carrier. Most of the patients have suppressed HBV DNA replication. Significant numbers of patients have cirrhosis.

Hepatitis delta virus: From infection to new therapeutic strategies

The hepatitis delta virus(HDV)is a small RNA virus that encodes a single protein and which requires the hepatitis B virus(HBV)-encoded hepatitis B surface antigen(HBsAg)for its assembly and transmission.HBV/HDV co-infections exist worldwide and show a higher prevalence among selected groups of HBV-infected populations,specifically intravenous drug users,practitioners of high-risk sexual behaviours,and patients with cirrhosis and hepatocellular carcinoma.The chronic form of HDV-related hepatitis is usually severe and rapidly progressive.Patterns of the viral infection itself,including the status of co-infection or super-infection,virus genotypes(both for HBV and HDV),and persistence of the virus’replication,influence the outcome of the accompanying and manifested liver disease.Unfortunately,disease severity is burdened by the lack of an effective cure for either virus type.For decades,the main treatment option has been interferon,administered as mono-therapy or in combination with nucleos(t)ide analogues.While its efficacy has been reported for different doses,durations and courses,only a minority of patients achieve a sustained response,which is the foundation of eventual improvement in related liver fibrosis.The need for an efficient therapeutic alternative remains.Research efforts towards this end have led to new treatment options that target specific steps in the HDV life cycle;the most promising among these are myrcludex B,which inhibits virus entry into hepatocytes,lonafarnib,which inhibits farnesylation of the viral-encoded LHDAg large hepatitis D antigen,and REP-2139,which interferes with HBsAg release and assembly.

Host RNA circles and the origin of hepatitis delta virus

Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases.In some cases,such circles actually bind host microRNAs.Such depletion of available microRNAs appears to have biological roles;for instance,in homeostasis and disease.These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus,hepatitis delta virus(HDV),an agent with a small circular RNA genome;specifically,it is proposed that in hepatocytes infected with hepatitis B virus(HBV),some viral RNA species are processed to circular forms,which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled,using HBV envelope proteins,to form particles some of which are infectious.Such a model also may provide some new insights into the potential pathogenic potential of HDV infections.In return,new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.

Detection of Antibody to Hepatitis Delta Virus in Human Serum by Double Antigen Sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

Structural and nucleic acid binding properties of hepatitis delta virus small antigen

AIM To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195(?60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization.METHODS Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ?60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS Results showed that the truncated ?60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ?60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding.CONCLUSION Taken together these results allowed concluding that ?60HDAg is intrinsically disordered but compact; ?60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.

Molecular mechanisms of viral hepatitis induced hepatocellular carcinoma

Chronic infection with viral hepatitis affects half a billion individuals worldwide and can lead to cirrhosis,cancer,and liver failure.Liver cancer is the third leading cause of cancer-associated mortality,of which hepatocellular carcinoma(HCC)represents 90%of all primary liver cancers.Solid tumors like HCC are complex and have heterogeneous tumor genomic profiles contributing to complexity in diagnosis and management.Chronic infection with hepatitis B virus(HBV),hepatitis delta virus(HDV),and hepatitis C virus(HCV)are the greatest etiological risk factors for HCC.Due to the significant role of chronic viral infection in HCC development,it is important to investigate direct(viral associated)and indirect(immune-associated)mechanisms involved in the pathogenesis of HCC.Common mechanisms used by HBV,HCV,and HDV that drive hepatocarcinogenesis include persistent liver inflammation with an impaired antiviral immune response,immune and viral protein-mediated oxidative stress,and deregulation of cellular signaling pathways by viral proteins.DNA integration to promote genome instability is a feature of HBV infection,and metabolic reprogramming leading to steatosis is driven by HCV infection.The current review aims to provide a brief overview of HBV,HCV and HDV molecular biology,and highlight specific viral-associated oncogenic mechanisms and common molecular pathways deregulated in HCC,and current as well as emerging treatments for HCC.

Hepatitis B virus/hepatitis D virus epidemiology: Changes over time and possible future influence of the SARS-CoV-2 pandemic

Hepatitis D virus(HDV)is a defective liver-tropic virus that needs the helper function of hepatitis B virus(HBV)to infect humans and replicate.HDV is transmitted sexually or by a parenteral route,in co-infection with HBV or by super-infection in HBV chronic carriers.HDV infection causes acute hepatitis that may progress to a fulminant form(7%-14%by super-infection and 2%-3%by HBV/HDV co-infection)or to chronic hepatitis(90%by HDV super-infection and 2%-5%by HBV/HDV co-infection),frequently and rapidly progressing to cirrhosis or hepatocellular carcinoma(HCC).Peg-interferon alfa the only recommended therapy,clears HDV in only 10%-20%of cases and,consequently,new treatment strategies are being explored.HDV endemicity progressively decreased over the 50 years from the identification of the virus,due to improved population lifestyles and economic levels,to the use of HBV nuclei(t)side analogues to suppress HBV replication and to the application of universal HBV vaccination programs.Further changes are expected during the severe acute respiratory syndrome coronavirus-2 pandemic,unfortunately towards increased endemicity due to the focus of healthcare towards coronavirus disease 2019 and the consequently lower possibility of screening and access to treatments,lower care for patients with severe liver diseases and a reduced impulse to the HBV vaccination policy.

Hepatitis D virus and liver transplantation: Indications and outcomes

Hepatitis D virus(HDV)is a dependent virus that relies on hepatitis B virus for its replication and transmission.Chronic hepatitis D is a severe form of viral hepatitis that can result in end stage liver disease.Currently,pegylated interferon alpha is the only approved therapy for chronic HDV infection and is associated with significant side effects.Liver transplantation(LT)is the only treatment option for patients with end-stage liver disease,hepatocellular carcinoma,or fulminant hepatitis due to coinfection with HDV.As LT for HDV and hepatitis B virus coinfection is uncommon in the United States,most data on the long-term impact of LT on HDV are from international centers.In this review,we discuss the indications and results of LT with treatment options in HDV patients.

"Defective" mutations of hepatitis D viruses in chronic hepatitis D patients

AIM: To verify whether 'defective' mutations existed in hepatitis D virus (HDV).METHODS: Hepatitis delta antigen (HDAg)-codingsequences were amplified using Pfu DNA polymerases with proof-reading activities from sera of five patients with chronic hepatitis D. Multiple colonies were sequenced for each patient. Pfu analyzed a total of 270 HDV clones.Three representative defective HDV clones were constructed in expression plasmids and transfected into a human hepatoma cell line. Cellular proteins were extracted and analyzed by Western blot.RESULTS: Four of five cases (80%) showed defective HDV genomes in their sera. The percentage of defective genomes was 3.7% (10/270). The majority (90%) of the defective mutations were insertions or deletions that resulted in frameshift and abnormal stop translation of the HDAg. The predicted mutated HDAg ranged from 45amino acids to ＞214 amino acids in length. Various domains of HDAg associated with viral replication or packaging were affected in different HDV isolates. Western blot analysis showed defected HDAg in predicted positions.CONCLUSION: 'Defective' viruses do exist in chronic HDV infected patients, but represented as minor strains. The clinical significance of the 'defected' HDV needs further study to evaluate.

Performance of Hepatitis Delta Virus(HDV)RNA Testing for the Diagnosis of Active HDV Infection:Systematic Review and Meta-analysis

Background and Aims:Hepatitis delta virus(HDV)is a defective virus and causes severe liver disease.Several HDV RNA assays have been developed,however the diagnostic efficacy remains unclear.This systematic review and metaanalysis aims to evaluate the diagnostic accuracy of HDV RNA assays to aid in the diagnosis of active hepatitis D.Methods:The PubMed,Embase,and Cochrane Library databases were systematically searched from the beginning to June 31,2022.Information on the characteristics of the literature and data on sensitivity,specificity,and area under curve(AUC)of the receiver operating characteristic(ROC)were extracted.Stata 14.0 was used for meta-analysis of the combined sensitivity,specificity,positive likelihood ratio,and negative likelihood ratio.Results:A total of 10 studies were included in the meta-analysis.The summary sensitivity,specificity,positive likelihood ratio,negative likelihood ratio,and diagnostic odds ratio of HDV RNA assays for HDV diagnosis were 0.92(95%CI:0.87-0.95),0.90(95%CI:0.86-0.93),7.74(95%CI:5.31-11.29),0.10(95%CI:0.06-0.18)and 99.90(95%CI:47.08-211.99),respectively.The AUC of the pooled ROC curve was 0.95(95%CI:0.92-0.96).Conclusions:The results show that HDV RNA assays had high diagnostic performance.However,that is limited by the number and quality of studies.Standard protocols for the development of assays by manufacturers and larger studies on the use of the assays are needed.

Folding Kinetics of HDV Ribozyme with C13A:G82U and A16U:U79A Mutations

Gene mutations influence the folding kinetics of hepatitis delta virus （HDV） ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation method combined with RNA folding free energy landscape, we predict the folding kinetics of C13A：G82U and A16U：U79A mutated HDV sequences. Their folding pathways are identified by recursively searching the states with high net flux-in（out） population starting from the native state. The results indicate that the folding kinetics of C 13A：G82U mutation sequence is bi-phasic, which is similar to the wild type （wtHDV） sequence. While the folding kinetics of A16U：U79A mutation sequence is mono-phasic, it quickly folds to the native state in 30 s. Thus, the folding kinetics of double mutated HDV ribozyme depends on the mutation sites.