Rubella virus-associated uveitis at a tertiary care hospital in Germany between 2013 and 2019.

Uveitis is a process of intraocular inflammation that may involve different sections of the uveal tract. Apart from systemic or localized immune-mediated diseases, infections are key players in the etiology of uveitis and entail different treatment strategies. Rubella virus (RuV) is a recognized causative agent for the development of Fuchs uveitis, representing a major cause of virus-associated intraocular inflammation. A cohort of 159 patients diagnosed with different forms of uveitis between 2013 and 2019 was subjected to diagnostic antibody testing of the aqueous or vitreous humor. The diagnostic panel included RuV, cytomegalovirus, herpes simplex virus, varicella-zoster virus, and toxoplasmosis. Within this cohort, 38 RuV-associated uveitis (RAU) patients were identified based on a pathologic Goldman-Witmer coefficient indicative of an underlying RuV infection. With a mean age of 45.9 years, the RAU patients were younger than the non-RAU patients (56.3, p < 0.001). The evaluation of clinical parameters revealed a predominance of anterior uveitis and late sequalae such as cataract and glaucoma among the RAU patients. In 15 of the patients a history of prior RuV infections could be confirmed. The study underlines the importance of long-term surveillance of RuV associated diseases that originate from infections before the introduction of RuV vaccination programs.

Human Rhinoviruses in Pediatric Patients in a Tertiary Care Hospital in Germany: Molecular Epidemiology and Clinical Significance.

Rhinoviruses (RVs) constitute a substantial public health burden. To evaluate their abundance and genetic diversity in pediatric patients, RV RNA in respiratory samples was assessed using real-time RT-PCR and partial nucleic acid sequencing of viral genomes. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of pediatric RV infections. In total, the respiratory specimens of 776 patients (<18 years), collected from 2013 to 2017, were analyzed. Infections occurred throughout the entire year, with peaks occurring in fall and winter, and showed remarkably high intra- and interseasonal diversity for RV genotypes. RV species were detected in the following frequencies: 49.1% RV-A, 5.9% RV-B, and 43.6% RV-C. RV-C was found to be more frequently associated with asthma (p = 0.04) and bronchiolitis (p < 0.001), while RV-A was more frequently associated with fever (p = 0.001) and pneumonia (p = 0.002). Additionally, 35.3% of the patients had co-infections with other pathogens, which were associated with a longer hospital stay (p < 0.001), need for ventilation (p < 0.001), and pneumonia (p < 0.001). Taken together, this study shows pronounced RV genetic diversity in pediatric patients and indicates differences in RV-associated pathologies, as well as an important role for co-infections.

Photodynamic Inactivation of SARS-CoV-2 Infectivity and Antiviral Treatment Effects In Vitro.

Despite available vaccines, antibodies and antiviral agents, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic still continues to cause severe disease and death. Current treatment options are limited, and emerging new mutations are a challenge. Thus, novel treatments and measures for prevention of viral infections are urgently required. Photodynamic inactivation (PDI) is a potential treatment for infections by a broad variety of critical pathogens, including viruses. We explored the infectiousness of clinical SARS-CoV-2 isolates in Vero cell cultures after PDI-treatment, using the photosensitizer Tetrahydroporphyrin-tetratosylate (THPTS) and near-infrared light. Replication of viral RNA (qPCR), viral cytopathic effects (microscopy) and mitochondrial activity were assessed. PDI of virus suspension with 1 µM THPTS before infection resulted in a reduction of detectable viral RNA by 3 log levels at day 3 and 6 after infection to similar levels as in previously heat-inactivated virions (<99.9%; p < 0.05). Mitochondrial activity, which was significantly reduced by viral infection, was markedly increased by PDI to levels similar to uninfected cell cultures. When applying THPTS-based PDI after infection, a single treatment had a virus load-reducing effect only at a higher concentration (3 µM) and reduced cell viability in terms of PDI-induced toxicity. Repeated PDI with 0.3 µM THPTS every 4 h for 3 d after infection reduced the viral load by more than 99.9% (p < 0.05), while cell viability was maintained. Our data demonstrate that THPTS-based antiviral PDI might constitute a promising approach for inactivation of SARS-CoV-2. Further testing will demonstrate if THPTS is also suitable to reduce the viral load in vivo.

Analysis and optimization of a Caco-2 cell culture model for infection with human norovirus.

Human noroviruses (hNoVs) are an important cause of acute gastroenteritis worldwide. However, the lack of a reproducible in vitro cell culture system has impaired research and the development of preventive measures, therapeutic drugs, and vaccines. The aim of this study was to analyze and optimize a suitable cell line for in vitro cultivation of hNoV. The Caco-2 cell line, which is of colorectal origin and differentiates spontaneously into intestinal enterocyte-like cells, was chosen as a model. It was found that differentiated cells were more susceptible to infection with hNoV, resulting in a higher virus yield. This was accompanied by an increase in H type 1 antigen in the cell membrane during differentiation, which functions as an attachment factor for hNoV. Induced overexpression of H type 1 antigen in undifferentiated Caco-2 cells resulted in an increase in viral output to a level similar to that in differentiated cells. However, the relatively low level of viral output, which contrasts with what is observed in vivo, shows that the viral replication cycle is restricted in this model. The results indicate that there is a block at the level of viral release.

Molecular epidemiology of hepatitis B virus among HIV co-infected and mono-infected cohorts in Northwest Ethiopia.

BACKGROUND: Hepatitis B virus (HBV) infection is a particular concern in human immunodeficiency virus (HIV) infected individuals. In Ethiopia, detailed clinical and virological descriptions of HBV prevailing during HIV co-infection and symptomatic liver disease patients are lacking. The aim of this study was to investigate HBV virological characteristics from Ethiopian HBV/HIV co-infected and HBV mono-infected individuals. METHODS: A total of 4105 sera from HIV positive individuals, liver disease patients, and blood donors were screened serologically for HBV. The overlapping polymerase/surface genome region of HBV from 180 infected individuals was extracted, amplified, and sequenced for genotypic analysis. RESULTS: The HBsAg seroprevalence was detected 43% in liver disease patients, 8.4% in blood donors, and 6.7% in HIV/HBV co-infected individuals. The occult HBV prevalence was 3.7% in HIV/HBV co-infected individuals and 2.8% in blood donors with an overall prevalence rate of 3.4%. A phylogenetic analysis showed three HBV genotypes; A (61.1%), D (38.3%) and E (0.6%). Genotype A belongs to subtypes A1 (99.1%) and A9 (0.9%), but genotype D showed heterogeneous subtypes; D2 (63.8%) followed by D4 (21.7%), D1 (8.7%), D3 (4.3%), and D10 (1.4%). CONCLUSIONS: The HIV/HBV co-infected individuals and blood donors showed lower HBsAg seroprevalence compared to liver diseases patients. Occult HBV prevalence showed no difference between HIV/HBV co-infected and blood donor groups. This study demonstrated predominance distribution of HBV subtypes A1 and D2 in northwest Ethiopia. The observed virological characteristics could contribute for evidence-based management of viral hepatitis in Ethiopia where antiretroviral therapy guidelines do not cater for viral hepatitis screening during HIV co-infection.

Human Rhinoviruses in Adult Patients in a Tertiary Care Hospital in Germany: Molecular Epidemiology and Clinical Significance.

Rhinoviruses (RVs) constitute a substantial public health burden. To evaluate their abundance and genetic diversity in adult patients, RV RNA in respiratory samples was assessed using real-time RT-PCR and the partial nucleic acid sequencing of viral genomes. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of adult RV infections. In total, the respiratory specimens of 284 adult patients (18-90 years), collected from 2013 to 2017, were analyzed. Infections occurred throughout the entire year, with peaks occurring in fall and winter, and showed a remarkably high intra- and interseasonal diversity of RV genotypes. RV species were detected in the following ratios: 60.9% RV-A 173, 12.7% RV-B, and 26.4% RV-C. No correlations between RV species and underlying comorbidities such as asthma (p = 0.167), COPD (p = 0.312) or immunosuppression (p = 0.824) were found. However, 21.1% of the patients had co-infections with other pathogens, which were associated with a longer hospital stay (p = 0.024), LRTI (p < 0.001), and pneumonia (p = 0.01). Taken together, this study shows a pronounced genetic diversity of RV in adults and underlines the important role of co-infections. No correlation of specific RV species with a particular clinical presentation could be deduced.

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Comprehensive evaluation of eight commercial SARS-CoV-2 IgG assays.

Sensitivity and specificity of serological assays are key parameters for the accurate estimation of SARS-CoV-2 sero-prevalence. The aim of this study was to compare 8 readily available IgG antibody tests using a panel of well-defined serum samples of prepandemic and pandemic origin. A cross-reaction panel included samples of patients with recent infection with either of the endemic Coronaviruses 229E, NL63, HKU1, or OC43. Additionally, samples with high antibody levels against influenza virus, adenovirus, and during acute EBV infection were included. Previous infection with endemic coronaviruses caused a significant amount of cross-reactivity in two of the assays. In contrast, the confidence intervals for the assays of Abbott, DiaSorin, Euroimmun and Roche encompassed the value of 98% for samples with a previous endemic HCoV infection. For all assays, sensitivities were between 91.3% and 98.8%. Assay performance was independent of the usage of either nucleocapsid or spike proteins.

Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19.

In coronavirus disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying causes and the effects of the main anti-hypertensive therapies-angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)-remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical COVID-19 progression. ACEI treatment was associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic antiviral responses, whereas ARB treatment related to enhanced epithelial-immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.

Dynamics of nosocomial parainfluenza virus type 3 and influenza virus infections at a large German University Hospital between 2012 and 2019.

Nosocomial virus infections cause significant morbidity and mortality. Besides influenza viruses, the disease burden of parainfluenza virus type 3 (PIV-3) is comparatively high among hospitalized patients and severe disease courses can occur. PIV-3 showed the highest rates of nosocomial infections of a panel of respiratory viruses. Therefore, a retrospective observational study was conducted among patients with either PIV-3 or influenza viruses, which served as reference pathogen. The aim was to compare the seasonal dynamics and clinical characteristics of nosocomial infections with these highly transmittable viruses. Nosocomial infection occurred in 15.8% (n = 177) of all influenza cases, mainly in the first half of a season. About 24.3% (n = 104) of the PIV-3 cases were nosocomial and occurred mainly in the second half of a season. Both nosocomial rates of influenza and nosocomial rates of PIV-3 varied between the seasons. Community acquired and nosocomial cases differed in underlying medical conditions and immunosuppression. Knowledge of the baseline rates of nosocomial infections could contribute to the implementation of appropriate infection control measures.

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis.

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.

Vaccinations and Infections Are Associated With Unrelated Antibody Titers: An Analysis From the German Birth Cohort Study LISA.

The evidence for non-specific effects (NSE) of vaccinations on all-cause morbidity and mortality among children is growing. However, our understanding of the underlying mechanisms is still limited. One hypothesis is that NSE are mediated by antibody titers. We used data of 2,123 children from the population-based birth cohort study LISA conducted in Germany to explore whether routine childhood vaccinations and the individual infection history in the first 2 years of life are associated with unrelated antibody titers. We selected 19 exposures (infections and vaccinations) and investigated their association with levels of 12 IgG antibody titers at the age of 2 years. Based on univariable analyses (ANOVA), we identified 21 crude associations between exposures and titers (p < 0.05), while 11 (95%-CI: 6, 17) spurious associations were expected due to multiple testing. In exploratory multivariable analyses, we observed associations between seven investigated IgG titers and 10 exposures; either administered vaccines [e.g., higher anti-hRSV IgG titer in BCG-vaccinated children (regression-coefficient in standard-deviation-units: 0.38; 95%-CI: 0.12, 0.65)] or infections [e.g., higher anti-measles IgG titer in children with reported chickenpox (0.44; 95%-CI: 0.08, 0.80)]. Our results indicate the existence of associations between immunogenic exposures and unrelated antibody titers. Further studies investigating the underlying immunological mechanisms are required.

Correction: HIV-1C proviral DNA for detection of drug resistance mutations.

[This corrects the article DOI: 10.1371/journal.pone.0205119.].

HIV-1C proviral DNA for detection of drug resistance mutations.

BACKGROUND: Using HIV proviral DNA as a template may be suitable for initial detection of transmitted drug resistance mutations (TDRMs) as it is easy to handle and less expensive compared to RNA. However, existing literatures which are mainly focused on HIV-1B subtypes DNA extracted from PBMCs revealed controversial findings ranging from the detection of significantly lower or higher mutations in proviral DNA compared to historic viral RNA. Thus, to verify whether viral RNA or proviral DNA has improved sensitivity in detecting transmitted genotypic drug resistance mutations paired viral RNA and proviral DNA (which is directly extracted from stored whole blood) samples were tested from Ethiopian antiretroviral naive HIV-1C infected subjects. METHODS: In the present comparative study the frequency of TDR mutations was assessed in paired samples of viral RNA and proviral DNA (extracted directly from stored whole blood) of HIV-1C infected treatment naïve patients and interpreted using the 2009 WHO drug resistance surveillance mutation lists, Stanford University drug resistance data base and International Antiviral Society-USA mutation lists. RESULTS: High agreement in rate of TDR between the two compartments was observed using the WHO mutation lists. While mutations G190A and E138A were concurrently found in both compartments, others such as G73S on PR and A62V, M184I, M230I on RT were identified in proviral DNA only. All signature mutations seen in viral RNA were not missed in proviral DNA. CONCLUSIONS: The concordance of major genotype drug resistance mutation between RNA and proviral DNA in treatment naïve patients suggests that proviral DNA might be an alternative approaches for an initial assessment of drug resistance prior to initiation of antiretroviral therapy using the WHO mutations lists in resource-limited countries. However, the clinical importance of TDRMs observed only in proviral DNA in terms of being a risk factor for virologic failure and whether they limit future treatment options needs additional investigation using more sensitive sequencing approaches such as Next Generation Sequencing (NGS).

Epigenetic immune cell counting in human blood samples for immunodiagnostics.

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

Analysis of HBV basal core promoter/precore gene variability in patients with HBV drug resistance and HIV co-infection in Northwest Ethiopia.

BACKGROUND: We recently reported complex hepatitis B virus (HBV) drug resistant and concomitant vaccine escape hepatitis B surface antigen (HBsAg) variants during human immunodeficiency virus (HIV) co-infection and antiretroviral therapy (ART) exposure in Ethiopia. As a continuation of this report using the HBV positive sera from the same study participants, the current study further analyzed the HBV basal core promoter (BCP)/precore (PC) genes variability in patients with HBV drug resistance (at tyrosine-methionine-aspartate-aspartate (YMDD) reverse transcriptase (RT) motifs) and HIV co-infection in comparison with HBV mono-infected counterparts with no HBV drug resistant gene variants. MATERIALS AND METHODS: A total of 143 participants of HBV-HIV co-infected (n = 48), HBV mono-infected blood donors (n = 43) and chronic liver disease (CLD) patients (n = 52) were included in the study. The BCP/PC genome regions responsible for HBeAg expression from the EcoRI site (nucleotides 1653-1959) were sequenced and analyzed for the BCP/PC mutant variants. RESULTS: Among the major mutant variants detected, double BCP mutations (A1762T/G1764A) (25.9%), Kozak sequences mutations (nt1809-1812) (51.7%) and the classical PC mutations such as A1814C/C1816T (15.4%), G1896A (25.2%) and G1862T (44.8%) were predominant mutant variants. The prevalence of the double BCP mutations was significantly lower in HIV co-infected patients (8.3%) compared with HBV mono-infected blood donors (32.6%) and CLD patients (36.5%). However, the Kozak sequences BCP mutations and the majority of PC mutations showed no significant differences among the study groups. Moreover, except for the overall BCP/PC mutant variants, co-prevalence rates of each major BCP/PC mutations and YMDDRT motif associated lamivudine (3TC)/entecavir (ETV) resistance mutations showed no significant differences when compared with the rates of BCP/PC mutations without YMDD RT motif drug resistance gene mutations. Unlike HIV co-infected group, no similar comparison made among HBV mono-infected blood donors and CLD patients since none of them developed the YMDD RT motif associated 3TC/ETV resistance mutations. However, HBV mono-infected blood donors and CLD patients who had no any drug resistance gene variants developed comparable G1862T (60.6% vs. 65.1%) and G1896A (24.2% vs. 11.6%) PC gene mutations. CONCLUSION: No correlation observed between the BCP/PC genome variability and the YMDD RT motif associated HBV drug resistance gene variants during HIV co-infection. Nevertheless, irrespective of HIV co-infection status, the higher records of the BCP/PC gene variability in this study setting indicate a high risk of potential HBeAg negative chronic HBV infection in Northwest Ethiopia.

Upward trends of acquired drug resistances in Ethiopian HIV-1C isolates: A decade longitudinal study.

BACKGROUND: The emergence, accumulation and spread of HIV-1 drug resistance strains in Africa could compromise the effectiveness of HIV treatment programs. This study was aimed at determining the incidence of virological failure and acquired drug resistance mutations overtime and identifying the most common mutational pathways of resistance in a well characterized HIV-1C infected Ethiopian cohort. METHODS: A total of 320 patients (220 ART naïve and 100 on first lines ART) were included and followed. ART initiation and patients' monitoring was based on the WHO clinical and immunological parameters. HIV viral load measurement and genotypic drug resistance testing were done at baseline (T0-2008) and after on average at a median time of 30 months on ART at three time points (T1-2011, T2-2013, T3-2015). FINDINGS: The incidence of virological failure has increased overtime from 11 at T1 to 17 at T2 and then to 30% at T3. At all time point's almost all of the patients with virological failure and accumulated drug resistance mutations had not met the WHO clinical and immunologic failure criteria and continued the failing regimen. A steep increase in the incidence and accumulation of major acquired NRTI and NNRTI drug resistance mutations have been observed (from 40% at T1 to 64% at T2 and then to 66% at T3). The most frequent NRTIs drug resistance associated mutations are mainly the lamivudine-induced mutation M184V which was detected in 4 patients at T1 and showed a 2 fold increase in the following time points (T2: n = 8) and at (T3: n = 12) and the thymidine analogue mutations (such as D67N, K70R and K219E) which were not-detected at baseline T0 and T1 but were increased progressively to 10 at T2 and to 17 at T3. The most frequent NNRTIs associated mutations were K103N, V106M and Y188C. CONCLUSIONS: An upward trend in the incidence of virological failure and accumulation of NRTI and NNRTI associated acquired antiretroviral drug resistance mutations are observed. The data suggest the need for virological monitoring, resistance testing for early detection of failure and access for TDF and PI containing drugs. Population-level and patient targeted interventions to prevent the spread of mutant variants is warranted.

Infection of iPSC Lines with Miscarriage-Associated Coxsackievirus and Measles Virus and Teratogenic Rubella Virus as a Model for Viral Impairment of Early Human Embryogenesis.

Human induced pluripotent stem cell (iPSC) lines are a promising model for the early phase of human embryonic development. Here, their contribution to the still incompletely understood pathogenesis of congenital virus infections was evaluated. The infection of iPSC lines with miscarriage-associated coxsackievirus B3 (CVB3) and measles virus (MV) was compared to the efficient teratogen rubella virus (RV). While CVB3 and MV were found to be cytopathogenic on iPSC lines, RV replicated without impairment of iPSC colony morphology and integrity. This so far outstanding course of infection enabled maintenance of RV-infected iPSC cultures over several passages and their subsequent differentiation to ectoderm, endoderm, and mesoderm. A modification of the metabolic profile of infected iPSC lines was the only common aspect for all three viruses. This study points toward two important aspects. First, iPSC lines represent a suitable cell culture model for early embryonic virus infection. Second, metabolic activity represents an important means for evaluation of pathogen-associated alterations in iPSC lines.

Clade homogeneity and low rate of delta virus despite hyperendemicity of hepatitis B virus in Ethiopia.

BACKGROUND: Although hepatitis B virus (HBV) is hyperendemic and heterogeneous in its genetic diversity in Ethiopia, little is known about hepatitis D virus (HDV) circulating genotypes and molecular diversity. METHODS: A total of 321 hepatitis B surface antigen (HBsAg) positives (125 HIV co-infected, 102 liver disease patients and 94 blood donors) were screened for anti-HDV antibody. The anti-HDV positive sera were subjected to Real time PCR for HDV-RNA confirmation. The non coding genome region (spanning from 467 to 834 nucleotides) commonly used for HDV genotyping as well as complete HDV genome were sequenced for genotyping and molecular analysis. RESULTS: The anti-HDV antibody was found to be 3.2% (3) in blood donors, 8.0% (10) in HIV co-infected individuals and 12.7% (13) in liver disease patients. None of the HIV co-infected patients who revealed HBV lamivudine (3TC) resistance at tyrosine-methionine/isoleucine-aspartate-aspartate (YM(I)DD) reverse transcriptase (RT) motif with concomitant vaccine escape gene mutants was positive for anti-HDV antibody. The HDV viremia rate was 33.3%, 30.0% and 23.1% in respect to the above study groups. All the six isolates sequenced were phylogenetically classified as HDV genotype 1 (HDV-1) and grouped into two monophyletic clusters. Amino acid (aa) residues analysis of clathrin heavy chain (CHC) domain and the isoprenylation signal site (Py) at 19 carboxyl (C)-terminal amino acids (aa 196-214) and the HDV RNA binding domain (aa 79-107) were highly conserved and showed a very little nucleotide variations. All the sequenced isolates showed serine at amino acid position 202. The RNA editing targets of the anti-genomic HDV RNA (nt1012) and its corresponding genomic RNA (nt 580) showed nucleotides A and C, respectively. CONCLUSIONS: The low seroprevalence and viraemic rates of HDV in particular during HIV-confection might be highly affected by HBV drug resistance selected HBsAg mutant variants in this setting, although HDV-1 sequences analysis revealed clade homogeneity and highly conserved structural and functional domains. Thus, the potential role of HBV drug resistance associated polymerase mutations and concomitant HBsAg protein variability on HDV viral assembly, secretion and infectivity needs further investigation.