Understanding the temporal dynamics of invasive late blight populations in India for improved management practices.

The microbial oomycete pathogen, Phytophthora infestans causes severe epidemics of potato late blight in crops globally. Disease management benefits from an understanding of the diversity of pathogen populations. In this study, we explore the dynamics of P. infestans populations in the late blight-potato agro-ecosystem across the Indian subcontinent. Investigations of the macroecological observations at the field level and microbial ecological principles provided insights into future pathogen behaviour. We use a comprehensive simple sequence repeat allele dataset to demonstrate that an invasive clonal lineage called EU_13_A2 has dominated populations over 14 years across India, Bangladesh, and Pakistan. Increasing levels of sub-clonal variation were tracked over time and space and, for the first time, populations in Asia were also compared to the source populations from Europe. Within India, a regional pathogen population structure was observed with evidence for local migration, cross-border movement between surrounding countries, and introductions via imports. There was also evidence of genetic drift and between-season transmission of more strongly pathogenic sub-clones with a complete displacement of some sub-clonal types. The limited introduction of novel genotypes and the use of resistant potato cultivars could contribute to the dominance of the 13_A2 lineage. The insights will contribute to the management of the pathogen in these key global potato production regions.

Phylogenetic Characterization of a Novel Insect-Specific Flavivirus Detected in a Culex Pool, Collected from Assam, India.

OBJECTIVE: We report the phylogenetic characterization of a unique flavivirus sequence detected in a wild Culex tritaeniorhynchus mosquito pool, collected from the northeast Indian state of Assam. METHODS: DNA and RNA were extracted from field-collected mosquito pools. Extracts were subjected to PCR and reverse transcriptase PCR amplification using universal and type-specific primers for direct detection of flavivirus-specific viral nucleic acids. An amplified flavivirus nonstructural protein 5 (NS5) genetic region was sequenced and BLAST searched, and phylogenetic analyses performed with reference sequences retrieved from GenBank. RESULTS: Phylogenetic analyses revealed the sequence to be related to insect-specific flaviviruses (ISFs) of the genus Flavivirus, family Flaviviridae. Despite being related to the Palm Creek virus (PCV; an ISF very recently reported from Northern Australia), the present sequence (provisionally named Assam virus) was found to be highly divergent from PCV and other ISF sequences available in GenBank. The partial NS5 sequence analysis demonstrated low nucleotide sequence identity (66-77%) with known ISFs reported from other parts of the globe. CONCLUSION: Findings of this study suggest the presence of a candidate novel ISF - the first to be reported from India.

Detection of human papillomavirus in women attending Pap cervical screening camp at a peripheral hospital of North-Eastern India.

Human papillomavirus (HPV) associated cervical cancer is the leading cause of deaths in India. However, cytological/HPV screening may result in early detection of cervical cancer, resulting in early treatment and reduced mortality. Although reports related to general population is available, data on HPV prevalence among women attending AFMS health care facilities is scarce. Cervical samples were collected for cytological staining by Pap test and molecular detection by PCR, genotyping by HPV specific primers and sequencing. Apart from finding of atypical cells of undetermined significance (ASCUS) in one subject, no evidence of malignancy was observed. A high prevalence of HPV was found in this study group, which was intermediate between previous reports from general population and cervical cancer patients. All the subjects had infection of high risk HPV type16. HPV prevalence was found similar between different age groups. Although, none of the study subjects had malignant changes, but due to high prevalence of high risk HPV infection and other associated risk factors, these subjects might be at an elevated risk of developing cervical cancer. Regular follow-up of these patients who were detected HPV positive are required to screen for cervical malignancy.

Tumor suppressor micro RNA miR-145 and onco micro RNAs miR-21 and miR-222 expressions are differentially modulated by hepatitis B virus X protein in malignant hepatocytes.

BACKGROUND: Hepatitis B Virus (HBV) X protein (HBx) is known to be involved in the initiation and progression of hepatocellular carcinoma (HCC) through modulation of host gene response. Alterations in miRNA expressions are frequently noted in HCC. This study is aimed to examine the role of HBx protein in the modulation of oncogenic miRNA-21, miRNA-222 and tumor suppressor miRNA-145 in malignant hepatocytes. METHODS: Expressions of miRNA-21, miRNA-222 and miRNA-145 were measured in HepG2 cells transfected with HBx-plasmid (genotype D) and with full length HBV genome (genotype D) and also in stably HBV producing HepG2.2.15 cells using real time PCR. Their target mRNAs and proteins - PTEN, p27 and MAP3K - were analyzed by real time PCR and western blot respectively. miRNA expressions were measured after HBx/D mRNA specific siRNA treatment. The expressions of these miRNAs were analyzed in liver cirrhosis and HCC patients also. RESULTS: The study revealed a down-regulation of miRNA-21 and miRNA-222 expressions in HBx transfected HepG2 cells, pUC-HBV 1.3 plasmid transfected HepG2 cells as well as in HepG2.2.15 cells. Down regulation of miRNA-21 and miRNA-222 expression was observed in patient serum samples. Down regulation of miRNA-145 expression was observed in HepG2 cells transiently transfected with HBx and pUC-HBV1.3 plasmid as well as in patient samples but the expression of miRNA-145 was increased in HepG2.2.15 cells. Target mRNA and protein expressions were modulated in HepG2 cells and in HepG2.2.15 cell line consistent with the modulation of miRNA expressions. CONCLUSION: Thus, HBx protein differentially modulated the expression of miRNAs. The study throws light into possible way by which HBx protein acts through microRNA and thereby regulates host functioning. It might suggest new therapeutic strategies against hepatic cancer.

Characterization of the occult hepatitis B virus variants circulating among the blood donors from eastern India.

A previous study from West Bengal documented very high rate of occult HBV infection (OBI) among the HBsAg negative blood donors. This study was aimed to characterize the OBI strains circulating among the blood donors and to estimate the risk associated with the prevailing viral variants/mutants. Blood samples from 2195 voluntary blood donors were included in the study. HBsAg, HBeAg, anti-HBc, and anti-HBs statuses of the samples were done by ELISA based detection. PCR amplification and sequencing were done to determine HBV genotypes, basal core promoter (BCP), and precore (Pre-C) mutations. Among the study samples, 268 were anti-HBc positive/HBsAg negative, among which 65 (24.25%) were HBV DNA positive. Phylogenetic analysis revealed the presence of HBV/D (87.23%), HBV/A (8.51%), and HBV/C (4.26%) (P < 0.0001). HBV/D3 (65.85%) was the significantly prevalent subgenotype over HBV/D2 (26.83%) and HBV/D1 (7.31%) (P = 0.0003). Considerable prevalence of differential BCP (1752C, 1753C, 1762T/1764A, 1753C+1762T/1764A, 1773C, and 1814C) and reverse transcriptase (rt) gene (rtI91L, rtL93P, rtS106C, rtR110G, rtN118T, rtS119T, rtY126H, rtG127W/R, rtC136R, and rtY158H) mutations was identified. Association of specific HBV subgenotypes with OBI was interesting and needs further study. Clinically relevant mutations were prevalent among the OBI strains which are of serious concern.

Variations in the functional domain of basal core promoter of hepatitis B virus among Eastern Indian patients with prevalence of genotypes A, C, and D among the same ethnic population.

Mutations in the basal core promoter (BCP) and precore (PC) regions are associated with persistent and intermittently high hepatitis B virus (HBV) replication in several patients. The variability in the functional domains of BCP and PC region of HBV and their association with disease progression and clinical outcome were assessed in Eastern India, an unique region where three HBV genotypes, A, D, and C are prevalent among the same ethnic group. PCR amplification and direct sequencing of BCP and PC region was done on sera obtained from 130 HBsAg positive subjects with different clinical presentations. Associations of the apparent risk factors with clinical advancement were evaluated by statistical methods including multiple logistic regression analyses (MLR). HBV genotype A was present in 33.08%, C in 25.38%, and D in 41.54% cases. Genotypes A and C were associated with higher rate of T1762/A1764 mutations than the most predominant genotype D. HBeAg negative state was associated with considerably higher rate of C1753 mutation. T1762/A1764 along with C1753 was common among cirrhosis and T1762/A1764 without C1753 was frequent among chronic liver disease cases. No significant association was found between A1896 point mutation and clinical status. Multivariate analysis revealed that T1762/A1764 double mutation, HBV/A, age ≥25 years, C1753 and A1899 were critical factors for clinical advancement while age ≥25 years and C1753 as significant predictor for cirrhosis in comparison with chronic liver disease. In conclusion, the analysis of the BCP variability may help in monitoring the progression towards advanced liver disease in Eastern Indian patients.

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1.

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE-luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa.

In recent years, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) bacteria including Pseudomonas aeruginosa has drawn considerable attention, globally. In this work, we report the isolation and detailed characterization of a highly lytic Pseudomonasphage DRL-P1 isolated from wastewater. Under TEM, DRL-P1 appeared as a member of the phage family Myoviridae. DRL-P1 featured rapid adsorption (~ 5 min), short-latency (~ 30 min), and large burst size (~ 100 PFU per infected cell). DRL-P1 can withstand a wide temperature range (4 °C to 40 °C) and pH (5.0 to 10.0) conditions. The 66,243 bp DRL-P1 genome (MN564818) encodes at least 93 ORFs, of which 36 were functionally annotated based on homology with similar phage proteins available in the databases. Comparative analyses of related genomes suggest an independent evolutionary history and discrete taxonomic position of DRL-P1 within genus Pbunavirus. No toxin or antibiotic resistance genes was identified. DRL-P1 is tolerant to lyophilization and encapsulation techniques and retained lytic activity even after 18 months of storage. We also demonstrated decontaminating potentials of DRL-P1 in vitro, on an artificially contaminated cover-slip model. To the best of our knowledge, this is the first Pbunavirus to be reported from India. Our study suggests DRL-P1 as a potential candidate for various applications.

Genetic characterization of hepatitis B virus in peripheral blood leukocytes: evidence for selection and compartmentalization of viral variants with the immune escape G145R mutation.

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

BACKGROUND: Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. METHODS: A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. RESULTS: A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. CONCLUSION: Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.

Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway.

BACKGROUND: The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated. RESULTS: Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones. CONCLUSIONS: The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.

Excavating new facts from ancient Hepatitis B virus sequences.

Recently, two independent studies discovered 15 ancient Hepatitis B virus (aHBV) sequences, of which 7 dated back to the Neolithic age (NA) and the Bronze Age (BA). In the present research, all the available aHBV sequences were collectively re-analysed with reference to extant HBV diversity to understand the role of these aHBV genotypes in evolution of extant HBV genetic diversity. Several intergenotype recombination events were documented, which corroborated well with population admixture and ancient human migration. Present analyses suggested replacement of HBV genotype associated with early Neolithic European farming cultures by the migrating steppe people, during Bronze Age Steppe migration. Additionally, detailed analyses of recombinations revealed evolution of a number of extant genotypes and suggested their possible site of origin. Through this manuscript, novel and important findings of the analyses are communicated.

Frequency and distribution of hepatitis B virus genotypes among eastern Indian voluntary blood donors: Association with precore and basal core promoter mutations.

AIM: To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762/A1764) and precore (PreC; A1896) mutations among the HBV surface antigen (HBsAg) positive voluntary blood donors in eastern India. METHODS: HBV genotypes, BCP and PreC mutations of 141 HBsAg positive voluntary blood donors were determined by the restriction fragment length polymorphism (RFLP) method and a phylogenetic tree was constructed from surface (S) gene region sequences of representative HBsAg positive donors to confirm the results. RESULTS: HBV/D was the most predominant (79, 56.0%) genotype followed by HBV/C (33, 23.4%) and HBV/A (29, 20.6%). HBV/C infected blood donors are mostly young (18-25 years). The occurrence of BCP mutation was found to be significantly higher in HBV/C (24, 72.7%) than in HBV/A (7, 24.1%, P < 0.001) and HBV/D (17, 21.5%, P < 0.001), whereas PreC mutation was more frequent in HBV/D (28, 35.4%) than in HBV/C (9, 27.3%). However, the simultaneous presence of BCP and PreC mutations was more common in HBV/C (8/33, 24.2%), followed by HBV/D (6/79, 7.6%). CONCLUSION: In addition to HBV/D and HBV/A, a significant proportion of HBV/C (23.4%) was also present among the voluntary blood donors from eastern India, most frequently in the 18-25 year age group. BCP mutation was more common in HBV/C infected donors.

Bioethanol production from waste lignocelluloses: A review on microbial degradation potential.

Tremendous explosion of population has led to about 200% increment of total energy consumptions in last twenty-five years. Apart from conventional fossil fuel as limited energy source, alternative non-conventional sources are being explored worldwide to cater the energy requirement. Lignocellulosic biomass conversion for biofuel production is an important alternative energy source due to its abundance in nature and creating less harmful impacts on the environment in comparison to the coal or petroleum-based sources. However, lignocellulose biopolymer, the building block of plants, is a recalcitrant substance and difficult to break into desirable products. Commonly used chemical and physical methods for pretreating the substrate are having several limitations. Whereas, utilizing microbial potential to hydrolyse the biomass is an interesting area of research. Because of the complexity of substrate, several enzymes are required that can act synergistically to hydrolyse the biopolymer producing components like bioethanol or other energy substances. Exploring a range of microorganisms, like bacteria, fungi, yeast etc. that utilizes lignocelluloses for their energy through enzymatic breaking down the biomass, is one of the options. Scientists are working upon designing organisms through genetic engineering tools to integrate desired enzymes into a single organism (like bacterial cell). Studies on designer cellulosomes and bacteria consortia development relating consolidated bioprocessing are exciting to overcome the issue of appropriate lignocellulose digestions. This review encompasses up to date information on recent developments for effective microbial degradation processes of lignocelluloses for improved utilization to produce biofuel (bioethanol in particular) from the most plentiful substances of our planet.

Pro-oncogenic, intra host viral quasispecies in Diffuse large B cell lymphoma patients with occult Hepatitis B Virus infection.

Non Hodgkin lymphoma, predominantly Diffuse Large B-cell Lymphoma (DLBCL) has been reported to have a significant association with Hepatitis B virus (HBV). We investigated the presence of different gene segments of HBV in plasma, B-cells and tumor tissues from DLBCL patients and explored the genetic variability of HBV within and across different compartments in a host using Next Generation Sequencing. Despite all 40 patients being HBV seronegative, 68% showed evidence of occult HBV. Sequencing of these gene segments revealed inter-compartment viral variants in 26% of them, each with at least one non-synonymous mutation. Between compartments, core gene variants revealed Arg94Leu, Glu86Arg and Ser41Thr while X gene variants revealed Phe73Val, Ala44Val, Ser146Ala and Ser147Pro. In tumor compartments per se, several mis-sense mutations were detected, notably the classic T1762A/A1764G mutation in the basal core promoter. In addition, a virus surface antigen mis-sense mutation resulting in M125T was detected in all the samples and could account for surface antigen negativity and occult HBV status. It would be interesting to further explore if a temporal accumulation of viral variants within a favored niche, like patients' lymphocytes, could bestow survival advantage to the virus, and if certain pro-oncogenic HBV variants could drive lymphomagenesis in DLBCL.

An overview of molecular epidemiology of hepatitis B virus (HBV) in India.

Hepatitis B virus (HBV) is one of the major global public health problems. In India, HBsAg prevalence among general population ranges from 2% to 8%, placing India in intermediate HBV endemicity zone and the number of HBV carriers is estimated to be 50 million, forming the second largest global pool of chronic HBV infections. India is a vast country, comprised of multiracial communities with wide variations in ethnicity and cultural patterns, which is attributable to its geographical location, gene influx due to invasion and/or anthropological migrations in the past. Moreover, recent increase in trade, trafficking and use of illicit drugs has also considerably influenced the epidemiology of HBV, specifically in the eastern and north eastern parts of India. However, data on the molecular epidemiology of HBV in India is scanty. HBV genotypes A and D have been well documented from different parts of mainland India. Interestingly, in addition to genotypes A and D, genotype C having high nucleotide similarity with south East Asian subgenotype Cs/C1 strain, have been detected exclusively from eastern Indian HBV carriers, suggesting a recent introduction. Thus, compared to other parts of India, the molecular epidemiology of HBV is naturally distinct in eastern India. Very recently, taking the advantage of circulation of three distinct HBV genotypes within the population of eastern India, different aspects of HBV molecular epidemiology was studied that revealed very interesting results. In this study, the clinical significance of HBV genotypes, core promoter and precore mutations, possible routes of introduction of HBV genotype C in eastern India, the clinical implications of x gene variability, prevalence of the AFB1 induced p53 gene codon 249 mutation, the transmission potentiality of HBV among asymptomatic/inactive or occult HBV carriers and the genetic variability of HBV persisting in the PBL was investigated. In this manuscript, the information available on the molecular epidemiology of HBV in India has been reviewed and the results of studies among the eastern Indian population have been summarised.

Molecular epidemiology and clinical significance of hepatitis B virus genotypes, core promoter and precore mutations in eastern India.

OBJECTIVES: This unmatched case-control study aimed at determining the molecular epidemiology and clinical significance of HBV genotypes, core promoter (CP) and precore (PC) mutations in Eastern India. METHODS: Serological, biochemical and molecular assays were used to examine antigens, ALT, genotypes, mutations and viremia among 106 inactive carriers and 183 chronic liver disease (CLD) patients. RESULTS: Male gender (p < 0.001), HBeAg positivity (p = 0.050), high ALT (p < 0.001), high viremia (p < 0.001), CP mutations (p < 0.001), and genotypes A (p < 0.001) and C (p = 0.027) were significantly associated with CLD. Subjects infected with genotypes A and C had significantly higher prevalence of BCP mutations (p < 0.001), and low incidence of PC mutation (p < 0.001 and p = 0.047, respectively). Prevalence of genotype D was significantly higher among subjects with history of familial/childhood jaundice, while genotypes A and C were frequent among subjects with possible percutaneous exposure. CONCLUSIONS: Significant differences in risk factors and disease manifestation do exist among patients infected with different HBV genotypes. Genotypes A and C are frequently found among chronic liver disease patients, while genotype D is associated with inactive HBeAg-negative infections. This evaluation of clinical relevance of HBV genotypes, mutations and risk factors may be useful in disease prognosis, management and prevention strategies.

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

BACKGROUND: In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. RESULTS: In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. CONCLUSION: The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.