A Rare HBV Subgenotype D4 with Unique Genomic Signatures Identified in North-Eastern India –An Emerging Clinical Challenge?

BACKGROUND/AIMS: HBV has been classified into ten genotypes (A-J) and multiple subgenotypes, some of which strongly influence disease outcome and their distribution also correlate with human migration. HBV infection is highly prevalent in India and its diverse population provides an excellent opportunity to study the distinctiveness of HBV, its evolution and disease biology in variegated ethnic groups. The North-East India, having international frontiers on three sides, is one of the most ethnically and linguistically diverse region of the country. Given the paucity of information on molecular epidemiology of HBV in this region, the study aimed to carry out an in-depth genetic characterization of HBV prevailing in North-East state of Tripura. METHODS: From sera of chronically HBV infected patients biochemical/serological tests, HBV DNA quantification, PCR-amplification, sequencing of PreS/S or full-length HBV genomes were done. HBV genotype/subgenotype determination and sequence variability were assessed by MEGA5-software. The evolutionary divergence times of different HBV subgenotypes were estimated by DNAMLK/PHYLIP program while jpHMM method was used to detect any recombination event in HBV genomes. RESULTS: HBV genotypes D (89.5%), C (6.6%) and A (3.9%) were detected among chronic carriers. While all HBV/A and HBV/C isolates belonged to subgenotype-A1 and C1 respectively, five subgenotypes of HBV/D (D1-D5) were identified including the first detection of rare D4. These non-recombinant Indian D4 (IndD4) formed a distinct phylogenetic clade, had 2.7% nucleotide divergence and recent evolutionary radiation than other global D4. Ten unique amino acids and 9 novel nucleotide substitutions were identified as IndD4 signatures. All IndD4 carried T120 and R129 in ORF-S that may cause immune/vaccine/diagnostic escape and N128 in ORF-P, implicated as compensatory Lamivudine resistance mutation. CONCLUSIONS: IndD4 has potential to undermine vaccination programs or anti-viral therapy and its introduction to North-East India is believed to be linked with the settlement of ancient Tibeto-Burman migrants from East-Asia

SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition

Background Following SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. Methods The study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. Results The frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. Conclusions The antibody responses to the spike protein, but not the anti-RBD B cell responses diverge between convalescent males and females who develop PCC. Our findings also suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens

SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition

BackgroundFollowing SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic.MethodsThe study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA.ResultsThe frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups.ConclusionsThe antibody responses to the spike protein, but not the anti-RBD B cell responses diverge between convalescent males and females who develop PCC. Our findings also suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens

IL-15 Prevents the Development of T-ALL from Aberrant Thymocytes with Impaired DNA Repair Functions and Increased NOTCH1 Activation

We previously reported that NOD.Scid mice lacking interleukin-15 (IL-15), or IL-15 receptor alpha-chain, develop T-acute lymphoblastic leukemia (T-ALL). To understand the mechanisms by which IL-15 signaling controls T-ALL development, we studied the thymocyte developmental events in IL-15-deficient Scid mice from NOD and C57BL/6 genetic backgrounds. Both kinds of mice develop T-ALL characterized by circulating TCR-negative cells expressing CD4, CD8 or both. Analyses of thymocytes in NOD.Scid.Il15−/− mice prior to T-ALL development revealed discernible changes within the CD4−CD8− double-negative (DN) thymocyte developmental stages and increased frequencies of CD4+CD8+ double-positive cells with a high proportion of TCR-negative CD4+ and CD8+ cells. The DN cells also showed elevated expressions of CXCR4 and CD117, molecules implicated in the expansion of DN thymocytes. T-ALL cell lines and primary leukemic cells from IL-15-deficient NOD.Scid and C57BL/6.Scid mice displayed increased NOTCH1 activation that was inhibited by NOTCH1 inhibitors and blockers of the PI3K/AKT pathway. Primary leukemic cells from NOD.Scid.Il15−/− mice survived and expanded when cultured with MS5 thymic stromal cells expressing Delta-like ligand 4 and supplemented with IL-7 and FLT3 ligand. These findings suggest that IL-15 signaling in the thymus controls T-ALL development from aberrant thymocytes with an impaired DNA repair capacity and increased NOTCH1 activation

IL-15 Prevents the Development of T-ALL from Aberrant Thymocytes with Impaired DNA Repair Functions and Increased NOTCH1 Activation

We previously reported that NOD.Scid mice lacking interleukin-15 (IL-15), or IL-15 receptor alpha-chain, develop T-acute lymphoblastic leukemia (T-ALL). To understand the mechanisms by which IL-15 signaling controls T-ALL development, we studied the thymocyte developmental events in IL-15-deficient Scid mice from NOD and C57BL/6 genetic backgrounds. Both kinds of mice develop T-ALL characterized by circulating TCR-negative cells expressing CD4, CD8 or both. Analyses of thymocytes in NOD.Scid.Il15−/− mice prior to T-ALL development revealed discernible changes within the CD4−CD8− double-negative (DN) thymocyte developmental stages and increased frequencies of CD4+CD8+ double-positive cells with a high proportion of TCR-negative CD4+ and CD8+ cells. The DN cells also showed elevated expressions of CXCR4 and CD117, molecules implicated in the expansion of DN thymocytes. T-ALL cell lines and primary leukemic cells from IL-15-deficient NOD.Scid and C57BL/6.Scid mice displayed increased NOTCH1 activation that was inhibited by NOTCH1 inhibitors and blockers of the PI3K/AKT pathway. Primary leukemic cells from NOD.Scid.Il15−/− mice survived and expanded when cultured with MS5 thymic stromal cells expressing Delta-like ligand 4 and supplemented with IL-7 and FLT3 ligand. These findings suggest that IL-15 signaling in the thymus controls T-ALL development from aberrant thymocytes with an impaired DNA repair capacity and increased NOTCH1 activation

Primers used in this study.

<p>**  =  nucleotide [nt] positions are given according to HBV sequence with accession no. AF121242 obtained from GenBank.</p>ψ<p>Gunther S <i>et al</i>. J. Virol. 1995; 69: 5437–44.</p><p>Primers used in this study.</p

Estimation of divergence times of non-recombinant HBV subgenotypes D1–D7 including Indian HBV/D4 (IndD4).

<p>The tree was constructed by the DNAMLK program of the PHYLIP package, using indel-free HBV sequences of different subgenotypes of D, derived from GenBank, along with 12 IndD4 sequences. Reference sequences are indicated by their specific subgenotypes followed by accession numbers and the IndD4 sequences are indicated by respective isolate numbers beginning with “TP.”</p