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Leishmania sp. belong to the trypanosomatid group of protozoan parasites and are responsible for a wide variety of diseases collectively known as leishmaniasis. They continue to be a major health problem of the world threatening ~ 350 million people, majority of whom are from the impoverished sections of the community. Unavailability of a vaccine and increasing signs of resistance to few existing drugs warrants consolidated effort towards discovery of novel antileishmanial drug targets. In recent times, carbonic anhydrases (CAs) have been studied in a number of pathogenic microorganisms and preliminary evidences suggest that it can be a promising drug target. We discovered that Leishmania major expresses two CAs (LmCAs), namely a β-family LmCA1 and an α-family LmCA2. Our preliminary pharmacological inhibition studies, using an established CA inhibitor, suggested that LmCA activity is important for parasite growth and survival. Coupling pharmacological inhibition data with structural insights obtained from homology model of LmCA1, we arrived at the conclusion that LmCAs, especially LmCA1, is structurally distinct from its human α-CA counterparts. These encouraging results prompted us to investigate the role of LmCA1 in parasite physiology. Subcellular localization studies confirmed that LmCA1 is localized in the cytosol of the parasite. LmCA1-/- knockout strain could not be generated in repeated attempts, suggesting its functional indispensability to the parasite. However, we were able to generate LmCA1+/- heterozygous strain, which grew normally in neutral medium but suffered from intracellular acidosis-induced cell cycle arrest, growth retardation and morphological abnormalities in acidic pH. This strain also showed reduced parasite load and infectivity in macrophage cell line and mice. Thus, LmCA1 is crucial for cytosolic buffering of L. major and plays a determining role in parasite proliferation in acidic milieu in vitro as well as within phagolysosomes of host macrophages. In addition to acid acclimatization, LmCA1 plays an indispensable role in the gluconeogenic pathway of the parasite. Under low glucose stress, LmCA1+/- heterozygous strain suffered from cell cycle arrest, growth retardation and morphological abnormalities. These phenotypes were due to depleted intracellular glucose pool, which ultimately resulted in faulty ATP production in the mutant parasite. Thus, indispensability of LmCA1 in such crucial physiological and metabolic processes of Leishmania highlights LmCA1 as a potential antileishmanial drug target.

Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Günther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq™ sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants.