Mapping of long stretches of highly conserved sequences in over 6 million SARS-CoV-2 genomes.

We identified 11 conserved stretches in over 6.3 million SARS-CoV-2 genomes including all the major variants of concerns. Each conserved stretch is ≥100 nucleotides in length with ≥99.9% conservation at each nucleotide position. Interestingly, six of the eight conserved stretches in ORF1ab overlapped significantly with well-folded experimentally verified RNA secondary structures. Furthermore, two of the conserved stretches were mapped to regions within the S2-subunit that undergo dynamic structural rearrangements during viral fusion. In addition, the conserved stretches were significantly depleted for zinc-finger antiviral protein (ZAP) binding sites, which facilitated the recognition and degradation of viral RNA. These highly conserved stretches in the SARS-CoV-2 genome were poorly conserved at the nucleotide level among closely related ß-coronaviruses, thus representing ideal targets for highly specific and discriminatory diagnostic assays. Our findings highlight the role of structural constraints at both RNA and protein levels that contribute to the sequence conservation of specific genomic regions in SARS-CoV-2.

Antibacterial and cytocompatibility study of modified Ti6Al4V surfaces through thermal annealing.

Silver coating of different thicknesses ranging from 5 to 20 nm was deposited on the Ti6Al4V substrate using DC sputtering followed by thermal annealing at 750 °C for 15 min in an ambient environment. The surface topography and elemental composition of annealed samples were analyzed using different characterization techniques. The silver ions (Ag+) concentration released from the modified titanium surface was calculated through inductive coupled plasma mass spectroscopy (ICP-MS). The plate counting method was used to quantify the bacteria-killing potential of modified titanium surface against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Fluoroquinolones-resistant Salmonella typhi (FRST) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria. The cell membrane integrity study of E. coli and S. aureus bacterium was done qualitatively using scanning electron microscopy and further confirmed with fluorescence microscopy. Due to thermal annealing, polygonal shaped oxide nanoparticles were formed on the titanium substrate. Moreover, the surface topography of modified titanium surface changes with the thickness of the silver film. In order to check the cytotoxic effect of modified titanium surface, mouse fibroblast cells (NIH3T3) were used for 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay. The limited (<35 ppb) Ag+ ion release was noticed for 15 nm silver film which has shown the good bactericidal property and significant growth of fibroblast cells. This study proposes a simple and efficient method to enhance the antibacterial property of Ti6Al4V surfaces to avoid implant-related infection.

A simple and rapid method for the quantitation of secreted hepatitis B virions in cell culture models.

Cell culture models for hepatitis B virus (HBV) remain the mainstay for screening and testing the efficacy of anti-hepatitis B virus agents. Gradient-based ultracentrifugation followed by Southern Blotting is used for hepatitis B virion estimation in cell culture; this method has several limitations. We report the development of an assay using a commercially available HBsAg-ELISA plate for immunocapture followed by real-time PCR for quantification of hepatitis B virions in cell cultures. This assay is rapid, highly sensitive (50 copies/reaction) and highly specific for virion-associated DNA. In addition, the assay requires only 20 µL of supernatant, allowing scaling down of transfections.

Hepatitis B viral DNA is methylated in liver tissues.

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.

Protective effect of picroliv against hydrazine-induced hyperlipidemia and hepatic steatosis in rats.

The protective effect of picroliv (PIC) obtained from Picrorhiza kurroa (family: Scrophulariaceae) against hydrazine (Hz)-induced hyperlipidemia was evaluated in rats. Hz administration (50 mg/kg, i.p.) caused an increase in triglyceride (TG), cholesterol (CHO), free fatty acids (FFA), and total lipids (TL) in both the plasma and liver tissue of rats accompanied by a fall in phospholipids (PL) in the liver tissue 24 h after its administration, indicating its hyperlipidemic property. The above abnormality was prevented by simultaneous treatment of PIC (50 mg/kg, p.o.) with Hz. Hz treatment also caused an increase in the mobility of TG and TL from adipose tissue, and these results indicate that Hz administration could cause hepatic steatosis by nonhepatocellular factors (such as mobilization of depot fats). This effect was also prevented by simultaneous treatment of PIC with Hz. PIC-alone treatment, however, did not produce any change in the status of all the lipid parameters evaluated in plasma, liver, and adipose tissues. These results indicate that increased mobilization of depot fats from adipose tissue may contribute to the development of hepatic steatosis in addition to decreased lipoprotein secretion, increased hepatic TG biosynthesis, and increased hepatic uptake of FFA. These have been reported as the mechanism responsible for the development of Hz-induced hepatic steatosis. PIC prevents Hz-induced hyperlipidemia, hepatic steatosis, and mobilization of lipids from depot fats, but the mechanism behind the protective effect of PIC remains to be elucidated.

Significance of the hepatitis C virus (HCV) core antigen as an alternative plasma marker of active HCV infection.

PURPOSE: To evaluate the role of core antigen (Ortho trak-C assay) as a marker of active HCV infection in comparison to HCV RNA as detected by reverse transcription polymerase chain reaction (RT-PCR). METHODS: This evaluation was carried out during January 2000 to December 2003 in HCV infected individuals who were treatment naomicronve or were on anti-viral therapy. Additionally, sequential plasma samples from patients on clinical follow-up were included in this study. A total of 167 samples from 61 patients were tested by trak-C and RT-PCR. HCV RNA detection was achieved by a RT-PCR. Trak-C assay results were also compared in a limited proportion of these samples with known HCV viral load and genotype. RESULTS: Of 167 samples tested, 56.9% were RNA positive and 43.1% were RNA negative while 50.3% were trak-C positive and 49.7% were trak-C negative, yielding a sensitivity of 85.3% and a specificity of 95.8% for the trak-C assay (Kappa co-efficient = 0.8). The concentration of HCVcAg and HCV RNA showed significant correlation (n=38, r=0.334, P =0.04). The trak-C assay detected the most prevalent HCV genotypes in India without significant difference (P =0.335). The difference between mean absorbance values of HCV RNA positive samples compared to HCV RNA negative samples in the trak-C assay was highly significant (P < 0.000). Qualitative results of trak-C assay and RT-PCR were comparable in 93% of follow-up samples. CONCLUSIONS: Trak-C assay can be recommended for confirmation of HCV infection and follow-up in laboratories with resource-poor facilities.