A comprehensive account of SARS-CoV-2 genome structure, incurred mutations, lineages and COVID-19 vaccination program.

This review collates information on the onset of COVID-19, SARS-CoV-2 genome architecture, emergence of novel viral lineages that drove multiple waves of infection around the world and standard and fast track development of vaccines. With the passage of time, the continuously evolving SARS-CoV-2 has acquired an expanded mutational landscape. The functional characterization of spike protein mutations, the primary target of diagnostics, therapeutics and vaccines has revealed increased transmission, pathogenesis and immune escape potential in the variant lineages of the virus. The incurred mutations have also resulted in substantial viral neutralization escape to vaccines, monoclonal, polyclonal and convalescent antibodies presently in use. The present situation suggests the need for development of precise next-generation vaccines and therapeutics by targeting the more conservative genomic viral regions for providing adequate protection.

Evaluation of vascular effect of arsenic using in vivo assays.

Arsenic (As) is an abundant toxicant present in groundwater and soil in various parts of the world including eastern part of India. The epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. Placenta is known to utilize vasculogenesis to develop its vasculature circulation. The effects of four different doses of sodium meta-arsenite (0, 10, 20, 75, and 150 ppm) were assessed on the vascular structure using two different in vivo models, i.e., Matrigel and chorioallantoic membrane (CAM) assay. For the Matrigel assay, mice were exposed to different doses of arsenic through drinking water for 1 month. Placenta and Matrigel plug (which was inserted on gestational day (GD 0.5)) were removed on GD 14. Similar arsenic concentration was used in CAM assay to observe the effect of vessel development in hen's eggs. The CAM assay outcome evaluated by Angiosys software showed that arsenic exposure reduced the total and mean tubule length in all the arsenic-treated groups. The percentage tubule inhibition was declined significantly in 20, 75, and 150 ppm arsenic-treated groups as evaluated by ImageJ software. Analysis of the CAM outcome by both the image analysis software indicated the adverse effect of arsenic on the tubules. Further, a significant higher blood vessel density in 10 ppm and lower vessel density in 20, 75, and 150 ppm arsenic-exposed mice were also observed in Matrigel plug assay. The placental hypertrophy and dysplasia especially in the labyrinth zone (vasculature) were noted in placenta of arsenic-treated mice. The study indicated that higher arsenic exposures inhibited the angiogenesis which was dose-dependent in both CAM and Matrigel assay and altered structural morphology of placenta. However, no inhibition of blood vessels was noted at lower, i.e., 10 ppm of arsenic-treated group.

Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against Chikungunya virus in vero cells.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented and explosive epidemics in India and the Indian Ocean islands after a gap of 32 years is a major public health concern. Currently, there is no specific therapy available to treat CHIKV infection. In the present study, the in vitro prophylactic and therapeutic effects of chloroquine on CHIKV replication in Vero cells were investigated. Inhibitory effects were observed when chloroquine was administered pre-infection, post-infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. The inhibitory effects were confirmed by performing a plaque reduction neutralization test (PRNT), real-time reverse transcriptase (RT)-PCR analysis of viral RNA levels, and cell viability assays. Chloroquine diminished CHIKV infection in a dose-dependent manner, with an effective concentration range of 5-20 microM. Concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by >/=99.99%. The maximum inhibitory effect of chloroquine was observed within 1-3 hr post-infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. The mechanism of inhibition of virus activity by chloroquine involved impaired endosomal-mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent.

Antibodies against recombinant shiga toxin subunit B neutralize shiga toxin toxicity in HeLa cells.

Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin 1 (Stx) and Shiga toxin1 (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticaly-active A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti-StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.

Synthesis and antimalarial evaluation of 1, 3, 5-trisubstituted pyrazolines.

A series of 1,3,5-trisubstituted pyrazolines were synthesized and evaluated for in vitro antimalarial efficacy against chloroquine sensitive (MRC-02) as well as chloroquine resistant (RKL9) strains of Plasmodium falciparum. The activity was at nano molar concentration. beta-hematin formation inhibition activity (BHIA(50)) of the pyrazolines were determined and correlated with antimalarial activity. A reasonably good correlation (r=0.62) was observed between antimalarial activity (IC(50)) and BHIA(50). This suggests that antimalarial mode of action of this class of compounds appears to be similar to that of chloroquine and involves the inhibition of hemozoin formation. Some of the compounds were showing better antimalarial activity than chloroquine against resistant strain of P. falciparum and were also found active in the in vivo experiment.

Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya.

The resurgence of chikungunya (CHIK) in the form of unprecedented explosive epidemic after a gap of 3 decades in India and Indian Ocean islands is a point of major public health concern. The laboratory diagnosis is essentially based on virus isolation, IgM ELISA, and reverse transcriptase polymerase chain reaction (RT-PCR). Although PCR-based methods are used for early and accurate diagnosis, the high cost of the assay and requirement of thermal cycler limit its application only to referral laboratories. The antibody-based IgM ELISA is found to be cost-effective, but it takes 5 to 6 days for the patient to develop antibody and, thus, has less implication for early clinical diagnosis and patient management. Therefore, a simple rapid, sensitive, and specific antigen detection system is reported for early and reliable clinical diagnosis as well as effective surveillance of CHIK. A double antibody sandwich system was designed for antigen capture ELISA, employing rabbit and mouse anti-CHIK IgG antibodies as capture and detector antibodies, respectively. An optimal assay condition with 0 background was established having no reactivity with healthy human serum and Cerebro spinal fluid (CSF) samples. The comparative evaluation with SYBR Green I-based real-time RT-PCR revealed an accordance of 96% with a sensitivity and specificity of 95% and 97%, respectively. The specificity of this assay was confirmed through cross-reactivity studies with confirmed dengue and Japanese encephalitis (JE) patient serum and CSF samples. The antigen capture ELISA reported in this study was able to detect the presence of viral antigen as early as the second day of fever and, thus, can be very useful for early clinical diagnosis of CHIK with acute phase patient serum and CSF samples. This can also be used for rapid screening of large numbers of clinical samples in endemic areas during epidemics.

Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus.

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6 -- 8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.

RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells.

Chikungunya has emerged as one of the most important arboviral infection of public health significance. Recently several parts of Indian Ocean islands and India witnessed explosive, unprecedented epidemic. So far, there is no effective antiviral or licensed vaccine available against Chikungunya infection. RNA interference mediated inhibition of viral replication has emerged as a promising antiviral strategy. In this study, we examined the effectiveness of small interfering RNAs (siRNAs) against the inhibition of Chikungunya virus replication in Vero cells. Two siRNAs against the conserved regions of nsP3 and E1 genes of Chikungunya virus were designed. The siRNA activity was assessed by detecting both the infectious virus and its genome. The results indicated a reduction of virus titer up to 99.6% in siRNA transfected cells compared to control. The viral inhibition was most significant at 24h (99%), followed by 48 h (65%) post infection. These results were also supported by the quantitative RT-PCR assay revealing similar reduction in Chikungunya viral genomic RNA. The siRNAs used had no effect on the expression of house keeping gene indicating non-interference in cellular mechanism. The specific and marked reduction in viral replication against rapidly replicating Chikungunya virus achieved in this study offers a potential new therapeutic approach. This is the first report demonstrating the effectiveness of siRNA against in vitro replication of Chikungunya virus.

Mushroom cryopreservation and its effect on survival, yield and genetic stability.

Mycelial stock cultures of Agaricus bisporus, A. Bitorquis, Pleurotus flabellatus, P. Sajor-caju, P. Ostreatus, P. Sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15 percent glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.