First detection of SARS-CoV-2 Delta variant (B.1.617.2) in the wastewater of (Ahmedabad), India

Contrary to the conventional genomic surveillance based on clinical samples (symptomatic patients), the wastewater-based genomic surveillance can identify all the variants shed by the infected individuals in the population, as it does also include RNA fragmented shredded by clinically escaped asymptomatic patients. We analyzed four samples to detect key mutations in the SARS-CoV-2 genome and track circulating variants in Ahmedabad during the first wave (Sep/ Nov 2020) and before the second wave (in Feb 2021) of COVID-19 in India. The analysis showed a total of 35 mutations in the spike protein across four samples categorized into 23 types. We noticed the presence of spike protein mutations linked to the VOC-21APR-02; B.1.617.2 lineage (Delta variant) with 57% frequency in wastewater samples of Feb 2021. The key spike protein mutations were T19R, L452R, T478K, D614G, & P681R and deletions at 22029 (6 bp), 28248 (6 bp), & 28271 (1 bp). Interestingly, these mutations were not observed in the samples of Sep and Nov 2020 but appeared before the devastating second wave of COVID-19, which started in early April 2021 in India, caused rapid transmission and deaths all over India. We found the genetic traces of the B.1.617.2 in samples of early Feb 2021 i.e., more than a month before the first clinically confirmed case of the same variant in March 2021 in Ahmedabad, Gujarat. The present study tells about the circulating variants in Ahmedabad and suggests early prediction VOCs employing the wastewater genomic surveillance approach that must be exploited at a large scale for effective COVID-19 management. HighlightsO_LIWhole-genome sequencing of SARS-CoV-2 from the WW samples was carried out. C_LIO_LIVariant of Concern (VoC: VOC-21APR-02; B.1.617.2) were detected in WW samples. C_LIO_LIWBE may detect prevalent SARS-CoV-2 variants and monitor their cryptic transmission C_LIO_LIWW genomic surveillance can aid the decision-making system for public health policies. C_LI

Unravelling the early warning capability of wastewater surveillance for COVID-19: A temporal study on SARS-CoV-2 RNA detection and need for the escalation

Wastewater-based Epidemiological (WBE) surveillance offers a promising approach to assess the pandemic situation covering pre-symptomatic and asymptomatic cases in highly populated area under limited clinical tests. In the present study, we analysed SARS-CoV-2 RNA in the influent wastewater samples (n = 43) from four wastewater treatment plants (WWTPs) in Gandhinagar, India, during August 7th to September 30th, 2020. A total of 40 samples out of 43 were found positive i.e. having at least two genes of SARS-CoV-2. The average Ct values for S, N, and ORF 1ab genes were 32.66, 33.03, and 33.95, respectively. Monthly variation depicted a substantial rise in the average copies of N ([~]120%) and ORF 1ab ([~]38%) genes in the month of September as compared to August, while S-gene copies declined by 58% in September 2020. The SARS-CoV-2 genome concentration was higher in the month of September ([~]924.5 copies/ L) than August ([~]897.5 copies/ L), corresponding to a [~] 2.2-fold rise in the number of confirmed cases during the study period. Further, the percentage change in genome concentration level on a particular date was found in the lead of 1-2 weeks of time with respect to the official confirmed cases registered based on clinical tests on a temporal scale. The results profoundly unravel the potential of WBE surveillance to predict the fluctuation of COVID-19 cases to provide an early warning. Our study explicitly suggests that it is the need of hour that the wastewater surveillance must be included as an integral part of COVID-19 pandemic monitoring which can not only help the water authorities to identify the hotspots within a city but can provide up to 2 weeks of time lead for better tuning the management interventions. HIGHLIGHTS{square} Study unravels the early warning potential of wastewater based surveillance of COVID-19. {square}Adequate SARS-CoV-2 RNA were detected despite of limited reported case in the vicinity. {square}Up to 2 weeks of lead is possible from a regular wastewater based COVID-19 surveillance. {square}SARS-CoV-2 RNA was higher in September than August in response to a [~] 2.2-fold rise in COVID-19 active cases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/20248744v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@15d8cc7org.highwire.dtl.DTLVardef@c57729org.highwire.dtl.DTLVardef@1246feorg.highwire.dtl.DTLVardef@1f2e760_HPS_FORMAT_FIGEXP M_FIG C_FIG

The first proof of the capability of wastewater surveillance for COVID-19 in India through the detection of the genetic material of SARS-CoV-2

we made the first ever successful effort from India to detect the genetic material of SARS-CoV-2 viruses to understand the capability and application of WBE surveillance in India. Sampling was carried out on 8 and 27 May, 2020 from Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat with 106 million liters per day (MLD) capacity receiving effluent of Civil Hospital treating COVID-19 patient. All three i.e. ORF1ab, N and S genes of SARS-CoV-2 were discerned in the influents with no gene spotted in the effluent collected on 8 and 27 May 2020. Temporal difference between 8 and 27 May 2020 samples was of 10x in gene copy loading with corresponding change of 2x in the number active COVID-19 patient in the city. Number of gene copies was found comparable to that reported in the untreated wastewaters of Australia, China and Turkey and lower than that of the USA, France and Spain. This study, being the first from India and probably among the first ten reports in the world of gene detection of SARS-CoV-2 in the environmental samples, aims to assist concerned authorities and policymakers to formulate and/or upgrade the COVID-19 surveillance to have explicit picture of phase of the pandemic. While infectious SARS-CoV-2 has yet to be identified in the aquatic environment, the virus potentially enters the wastewater stream from patient excretions and thus can be a great tool for pandemic monitoring. HIGHLIGHTS{square} First ever report of the presence of gene of SARS-CoV-2 in the wastewater in India. {square}CT value is explicitly indicative of the increase of COVID-19 patient in the vicinity. {square}All three i.e. ORF1ab, N and S genes of SARS-CoV-2 were discerned in the influents. {square}None of three genes were spotted in the effluent collected on 8 and 27 May 2020. {square}Wastewater surveillance conclusively specified temporal difference in COVID-19 load. {square}Temporal difference was 10x and 2x in gene copies and COVID-19 patient, respectively.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective:To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques.Methods:We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS andResults:Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS andConclusions:It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS and

Cytokine expression pattern in milk somatic cells of subclinical mastitis-affected cattle analyzed by real time PCR / 대한수의학회지

The expression profiles of inflammatory cytokines viz. interleukins (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon-gamma and tumor necrosis factor-alpha in response to subclinical mastitis in indigenous cattle breed Kankrej (n = 6), Gir (Bos indicus) (n = 12) and crossbred (Bos taurus x Bos indicus) (n = 7) were investigated using quantitative real time PCR. Significant correlation (p < 0.05) was observed between total bacterial load and somatic cell count (SCC) in all three breeds of cattle. All the cytokines were observed to be up-regulated compared to cows with healthy quarters, however, level of their expression varied among three breeds of cattle. In Kankrej most cytokines were found to be transcribed to higher levels than in other two breeds; the milk had higher load of bacteria but not so high SCC, implying that Kankrej has a higher inherent resistance against mastitis. The results of present study indicated that mammary glands of crossbred cattle are more sensitive to bacterial infection than indigenous breed of cattle as they elicit immune response at lower bacterial load and result into higher SCC. Research on identification of factors responsible for differentially expressed cytokines profiles and use of cytokines as immunomodulatory tools can pave way for formulating control strategies against bovine mastitis.

Comparative evaluation of phenobarbital-induced CYP3A and CYP2H1 gene expression by quantitative RT-PCR in Bantam, Bantamized White Leghorn and White Leghorn chicks

The present work was to study induction of cytochrome P450 (CYP)3A and CYP2H1 gene by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RTPCR in Bantam, Bantamized White Leghorn and White Leghorn chicks. Out of 18 chicks total 3 from each group (Bantam, Bantamized White Leghorn and White Leghorn) were treated intraperitoneal with phenobarbital at the dose rate of 12 mg/100 g (body weight) while the control group was treated with the saline. Total RNA was extracted from the liver samples using Tri Reagent based method. First strand cDNA was synthesized using one step RT-PCR kit. The PCR was performed and the product was subjected to agarose gel electrophoresis. Quantitative RT-PCR was conducted to quantify gene expression level of CYP3A and CYP2H1 genes. Relative expression ratio of CYP3A and CYP2H1 genes was calculated using relative expression software tool (REST). It was found that CYP3A is up regulated by factor of 1.34, 14.51 and 1.00 in Bantam, Bantamized White Leghorn and White Leghorn chicks, respectively. In Bantam and Bantamized White Leghorn chicks CYP2H1 gene was up regulated by factor 1.50 and 80.87, respectively but down regulated by a factor of 1.97 in White Leghorn chicks. The PCR efficiency ranged from 1.30 to 1.70, 0.86 to 1.70 and 0.91 to 1.58 for CYP3A, CYP2H1 and beta-actin, respectively in Bantam, Bantamized White Leghorn and White Leghorn chicks.