Search

Summary: Background: Environmental surveillance (ES) of a pathogen is crucial for understanding the community load of disease. As an early warning system, ES for SARS-CoV-2 has complemented routine diagnostic surveillance by capturing near real-time virus circulation at a population level. Methods: In this longitudinal study conducted between January 2022 and June 2022 in 28 sewershed sites in Bengaluru city (∼11 million inhabitants), we quantified weekly SARS-CoV-2 RNA concentrations to track infection dynamics and provide evidence of change in the relative abundance of emerging variants. Findings: We describe an early warning system using the exponentially weighted moving average control chart and demonstrate how SARS-CoV-2 RNA concentrations in wastewater correlated with clinically diagnosed new COVID-19 cases, with the trends appearing 8–14 days earlier in wastewater than in clinical data. This was further corroborated by showing that the estimated number of infections is strongly correlated with SARS-CoV-2 RNA copies detected in the wastewater. Using a deconvolution matrix, we detected emerging variants of concern up to two months earlier in wastewater samples. In addition, we found a huge diversity in variants detected in wastewater compared to clinical samples. The findings from this study have been discussed regularly with local authorities to inform policy-making decisions. Interpretation: Our study highlights that quantifying viral titre, correlating it with a known number of cases in the area, and combined with genomic surveillance helps in tracking variants of concern (VOC) over time and space, enabling timely and making informed policy decisions. Funding: This work has been supported by funding from the Rockefeller Foundation grant to National Centre for Biological Sciences, TIFR) and the Indian Council of Medical Research grant to (FI) Tata Institute for Genetics and Society and Tata Trusts.

Abstract Anopheles stephensi is the most menacing malaria vector to watch for in newly urbanising parts of the world. Its fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions in the genome of malaria vectors are implicated in adaptation. We report the genome assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome reported here with a L50 of 4, completes the trilogy of high-resolution genomes of strains with respect to a 16.5 Mbp 2Rb genotype in An. stephensi known to be associated with adaptation to environmental heterogeneity. Unlike the reported genomes of two other strains, STE2 (2R+b/2Rb) and UCI (2Rb/2Rb), IndCh is found to be homozygous for the standard form (2R+b/2R+b). Comparative genome analysis revealed base-level details of the breakpoints and allowed extraction of 22,650 segregating SNPs for typing this inversion in populations. Whole genome sequencing of 82 individual mosquitoes from diverse geographical locations reveal that one third of both wild and laboratory populations maintain the heterozygous genotype of 2Rb. The large number of SNPs can be tailored to 1740 exonic SNPs enabling genotyping directly from transcriptome sequencing. The genome trilogy approach accelerated the study of fine structure and typing of an important inversion in An. stephensi, putting the genome resources for this understudied species on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse geographical locations cluster with IndCh, pointing to significant convergence resulting from travel and commerce between cities, perhaps, contributing to the survival of the fittest strain.

Abstract Background The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector’s genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution. Results To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role. Conclusion Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.

Malaria remains a major healthcare risk to growing economies like India, and a chromosome-level reference genome of Anopheles stephensi is critical for successful vector management and understanding of vector evolution using comparative genomics. We report chromosome-level assemblies of an Indian strain, STE2, and a Pakistani strain SDA-500 by combining draft genomes of the two strains using a homology-based iterative approach. The resulting assembly IndV3/PakV3 with L50 of 9/12 and N50 6.3/6.9 Mb had scaffolds long enough for building 90% of the euchromatic regions of the three chromosomes, IndV3s/PakV3s, using low-resolution physical markers and enabled the generation of the next version of genome assemblies, IndV4/PakV4, using HiC data. We have validated these assemblies using contact maps against publicly available HiC raw data from two strains including STE2 and another lab strain of An. stephensi from UCI and compare the quality of the assemblies with other assemblies made available as preprints since the submission of the manuscript. We show that the IndV3s and IndV4 assemblies are sensitive in identifying a homozygous 2Rb inversion in the UCI strain and a 2Rb polymorphism in the STE2 strain. Multiple tandem copies of CYP6a14, 4c1, and 4c21 genes, implicated in insecticide resistance, lie within this inversion locus. Comparison of assembled genomes suggests a variation of 1 in 81 positions between the UCI and STE2 lab strains, 1 in 82 between SDA-500 and UCI strain, and 1 in 113 between SDA-500 and STE2 strains of An. stephensi, which are closer than 1 in 68 variations among individuals from two other lab strains sequenced and reported here. Based on the developmental transcriptome and orthology of all the 54 olfactory receptors (ORs) to those of other Anopheles species, we identify an OR with the potential for host recognition in the genus Anopheles. A comparative analysis of An. stephensi genomes with the completed genomes of a few other Anopheles species suggests limited inter-chromosomal gene flow and loss of synteny within chromosomal arms even among the closely related species.