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Additional file 2: Additional Methods.

Reinfection of SARS-CoV-2 is an apparently rare entity and only a few cases have been reported from across the world with the genetic characterization of the virus, differentiating reinfection from persistent virus shedding. These cases, therefore, provide unique insights into the long term protective immunity to SARS-CoV-2. The earlier reports suggest that patients were symptomatic in either one or both the episodes of infection. Here we report a unique case of asymptomatic SARS-CoV-2 reinfection in two healthcare workers from India identified in routine surveillance. Genome sequencing of the virus suggests that genetically distinct SARS-CoV-2 caused the infections. Our analysis demonstrates that asymptomatic reinfection could potentially be an under-reported entity with implications in long term surveillance of SARS-CoV-2 infections. This report also highlights the need for genomic surveillance of healthcare workers who are potentially not only at higher risk for primary infections but also for reinfections.

Induction of triploidy and tetraploidy was attempted in Heteropneustes fossilis using heat shock. The optimal age of zygote, temperature level and duration of thermal shock required for effective induction of triploidy and tetraploidy was investigated in a series of experiments. A maximum of 82±7% triploids (3n=87) were obtained when fertilized eggs (2.5-min old) were heat shocked at 40°C for 4-min duration. A maximum of 40±8% tetraploids (4n=116) was obtained when the fertilized eggs (30-min old) were heat shocked at 40°C for 4-min duration. The triploid and tetraploid red blood cells (RBCs) nucleus volumes were 1.4 and 2.1 times greater, respectively, than that of the diploid RBC nucleus.

The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we havefound that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in theevolution. Homology modeling and docking with the similar structure and Ca 2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.