Biology subjects

Mohan, C. V.

Publications and source records attributed to Mohan, C. V..

5 recordsLinked to original sources

bioRxiv2023

A multiplexed RT-PCR Assay for Nanopore Whole Genome Sequencing of Tilapia lake virus (TiLV)

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

genomics↗

bioRxiv2021

Relationships between pond water and tilapia skin microbiomes in aquaculture ponds in Malawi

Intensification of fish farming practices is being driven by the demand for increased food production to support a rapidly growing global human population, particularly in lower-middle income countries. Intensification of production, however, increases the risk of disease outbreaks and thus the likelihood for crop losses. The microbial communities that colonise the skin mucosal surface of fish are poorly understood, but are important in maintaining fish health and resistance against disease. This skin microbial community is susceptible to disruption through stressors associated with transport, handling and the environment of intensive practices, and this risks the propagation of disease-causing pathogens. In this study, we characterised the microbial assemblages found on tilapia skin -- the most widely farmed finfish globally -- and in the surrounding water of seven earthen aquaculture ponds from two pond systems in distinct geographic regions in Malawi. Metabarcoding approaches were used to sequence the prokaryotic and microeukaryotic communities. We found 92% of prokaryotic amplicon sequence variants were common to both skin and water samples. Differentially enriched and core taxa, however, differed between the skin and water samples. In tilapia skin, Cetobacterium, Paucibacter, Pseudomonas and Comamonadaceae were enriched, whereas, the cyanobacteria Cyanobium, Microcystis and/or Synechocystis, and the diatom Cyclotella, were most prevalent in pond water. Ponds that clustered together according to their water prokaryotic communities also had similar microeukaryotic communities indicating strong environmental influences on prokaryotic and microeukaryotic community structures. While strong site-specific clustering was observed in pond water, the grouping of tilapia skin prokaryotes by pond site was less distinct, suggesting fish microbiota have a greater buffering capacity against environmental influences. The characterised diversity, structure and variance of microbial communities associated with tilapia culture in Malawi provide the baseline for studies on how future intensification practices may lead to microbial dysbiosis and disease onset. HighlightsO_LIFish skin and pond water communities differ structurally, but share common taxa C_LIO_LIPond locations have a stronger influence on water versus fish skin microbiome community structure C_LIO_LISelected skin-associated taxa could be used to monitor dysbiotic events in aquaculture C_LIO_LITaxa with opportunistic pathogen potential were identified at low abundance C_LI

microbiology↗

bioRxiv2021

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per {micro}L of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 {+/-} 3.3% of virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 x 102 to 2.79 x 104 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 x 102 to 3.53 x 103 copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.

molecular biology↗

bioRxiv2020

A novel RNA virus, Macrobrachium rosenbergii Golda virus (MrGV), linked to mass mortalities of the larval giant freshwater prawn in Bangladesh

1Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single-stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of MrGV suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

molecular biology↗

bioRxiv2019

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

The gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00-99.94 and 99.00-100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) monophyletic Israel-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013-2019 yielded positive test results for all samples tested, confirming that our newly designed primers and detection protocol against TiLV segment 1, have a potential application for detection of all current genetic variants of TiLV.

genetics↗