Oral feed-based administration of phage cocktail protects rohu fish (Labeo rohita) against Aeromonas hydrophila infection.

Aeromonas hydrophila is one of the major freshwater fish pathogens. In the current study, a cocktail of D6 and CF7 phages was given orally to Labeo rohita to assess phage survival in fish organs as well as to determine the therapeutic efficacy of phage treatment against fish mortality caused by A. hydrophila. In the phage-coated feed, prepared by simple spraying method, phage counts were quite stable for up to 2 months with a decline of ≤ 0.23 log10 and ≤ 1.66 log10 PFU/g feed during 4 oC and room temperature storage. Throughout the experimental period of 7 days, both phages could be detected in the gut of fish fed with phage-coated feed. Besides, both CF7 and D6 phages were also detected in fish kidneys indicating the ability of both the phage to cross the intestinal barrier. During challenge studies with LD50 dose of A. hydrophila, phage cocktail doses of 1 × 106 - 1 × 108 PFU/g feed prevented the mortality in L. rohita with relative percentage survival (RPS) of 8.7-65.2. When challenged with LD90 dose of A. hydrophila, an RPS value of 28.6 was obtained at a phage cocktail dose of 1 × 108 PFU/g feed. The RPS data showed that orally-fed phage cocktail protected the fish against the mortality caused by A. hydrophila in a dose-dependent manner. Simple practical approaches for phage cocktail development, medicated feed preparation and oral administration along with phage survival and protection data make the current study useful for farmer-level application.

Gelation characteristics of partially purified myofibrillar proteins extracted from commercially harvested Indian mackerel and threadfin bream.

Gelling behaviors of partially purified myofibrillar proteins (PPMP) extracted from Indian mackerel (IM) and threadfin bream (TB) as a function of heating temperatures (20-75°C) were comparatively studied. PPMP obtained from IM (IM-MP) showed lower turbidity and surface hydrophobicity as compared to those extracted from TB (TB-MP). Moreover, lower disulfide bond content was noticed in IM-MP (7.7-9.46 mol/106  g protein) as compared to TB-MP (10.99-13.95 mol/106  g protein) during the heating process. There was no major difference in the amino acid profile noticed between PPMP from both the species, except lysine and glutamine contents, which were higher in TB-MP. Structural analysis, FTIR spectra, amide I band, and fluorescence intensity substantiated those changes. The protein pattern also revealed autolysis of IM-MP. The transmission analysis also showed lower aggregation and crosslinking ability of IM-MP than TB-MP. Therefore, poorer gelling behavior of IM-MP reconfirmed the inferior gel property of surimi gel from IM to gel from TB. Potential development is still required for the improvement of the gel properties of dark-fleshed fish surimi such as IM. PRACTICAL APPLICATION: Indian mackerel (IM) is an abundant and widely captured fish species. Due to overexploitation of lean fish, pelagic fish could be explored as a potential raw material for surimi production. However, poor gelling properties of IM limit its use in the surimi industry. This study provides an insight into the gelling behavior of myofibrillar proteins from IM during the gelation process in comparison with the lean fish (threadfin bream). Overall, structural and rheological changes of myofibrillar proteins play a role in gelation, thus affecting gel properties between two species. Further improvement of the gel of IM is still required.

Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms / Prevalencia y resistencia a los antimicrobianos de especies de Vibrio relacionadas con la inocuidad de los alimentos en granjas de cultivo de camarones de aguas saladas continentales

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.(AU)

Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms.

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.

Probiotic Supplementation as an Emerging Alternative to Chemical Therapeutics in Finfish Aquaculture: a Review.

Aquaculture is a promising food sector to fulfil nutritional requirements of growing human population. Live weight aquaculture production reached up to 114.5 million tonnes in 2018 and it is further expected to grow by 32% by year 2030. Among total aquaculture production, major product harvested is finfish and its contribution has reached 46% in recent years. Frequent outbreaks of infectious diseases create obstacle in finfish production, result in economic losses to the farmers and threaten the sustainability of aquaculture industry itself. In spite of following the best management practices, the use of antibiotics, chemotherapeutics and phytochemicals often become the method of choice in finfish culture. Among these, phytochemicals have shown lesser effect in animal welfare while antibiotics and other chemotherapeutics have led to negative consequences like emergence of drug-resistant bacteria, and accumulation of residues in host and culture system, resulting in quality degradation of aqua products. Making use of probiotics as viable alternative has paved a way for sustainable aquaculture and minimise the use of antibiotics and other chemotherapeutics that pose adverse effect on host and culture system. This review paper elucidates the knowledge about antibiotics and other chemicals, compilation of probiotics and their effects on health status of finfish as well as overall culture environment. Besides, concoction of probiotics and prebiotics for simultaneous application has also been discussed briefly.

Vibrio parahaemolyticus Isolates from Asian Green Mussel: Molecular Characteristics, Virulence and Their Inhibition by Chitooligosaccharide-Tea Polyphenol Conjugates.

Fifty isolates of Vibrio parahaemolyticus were tested for pathogenicity, biofilm formation, motility, and antibiotic resistance. Antimicrobial activity of chitooligosaccharide (COS)-tea polyphenol conjugates against all isolates was also studied. Forty-three isolates were randomly selected from 520 isolates from Asian green mussel (Perna viridis) grown on CHROMagarTM Vibrio agar plate. Six isolates were acquired from stool specimens of diarrhea patients. One laboratory strain was V. parahaemolyticus PSU.SCB.16S.14. Among all isolates tested, 12% of V. parahaemolyticus carried the tdh+trh- gene and were positive toward Kanagawa phenomenon test. All of V. parahaemolyticus isolates could produce biofilm and showed relatively strong motile ability. When COS-catechin conjugate (COS-CAT) and COS-epigallocatechin-3-gallate conjugate (COS-EGCG) were examined for their inhibitory effect against V. parahaemolyticus, the former showed the higher bactericidal activity with the MBC value of 1.024 mg/mL against both pathogenic and non-pathogenic strains. Most of the representative Asian green mussel V. parahaemolyticus isolates exhibited high sensitivity to all antibiotics, whereas one isolate showed the intermediate resistance to cefuroxime. However, the representative clinical isolates were highly resistant to nine types of antibiotics and had multiple antibiotic resistance (MAR) index of 0.64. Thus, COS-CAT could be used as potential antimicrobial agent for controlling V. parahaemolyticus-causing disease in Asian green mussel.

Determining the efficacy of ginger Zingiber officinale as a potential nutraceutical agent for boosting growth performance and health status of Labeo rohita reared in a semi-intensive culture system.

A 120-day feeding trial was conducted in a pilot field setting to study the nutraceutical properties of ginger powder (GP), focusing on the growth performance and health status of Indian major carp L. rohita reared under a semi-intensive culture system. L. rohita fingerlings (average weight: 20.5 g) were divided into five groups and fed a diet with no GP supplementation (control), or a diet supplemented with GP at 5 g (GP5), 10 g (GP10), 15 g (GP15), and 20 g (GP20) per kg of feed. The study was carried out in outdoor tanks (20 m2) following a complete randomized design with three replicates for each experimental group. Dietary supplementation of GP at 15 g·kg-1 (GP15) of feed caused a significant increase in the growth performances of the fish. Results also showed that feeding of GP15 diet led to a significant improvement in the health status of fish as indicated by a marked change in the tested haematological indices (i.e., higher RBC, WBC, Hb, and Ht values), oxidative status (increased SOD and decreased LPO levels), biochemical parameters (increased HDL, decreased cholesterol, and triglycerides levels), and activities of the liver enzymes (decreased AST and ALT). Overall results suggested that dietary supplementation of GP could positively influence the growth and health status of L. rohita fingerlings, and hence could be an important natural nutraceutical for sustainable farming of carp.

Genome dynamics, codon usage patterns and influencing factors in Aeromonas hydrophila phages.

In the present study, genome characteristics and codon usage patterns of 44 Aeromonas hydrophila phages were studied. Phage genomes varied from 30.8 to 262.0 kb with mean±SD and median values of 111.3 ± 81.4 kb and 79.4 kb, respectively. Though the great variation in phage GC contents (35.1-62.2%) was observed, GC contents of all phages (except two phages) were significantly less than the GC content (62.4 ± 5.6%) of the host. The effective number of codons (ENC) values of phage genes ranged from 27.7 to 61 with a mean±SD value of 47.4 ± 6.8. Out of a total 5773 phage genes, 207 (3.6%), 3,528 (61.1%) and 2,012 (34.9%) genes had strong (ENC < 35), moderate (35 < ENC < 50) and low (ENC ≥ 50) codon usage bias, respectively. During relative synonymous codon usage (RSCU) analysis, shared usage of preferred codons was also observed between the phages and host. During codon adaptation index (CAI) analysis, 1028 (17.8%) phage genes showed significant adaptation towards the host. Among these genes, 797 (78.0%) genes encoded hypothetical proteins or proteins of unknown function; whereas 118 (12%) genes encoded the phage structural and packaging proteins. Segregation of ENC, RSCU and CAI analysis results based on genome size also indicated that codon usage bias was more prominent in phages with small genomes. Correlation, neutrality and GC3 versus ENC analyzes indicated a more dominant role of natural selection in shaping the codon usage patterns of A. hydrophila phages. The findings of the current study could be useful from evolutionary and host-pathogen interaction perspectives leading to efficient utilization of phages for therapeutic and other applications.

Complete genome sequencing and characterization of single-stranded DNA Vibrio parahaemolyticus phage from inland saline aquaculture environment.

Despite their evolutionary, molecular biology and biotechnological significance, relatively fewer numbers of single-stranded DNA (ssDNA) filamentous phages belonging to the family Inoviridae have been discovered and characterized to date. The present study focused on genome sequencing and characterization of an ssDNA Vibrio parahaemolyticus phage V5 previously isolated from an inland saline shrimp culture farm. The complete circular genome of phage V5 consisted of 6658 bp with GC content of 43.7%. During BLASTn analysis, only 36% of phage V5 genome matched with other Vibrio phage genomes in the NCBI database with a sequence identity value of 79%. During the phylogenetic analysis, phage V5 formed a separate branch in the minor clade. These features indicate the novel nature of the phage V5 genome. Among 10 predicted open reading frames (ORFs) in the phage V5 genome, 6 encoded for the proteins of known biological functions, whereas the rest were classified as hypotheticals. Proteins involved in replication and structural assembly were encoded by the phage genome. However, the absence of genes encoding for DNA/RNA polymerases and tRNAs signified that phage V5 is dependent on the host`s molecular machinery for its propagation. As per our knowledge, this is the first study describing the novel genome sequence of an ssDNA V. parahaemolyticus phage from the inland saline environment.

Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach.

Biofilm vaccine has been recognised as one of the successful strategy to reduce the Aeromonas hydrophila infection in fish. But, the vaccine contains the protective and non-protective proteins, which may lead to show altered heterologous adaptive immunity response. Moreover, cross protection and effectiveness of previously developed biofilm vaccine was not tested against different geographical A. hydrophila isolates. Therefore, in the present study, whole-cell A. hydrophila biofilm vaccine was evaluated in rohu, vaccinated group showed increased antibody titer and protection against the different geographical A. hydrophila isolates namely KAH1 and AAH2 with 78.9% and 84.2% relative percentage survival, respectively. In addition, by using the immune sera of biofilm vaccinated group, a total of six protective proteins were detected using western blot assay. Further, the same proteins were identified by nano LC-MS/MS method, a total of fourteen candidate proteins showing the immunogenic property including highly expressed OMP's tolC, bamA, lamb, AH4AK4_2542, AHGSH82_029580 were identified as potential vaccine candidates. The STRING analysis revealed that, top candidate proteins identified may potentially interact with other intracellular proteins; involved in ribosomal and (tricarboxylic acid) TCA pathway. Importantly, all the selected vaccine candidate proteins contain the B-cell epitope region. Finally, the present study concludes that, whole-cell A. hydrophila biofilm vaccine able to protect the fish against the different geographical A. hydrophila isolates. Further, through reverse vaccinology approach, a total of fourteen proteins were identified as potential vaccine candidates against A. hydrophila pathogen.

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp.

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3-45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00934-6.

Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Aeromonas hydrophila is an important finfish pathogen, besides being an opportunistic human pathogen. In the present study, the genomes of three A. hydrophila-specific phages, CF8, PS1, and PS2, were isolated, characterized and sequenced. Transmission electron microscopy showed that all three phages had typical Myoviridae morphology. The linear dsDNA genomes of CF8, PS1, and PS2 were 238,150 bp, 237,367 bp, and 240,447 bp in length, with a GC content of 42.2%, 38.8%, and 38.8%, respectively. The low sequence similarity (67.6% - 69.8% identity with 27.0% - 29.0% query coverage) to other phage genomes in the NCBI database indicated the novel nature of the CF8, PS1, and PS2 genomes. A total of 244, 247, and 250 open reading frames (ORFs) were predicted in the CF8, PS1, and PS2 genome, respectively. During the annotation process, functional predictions were made for 28-31 ORFs, while the rest were classified as "hypothetical proteins" with yet unknown functions. Genes for tRNAs were also detected in all phage genomes. As all three phages in the present study had a very narrow host range with lytic activity against only one strain of A. hydrophila, these phages could be good candidates for phage typing applications. Moreover, the endolysin- and lytic-transglycosylase-encoding genes could be used for recombinant cloning and expression of anti-microbial proteins.

Correction to: Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Authors would like to correct the incorrect version.

Shotgun metagenomics offers novel insights into taxonomic compositions, metabolic pathways and antibiotic resistance genes in fish gut microbiome.

Gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. With the presence of 36 phyla, 326 families and 985 genera, the fish gut microbiota was found to be quite diverse in nature. However, at the phylum level, more than three-fourths of gut microbes belonged to Proteobacteria. Very low prevalence of commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) in fish gut suggested the need to search for alternative probiotics for aquaculture use. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy metabolism (4%) and fermentation (2%). In conformity with herbivorous feeding habit of L. rohita, gut microbiome also had pathways for the degradation of cellulose, hemicellulose, chitin, pectin, starch, and other complex carbohydrates. High prevalence of Actinobacteria and antibiotic biosynthesis pathways in the fish gut microbiome indicated its potential for bioprospecting of potentially novel natural antibiotics. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment. The role of ARGs in fish gut microbiome needs further investigations.

Computational modeling of early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) to identify personalized therapy using genomics.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematological malignancy for which optimal therapeutic approaches are poorly characterized. Using computational biology modeling (CBM) in conjunction with genomic data from cell lines and individual patients, we generated disease-specific protein network maps that were used to identify unique characteristics associated with the mutational profiles of ETP-ALL compared to non-ETP-ALL (T-ALL) cases and simulated cellular responses to a digital library of FDA-approved and investigational agents. Genomics-based classification of ETP-ALL patients using CBM had a prediction sensitivity and specificity of 93% and 87%, respectively. This analysis identified key genomic and pathway characteristics that are distinct in ETP-ALL including deletion of nucleophosmin-1 (NPM1), mutations of which are used to direct therapeutic decisions in acute myeloid leukemia. Computational simulations based on mutational profiles of 62 ETP-ALL patient models identified 87 unique targeted combination therapies in 56 of the 62 patients despite actionable mutations being present in only 37% of ETP-ALL patients. Shortlisted two-drug combinations were predicted to be synergistic in 11 profiles and were validated by in vitro chemosensitivity assays. In conclusion, computational modeling was able to identify unique biomarkers and pathways for ETP-ALL, and identify new drug combinations for potential clinical testing.

Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene.

DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression. These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing, validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments. Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant.

Genome dynamics and evolution of codon usage patterns in shrimp viruses.

We analysed the genomes and codon usage patterns of seven small (DNA and RNA) shrimp viruses. Effective number of codon (ENC) values indicated moderate (35 < ENC < 50) codon usage bias in shrimp viruses. Correlation analysis between GC compositions at non-synonymous codon and synonymous codon positions (GC1, 2 and GC3) as well as GC3 versus ENC curves indicated varying influences of mutational pressure on codon usage. The presence of deoptimized codons and host-antagonistic codon usage trends in shrimp viruses suggested the adaptation of a slow replication strategy by these viruses to avoid host defences. Low CpG frequencies indicated that shrimp viruses have evolved with underrepresentation of CpGs to avoid the host's immune response.

A detailed analysis of codon usage patterns and influencing factors in Zika virus.

Recent outbreaks of Zika virus (ZIKV) in Africa, Latin America, Europe, and Southeast Asia have resulted in serious health concerns. To understand more about evolution and transmission of ZIKV, detailed codon usage analysis was performed for all available strains. A high effective number of codons (ENC) value indicated the presence of low codon usage bias in ZIKV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations between nucleotide compositions at third codon positions and ENCs. Correlation analysis between Gravy values, Aroma values and nucleotide compositions at third codon positions also indicated some influence of natural selection. However, the low codon adaptation index (CAI) value of ZIKV with reference to human and mosquito indicated poor adaptation of ZIKV codon usage towards its hosts, signifying that natural selection has a weaker influence than mutational pressure. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.

Characterization of codon usage pattern and influencing factors in Japanese encephalitis virus.

Recently, several outbreaks of Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV), have been reported and it has become cause of concern across the world. In this study, detailed analysis of JEV codon usage pattern was performed. The relative synonymous codon usage (RSCU) values along with mean effective number of codons (ENC) value of 55.30 indicated the presence of low codon usages bias in JEV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations of A3s, U3s, G3s, C3s, GC3s, ENC values, with overall nucleotide contents (A%, U%, G%, C%, and GC%). The correlation analysis of A3s, U3s, G3s, C3s, GC3s, with axis values of correspondence analysis (CoA) further confirmed the role of mutational pressure. However, the correlation analysis of Gravy values and Aroma values with A3s, U3s, G3s, C3s, and GC3s, indicated the presence of natural selection on codon usage bias in addition to mutational pressure. The natural selection was further confirmed by codon adaptation index (CAI) analysis. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.