Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Transmission of African swine fever virus to the wild boars of Northeast India.

BACKGROUND: India recorded the first outbreak of African swine fever (ASF) in North-eastern region (NER) in the year 2020. AIM: The current study was undertaken to investigate the transmission of African swine fever virus (ASFV) in the wild boars of Northeast India, particularly of Assam. MATERIAL AND METHODS: ASF suspected mortal tissue remains and blood samples of wild boars collected from different locations of Assam were screened for molecular detection of swine viruses which includes Classical swine fever virus, Porcine Circovirus 2, Porcine reproductive and respiratory syndrome virus and ASFV. RESULTS: One sample each from Manas and Nameri National Parks was detected positive for ASFV. Besides this, one of the samples was positive for CSFV and one of the ASFV positive samples was also positive for PCV2. Several striking gross and microscopic alterations were noticed in different organs of ASFV infected animals. Sequencing and phylogenetic analysis of B646L gene confirmed the presence of ASFV genotype-II in wild boars. Circulation of similar genotype in domestic pigs of NER in the contemporary period as well as locations near to the aforementioned national parks indicates the transmission of ASFV from domestic to wild boars. CLINICAL RELEVANCE: The detection of ASFV in the wild boars of Assam is alarming as it is an impending threat to pig population and other endangered species (particularly Pygmy hog), making it increasingly daunting to control the disease. CONCLUSION: Chances are high for ASFV to become endemic in Assam region if stringent measures are not taken at proper time.

Computational analysis of immunogenic epitopes in the p30 and p54 proteins of African swine fever virus.

African swine fever (ASF) is a highly infectious viral disease of pigs, which causes acute fatal haemorrhage and is a severe concern to the global pork industry. The present study followed computational approaches to identify B- and T-cell epitopes for the p30 and p54 proteins of the African swine fever virus (ASFV) by interacting with the swine leukocyte antigen (SLA) alleles. The amino acid sequences of p30 and p54 were analysed for variability and relative solvent accessibility, and their three-dimensional structures were predicted and validated. Molecular dynamics simulation was performed to study the structural and dynamic properties of the protein. Six and five linear B-cell epitopes have been predicted for p30 and p54, respectively. Four and three discontinuous B-cell epitopes have been predicted for p30 and p54, respectively. Further, the top five T-cell epitopes for SLA-1, 2, and 3 have been listed for both proteins. These results can help us to understand the immunodominant regions in the p30 and p54 proteins of ASFV and potentially assist in designing peptide-based diagnostics and vaccines. Also, the identified T-cell epitopes may be considered for peptide-based vaccine design against ASFV.Communicated by Ramaswamy H. Sarma.

Molecular characterization of porcine circovirus 2 circulating in Assam and Arunachal Pradesh of India.

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of cap gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.

Draft Genome Sequence Analysis of the Genotype II African Swine Fever Virus from India.

African swine fever virus (ASFV) entered the northeastern (NE) part of India early in 2020, causing huge economic loss to the piggery sector. Here, we are presenting a brief report on the draft genome sequence of an ASFV strain ABTCVSCK_ASF007 from Assam state of NE India belonging to genotype II.

Whole-Genome Sequence of a Porcine Circovirus Type 2 Strain Detected in Assam, India.

Porcine circovirus-associated disease caused by porcine circovirus type 2 (PCV2) is a vital threat to the global pig industry. In this study, we have characterized the complete genome sequence of a PCV2 isolate, namely, Assam-01, belonging to the genotype PCV2d.

Organoid Technology: A Reliable Developmental Biology Tool for Organ-Specific Nanotoxicity Evaluation.

Engineered nanomaterials are bestowed with certain inherent physicochemical properties unlike their parent materials, rendering them suitable for the multifaceted needs of state-of-the-art biomedical, and pharmaceutical applications. The log-phase development of nano-science along with improved "bench to beside" conversion carries an enhanced probability of human exposure with numerous nanoparticles. Thus, toxicity assessment of these novel nanoscale materials holds a key to ensuring the safety aspects or else the global biome will certainly face a debacle. The toxicity may span from health hazards due to direct exposure to indirect means through food chain contamination or environmental pollution, even causing genotoxicity. Multiple ways of nanotoxicity evaluation include several in vitro and in vivo methods, with in vitro methods occupying the bulk of the "experimental space." The underlying reason may be multiple, but ethical constraints in in vivo animal experiments are a significant one. Two-dimensional (2D) monoculture is undoubtedly the most exploited in vitro method providing advantages in terms of cost-effectiveness, high throughput, and reproducibility. However, it often fails to mimic a tissue or organ which possesses a defined three-dimensional structure (3D) along with intercellular communication machinery. Instead, microtissues such as spheroids or organoids having a precise 3D architecture and proximate in vivo tissue-like behavior can provide a more realistic evaluation than 2D monocultures. Recent developments in microfluidics and bioreactor-based organoid synthesis have eased the difficulties to prosper nano-toxicological analysis in organoid models surpassing the obstacle of ethical issues. The present review will enlighten applications of organoids in nanotoxicological evaluation, their advantages, and prospects toward securing commonplace nano-interventions.

Analysis of codon usage of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) and its adaptability in dog.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is recognized as one of the life-threatening viruses causing the most destructive pandemic in this century. The genesis of this virus is still unknown. To elucidate its molecular evolution and regulation of gene expression, the knowledge of codon usage is a pre-requisite. In this study, an attempt was made to document the genome-wide codon usage profile and the various factors influencing the codon usage patterns of SARS-CoV-2 in human and dog. The SARS-CoV-2 genome showed relative abundance of A and U nucleotides and relative synonymous codon usage analysis revealed that the preferred synonymous codons mostly end with A/U. The analysis of ENc-GC3s, Neutrality and Parity rule 2 plots indicated that natural selection and other undefined factors dominate the overall codon usage bias in SARS-CoV-2 whereas the impact of mutation pressure is comparatively minor. The codon adaptation index and relative codon deoptimization index of SARS-CoV-2 deciphered that human is more favoured host for adaptation compared to dog. These results enhance our understanding of the factors involved in evolution of the novel human SARS-CoV-2 and its adaptability in dog.

Identification of serum protein markers for early diagnosis of pregnancy in buffalo.

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science.

Hemato-biochemical and hormonal profiles in post-partum water buffaloes (Bubalus bubalis).

AIM: The objective of the present study was to compare serum as well as follicular fluid (FF) biochemical and hormonal profiles along with hematological parameters in postpartum estrus, anestrus, and cystic buffaloes. MATERIALS AND METHODS: Postpartum buffaloes were selected in three different groups (within 40-60 days of parturition at estrus-Group-I, postpartum >90 days at anestrum-Group-II, and postpartum cystic buffaloes in Group III). The animals selected were examined for follicular wave dynamics by routine trans-rectal ultrasonography and FF was collected by transvaginal ultrasound-guided ovum pick up technique. All hematological and biochemical parameters were analyzed by automatic analyzers while hormonal profiles analyzed by commercially available ELISA kits. RESULTS: In the present investigation, estrum and anestrum animal differ significantly in hemoglobin levels. Serum estradiol differs significantly in estrus and anestrus while no significant difference in progesterone concentration was noted among all three stages. The results of our study suggest that significant higher increase in total protein (TP), calcium and glucose values in estrum while urea, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase significantly higher in anestrum animals. CONCLUSION: The conclusion of the present study is that TP and albumin, calcium, urea, glucose affects oocyte development and quality.