Genomic characterization of Lumpy Skin Disease virus (LSDV) from India: Circulation of Kenyan-like LSDV strains with unique kelch-like proteins.

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naïve countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from an LSD affected calf in 2019 determined by Illumina next-generation sequencing (NGS). The LSDV-WB/IND/19 has a genome size of 150,969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10-12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimen. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.

SARS-CoV-2 Spike Glycoprotein and ACE2 Interaction Reveals Modulation of Viral Entry in Wild and Domestic Animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral pathogen causing life-threatening diseases in humans. Interaction between the spike protein of SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2) is a potential factor in the infectivity of a host. In this study, the interaction of SARS-CoV-2 spike protein with its receptor, ACE2, in different hosts was evaluated to predict the probability of viral entry. Phylogeny and alignment comparison of the ACE2 sequences did not lead to any meaningful conclusion on viral entry in different hosts. The binding ability between ACE2 and the spike protein was assessed to delineate several spike binding parameters of ACE2. A significant difference between the known infected and uninfected species was observed for six parameters. However, these parameters did not specifically categorize the Orders into infected or uninfected. Finally, a logistic regression model constructed using spike binding parameters of ACE2, revealed that in the mammalian class, most of the species of Carnivores, Artiodactyls, Perissodactyls, Pholidota, and Primates had a high probability of viral entry. However, among the Proboscidea, African elephants had a low probability of viral entry. Among rodents, hamsters were highly probable for viral entry with rats and mice having a medium to low probability. Rabbits have a high probability of viral entry. In Birds, ducks have a very low probability, while chickens seemed to have medium probability and turkey showed the highest probability of viral entry. The findings prompt us to closely follow certain species of animals for determining pathogenic insult by SARS-CoV-2 and for determining their ability to act as a carrier and/or disseminator.

Effect of cryopreservation on therapeutic potential of canine bone marrow derived mesenchymal stem cells augmented mesh scaffold for wound healing in guinea pig.

The objective of this study was to compare the therapeutic potential of canine bone marrow derived mesenchymal stem cells (BM MSCs) augmented mesh scaffold for wound healing potential in guinea pig before and after cryopreservation. Bone marrow aspirate was obtained from healthy dogs and culture was expanded in vitro. MSCs augmented mesh scaffold were cryopreserved for 30 days and then used for therapeutic purposes. Both fresh and frozen thaw MSCs augmented mesh scaffold along with fresh MSCs were used for therapeutic purposes in guinea pig. No significant (P > 0.05) difference was observed in population doubling time (PDT) among fresh and frozen thawed BM MSCs. Both fresh and frozen thawed BM MSCs expressed cell surface markers (CD73, CD90, and CD105), and did not express CD34 as was confirmed by Immunocytochemistry and Real-Time Polymerase Chain Reaction. The fresh and frozen thawed BM MSCs successfully differentiated into osteogenic, chondrogenic and adipogenic lineages. Therapeutic results revealed that the percent wound contraction on day 14 was more than 65 % for the mesh augmented with MSCs as well as freshly injected MSCs group as against 33-34 % in the control group. Healed wound quality parameters viz. surface epithelium, neovascularization, and collagen characteristics were better for the mesh augmented with MSCs as well as freshly injected MSCs group compared to the control group. No significant difference was noted among fresh and frozen thawed BM MSCs group and fresh MSCs injected group. Thus, it is concluded from this study that canine BM MSCs augmented mesh scaffold both fresh and frozen thaw can be used for quality wound healing.

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells.

This study was conducted to characterize canine bone marrow-derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell-treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell-based therapies, especially for diseases like SCI, where the conventional medication is not so promising.

Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes.

Background & objectives: The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes. Methods: All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ≥6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential. Results: Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-τ, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group. Interpretation & conclusions: The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 µM roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs.

GREM1, EGFR, and HAS2; the oocyte competence markers for improved buffalo embryo production in vitro.

Selection of competent oocytes is crucial for successful in vitro embryo production and correlating expression profile of oocyte competence markers in cumulus cells with oocyte quality is an important preamble. In the present study, expression profile of oocyte competence markers (GREM1, EGFR, HAS2, and TNFAIP6) was correlated through brilliant cresyl blue (BCB) staining and subsequently with in vitro embryo production. Excellent to good quality buffalo, cumulus oocyte complexes (COCs) were stained with BCB (26 µM) and based on the blue coloration of cytoplasm COCs were divided in two groups as BCB(+ve) and BCB(-ve). Mean percentage of BCB(+ve) oocytes was significantly higher (P < 0.05) than BCB(-ve) oocytes (56.79 ± 1.22 vs. 43.20 ± 1.22), the mean oocyte diameter was also significantly larger (P < 0.05) for the BCB(+ve) group than that of BCB(-ve) group (145.7 ± 1.8 µm vs. 132.7 ± 1.9 µm). The cleavage rate (%), blastocyst rate (%), and total cell number was significantly (P < 0.05) higher in BCB(+ve) than BCB(-ve) group (71.15 ± 2.17 vs. 52.89 ± 2.65; 31.58 ± 1.11 vs. 7.73 ± 0.97, and 93.14 ± 2.42 vs. 71.42 ± 2.09, respectively). Relative mRNA expression of marker genes in cumulus cells increased significantly (P < 0.05) after maturation viz. GREM1 (10.13 folds), EGFR (9.04 folds), HAS2 (27.91 folds), and TNFAIP6 (64.81 folds). Brilliant cresyl blue positive oocytes showed significantly higher (P < 0.05) expression of GREM1, EGFR, and HAS2 transcripts than BCB(-ve) oocytes, however, no significant change in the expression of TNFAIP6 gene was observed. It is concluded from the study that GREM1, EGFR, and HAS2 could be used as molecular markers of oocyte competence for an improved buffalo embryo production in vitro.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

In vivo differentiation potential of buffalo (Bubalus bubalis) embryonic stem cell.

Embryonic stem cells (ESCs) derived from inner cell mass (ICM) of mammalian blastocyst are having indefinite proliferation and differentiation capability for any type of cell lineages. In the present study, ICMs of in vitro-derived buffalo blastocysts were cultured into two different culture systems using buffalo fetal fibroblast as somatic cell support and Matrigel as synthetic support to obtain pluripotent buffalo embryonic stem cell (buESC) colonies. Pluripotency of the ESCs were characterised through pluripotency markers whereas, their differentiation capability was assessed by teratoma assay using immuno-compromised mice. Cumulus ooccyte complexes from slaughter house-derived ovaries were subjected to in vitro maturation, in vitro fertilization and in vitro culture to generate blastocysts. Total 262 blastocysts were derived through IVEP with 11.83 % (31/262) hatching rate. To generate buESCs, 15 ICMs from hatched blastocysts were cultured on mitomycin-C-treated homologous fetal fibroblast feeder layer, whereas the leftover 16 ICMs were cultured on extra-cellular matrix (Matrigel). No significant differences were observed for primary ESCs colony formation between two culture systems. Primary colonies as well as passaged ESCs were characterised by alkaline phosphatase staining, karyotyping and expression of transcription-based stem cell markers, OCT-4 and cell surface antigens SSEA-4 and TRA-1-60. Batch of ESCs found positive for pluripotency markers and showing normal karyotype after fifteenth passage were inoculated into eight immuno-compromised mice through subcutaneous and intramuscular route. Subcutaneous route of inoculation was found to be better than intramuscular route. Developed teratomas were excised surgically and subjected to histological analysis. Histological findings revealed presence of all the three germinal layer derivatives in teratoma sections. Presence of germinal layer derivatives were further confirmed by reverse transcriptase-polymerase chain reaction for the presence of differentiation markers like nerve cell adhesion molecule, fetal liver kinase-1 and alpha-feto protein for ectoderm, mesoderm and endoderm, respectively.

Localization and expression of follicle-stimulating hormone receptor gene in buffalo (Bubalus bubalis) pre-antral follicles.

Follicle-stimulating hormone (FSH) stimulates antral follicles to grow, but its role in earlier stages (pre-antral) of follicle development, if any, is obscure. Aim of this study was to study the expression of follicle-stimulating hormone receptor (FSHR) gene in different sizes of pre-antral follicles (PFs) (<150, 200, 250, 300, 350, 400 µm) and to find out an optimum dose of FSH for better growth, development and steroidogenesis of PFs in vitro. Buffalo ovaries were collected from a local abattoir, and PFs were isolated by mechanical method. A semi-quantitative RT-PCR amplification strategy was used for mRNA expression, while FSHR protein was localized by immunohistochemistry. Isolated pre-antral follicles (80-85 µm) were cultured in TCM-199 supplemented with 10% foetal bovine serum, 1% ITS and 30 ng/ml EGF served as control medium. Addition of three different doses of FSH (0.5, 1.0, 2.0 µg/ml) in control medium was considered as treatment groups. A single 2.184-kb receptor mRNA transcript was present in all sizes (<150-400 µm) of follicles. Follicle-stimulating hormone receptor was also localized immunohistochemically in granulosa cells of all sizes of follicles. Survival and growth rate of follicles significantly (p<0.05) increased following supplementation of FSH at a concentration of 1.0 µg/ml and the culture medium also showed a significantly (p<0.05) greater accumulation of oestradiol and progesterone. In conclusion, FSHR is expressed in all sizes of PFs and in vitro survival, growth and steroidogenesis of follicles are optimally stimulated by 1.0 µg/ml FSH. These findings demonstrate that FSH has an important role during the recruitment, growth and development of buffalo ovarian PFs.

Effects of IGF-1, TGF-alpha plus TGF-beta1 and bFGF on in vitro survival, growth and apoptosis in FSH-stimulated buffalo (Bubalis bubalus) preantral follicles.

OBJECTIVE: Investigate the effect of various growth factors viz. IGF-I, TGF-alpha + TGF-beta1 and bFGF either alone or in combination, with FSH on in vitro growth, survival, antrum formation, steroidogenesis and apoptosis of buffalo preantral follicles (PFs). METHODS: Buffalo ovaries were collected from abattoir; PFs were isolated and divided into five treatment groups. TCM-199 supplemented with 10% FBS, 1% ITS+EGF+FSH control (group a), control+IGF-I (group b), control + TGF-alpha + TGF-beta1 (group c), control + IGF-I + TGF-alpha + TGF-beta1 (group d) and control+bFGF (group e). Progesterone (P4) and 17beta-estradiol (E2) concentrations were evaluated by RIA and apoptosis by TUNEL assay. RESULTS: TGF-alpha + TGF-beta1 inhibited follicular survival and induced oocyte apoptosis, while IGF-I + TGF-alpha + TGF-beta1 suppressed this inhibitory action. IGF-I significantly (P < 0.05) enhanced the follicle survival, growth and induced antrum formation. FGF had greater effects on both survival and growth rate of oocytes than other treatment groups. Progesterone and estradiol accumulation was significantly (P < 0.05) greater in presence of FGF and IGF-I than TGF-alpha + TGF-beta1. CONCLUSION: Survival, growth, antrum formation and steroidogenesis are stimulated by IGF-I and bFGF, whereas TGF-alpha + TGF-beta1 inhibited growth and survival of PFs which led to induced oocyte apoptosis in buffalo PFs.

Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro.

The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.

Neurocysticercosis in free roaming pigs--a slaughterhouse survey.

To investigate the prevalence of neurocysticercosis among free ranging pigs and to study the type of pathomorphological lesions in affected brains, a total of 200 brains were collected from pigs slaughtered at a local abattoir, between August, 2005 to March, 2006. Gross and histopathological examination revealed 3% (6/200) occurrence of neurocysticercosis in pigs. Taenia solium cysticercosis is an under-rated zoonosis and is a leading cause of epilepsy due to neurocysticercosis in human population of India. The prevailing situation warrants immediate implementation of effective control measures for this dreaded disease.