Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Balanced spatiotemporal arrangements of histone H3 and H4 posttranslational modifications are necessary for meiotic prophase I chromosome organization.

Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate (heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.

Changes in m6A RNA methylation of goat lung following PPRV infection.

Peste des petits ruminants (PPR) is an acute, highly contagious viral disease of goats and sheep, caused by the Peste des petits ruminants virus (PPRV). Earlier studies suggest the involvement of diverse regulatory mechanisms in PPRV infection. Methylation at N6 of Adenosine called m6A is a type RNA modification that influences various physiological and pathological phenomena. As the lung tissue represents the primary target organ of PPRV, the present study explored the m6A changes and their functional significance in PPRV disease pathogenesis. m6A-seq analysis revealed 1289 m6A peaks to be significantly altered in PPRV infected lung in comparison to normal lung, out of which 975 m6A peaks were hypomethylated and 314 peaks were hypermethylated. Importantly, hypomethylated genes were enriched in Interleukin-4 and Interleukin-13 signaling and various processes associated with extracellular matrix organization. Further, of the 843 differentially m6A-containing cellular transcripts, 282 transcripts were also found to be differentially expressed. Functional analysis revealed that these 282 transcripts are significantly enriched in signaling by Interleukins, extracellular matrix organization, cytokine signaling in the immune system, signaling by receptor tyrosine kinases, and Toll-like Receptor Cascades. We also found m6A reader HNRNPC and the core component of methyltransferase complex METTL14 to be highly upregulated than the m6A readers - HNRNPA2B1 and YTHDF1 at the transcriptome level. These findings suggest that alteration in the m6A landscape following PPRV is implicated in diverse processes including Interleukin signaling.

First report of whole genome sequence of septicemic Pasteurella multocida serovar B:2 'Soron' strain isolated from swine.

Pig pasteurellosis, caused by Pasteurella multocida, is an acute infection that also has economic implications for pig farmers. We report the complete genome sequence of a P. multocida, serovar B:2 'Soron' strain isolated from the blood of a pig that had died of pasteurellosis in India. The isolate was not found to be haemorrhagic septicaemia (HS) specific B:2 by the PCR assay. The genome of 'Soron' strain is a single circular chromosome of 2,272,124 base pairs in length and contains 2014 predicted coding regions, 4 ribosomal RNA operons, and 52 tRNAs. It has 1812 protein-coding genes that were also found in reference sequence PmP52Vac. Phylogenetic analysis revealed that Pm_P52VAc and P. multocida 'Soron' serovar B:2 were clustered in different clades. Pasteurella multocida 'Soron' serovar B:2 was found to cluster with the same ancestor of Pm70, which is of avian origin. The genome was found to contain regions that encode proteins which may confer resistance to various antibiotics including cephalosporin, which is used to treat pasteurellosis. The isolate was also found to harbour a phage region. This strain represents a novel multi-locus sequence type (MLST) that has not been previously identified, as all of the alleles used for MLST were found, but did not match any of the alleles in the database with 100% nucleotide identity. The most closely related ST was ST221. This is the first whole-genome sequence from P. multocida serovar B:2 of pig origin.

Genome-wide expression analysis reveals different heat shock responses in indigenous (Bos indicus) and crossbred (Bos indicus X Bos taurus) cattle.

Environmental heat stress in dairy cattle leads to poor health, reduced milk production and decreased reproductive efficiency. Multiple genes interact and coordinate the response to overcome the impact of heat stress. The present study identified heat shock regulated genes in the peripheral blood mononuclear cells (PBMC). Genome-wide expression patterns for cellular stress response were compared between two genetically distinct groups of cattle viz., Hariana (B. indicus) and Vrindavani (B. indicus X B. taurus). In addition to major heat shock response genes, oxidative stress and immune response genes were also found to be affected by heat stress. Heat shock proteins such as HSPH1, HSPB8, FKB4, DNAJ4 and SERPINH1 were up-regulated at higher fold change in Vrindavani compared to Hariana cattle. The oxidative stress response genes (HMOX1, BNIP3, RHOB and VEGFA) and immune response genes (FSOB, GADD45B and JUN) were up-regulated in Vrindavani whereas the same were down-regulated in Hariana cattle. The enrichment analysis of dysregulated genes revealed the biological functions and signaling pathways that were affected by heat stress. Overall, these results show distinct cellular responses to heat stress in two different genetic groups of cattle. This also highlight the long-term adaptation of B. indicus (Hariana) to tropical climate as compared to the crossbred (Vrindavani) with mixed genetic makeup (B. indicus X B. taurus).

Anti-spike Antibody Status in Pre-vaccinated Healthy Participants and Rheumatoid Arthritis Patients During the Third Wave of COVID-19.

Introduction Anti-spike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies produced after infection with the coronavirus disease of 2019 (COVID-19) will offer protection and prevent re-infection for a few months. Seroprevalence studies measuring the SARS­CoV-2 immunoglobulin G (IgG) levels will be helpful to know the herd immunity level that prevents community transmission. Very few studies have addressed the antibody titer among healthy participants and rheumatoid arthritis (RA) patients. The present study was conducted to determine the anti-spike SARS-CoV-2 antibody (Ab) status before COVID-19 vaccination in healthy participants and RA patients. Methodology A cross-sectional study was conducted at a tertiary care hospital to estimate the serum anti-spike antibody levels against COVID-19 among the pre-vaccinated healthy participants and patients with RA during the third wave of COVID-19. After receiving written informed consent, participants were recruited as per the inclusion and exclusion criteria. Demographic details, co-morbid status, and medication details were collected. Five milliliters of blood samples were collected, and anti-spike antibodies were estimated. The SARS-CoV-2 Ab positivity rate was expressed in percentage and was correlated with gender and age groups. Ab-positive participants were classified into three categories based on the neutralizing antibody titers (NAT). Results A total of 58 participants (49 healthy volunteers and nine RA patients) were recruited. Out of 58 participants, 40 were males, nine were females among healthy participants, and one male and eight females in the RA group were enrolled. Among the RA patients, one participant was found to have the chronic obstructive pulmonary disease (COPD), and two participants with hypothyroidism. Antibody positivity was found to be 83.6% among the healthy volunteers and 100% in the RA patients. About 48% had NAT between 50 and 90%. There was no significant difference for age and gender-specific positivity for SARS-CoV-2 neutralizing antibodies and neutralizing antibody titers among healthy participants. Conclusion Our study showed 84% positivity for anti-spike SARS-CoV-2 antibodies around the third wave (between November 2021 and February 2022). The majority had high neutralizing antibody titers. The probable reason for the SARS-CoV-2 antibody positivity before vaccination was either asymptomatic infection or herd immunity.

Non-Synonymous Variants in Fat QTL Genes among High- and Low-Milk-Yielding Indigenous Breeds.

The effect of breed on milk components-fat, protein, lactose, and water-has been observed to be significant. As fat is one of the major price-determining factors for milk, exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. Here, on whole-genome sequencing, 25 differentially expressed hub or bottleneck fat QTLs were explored for variations across indigenous breeds. Out of these, 20 genes were identified as having nonsynonymous substitutions. A fixed SNP pattern in high-milk-yielding breeds in comparison to low-milk-yielding breeds was identified in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E and, vice versa, in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The identified SNPs were ratified by pyrosequencing to prove that key differences exist in fat QTLs between the high- and low-milk-yielding breeds.

N6-Methyladenosine RNA Modification in Host Cells Regulates Peste des Petits Ruminants Virus Replication.

N6-methyladenosine (m6A) modification is a major RNA epigenetic regulatory mechanism. The dynamics of m6A levels in viral genomic RNA and their mRNAs have been shown to have either pro- or antiviral functions, and therefore, m6A modifications influence virus-host interactions. Currently, no reports are available on the effect of m6A modifications in the genome of Peste des petits ruminants virus (PPRV). In the present study, we took PPRV as a model for nonsegmented negative-sense single-stranded RNA viruses and elucidate the role of m6A modification on viral replication. We detected m6A-modified sites in the mRNA of the virus and host cells, as well as the PPRV RNA genome. Further, it was found that the level of m6A modification in host cells alters the viral gene expression. Knockdown of the METTL3 and FTO genes (encoding the m6A RNA modification writer and eraser proteins, respectively) results in alterations of the levels of m6A RNA modifications in the host cells. Experiments using these genetically modified clones of host cells infected with PPRV revealed that both higher and lower m6A RNA modification in the host cells negatively affect PPRV replication. We found that m6A-modified viral transcripts had better stability and translation efficiency compared to the unmodified mRNA. Altogether, from these data, we conclude that the m6A modification of RNA regulates PPRV replication. These findings contribute toward a way forward for developing novel antiviral strategies against PPRV by modulating the dynamics of host m6A RNA modification. IMPORTANCE Peste des petits ruminants virus (PPRV) causes a severe disease in sheep and goats. PPRV infection is a major problem, causing significant economic losses to small ruminant farmers in regions of endemicity. N6-methyladenosine (m6A) is an important RNA modification involved in various functions, including virus-host interactions. In the present study, we used stable clones of Vero cells, having knocked down the genes encoding proteins involved in dynamic changes of the levels of m6A modification. We also used small-molecule compounds that interfere with m6A methylation. This resulted in a platform of host cells with various degrees of m6A RNA modification. The host cells with these different microenvironments were useful for studying the effect of m6A RNA modification on the expression of viral genes and viral replication. The results pinpoint the level of m6A modifications that facilitate the maximum replication of PPRV. These findings will be useful in increasing the virus titers in cultured cells needed for the economical development of the vaccine. Furthermore, the findings have guiding significance for the development of novel antiviral strategies for limiting PPRV replication in infected animals.

Genotypic characterization of Japanese encephalitis virus circulating in swine population of India: Genotype-III still in dominance.

Swine is considered as a suitable sentinel to predict Japanese encephalitis virus (JEV) outbreaks in humans. The present study was undertaken to determine the circulating genotypes of JEV in swine population of India. A total of 702 swine serum samples from four states of western, northern, northern-temperate, and north-eastern zones of India were screened by real-time RT-PCR targeting envelope gene of JEV, which showed positivity of 35.33%. The viral copy number ranged from 3 copies to 6.3 × 104 copies/reaction. Subsequently, the capsid/prM structural gene region of JEV positive samples was amplified by nested RT-PCR, sequenced, and genetically characterized. The phylogenetic analysis of the partial sequences of the capsid gene of 42 JEV positive samples showed that they all belonged to genotype-III (G-III) of JEV. Notably, JEV positive swine samples showed high nucleotide identity with human isolates from China and Nepal which explains the probable spillover of infection between neighboring countries probably by migratory birds. The novel mutations were observed in JEV positive sample B8 at C54 position (Phe → Ser), and JEV positive sample K50 at C62 (Thr → Ala) and C65 (Leu → Pro) positions which were absent from other JEV isolates reported till now. The mutation at the C66 position (Leu → Ser) observed in live attenuated vaccine SA14-14-2 strain was not found in JEV positive samples of our study. The detection of the G-III JE virus from climatically diverse states of India reinforces the need to continue the ongoing human vaccination program in India by extending vaccine coverage in temperate states.

Comparative gene expression profile in circulating PBMCs of Bos indicus and crossbred cattle to understand disease tolerance mechanism.

The present investigation was performed to compare the global gene expression profile in peripheral blood mononuclear cells (PBMCs) of Bos indicus and crossbred (Bos taurus × B. indicus) cattle. Previously, several studies revealed the disease tolerance potential of B. indicus cattle but underlying genetic mechanism is still not fully explored. The PBMCs model was used for this investigation as it plays crucial role in the immune system regulation. Transcriptomic analysis revealed total 6767 significantly differentially expressed transcripts (fold change (absolute) >2.0, p < .05). In addition, 4149 transcripts were upregulated, 2618 transcripts were downregulated and fold change (absolute) of differentially expressed transcript varied from -223.32 to 213.63. Functional annotation analysis of differentially expressed genes confirmed their role in various molecular pathways viz. innate immune response, antigen processing and presentation, MHC protein complex, defense response to bacterium, regulation of immune response, positive regulation of JAK-STAT cascade, cytoskeletal protein binding, etc. Protein-protein interaction network analysis provided understanding of inter-relationship of immune genes with differentially expressed genes. In conclusion, this study could provide comprehensive information about the dysregulated genes and biological pathways in PBMCs which might be responsible for disease tolerance in B. indicus cattle.

Adverse events following remdesivir administration in moderately ill COVID-19 patients - A retrospective analysis.

Introduction: Remdesivir, an antiviral drug, received an emergency use authorization for treating coronavirus disease 2019 (COVID-19) patients. Though many studies have reported the safety aspects of this antiviral agent, most of them were observed in severely ill COVID-19 patients, making very less data available in the moderately ill patients. The present study was conducted with an objective of finding the adverse events (AEs) associated with remdesivir in moderately ill COVID-19 patients. Methodology: A retrospective observational study was conducted by collecting data of demographic details and details of remdesivir, laboratory investigations, and AEs from the patient medical records from May to July 2021 and analyzed by using the appropriate statistics. Results: Out of the 160 COVID-19 patients, 32 were moderately ill (males: 29, females: 03) and were treated with remdesivir along with steroids and low molecular weight heparin (LMW) heparin. The average number of administered remdesivir doses was 4, with a loading dose of 200 mg and a maintenance dose of 100 mg. A total of 41 AEs were observed out of which 17 were adverse drug reactions (ADRs) (a significant increase in the alanine transaminase (ALT) [P < 0.001]) and 23 AEs (a significant rise in random blood sugars, RBS [one of the AEs] [P = 0.007]). The AEs were more commonly seen in the hypertensive patients. An increased oxygen requirement was a major serious AE observed in four patients. Conclusion: Remdesivir caused a significant increase in the liver enzymes. Increased blood sugar levels were the most common AE and increased oxygen requirement was the major serious AE observed.

Immuno-chromatic probe based lateral flow assay for point-of-care detection of Japanese encephalitis virus NS1 protein biomarker in clinical samples using a smartphone-based approach.

Lateral flow assays (LFAs) are one of the most economical, point-of-care (PoC) diagnostic assays that exploit the colorimetric properties of gold nanoparticles (AuNPs). Up to the best of our knowledge, no rapid antigen-based LFA exists for Japanese Encephalitis Virus (JEV) detection. Herein, we have reported a novel portable sandwich-type LFA for on-site detection of the non-structural 1 (NS1) secretory protein of JEV. In-house JEV NS1 antibodies (Abs) were generated and labelled with AuNPs as immunoprobes. A glass fibre membrane conjugate pad was soaked with AuNPs-Ab solution, while the JEV NS1 Ab and anti-rabbit IgG 2° Ab were coated as the test and control lines, respectively, on a nitrocellulose (NC) membrane. Different layers of the LFA were fabricated and various parameters were standardised for optimum colour intensity development. JEV negative serum samples spiked with JEV NS1 Ags (linear range - 1 pg ml-1 to 1 µg ml-1) were applied onto the sample pad and the intensity of the red colour developed on the test line increased with increasing concentration of Ag. The visual limit of detection (LOD) determined from the LFA was 10 pg ml-1, which corresponded to the LOD determined by the graphical data obtained from ImageJ software and the Colorimeter smartphone application. Furthermore, the colorimetric based immunosensor showed minimal non-specific detection of other closely related flaviviral NS1 Ags in the spiked serum, provided a rapid result within 10 min, showed storage stability up to a month at 4 °C, successfully detected the JEV NS1 protein in clinically infected pig serum samples, and hence, may be developed into a PoC screening diagnostic kit for JEV.

Effect of acute heat shock on stress gene expression and DNA methylation in zebu (Bos indicus) and crossbred (Bos indicus × Bos taurus) dairy cattle.

Environmental temperature is one of the major factors to affect health and productivity of dairy cattle. Gene expression networks within the cells and tissues coordinate stress response, metabolism, and milk production in dairy cattle. Epigenetic DNA methylations were found to mediate the effect of environment by regulating gene expression patterns. In the present study, we compared three Indian native zebu cattle, Bos indicus (Sahiwal, Tharparkar, and Hariana) and one crossbred Bos indicus × Bos taurus (Vrindavani) for stress gene expression and differences in the DNA methylation patterns. The results indicated acute heat shock to cultured PBMC affected their proliferation, stress gene expression, and DNA methylation. Interestingly, expressions of HSP70, HSP90, and STIP1 were found more pronounced in zebu cattle than the crossbred cattle. However, no significant changes were observed in global DNA methylation due to acute heat shock, even though variations were observed in the expression patterns of DNA methyltransferases (DNMT1, DNMT3a) and demethylases (TET1, TET2, and TET3) genes. The treatment 5-AzaC (5-azacitidine) that inhibit DNA methylation in proliferating PBMC caused significant increase in heat shock-induced HSP70 and STIP1 expression indicating that hypomethylation facilitated stress gene expression. Further targeted analysis DNA methylation in the promoter regions revealed no significant differences for HSP70, HSP90, and STIP1. However, there was a significant hypomethylation for BDNF in both zebu and crossbred cattle. Similarly, NR3C1 promoter region showed hypomethylation alone in crossbred cattle. Overall, the results indicated that tropically adapted zebu cattle had comparatively higher expression of stress genes than the crossbred cattle. Furthermore, DNA methylation may play a role in regulating expression of certain genes involved in stress response pathways.

Selection and validation of suitable reference gene for qPCR gene expression analysis in lamb testis cells under Sheep pox virus infection.

Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.

Differential expression of long non-coding RNAs under Peste des petits ruminants virus (PPRV) infection in goats.

Peste des petits ruminants (PPR) characterized by fever, sore mouth, conjunctivitis, gastroenteritis, and pneumonia, is an acute, highly contagious viral disease of sheep and goats. The role of long non-coding RNAs (lncRNAs) in PPRV infection has not been explored to date. In this study, the transcriptome profiles of virulent Peste des petits ruminants virus (PPRV) infected goat tissues - lung and spleen were analyzed to identify the role of lncRNAs in PPRV infection. A total of 13,928 lncRNA transcripts were identified, out of which 170 were known lncRNAs. Intergenic lncRNAs (7625) formed the major chunk of the novel lncRNA transcripts. Differential expression analysis revealed that 15 lncRNAs (11 downregulated and 4 upregulated) in the PPRV infected spleen samples and 16 lncRNAs (13 downregulated and 3 upregulated) in PPRV infected lung samples were differentially expressed as compared to control. The differentially expressed lncRNAs (DElncRNAs) possibly regulate various immunological processes related to natural killer cell activation, antigen processing and presentation, and B cell activity, by regulating the expression of mRNAs through the cis- or trans-regulatory mechanism. Functional enrichment analysis of differentially expressed mRNAs (DEmRNAs) revealed enrichment of immune pathways and biological processes in concordance with the pathways in which correlated lncRNA-neighboring genes were enriched. The results suggest that a coordinated immune response is raised in both lung and spleen tissues of the goat through mRNA-lncRNA crosstalk.

Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep.

In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was "infectious diseases, antimicrobial response, and inflammatory response," which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.

Systems biology under heat stress in Indian cattle.

Transcriptome profiling of Vrindavani and Tharparkar cattle (n = 5 each) revealed that more numbers of genes were dysregulated in Vrindavani than in Tharparkar. A contrast in gene expression was observed with 18.9 % of upregulated genes in Vrindavani downregulated in Tharparkar and 17.8% upregulated genes in Tharparkar downregulated in Vrindavani. Functional annotation of genes differentially expressed in Tharparkar and Vrindavani revealed that the systems biology in Tharparkar is moving towards counteracting the effects due to heat stress. Unlike Vrindavani, Tharparkar is not only endowed with higher expression of the scavengers (UBE2G1, UBE2S, and UBE2H) of misfolded proteins but also with protectors (VCP, Serp1, and CALR) of naïve unfolded proteins. Further, higher expression of the antioxidants in Tharparkar enables it to cope up with higher levels of free radicals generated as a result of heat stress. In this study, we found relevant genes dysregulated in Tharparkar in the direction that can counter heat stress.

Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGß1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Transcriptomic analysis to infer key molecular players involved during host response to NDV challenge in Gallus gallus (Leghorn & Fayoumi).

Long non-coding RNAs (lncRNAs) are the transcripts of length longer than 200 nucleotides. They are involved in the regulation of various biological activities. Leghorn and Fayoumi breeds of Gallus gallus were known to be having differential resistance against Newcastle Disease Virus (NDV) infection. Differentially expressed genes which were thought to be involved in this pattern of resistance were already studied. Here we report the analysis of the transcriptomic data of Harderian gland of Gallus gallus for studying the lncRNAs involved in regulation of these genes. Using bioinformatics approaches, a total of 37,411 lncRNAs were extracted and 359 lncRNAs were differentially expressing. Functional annotation using co-expression analysis revealed the involvement of lncRNAs in the regulation of various pathways. We also identified 1232 quantitative trait loci (QTLs) associated with the genes interacting with lncRNA. Additionally, we identified the role of lncRNAs as putative micro RNA precursors, and the interaction of differentially expressed Genes with transcription factors and micro RNAs. Our study revealed the role of lncRNAs during host response against NDV infection which would facilitate future experiments in unravelling regulatory mechanisms of development in the genetic improvement of the susceptible breeds of Gallus gallus.

Systems Biology behind Immunoprotection of Both Sheep and Goats after Sungri/96 PPRV Vaccination.

Immune response is a highly coordinated cascade involving all the subsets of peripheral blood mononuclear cells (PBMCs). In this study, RNA sequencing (RNA-Seq) analysis of PBMC subsets was done to delineate the systems biology behind immune protection of the vaccine in sheep and goats. The PBMC subsets studied were CD4+, CD8+, CD14+, CD21+, and CD335+ cells from day 0 and day 5 of sheep and goats vaccinated with Sungri/96 peste des petits ruminants virus. Assessment of the immune response processes enriched by the differentially expressed genes (DEGs) in all the subsets suggested a strong dysregulation toward the development of early inflammatory microenvironment, which is very much required for differentiation of monocytes to macrophages, and activation as well as the migration of dendritic cells into the draining lymph nodes. The protein-protein interaction networks among the antiviral molecules (IFIT3, ISG15, MX1, MX2, RSAD2, ISG20, IFIT5, and IFIT1) and common DEGs across PBMC subsets in both species identified ISG15 to be a ubiquitous hub that helps in orchestrating antiviral host response against peste des petits ruminants virus (PPRV). IRF7 was found to be the key master regulator activated in most of the subsets in sheep and goats. Most of the pathways were found to be inactivated in B lymphocytes of both the species, indicating that 5 days postvaccination (dpv) is too early a time point for the B lymphocytes to react. The cell-mediated immune response and humoral immune response pathways were found more enriched in goats than in sheep. Although animals from both species survived the challenge, a contrast in pathway activation was observed in CD335+ cells.IMPORTANCE Peste des petits ruminants (PPR) by PPR virus (PPRV) is an World Organisation for Animal Health (OIE)-listed acute, contagious transboundary viral disease of small ruminants. The attenuated Sungri/96 PPRV vaccine used all over India against this PPR provides long-lasting robust innate and adaptive immune response. The early antiviral response was found mediated through type I interferon-independent interferon-stimulated gene (ISG) expression. However, systems biology behind this immune response is unknown. In this study, in vivo transcriptome profiling of PBMC subsets (CD4+, CD8+, CD14+, CD21+, and CD335+) in vaccinated goats and sheep (at 5 days postvaccination) was done to understand this systems biology. Though there are a few differences in the systems biology across cells (specially the NK cells) between sheep and goats, the coordinated response that is inclusive of all the cell subsets was found to be toward the induction of a strong innate immune response, which is needed for an appropriate adaptive immune response.