Hesperetin blocks poxvirus replication with a low tendency to select for drug-resistant viral variants.

In this study, we demonstrated the antiviral efficacy of hesperetin against multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus (VACV), and lumpy skin disease virus (LSDV). The time-of-addition and virus step-specific assays indicated that hesperetin reduces the levels of viral DNA, mRNA, and proteins in the target cells. Further, by immunoprecipitation (IP) of the viral RNA from BPXV-infected Vero cells and a cell-free RNA-IP assay, we demonstrated that hesperetin-induced reduction in BPXV protein synthesis is also consistent with diminished interaction between eukaryotic translation initiation factor eIF4E and the 5' cap of viral mRNA. Molecular docking and MD simulation studies were also consistent with the binding of hesperetin to the cap-binding pocket of eIF4E, adopting a conformation similar to m7GTP binding. Furthermore, in a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane and associated mortality in the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we, for the first time, demonstrated the antiviral activity of hesperetin against multiple poxviruses, besides providing some insights into its potential mechanisms of action.

Evaluation of the immune responses in buffaloes vaccinated with a live-attenuated lumpy skin disease vaccine (Lumpi-ProVacInd).

Since 2019, Lumpy skin disease (LSD) has suddenly spread in many Asian countries, including India. LSD primarily occurs in cattle. However, recent LSD outbreaks in India have also revealed significant morbidity and production losses in buffaloes. This has raised concerns about the role of buffaloes in the epidemiology and transmission of LSD and necessitates the inclusion of buffaloes in the mass vaccination program for the prevention and control of the disease in the country. However, there is no significant data on the immune response in buffaloes following vaccination with the LSD vaccine. In this study, we evaluated antibody- and cell-mediated immune responses following vaccination with a newly developed live-attenuated LSD vaccine (Lumpi-ProVacInd). The detectable amount of anti-LSDV antibodies was observed at 1-2 months following vaccination, with a peak antibody titer at 3 months. Upon stimulation of the peripheral blood mononuclear cells (PBMCs) with the UV-inactivated LSDV antigen, there was a significant increase in CD8 + T cell counts in vaccinated animals as compared to the unvaccinated animals. Besides, vaccinated animals also showed a significant increase in IFN-γ levels upon antigenic stimulation of their PBMCs with LSDV antigen. In conclusion, the buffaloes also mount a potent antibody- and cell-mediated immune response following vaccination with Lumpi-ProVacInd.

Resistance Evolution against Host-directed Antiviral Agents: Buffalopox Virus Switches to Use p38-ϒ under Long-term Selective Pressure of an Inhibitor Targeting p38-α.

Host-dependency factors have increasingly been targeted to minimize antiviral drug resistance. In this study, we have demonstrated that inhibition of p38 mitogen-activated protein kinase (a cellular protein) suppresses buffalopox virus (BPXV) protein synthesis by targeting p38-MNK1-eIF4E signaling pathway. In order to provide insights into the evolution of drug resistance, we selected resistant mutants by long-term sequential passages (P; n = 60) in the presence of p38 inhibitor (SB239063). The P60-SB239063 virus exhibited significant resistance to SB239063 as compared to the P60-Control virus. To provide mechanistic insights on the acquisition of resistance by BPXV-P60-SB239063, we generated p38-α and p38-ϒ (isoforms of p38) knockout Vero cells by CRISPR/Cas9-mediated genome editing. It was demonstrated that unlike the wild type (WT) virus which is dependent on p38-α isoform, the resistant virus (BPXV-P60-SB239063) switches over to use p38-ϒ so as to efficiently replicate in the target cells. This is a rare evidence wherein a virus was shown to bypass the dependency on a critical cellular factor under selective pressure of a drug.

miRNA profiling of primary lamb testicle cells infected with lumpy skin disease virus.

In this study, miRNA profiling of cells infected with lumpy skin disease virus (LSDV) was conducted for the first time. When compared to mock-infected cells, LSDV-infected primary lamb testicle (LT) cells showed dysregulation of 64, 85, and 85 miRNAs at 12 hours postinfection (hpi), 48 hpi, and 72 hpi, respectively. While some of these miRNAs were found to be dysregulated at a particular time point following LSDV infection, others were dysregulated at all three time points. Analysis of the differentially expressed miRNA-mRNA interaction networks, Gene Ontology analysis of the predicted targets, and KEGG analysis of highly enriched pathways revealed several cellular factors/pathways involved in protein/ion/enzyme binding, cell differentiation, movement of subcellular components, calcium reabsorption, aldosterone synthesis and secretion, and melanogenesis. Some selected upregulated (oar-mir-379-5p, oar-let-7d, Chr10-18769, Chr2_5162 and oar-miR-493-5p) and downregulated (ChrX-33741, Chr3_8257 and Chr26_32680) miRNAs were further confirmed by quantitative real-time PCR. These findings contribute to our understanding of virus replication, virus-host interactions, and disease pathogenesis, and the differentially expressed miRNAs and their cellular targets may serve as biomarkers as well as novel targets for therapeutic intervention against LSDV.

Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is a global health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been a continuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids.

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

Host-Directed Antiviral Therapy.

Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

16S rDNA and ITS Sequence Diversity of Burkholderia mallei Isolated from Glanders-Affected Horses and Mules in India (2013-2019).

Glanders is a highly contagious and fatal infection of equids caused by the bacteria known as Burkholderia mallei. It is one of the notifiable equine diseases and is still present in Asia, South America and Africa. In India, glanders re-emerged in 2006, and thereafter, increasing numbers of cases were reported in different regions of the country. Between 2013 and 2019, 39 B. mallei were isolated from glanders-affected horses (n = 30) and mules (n = 9) from seven states of India such as Uttar Pradesh, Haryana, Delhi, Himachal Pradesh, Gujarat, Maharashtra and Tamil Nadu. In this study, the phylogenetic relationships of these isolates were assessed by sequence analysis of 16S rDNA gene and ITS region. Purified PCR-amplified products of 16S rDNA gene and ITS region were sequenced, aligned and phylogenetic trees were constructed using MEGA 11 software. Additionally, B. mallei 16S rDNA (n = 36) and ITS (n = 18) sequences available in the GenBank were also included for analysis to determine the diversity of older B. mallei isolates with recent Indian isolates. Both the phylogeny showed that the majority of the recent isolates from India are closely related to each other, but are genetically diverse from older isolates that originated from India. Nucleotide substitutions were also observed in a single and double position in 12 recent and two old Indian isolates. The study also indicates that similar B. mallei strains were responsible for glanders outbreaks in different states (Uttar Pradesh- Himachal Pradesh and Uttar Pradesh- Haryana) and this is due to the migration of infected animals from one state to another state. This study implies that 16S rDNA and ITS region may be used for molecular characterization of B. mallei associated with glanders in resource-limited settings.

Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses.

Phage display is one of the important and effective molecular biology techniques and has remained indispensable for research community since its discovery in the year 1985. As a large number of nucleotide fragments may be cloned into the phage genome, a phage library may harbour millions or sometimes billions of unique and distinctive displayed peptide ligands. The ligand-receptor interactions forming the basis of phage display have been well utilized in epitope mapping and antigen presentation on the surface of bacteriophages for screening novel vaccine candidates by using affinity selection-based strategy called biopanning. This versatile technique has been modified tremendously over last three decades, leading to generation of different platforms for combinatorial peptide display. The translation of new diagnostic tools thus developed has been used in situations arising due to pathogenic microbes, including bacteria and deadly viruses, such as Zika, Ebola, Hendra, Nipah, Hanta, MERS and SARS. In the current situation of pandemic of Coronavirus disease (COVID-19), a search for neutralizing antibodies is motivating the researchers to find therapeutic candidates against novel SARS-CoV-2. As phage display is an important technique for antibody selection, this review presents a concise summary of the very recent applications of phage display technique with a special reference to progress in diagnostics and therapeutics for coronavirus diseases. Hopefully, this technique can complement studies on host-pathogen interactions and assist novel strategies of drug discovery for coronaviruses.

Virological and Immunological Outcomes of Coinfections.

Coinfections involving viruses are being recognized to influence the disease pattern that occurs relative to that with single infection. Classically, we usually think of a clinical syndrome as the consequence of infection by a single virus that is isolated from clinical specimens. However, this biased laboratory approach omits detection of additional agents that could be contributing to the clinical outcome, including novel agents not usually considered pathogens. The presence of an additional agent may also interfere with the targeted isolation of a known virus. Viral interference, a phenomenon where one virus competitively suppresses replication of other coinfecting viruses, is the most common outcome of viral coinfections. In addition, coinfections can modulate virus virulence and cell death, thereby altering disease severity and epidemiology. Immunity to primary virus infection can also modulate immune responses to subsequent secondary infections. In this review, various virological mechanisms that determine viral persistence/exclusion during coinfections are discussed, and insights into the isolation/detection of multiple viruses are provided. We also discuss features of heterologous infections that impact the pattern of immune responsiveness that develops.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.

Characterization of isolates of Bordetella bronchiseptica from horses.

Bordetella bronchiseptica is a well-known Gram-negative bacterial pathogen causing a plethora of diseases in different animals. Although its infection has been reported from pigs and dogs in India, no report of B. bronchiseptica from horses is described. We report for the first time, isolation, identification and characterization of strains of B. bronchiseptica from respiratory infection in horses from different states in India. The antimicrobial susceptibility testing showed resistance to penicillins, ceftazidime, and chloramphanicol. The virulence capability of the strains was confirmed by sequencing genes such as adenylate cyclase toxin (cyaA), bordetella virulence gene (bvgA) and by PCR detection of flagellin gene (fla). We demonstrate the involvement of B. bronchiseptica strains in respiratory tract infection in horses in India.

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution.

BACKGROUND: Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. RESULTS: The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. CONCLUSIONS: Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

Abundance of antibiotic resistance genes in environmental bacteriophages.

The ecosystem is continuously exposed to a wide variety of antimicrobials through waste effluents, agricultural run-offs and animal-related and anthropogenic activities, which contribute to the spread of antibiotic resistance genes (ARGs). The contamination of ecosystems with ARGs may create increased opportunities for their transfer to naive microbes and eventually lead to entry into the human food chain. Transduction is a significant mechanism of horizontal gene transfer in natural environments, which has traditionally been underestimated as compared to transformation. We explored the presence of ARGs in environmental bacteriophages in order to recognize their contribution in the spread of ARGs in environmental settings. Bacteriophages were isolated against environmental bacterial isolates, purified and bulk cultured. They were characterized, and detection of ARG and intI genes including blaTEM, blaOXA-2, intI1, intI2, intI3, tetA and tetW was carried out by PCR. This study revealed the presence of various genes [tetA (12.7 %), intI1 (10.9 %), intI2 (10.9 %), intI3 (9.1 %), tetW (9.1 %) and blaOXA-2 (3.6 %)] and blaTEM in a significantly higher proportion (30.9 %). blaSHV, blaOXA-1, tetO, tetB, tetG, tetM and tetS were not detected in any of the phages. Soil phages were the most versatile in terms of ARG carriage. Also, the relative abundance of tetA differed significantly vis-à-vis source. The phages from organized farms showed varied ARGs as compared to the unorganized sector, although blaTEM ARG incidences did not differ significantly. The study reflects on the role of phages in dissemination of ARGs in environmental reservoirs, which may provide an early warning system for future clinically relevant resistance mechanisms.

Knowledge, awareness and perception about equine glanders among veterinarians and medical professionals in India.

Glanders is a highly infectious and notifiable disease of equines that occurs due to Burkholderia mallei. In India, glanders re-emerged in 2006 and thereafter regular outbreaks have been reported in various states (n = 14). Frequent and prolonged contact with equids with glanders may transmit B. mallei infection to humans. This study was designed to learn more about the Knowledge, Awareness and Perception (KAP) of veterinarians, para veterinarians, and physicians about equine glanders, which will help in enhancing the nation-wide glanders eradication programme. A total of 165 respondent's from 11 Indian states and one union territory were surveyed. Most of the respondents (n = 160) were from equine glanders affected or endemic states. Knowledge gap analysis revealed that 40.3 and 22% of the participants were not aware of government regulations and the transmission of glanders, respectively. These are major concerns given the wide spread occurrence of disease in the country. Awareness test on glanders revealed that 65(39.4%) participants would collect biological samples for laboratory confirmation, 67(40.6%) would inform the concerned authorities and 106 (64.2%) replied that they would eliminate the glanders infected equines. Analysis of perception towards equine glanders showed that majority of the participants (n = 113, 68.4%) observed that equine keepers were reluctant to disclose the clinical symptoms of B. mallei infection. Furthermore, non-co-operation and unwillingness by superiors (33.9%), financial (31%), administrative (28.4%), and technical limitations (27.8%) were major constraints under the perception analysis. This study reveals that veterinarians need to be educated on governmental policies and guidelines on equine glanders with regular training and awareness programs. Intersectoral co-ordination to investigate human glanders is also needed.

Exploring landscape of measles vaccination coverage: A step towards measles elimination goal in India.

INTRODUCTION: Measles remains a critical public health concern causing significant morbidity and mortality globally. Despite the success of measles vaccination programs, challenges persist, particularly in India. This study investigates dose-wise measles vaccination coverage and explores gaps in immunization focusing on zero-dose, one-dose, and two-dose coverage among children aged 24-35 months. DATA SOURCES AND METHODOLOGY: The National Family Health Survey 2019-21 (NFHS-5) served as the data source and the study analyzed information from 43,864 children aged 24-35 months. Sociodemographic variables such as birth order, wealth quintile, gender, social group, religion, residence, mother education, delivery-related factors, and media exposure were considered. Statistical analysis involved weighted estimates, chi-square tests, and multivariate multinomial logistic regression. RESULTS: The study revealed that challenges persist in achieving optimal measles vaccination coverage. Analysis by sociodemographic factors highlighted disparities in coverage, with variations in zero dose prevalence across states and districts. The percentage of zero-dose children was significantly higher, with 11.5% of children in India remaining to receive any measles vaccination. Factors influencing vaccine coverage include birth order, age, wealth quintile, social group, religion, residence, maternal education, place of delivery, media exposure, and mode of delivery. The findings from the spatial analysis show the clustering of zero-dose children is high in the northeastern states of India. DISCUSSION: Measles zero-dose children pose a significant obstacle to achieving elimination goals. Spatial analysis identifies clusters of unvaccinated populations guiding targeted interventions. The study aligns with global initiatives such as the Immunization Agenda 2030 emphasizing equitable vaccine access and discusses how India can tailor its strategies to achieve the goal. Lessons from polio eradication efforts inform strategies for measles elimination, stressing the importance of high-quality data and surveillance. The study underscores the urgency of addressing last-mile measles vaccination gaps in India. Spatially targeted interventions informed by sociodemographic factors can enhance immunization coverage. Achieving measles elimination requires sustained efforts and leveraging lessons from successful vaccination campaigns. The study findings have the potential to contribute to informed decision-making, supporting India's roadmap for the measles and rubella elimination goal.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle.

Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.

Rapid detection of Corynebacterium pseudotuberculosis in clinical samples from sheep.

Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31%) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48%) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98-100% homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31%, 1.1% and 1.29% based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.