Production of highly soluble and immuno-reactive recombinant flagellin protein of Clostridium chauvoei.

OBJECTIVE: Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. METHODS: Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. RESULTS: rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). CONCLUSION: The study indicated the production of conformational recombinant flagellin-rFliA(C)-antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

Expression profiles of toll like receptors, MHC and cytokine genes along with viral load in organs of ducklings infected with an Indian isolate of duck enteritis virus.

A comprehensive study on the pathogenicity and host immune response was conducted in White Pekin ducklings after experimental infection with an Indian isolate of duck enteritis virus (DEV). The virus was found to be highly pathogenic and pantropic, which rapidly multiplied in various organs, mainly in the spleen and liver showing higher viral load with severe pathological lesions and caused 100% mortality. Expression profiles of immune gene transcripts in tissues (liver, spleen, brain) revealed upregulation of proinflammatory cytokines IFN-α, IFN- ß, IL-1ß, IL-6 and also iNOS with stimulation of TLRs (TLR-2, 3, 21). IFN-α was robustly upregulated (p < 0.05) especially in liver, might be playing role in antiviral innate immunity. Further, massive upregulation of MHC class-I (p < 0.01), expression of Th1 cytokines (IFN-γ & IL-2) and certain Th2 cytokines (IL-4 & IL-10) suggests stimulation of cell mediated as well as humoral immunity. To our knowledge, we are reporting first time about the robust upregulation of MHC class-I in spleen, liver and brain along with expression of certain cytokines in the peripheral blood mononuclear cells (PBMCs) during experimental DEV infection.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Immunogenicity and protective efficacy of an inactivated Newcastle disease virus vaccine encapsulated in poly-(lactic-co-glycolic acid) nanoparticles.

An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NP) to evaluate its immunogenicity and protective efficacy. The NDV vaccine was prepared by inactivating one virulent Indian strain of NDV belonging to Genotype VII by using beta-propiolactone. PLGA nanoparticles encapsulating inactivated NDV were prepared by the solvent evaporation method. Scanning electron microscopy and zeta sizer analysis revealed that the (PLGA+NDV) NP were spherical, with an average size of 300 nm, having a zeta potential of -6 mV. The encapsulation efficiency and loading efficiency were 72% and 2.4%, respectively. On immunization trial in chicken, the (PLGA+NDV) NP induced significantly (P < 0.0001) higher levels of HI and IgY antibodies with the peak HI titer of 28 and higher expression of IL-4 mRNA. The consistency of higher antibody levels suggests slow and pulsatile release of the antigens from the (PLGA+NDV) NP. The nano-NDV vaccine also induced cell mediated immunity with higher expression of IFN-γ indicating strong Th1 mediated immune responses in contrast to the commercial oil adjuvanted inactivated NDV vaccine. Further, the (PLGA+NDV) NP afforded 100% protection against the virulent NDV challenge. Our results suggested that PLGA NP have adjuvant potential on induction of humoral as well as Th1 biased cell mediated immune responses and also enhanced protective efficacy of the inactivated NDV vaccine. This study provides an insight for development of PLGA NP based inactivated NDV vaccine using the same genotype circulating in the field as well as for other avian diseases at exigencies.

Complete Genome Sequence of a Virulent Duck Enteritis Virus (DEV/India/IVRI-2016) Isolated from Southern India.

Molecular characterization of Indian isolates of duck enteritis virus (DEV) so far has been limited to a few selected genomic regions. Here, we report the complete genome sequence of an isolate, DEV/India/IVRI-2016, from southern India that is 158,091 bp in length.

Swine coronaviruses (SCoVs) and their emerging threats to swine population, inter-species transmission, exploring the susceptibility of pigs for SARS-CoV-2 and zoonotic concerns.

Swine coronaviruses (SCoVs) are one of the most devastating pathogens affecting the livelihoods of farmers and swine industry across the world. These include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV). Coronaviruses infect a wide variety of animal species and humans because these are having single stranded-RNA that accounts for high mutation rates and thus could break the species barrier. The gastrointestinal, cardiovascular, and nervous systems are the primary organ systems affected by SCoVs. Infection is very common in piglets compared to adult swine causing high mortality in the former. Bat is implicated to be the origin of all CoVs affecting animals and humans. Since pig is the only domestic animal in which CoVs cause a wide range of diseases; new coronaviruses with high zoonotic potential could likely emerge in the future as observed in the past. The recently emerged severe acute respiratory syndrome coronavirus virus-2 (SARS-CoV-2), causing COVID-19 pandemic in humans, has been implicated to have animal origin, also reported from few animal species, though its zoonotic concerns are still under investigation. This review discusses SCoVs and their epidemiology, virology, evolution, pathology, wildlife reservoirs, interspecies transmission, spill-over events and highlighting their emerging threats to swine population. The role of pigs amid ongoing SARS-CoV-2 pandemic will also be discussed. A thorough investigation should be conducted to rule out zoonotic potential of SCoVs and to design appropriate strategies for their prevention and control.

Prophylactic and therapeutic insights into trained immunity: A renewed concept of innate immune memory.

Trained immunity is a renewed concept of innate immune memory that facilitates the innate immune system to have the capacity to remember and train cells via metabolic and transcriptional events to enable them to provide nonspecific defense against the subsequent encounters with a range of pathogens and acquire a quicker and more robust immune response, but different from the adaptive immune memory. Reversing the epigenetic changes or targeting the immunological pathways may be considered potential therapeutic approaches to counteract the hyper-responsive or hypo-responsive state of trained immunity. The efficient regulation of immune homeostasis and promotion or inhibition of immune responses is required for a balanced response. Trained immunity-based vaccines can serve as potent immune stimuli and help in the clearance of pathogens in the body through multiple or heterologous effects and confer protection against nonspecific and specific pathogens. This review highlights various features of trained immunity and its applications in developing novel therapeutics and vaccines, along with certain detrimental effects, challenges as well as future perspectives.