Utišani gga-miR-142-3p u pilećem embriju: ekspresija profila XPO1.

Differential expression of gga-miR-142-3p microRNA of haemopoeitic origin during immune organ development and functional stages in chicken embryos creates new opportunities for understanding its pivotal role during embryonic developmental stages. To decipher the role of gga-miR-142-3p in-ovo knockdown was carried out with LNA modified anti-miR-gga-miR-142-3p via intravenous route at developmental and functional stages of the immune organs and other organs. Bioinformatic analysis of the genes targeted by gga-miRNA-142-3p revealed that the predicted gene XPO1 conserved binding sites at 3’UTR. The target gene XPO1 was evaluated as the validated target of gga-miR-142-3p by employing qPCR SYBR green based technology, which was evidenced by its increased expression in the tissues of gga-miR-142-3p knockdown chicken embryos. Histopathological alterations in the immune organs and visceral organs indicated that the systemic knockdown of gga-miR-124-3p led to over expression of the XPO1 gene during the embryonic stages, and changed the environment of the immune organs related to structural integrity, immune response, signal transduction and migration of B and T cells during the embryonic developmental stage in the chicken embryos. The results clearly indicated that these changes could alter the postnatal development and functions of these immune organs, and may lead to development of immuno-compromised chickens.Različita ekspresija gga-miR-142-3p mikroRNA hemopoetskog podrijetla daje nove mogućnosti razumijevanja njezine ključne uloge u embrionalnom razvoju limfnih organa i funkcionalnih zbivanja u pilećem zametku. Za otkrivanje uloge gga-miR-142-3p in ovo provedeno je utišavanje zaključanom nukleinskom kiselinom što preko anti-miR-gga-miR-142-3p preinačuje razvojne i funkcionalne sposobnosti limfnih i drugih organa. Bioinformatička analiza ciljnih gena za gga-miRNA-142-3p otkriva da gen XPO1 ima konzervirana mjesta vezanja na 3’UTR. Gen XPO1 bio je potvrđen kao cilj za gga-miR-142-3p uporabom tehnologije temeljene na qPCR SYBR zelenilu, što je bilo dokazano njegovom povećanom ekspresijom u tkivima pilećih zametaka s utišanim gga-miR-142-3p. Patohistološke promjene u imunosnim i unutarnjim organima pokazuju da sustavno utišavanje gga-miR-124-3p vodi do prevelike ekspresije gena XPO1 tijekom embrionalnog razvoja. To mijenja zadaću imunosnih organa s obzirom na strukturni integritet, imunosni odgovor, prijenos poruka te migraciju B i T limfocita tijekom razvoja pilećih zametaka. Rezultati jasno naznačuju da te promjene mogu preinačiti postnatalni razvoj i funkcije imunosnih organa te mogu dovesti do razvoja imunološki oslabljenih pilića

Identification and characterization of a novel infectious bursal disease virus from outbreaks in Maharashtra Province of India

Aim: The study was undertaken to isolate infectious bursal disease virus (IBDV) from clinical cases in broiler and cockerel flocks of Maharashtra state, India, and its molecular epidemiological investigation. Materials and Methods: The morbid bursal tissues were collected from flocks suspected for IBD. The samples were subjected for virus adaptation in primary chicken embryo fibroblast (CEF) cells followed by confirmation by reverse transcription polymerase chain reaction (RT-PCR) for partial VP2 sequence and phylogenetic analysis. Results: The isolation of IBDV from field samples took seven blind passages for adaptation in CEF. The cytopathic effects included rounding, aggregation, vacuolation, and detachment of the cells. The RT-PCR showed amplification of 627 bp amplicon specific to the primers for VP2 gene fragment which confirmed successful adaptation and isolation of IBDV using CEF. The nucleotide and deduced amino acids based on phylogeny clustered the current isolate in a distinct clade with classical virulent and antigenic variants. It showed divergence from very virulent (vv) and vaccine strains of Indian origin. The isolate showed unique amino acid substitution at A329V as compared to all other IBDVs. The variation in key amino acids was reported at A222, I242, Q249, Q253, A256, T270, N279, T284, I286, L294, N299, and V329. It shared conserved amino acids at position A222, I242, and Q253 as reported in vvIBDV isolates. However, the amino acids reported at position T270, N279, T284, L294, and N299 are conserved in classic, antigenic variant and attenuated strains of IBDV. The amino acids at positions N279 and T284 indicated that the isolate has key amino acids for cell culture replication. Conclusion: The IBDV field isolate does not reveal the full nucleotide sequence signature of vvIBDV as well as vaccine strains. Hence, we can conclude that it might not belong to vvIBDVs of Indian origin and the vaccine strain used in the region. This may be suggestive of the evolution of the IBDV in the field due to the coexistence of circulating field strains and live attenuated hot strains, resulting into morbidity and mortality, warranting the need for safer protective vaccines, and implementation of stringent biosecurity measures to minimize loss to farmers