Background: Although multiple studies have demonstrated a role for exosomes during virus infections, our understanding of the mechanisms by which exosome exchange regulates immune response during viral infections and affects viral pathogenesis is still in its infancy. In particular, very little is known for cytoplasmic single-stranded RNA viruses such as SARS-CoV-2 and Rift Valley fever virus (RVFV). We have used RVFV infection as a model for cytoplasmic single-stranded RNA viruses to address this gap in knowledge. RVFV is a highly pathogenic agent that causes RVF, a zoonotic disease for which no effective therapeutic or approved human vaccine exist. Results: We show here that exosomes released from cells infected with RVFV (designated as EXi-RVFV) serve a protective role for the host and provide a mechanistic model for these effects. Our results show that treatment of both naïve immune cells (U937 monocytes) and naïve non-immune cells (HSAECs) with EXi-RVFV induces a strong RIG-I dependent activation of IFN-B. We also demonstrate that this strong anti-viral response leads to activation of autophagy in treated cells and correlates with resistance to subsequent viral infection. Since we have shown that viral RNA genome is associated with EXi-RVFV, RIG-I activation might be mediated by the presence of packaged viral RNA sequences. Conclusions: Using RVFV infection as a model for cytoplasmic single-stranded RNA viruses, our results show a novel mechanism of host protection by exosomes released from infected cells (EXi) whereby the EXi activate RIG-I to induce IFN-dependent activation of autophagy in naïve recipient cells including monocytes. Because monocytes serve as reservoirs for RVFV replication, this EXi-RVFV-induced activation of autophagy in monocytes may work to slow down or halt viral dissemination in the infected organism. These findings offer novel mechanistic insights that may aid in future development of effective vaccines or therapeutics, and that may be applicable for a better molecular understanding of how exosome release regulates innate immune response to other cytoplasmic single-stranded RNA viruses.

Parasitic infections compromise livestock health, productivity, and profitability, while rising anthelmintic resistance restricts the effectiveness of conventional parasite control strategies. Bee pollen (BP) contains bioactive compounds with immunomodulatory and antimicrobial potential. This study investigated whether dietary BP supplementation reduces parasite burden and influences liver function, hematological and metabolic profiles, and circulating metabolomic signatures in goats. Thirty crossbred Boer × Spanish does were randomly assigned to either a BP-supplemented group (T-BP; n = 15) or a control group (T-CTL; n = 15). Over 49 days, T-BP goats received 10 g/day BP mixed with 50 g/day pelleted feed, while controls received the same feed without BP. Body weight, weekly blood samples, FAMACHA scores, and biweekly fecal egg counts (FEC) were collected. We assessed metabolomic profiles on days −8, 21, 42, and 56. Data were analyzed using mixed models and repeated measures. We identified strongyle-type eggs, presumably Haemonchus contortus, as the predominant parasite. No significant differences were observed between treatments for body weight, liver enzymes, metabolic biomarkers, or hematological profiles (p > 0.05). FEC varied over time (p < 0.001), with significantly lower FEC in BP-supplemented goats only on Day 42 (p < 0.05). Metabolomic differences were significant on Days 21 and 42 but not on Day 56. These differences involved metabolites annotated to lipid signaling, inflammatory processes, and cellular energy metabolism. In summary, BP supplementation demonstrated a potential antiparasitic effect against H. contortus but did not significantly affect liver, metabolic, or hematological parameters. Metabolomic findings should be considered exploratory and hypothesis-generating.

Goats in subtropical and temperate regions experience seasonal anestrus during long-day conditions. This study evaluated the effects of bee pollen (BP) supplementation on circulating metabolic and endocrine measures, plasma short-chain fatty acids (SCFAs), and reproductive outcomes. Thirty crossbred Spanish × Boer does were assigned to T-CTL (n = 15) or T-BP (n = 15). After a 7-day adaptation period, T-BP received 10 g/day BP for 42 days (approximately 0.31 g/kg initial body weight/day), while T-CTL received carrier feed alone. We measured plasma glucose, insulin, insulin-like growth factor 1 (IGF-1), progesterone (P4), and SCFAs. BP supplementation did not significantly (p > 0.05) alter plasma glucose, insulin, IGF-1, or overall P4 concentrations. Only five does became pregnant (T-BP, 2/15; T-CTL, 3/15); therefore, fertility, prolificacy, and reproductive rate were summarized descriptively. Most plasma SCFAs did not show a consistent treatment-dependent temporal response. Isovalerate showed a significant treatment × date interaction, whereas hexanoate differed overall between treatments without a significant treatment × date interaction. Under this single-dose protocol, BP did not consistently modify circulating metabolic–endocrine markers or improve reproductive performance during the suboptimal breeding season. Future studies should include dose-ranging, direct rumen measurements, and adequately powered reproductive endpoints.

Exosome-enriched EVs are vesicles released from various cell types into extracellular fluids such as plasma, urine, saliva, cell culture media, and other body fluids. Their diameters range from 30 nm to 150 nm. They have been implicated in different cellular processes, including cellular communications and immune modulation during infection. Based on these effects, they have been used to study the pathogenesis of several viruses. It is challenging to isolate Exosome-enriched EVs from virus-infected cell supernatants using current purification techniques. This is largely due to the overlap in size between certain viruses and Exosome-enriched EVs. Here, we aim to purify Exosome-enriched EVs from Dengue type 2-infected cell culture supernatant. Exosome-enriched EVs were isolated using a commercial exosome isolation kit. For the isolated Exosome-enriched EVs in this protocol, no infectious virus was detected by plaque assay under the conditions tested. The purified exosome-enriched EVs were characterized using western blotting for tetraspanins, nanoparticle tracking analysis, and transmission electron microscopy for size confirmation. We demonstrated that our exosome-enriched EVs are intact and within the normal exosome diameter range of 30-150 nm. Overall, our protocol provides an inexpensive method for purifying exosome-enriched EVs from virus-infected cell culture supernatant, which can be used for downstream functional assays.