Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long COVID (LC) are clinical conditions classified within the broader category of post-acute infection syndrome. No previous studies have explored the IgG isotype profile against the SARS-CoV-2 Spike-1 antigen in these populations induced by vaccination. This exploratory study aimed to analyze IgG isotyping in European patients with ME/CFS and LC who have received multiple doses of mRNA- and adenovirus-based vaccines, investigating the association between IgG isotypes and illness severity. Ninety-two convalescent COVID-19 patients, comprising 75 patients with ME/CFS, 10 patients with LC, and 7 healthy controls, were included. Serum levels of specific IgG isotypes of the anti-Spike-1-RBD antibody were measured using a previously optimized ELISA. Among the participants, 55.4% had received three or more doses of mRNA vaccines, 18.5% received two doses, and 4.3% received a single dose of mRNA vaccine. Additionally, 15.2% had received several doses of adenovirus-based vaccines combined with a booster of an mRNA vaccine, while 6.5% only received adenovirus-based vaccinations. We found that IgG4 was identified as the predominant antibody isotype in all seropositive individuals from the study participants, followed by IgG1 and IgG2 isotypes. No statistically significant differences were observed in the levels of IgG1, IgG2, or IgG4 in the participants. In the seropositive ME/CFS group, IgG4 was present in individuals who had received two or more doses of mRNA vaccines and sustained high levels during a median of 60 months post-vaccination, unaffected by subsequent breakthrough infections. Nonetheless, 37 ME/CFS subjects remained seronegative, regardless of the number of mRNA doses received. In contrast, 70% of LC subjects and 100% of controls showed prolonged persistence for IgG4 levels over time. Notably, subjects who received only adenovirus-based vaccines or those combined with a single mRNA booster tested negative for IgG4. This investigation suggests an immune dysregulation underlying an ineffective response to persistent antigenic stimuli, emphasizing the potential immune exhaustion in this population.

Introduction: Pathogens such as Toxoplasma gondii, lentiviruses (e.g., caprine arthritis- encephalitis,CAE), Hypoderma spp., Neospora caninum, Mycoplasma spp. and pestiviruses (Border disease, BD) play an important role in goat farming in Lithuania. Unfortunatelly, there is a lack of information on the prevalence and epidemiological situation of these pathogens in the Lithuanian goat population.

Material and Methods: A study covering multiple pathogens was conducted from 2021 to 2024. In total, 30 herds of goats from various regions of Lithuania were tested, and 380 blood samples from v. jugularis using blood collection tubes with clot activator were collected. Bood serums were analyzed for antibodies by Enzyme-Linked Immunosorbent Assays (ID.vet, Innovative Diagnostics, France). The final evaluation of the reaction was performed by measuring the optical density of the liquid with a spectrophotometer (BioTek Synergy Mx), and the presence or absence of antibodies in the sample was determined by differences in color intensity.

Results: Antibodies to toxoplasma (38.9%, 143/368) and CAE viruses (19.5%, 74/380) were most detected in the samples.
Antibodies to mycoplasma (0.3%, 1/368), hypoderma (3.8%, 7/184), and neospora (0.5%, 2/368) were detected in isolated cases.

Antibodies to border disease virus and Q fever were not detected. Mixed infections accounted for 7.6% of all tested samples, and only toxoplasmosis + CAE had a statistically significant association (χ2 = 19.05, p < 0.001). CAE was the only disease significantly affected by herd size (ꭓ2 = 7.913, df = 1, p < 0.05).

Conclusions: Toxoplasmosis and CAE are the most epidemiologically relevant diseases in this population, and toxoplasmosis is the most common component of mixed infections.

Acknowledgements: This research was granted by CAE-RAPID project, ID:39.

Poxviruses represent a large family of dsDNA viruses that can pose a significant threat to public health due to their ability to infect a broad range of hosts and spread in human populations. Vaccinia virus (VACV) has become a model to study poxvirus biology and pathogenicity. However, the diversity of this viral family remains overlooked and knowledge about other members is critically lacking. We decided to study the intricate structural details of three poxviruses using state-of-the-art cryo-electron tomography (cryoET) and advanced subvolume averaging processing. At IRBA, we developed a dedicated sample preparation workflow to be able to analyze native poxvirus samples of purified infectious particles in biological safety level 3 environment. By elucidating virion structures at high resolution and in the native environment, we aim at increasing our understanding of poxvirus diversity at the structural level to improve our preparedness and provide new targets for the future development of antiviral strategies.

Millions of people have or have had Long COVID. It is a disease whose exact mechanisms are unknown and for which many patients are unable to find medical help. Long COVID is defined as respiratory or extra-respiratory symptoms that appear or persist for several months after infection with SARS-CoV-2. We are particularly interested in neurological or neuropsychiatric symptoms such as anxiety, depression, and memory impairment. We have previously shown that SARS-CoV-2 is neuroinvasive, capable of infecting neurons and being transported along axons. Now, using the golden hamster model for long Covid that we developed, we show that the virus can persist in the brain for more than 80 days after the infection. This persistent infection is accompanied by dysregulation of several immuno-metabolic pathways in the brainstem, such as the dopaminergic and glutamatergic signaling, and energetic metabolism. Remarkably, using a set of different behavioral tests, we also demonstrate that the infected hamsters exhibit persistent or late-onset signs of anxiety, depression and memory impairment, reproducing some features of long Covid in humans.

Immuno-metabolic alterations and the viral persistence in the brain may partly explain the persistence or appearance of cognitive or neuropsychiatric symptoms. Our model enables a comprehensive virological, metabolic and behavioral study of a disease for which there are currently only symptomatic treatments. Identifying these fundamental mechanisms could be of great interest in understanding the cognitive and/or neuropsychiatric alterations observed with other neuroinvasive pathogens.

To date, more than twenty viruses require host-catalyzed de novo fatty acid biosynthesis for replication. In mammals, this pathway is driven by the enzyme fatty acid synthase (FASN), which condenses acetyl-CoA and malonyl-CoA to catalyze the production of the fatty acid palmitate. Palmitate serves as a precursor for various metabolic fates during viral infection, including lipid droplet formation for viral assembly, beta-oxidation for ATP generation, and/or post-translational modification of viral proteins. It was unknown if Mayaro virus (MAYV), an emerging mosquito-borne alphavirus that causes debilitating arthritogenic disease, requires FASN for replication. We found that pharmacological FASN inhibition with Fasnall, TVB-2640, and C75 significantly reduced MAYV infection in vitro. To determine the metabolic fate of palmitate, we pharmacologically inhibited the three pathways downstream of FASN and found that 2-bromopalmitate (2-BP), a protein palmitoylation inhibitor, led to a ~90% reduction in MAYV infection. Protein palmitoylation, or S-palmitoylation, is a post-translational modification in which palmitate is attached to sulfur atoms in cysteines residues. In Chikungunya virus , a related alphavirus, FASN-dependent palmitoylation of nonstructural protein 1 (nsP1) is essential for membrane association and formation of replication complexes. Based on this, we hypothesized that MAYV nsP1 is palmitoylated in a FASN-dependent manner. Protein labeling by click-chemistry confirmed that MAYV nsP1 is palmitoylated at conserved cysteine residues C417–C419. Using an alkyne acetate analog metabolized by FASN into alkyne palmitate, we observed specific labeling of wild-type nsP1 but not a cysteine-to-alanine mutant. Furthermore, treatment with FASN inhibitors or 2-BP abrogated alkyne acetate labeling of wild-type nsP1, reinforcing that MAYV protein palmitoylation is a FASN-dependent process. Our findings reveal a conserved mechanism of FASN-dependent protein palmitoylation in alphaviruses and highlight FASN as a potential therapeutic target for MAYV and related viruses.

Viral infections produce double-stranded (ds)RNAs or RNAs containing duplex regions that activate the protein kinase R (PKR), an interferon-induced protein. Activated PKR phosphorylates the eukaryotic translation initiation factor 2α (eIF2α), leading to translation inhibition, affecting viral replication of RNA viruses, and the modulation of the induction of innate immune responses. The regulation of PKR functions is not well known, so a better understanding is essential. Here, through co-immunoprecipitation analyses, we have identified for the first time an interaction between IFN alpha inducible protein 27 (IFI27) and PKR-activating protein (PACT or PRKRA) and with PKR, and these interactions are mediated by the presence of dsRNAs or RNAs containing duplex regions. Furthermore, we have shown that the interaction of IFI27 with PKR is PACT-dependent. The interaction of IFI27 with PKR and PACT, led to stronger activation of PKR and, therefore, a decrease in protein translation, both after treatment with the dsRNA analogue poly(I:C), and with different RNA viruses such as Severe Acute Respiratory virus 2 (SARS-CoV-2) and Vesicular Stomatitis virus (VSV). We also show that the function of IFI27 to activate PKR is dependent on its interaction with PACT. Moreover, we show that IFI27 expression increases the formation of stress granules, correlating with the increased PKR activation mediated by IFI27, as it has been shown that the translational arrest induced by activated PKR leads to the formation of stress granules. IFI27 positively modulates PKR activation in a PACT-dependent manner. This effect is broad, as it is shown for infections with SARS-CoV-2 and VSV, and with poly(I:C) treatment. Further understanding of the regulation of PKR activity will allow us to develop new antiviral drugs to modulate this signalling axis, which is crucial in RNA virus infections.

The transmembrane envelope (TM) proteins of retroviruses possess immunosuppressive properties. Both purified TM proteins and synthetic peptides corresponding to a highly conserved immunosuppressive (isu) domain have been shown to inhibit immune responses in vitro and in vivo. Immunization with TM proteins carrying mutations in the isu domain induces significantly stronger immune responses compared to immunization with the unmodified protein. The isu peptide of HIV-1 promotes increased secretion of IL-10, IL-6, and other cytokines from human peripheral blood mononuclear cells (PBMCs). Microarray analyses revealed that the expression of IL-6, IL-8, IL-10, MMP-1, TREM-1, and IL-1β is upregulated following exposure to this peptide. In a mouse tumor model, tumor cells that were normally non-tumorigenic in immunocompetent animals acquired tumorigenic capacity when expressing the TM protein or isu domain of various retroviruses on their surface. To further investigate these effects, we expressed the TM protein p15E of the porcine endogenous retrovirus (PERV) on the surface of human 293 cells and co-cultured them with human PBMCs. This interaction led to the release of IL-6 and IL-10 proteins and the modulation of multiple cytokines and other markers, including IL-6, IL-10, IFN-α, TNF-α, MMP1, and SEPP1. Moreover, p15E expression reduced MHC class I expression and conferred protection against cellular cytotoxicity. Based on these and previous findings, we propose that expression of the immunosuppressive p15E protein of PERV on the surface of pig xenotransplants could attenuate immune rejection and potentially reduce the need for pharmacological immunosuppression.

Respiratory Syncytial Virus (RSV) continues to cause severe respiratory illness, and effective antiviral treatments are still lacking. While many viruses rely on host‑cell signaling pathways to replicate, the role of the MEK–ERK1/2 cascade in RSV’s replication cycle has been unclear, with studies in cell lines showing mixed results. To address this, we tracked ERK1/2 activation over time and space in primary normal human bronchial epithelial (NHBE) cells. We created NHBE cells expressing a genetically encoded ERK Kinase Translocation Reporter (KTR) and used live‑cell imaging to follow ERK activity. In infected cells, RSV triggered a steady increase in ERK1/2 activation beginning about 3–4 hours after infection and lasting over 20 hours, in contrast to earlier reports of two separate activation phases.

Next, we used specific inhibitors and a recombinant RSV‑based FRET assay to look at how the pathway works. Blocking ERK1/2 with Ulixertinib stopped the virus from entering cells, revealing a new role for ERK1/2 in the first stage of infection. After entry, inhibiting MEK1/2 (with U0126) or ERK1/2 sharply reduced viral mRNA transcription and the release of infectious particles, while viral genome replication was unaffected. Further experiments showed that MEK1/2 inhibition prevented the viral phosphoprotein (P) from becoming hyper‑phosphorylated, which is vital for the viral polymerase to work.

Overall, our findings show that the MEK–ERK1/2 pathway is a key host dependency factor for RSV at multiple stages, controlling both entry and transcription. This makes MEK–ERK1/2 a strong candidate for host‑directed antiviral strategies.

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

The emergence of novel coronaviruses from animal reservoirs continues to pose significant zoonotic threats. Here, we investigate the evolutionary origins of a recently reported mink-derived HKU5-related coronavirus (nvHKU5r-CoV), as well as the virus’s structural and functional properties. Phylogenetic and recombination analyses reveal that nvHKU5r-CoV originated from bat HKU5-like viruses circulating in southeastern China. We characterize the spike loop2 region as a critical determinant of ACE2 receptor specificity and show that the bat merbecovirus with the closest loop2 sequence to nvHKU5r-CoV could already utilize mink ACE2. Targeted mutagenesis demonstrates that a single amino acid substitution (R548S) further enables robust entry via human ACE2, highlighting the zoonotic potential of HKU5r-CoVs. Molecular dating suggests prolonged period of circulation in bats prior to this virus’s transmission to mink, and viral entry assays using pseudovirus infectivity and full-length replication competent infectious clones confirm replication in mink ACE2-expressing cells. Using AlphaFold3, we predicted spike-ACE2 binding interfaces consistent with our experimental infectivity results, and instrumental in interpreting the structural basis of these interactions. These findings emphasize the evolutionary plasticity of HKU5r-CoV RBDs and the role of fur farming as potential hotspots for coronavirus emergence.