Feline infectious peritonitis (FIP) is a devastating and often fatal disease caused by feline coronavirus (FCoV). Currently, there is no widely used vaccine for FIP, and many attempts using a variety of platforms have been largely unsuccessful due to the disease\'s highly complicated pathogenesis. One such complication is antibody-dependent enhancement (ADE) seen in FIP, which occurs when sub-neutralizing antibody responses to viral surface proteins paradoxically enhance disease. A novel vaccine strategy is presented here that can overcome the risk of ADE by instead using a lipid nanoparticle-encapsulated mRNA encoding the transcript for the internal structural nucleocapsid (N) FCoV protein. Both wild type and, by introduction of silent mutations, GC content-optimized mRNA vaccines targeting N were developed. mRNA durability in vitro was characterized by quantitative reverse-transcriptase PCR and protein expression by immunofluorescence assay for one week after transfection of cultured feline cells. Both mRNA durability and protein production in vitro were improved with the GC-optimized construct as compared to wild type. Immune responses were assayed by looking at N-specific humoral (by ELISA) and stimulated cytotoxic T cell (by flow cytometry) responses in a proof-of-concept mouse vaccination study. These data together demonstrate that an LNP-mRNA FIP vaccine targeting FCoV N is stable in vitro, capable of eliciting an immune response in mice, and provides justification for beginning safety and efficacy trials in cats.

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.

Nucleocapsid antibody assays can be used to estimate SARS-CoV-2 infection prevalence in regions implementing spike-based COVID-19 vaccines. However, poor sensitivity of nucleocapsid antibody assays in detecting infection after vaccination has been reported. We derived a lower cutoff for identifying previous infections in a large blood donor cohort (N = 142,599) by using the Ortho VITROS Anti-SARS-CoV-2 Total-N Antibody assay, improving sensitivity while maintaining specificity >98%. We validated sensitivity in samples donated after self-reported swab-confirmed infections diagnoses. Sensitivity for first infections in unvaccinated donors was 98.1% (95% CI 98.0-98.2) and for infection after vaccination was 95.6% (95% CI 95.6-95.7) based on the standard cutoff. Regression analysis showed sensitivity was reduced in the Delta compared with Omicron period, in older donors, in asymptomatic infections, <30 days after infection, and for infection after vaccination. The standard Ortho N antibody threshold demonstrated good sensitivity, which was modestly improved with the revised cutoff.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC-capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.

While considerable attention has been devoted to respiratory manifestations, such as pneumonia and acute respiratory distress syndrome (ARDS), emerging evidence underlines the significance of extrapulmonary involvement. In this study, we examined 15 hospitalized patients who succumbed to severe complications following SARS-CoV-2 infection. These patients were admitted to the Sibiu County Clinical Emergency Hospital in Sibiu, Romania, between March and October 2021. All patients were ethnic Romanians. Conducted within a COVID-19-restricted environment and adhering to national safety protocols, autopsies provided a comprehensive understanding of the disease\'s multisystemic impact. Detailed macroscopic evaluations and histopathological analyses of myocardial, renal, hepatic, splenic, and gastrointestinal tissues were performed. Additionally, reverse transcription-quantitative polymerase chain reaction (rt-qPCR) assays and immunohistochemical staining were employed to detect the viral genome and nucleocapsid within the tissues. Myocardial lesions, including ischemic microstructural changes and inflammatory infiltrates, were prevalent, indicative of COVID-19\'s cardiac implications, while renal pathology revealed the chronic alterations, acute tubular necrosis, and inflammatory infiltrates most evident. Hepatic examination identified hepatocellular necroinflammatory changes and hepatocytic cytopathy, highlighting the hepatic involvement of SARS-CoV-2 infection. Splenic parenchymal disorganization was prominent, indicating systemic immune dysregulation. Furthermore, gastrointestinal examinations unveiled nonspecific changes. Molecular analyses detected viral genes in various organs, with immunohistochemical assays confirming viral presence predominantly in macrophages and fibroblasts. These findings highlighted the systemic nature of SARS-CoV-2 infection, emphasizing the need for comprehensive clinical management strategies and targeted therapeutic approaches beyond respiratory systems.

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

UNASSIGNED: We have previously demonstrated that older residents of long-term care facilities (LTCF) in the UK show levels of anti-spike antibodies that are comparable to the general population following primary series and booster vaccination for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, data on the humoral response to other SARS-CoV-2 proteins associated with natural infection are scarce in this vulnerable population.
UNASSIGNED: We measured quantitative levels of anti-nucleocapsid antibodies in blood samples taken from LTCF residents and staff after initial and repeat SARS-CoV-2 infections, between December 2020 and March 2023. Data on SARS-CoV-2 infection and vaccination were obtained through linkage to national datasets. Linear mixed effects models were used to investigate anti-nucleocapsid antibody levels, using log10 scale, in relation to time from most recent infection. This included evaluation of associations between repeat infection, staff/resident status, age, sex, Omicron infection and vaccination history and peak antibody level and slope of decline with time.
UNASSIGNED: We analysed 405 antibody observations from 220 residents and 396 observations from 215 staff. Repeat infection was associated with 8.5-fold (95%CI 4.9-14.8-fold) higher initial (peak) median anti-nucleocapsid antibody level, with steeper subsequent slope of decline. There were no significant differences in antibody level associated with resident (vs. staff) status or age, but Omicron infection was associated with 3.6-fold (95%CI 2.4-5.4-fold) higher levels. There was stronger evidence of waning of antibody levels over time in a sensitivity analysis in which observations were censored in cases with suspected undetected repeat infection.
UNASSIGNED: We found similar levels of anti-nucleocapsid antibody in residents and staff of LTCFs. Repeat infection and infection with an Omicron strain were associated with higher peak values. There was evidence of waning of anti-nucleocapsid antibody levels over time.
COVID-19 had a severe impact on care homes in the UK early in the pandemic. However, deaths and disease caused by the SARS-CoV-2 virus have decreased over time following successful introduction of vaccinations and resistance linked to prior infection. There has been a lot of research carried out on the body\'s immune response to the viral spike protein, which was used to create vaccines against the virus. Less is known about our immune response to other proteins produced by the virus, such as nucleocapsid, which have not been used in current vaccines. We evaluated antibody levels against the viral nucleocapsid protein in older care home residents following initial and repeat SARS-CoV-2 infection and compared these values to those observed in younger care home staff. This was done through a large established cohort study, in which residents and staff of participating homes could volunteer to provide blood samples for analysis. We found similar levels of antibody levels among staff and older residents of care homes. These findings are in line with previous studies, in which we have shown that care home residents who survive SARS-CoV-2 infection can develop robust immunity. Higher peak antibody levels were observed following repeat infection in both residents and staff.

According to the 2018 WHO R&D Blueprint, Nipah virus (NiV) is a priority disease, and the development of a vaccine against NiV is strongly encouraged. According to criteria used to categorize zoonotic diseases, NiV is a stage III disease that can spread to people and cause unpredictable outbreaks. Since 2001, the NiV virus has caused annual outbreaks in Bangladesh, while in India it has caused occasional outbreaks. According to estimates, the mortality rate for infected individuals ranges from 70 to 91%. Using immunoinformatic approaches to anticipate the epitopes of the MHC-I, MHC-II, and B-cells, they were predicted using the NiV glycoprotein and nucleocapsid protein. The selected epitopes were used to develop a multi-epitope vaccine construct connected with linkers and adjuvants in order to improve immune responses to the vaccine construct. The 3D structure of the engineered vaccine was anticipated, optimized, and confirmed using a variety of computer simulation techniques so that its stability could be assessed. According to the immunological simulation tests, it was found that the vaccination elicits a targeted immune response against the NiV. Docking with TLR-3, 7, and 8 revealed that vaccine candidates had high binding affinities and low binding energies. Finally, molecular dynamic analysis confirms the stability of the new vaccine. Codon optimization and in silico cloning showed that the proposed vaccine was expressed to a high degree in Escherichia coli. The study will help in identifying a potential epitope for a vaccine candidate against NiV. The developed multi-epitope vaccine construct has a lot of potential, but they still need to be verified by in vitro & in vivo studies.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.