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Respiratory infections are a major health burden worldwide. Respiratory syncytial virus (RSV) is among the leading causes of hospitalization in both young children and older adults. The onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the public health response had a profound impact on the normal seasonal outbreaks of other respiratory viruses. However, little is known about how a prior respiratory virus infection impacts SARS-CoV-2 disease outcomes. In this study, we examine the impact of a previous RSV infection on the disease severity of a subsequent SARS-CoV-2 challenge in BALB/c mice. Mice infected with RSV, followed by a SARS-CoV-2 challenge, 30 days later, exhibited decreased weight loss and increased survival as compared to control groups. Our results suggest a prior RSV infection can provide protection against a subsequent SARS-CoV-2 infection., Importance: Severe acute respiratory syndrome coronavirus 2 and respiratory syncytial virus are respiratory viruses that are a major health burden worldwide. Severe acute respiratory syndrome coronavirus 2 and respiratory syncytial virus frequently have peak seasonal outbreaks during the winter months, and are capable of causing severe respiratory disease, often leading to hospitalization. The 2019 pandemic brought attention to the importance of understanding how co-circulating viruses can impact the disease severity of other respiratory viruses. It is known that many hospitalized patients are undergoing multiple viral infections at once, yet not much has been studied to understand the impact this has on other respiratory viruses or patients. How co-circulating viruses impact one another can provide critical knowledge for future interventions of hospitalized patients and potential vaccination strategies., Competing Interests: The authors declare no conflict of interest.

Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, belonging to the genus beta-CoV, have caused outbreaks or pandemics. SARS-CoV-2 has evolved into many variants with increased resistance to the current vaccines. Spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are important vaccine targets; however, the RBD of the SARS-CoV-2 Omicron variant is highly mutated, rending neutralizing antibodies elicited by ancestral-based vaccines targeting this region ineffective, emphasizing the need for effective vaccines with broad-spectrum efficacy against SARS-CoV-2 variants and other CoVs with pandemic potential. This study describes a pan-beta-CoV subunit vaccine, Om-S-MERS-RBD, by fusing the conserved and highly potent RBD of MERS-CoV into an RBD-truncated SARS-CoV-2 Omicron S protein, and evaluates its neutralizing immunogenicity and protective efficacy in mouse models. Om-S-MERS-RBD formed a conformational structure, maintained effective functionality and antigenicity, and bind efficiently to MERS-CoV receptor, human dipeptidyl peptidase 4, and MERS-CoV RBD or SARS-CoV-2 S-specific antibodies. Immunization of mice with Om-S-MERS-RBD and adjuvants (Alum plus monophosphoryl lipid A) induced broadly neutralizing antibodies against pseudotyped MERS-CoV, SARS-CoV, and SARS-CoV-2 original strain, as well as T-cell responses specific to RBD-truncated Omicron S protein. Moreover, the neutralizing activity against SARS-CoV-2 Omicron subvariants was effectively improved after priming with an Omicron-S-RBD protein. Adjuvanted Om-S-MERS-RBD protein protected mice against challenge with SARS-CoV-2 Omicron variant, MERS-CoV, and SARS-CoV, significantly reducing viral titers in the lungs. Overall, these findings indicated that Om-S-MERS-RBD protein could develop as an effective universal subunit vaccine to prevent infections with MERS-CoV, SARS-CoV, SARS-CoV-2, and its variants., Importance: Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential., Competing Interests: The authors declare no financial interests. The authors have filed a patent application related to this study, with L.D., G.W., and F.L. as inventors.

Grasping the roles of epitopes in viral glycoproteins is essential for unraveling the structure and function of these proteins. Up to now, all identified epitopes have been found to either neutralize, have no effect on, or enhance viral entry into cells. Here, we used nanobodies (single-domain antibodies) as probes to investigate a unique epitope on the SARS-CoV-2 spike protein, located outside the protein's receptor-binding domain. Nanobody binding to this epitope enhances the cell entry of prototypic SARS-CoV-2, while neutralizing the cell entry of SARS-CoV-2 Omicron variant. Moreover, nanobody binding to this epitope promotes both receptor binding activity and post-attachment activity of prototypic spike, explaining the enhanced viral entry. The opposite occurs with Omicron spike, explaining the neutralized viral entry. This study reveals a unique epitope that can both enhance and neutralize viral entry across distinct viral variants, suggesting that epitopes may vary their roles depending on the viral context. Consequently, antibody therapies should be assessed across different viral variants to confirm their efficacy and safety., Competing Interests: The University of Minnesota has filed a patent on Nanosota-5, -6, and -7 with F.L., G.Y., F.B., and B.L. as inventors., (Copyright: © 2024 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has resulted in substantial morbidity and mortality. The basis of severe disease in humans is difficult to determine without the use of experimental animal models. Mice are resistant to infection with ancestral strains of SARS-CoV-2, although many variants that arose later in the pandemic were able to directly infect mice. In almost all cases, viruses that naturally infected mice or were engineered to enable mouse infection required mouse passage to become virulent. In most cases, changes in structural and nonstructural changes occurred during mouse adaptation. However, the mechanism of increased virulence in mice is not understood. Here, using a recently described strain of mouse-adapted SARS-CoV-2 (rSARS2-MA30 N501Y ), we engineered a series of recombinant viruses that expressed a subset of the mutations present in rSARS2-MA30 N501Y . Mutations were detected in the spike protein and in three nonstructural proteins (nsp4, nsp8, and nsp9). We found that infection of mice with recombinant SARS-CoV-2 expressing only the S protein mutations caused very mild infection. Addition of the mutations in nsp4 and nsp8 was required for complete virulence. Of note, all these recombinant viruses replicated equivalently in cultured cells. The innate immune response was delayed after infection with virulent compared to attenuated viruses. Further, using a lineage tracking system, we found that attenuated virus was highly inhibited in the ability to infect the parenchyma, but not the airway after infection. Together, these results indicate that mutations in both the S protein and nonstructural proteins are required for maximal virulence during mouse adaptation.IMPORTANCEUnderstanding the pathogenesis of coronavirus disease 2019 (COVID-19) requires the study of experimental animals after infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). For this purpose, several mouse-adapted SARS-CoV-2 strains have been developed. Here, using a strain of mouse-adapted virus that causes a range of diseases ranging from mild to severe, we show that mutations in both a structural protein [spike (S) protein] and nonstructural proteins are required for maximal virulence. Thus, changes in the S protein, the most widely studied viral protein, while required for mouse adaptation, are not sufficient to result in a virulent virus., Competing Interests: The authors declare no conflict of interest.

Upon SARS-CoV-2 infection, infected cells undergo necroptosis, whereas delayed apoptosis and pyroptosis occur in uninfected, bystander cells, thus providing a plausible explanation for the extensive injury among myriad uninfected cells.

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we perform a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identify mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we show that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines., (© 2024. The Author(s).)

Competing Interests: We all have a specialist interest in coronaviruses, with MP and SP involved in funded research on coronaviruses. AZ acknowledges support from the PANDORA-ID-NET (grant RIA2016E-1609) funded by the European and Developing Countries Clinical Trials Partnership (EDCTP2) under Horizon 2020, the EU's Framework Program for Research and Innovation; is in receipt of a National Institutes of Health Research senior investigator award, the Mahathir Science Award; is a EU-EDCTP Pascoal Mocumbi Prize Laureate; and is a member of Scientific Expert Committee of the EC-EDCTP-Global Health Program. SP is supported by grants paid to his institution from the US National Institutes of Health (NIH; RO1 AI129269) and is a member of the Vaccines and Related Biological Products Advisory Committee of the US Food and Drug Administration. MP is supported by grants paid to his institution from the NIH (U01 Al151810). ZAM declares no competing interests. We thank Noah Schuster for his help in designing the figure. Editorial note: The Lancet Group takes a neutral position with respect to territorial claims in institutional affiliations.

While the immunogenicity of SARS-CoV-2 vaccines has been well described in adults, pediatric populations have been less studied. In particular, children with type 1 diabetes are generally at elevated risk for more severe disease after infections, but are understudied in terms of COVID-19 and SARS-CoV-2 vaccine responses. We investigated the immunogenicity of COVID-19 mRNA vaccinations in 35 children with type 1 diabetes (T1D) and 23 controls and found that these children develop levels of SARS-CoV-2 neutralizing antibody titers and spike protein-specific T cells comparable to nondiabetic children. However, in comparing the neutralizing antibody responses in children who received 2 doses of mRNA vaccines (24 T1D; 14 controls) with those who received a third, booster dose (11 T1D; 9 controls), we found that the booster dose increased neutralizing antibody titers against ancestral SARS-CoV-2 strains but, unexpectedly, not Omicron lineage variants. In contrast, boosting enhanced Omicron variant neutralizing antibody titers in adults., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be nonneutralizing, we show that they confer protection in vivo through Fc-mediated effector functions. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines., Competing Interests: Competing interests statement:N.C.W. serves as a consultant for HeliXon. All authors declare no other competing interests.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8 + T cells are essential for combating severe infections, whereas CD4 + T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies., (© 2024. The Author(s).)

SARS-CoV-2 spike-based vaccines are used to control the COVID-19 pandemic. However, emerging variants have become resistant to antibody neutralization and further mutations may lead to full resistance. We tested whether T cells alone could provide protection without antibodies. We designed a T cell-based vaccine in which SARS-CoV-2 spike sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies, but strong specific T cell responses. We challenged mice with SARS-CoV-2 wild-type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by the SARS-CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with the SARS-CoV-2 wild-type strain or the Omicron variant were significantly reduced. Importantly, depletion of CD4+ or CD8+ T cells led to significant loss of the protection. Our analyses of spike protein sequences of the variants indicated that fewer than one-third presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in the subunit 1 region. As the subunit 2 region is conservative, the vaccines targeting spike protein are expected to protect against future variants due to the T cell responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic betacoronaviruses that continue to have a significant impact on public health. Timely development and introduction of vaccines and antivirals against SARS-CoV-2 into the clinic have substantially mitigated the burden of COVID-19. However, a limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections, respectively, calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. In this report, we examined the efficacy of two potent 3CLpro inhibitors, 5d and 11d , in fatal animal models of SARS-CoV-2 and MERS-CoV to demonstrate their broad-spectrum activity against both viral infections. These compounds significantly increased the survival of mice in both models when treatment started 1 day post infection compared to no treatment which led to 100% fatality. Especially, the treatment with compound 11d resulted in 80% and 90% survival in SARS-CoV-2 and MERS-CoV-infected mice, respectively. Amelioration of lung viral load and histopathological changes in treated mice correlated well with improved survival in both infection models. Furthermore, compound 11d exhibited significant antiviral activities in K18-hACE2 mice infected with SARS-CoV-2 Omicron subvariant XBB.1.16. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses.IMPORTANCEHuman coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) continue to have a significant impact on public health. A limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. We have previously reported a series of small-molecule 3C-like protease (3CLpro) inhibitors against human coronaviruses. In this report, we demonstrated the in vivo efficacy of 3CLpro inhibitors for their broad-spectrum activity against both SARS-CoV-2 and MERS-CoV infections using the fatal animal models. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses., Competing Interests: The authors declare no conflict of interest.

Background: Reevaluating response plans is essential to ensuring consistent readiness and resilience to the COVID-19 pandemic. The "During Action Review" and Tabletop (DART) methodology provides a retrospective and prospective assessment to inform the adaptive response. Israel introduced COVID-19 vaccinations in December 2020 and was the first country to implement booster vaccination to address waning immunity and surges caused by new variants. We assessed Israel's readiness and resilience related to COVID-19 response while capturing the pre-vaccination and vaccination periods., Methods: A DART analysis was conducted between December 2020 and August 2021 among experts involved in the management of the COVID-19 pandemic in Israel. During the retrospective stage, a role-based questionnaire and discussions were undertaken in a participant-led review of the response, focusing on epidemiology and surveillance, risk communication, and vaccines. The prospective stage included tabletop exercises to evaluate short to long-term simulated scenarios., Results: Participants emphasized the pivotal role of Israel globally by sharing experiences with the pandemic, and vaccination. Perceived strengths included multi-sectoral collaboration between the Ministry of Health, healthcare providers, academia, military, and others, stretching capacities, expanding laboratory workload, and establishing/maintaining surveillance. The vaccine prioritization plan and strong infrastructure, including computerized databases, enabled real-life assessment of vaccine uptake and impact. Challenges included the need to change case definitions early on and insufficient staffing. Quarantine of patients and contacts was particularly challenging among underprivileged communities. Risk communication approaches need to focus more on creating norms in behavior. Trust issues and limited cooperation were noted, especially among ethnic and religious minorities. To ensure readiness and resiliency, participants recommended establishing a nationally deployed system for bringing in and acting upon feedback from the field, especially concerning risk communication and vaccines., Conclusion: Our study appraised strengths and weaknesses of the COVID-19 pandemic response in Israel and led to concrete recommendations for adjusting responses and future similar events. An efficient response comprised multi-sectoral collaboration, policy design, infrastructure, care delivery, and mitigation measures, including vaccines, while risk communication, trust issues, and limited cooperation with minority groups were perceived as areas for action and intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Muhsen, Cohen, Glatman-Freedman, Husseini, Perlman and McNeil.)

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2 H353K ) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2 H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2 MA-H353K ) was prepared for detailed studies. In 7-11-month-old mACE2 H353K mice, a 10 4 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions., Competing Interests: The authors declare no conflict of interest.

Importance: The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics., Competing Interests: The University of Minnesota has filed a patent on Nanosota-2, -3 and -4 with F.L, G.Y., and B.L. as inventors. B.S. and B.E.W. are co-founders of Turkey Creek Biotechnology.

Background: As each country individually manages the COVID-19 pandemic, mass vaccination campaigns have varied considerably. Implementation campaigns often depend on nurses; however, nurses are not consistently involved in higher-level planning, prioritization, and policy development decisions. This study aimed to examine the involvement and engagement of nurses in country-level COVID-19 mass vaccination policies and practices in 10 Office of Economic Cooperation and Development countries, identify barriers and factors to enhancing the engagement of nurses in the evidence-informed mass vaccination decision-making processes, and suggest areas for improvement., Methods: A qualitative study using in-depth semi-structured interviews was conducted as a follow-up study to an International Council of Nurses survey. The study sample included a purposeful sample of 14 country-representative nurses from 10 Office of Economic Cooperation and Development countries. Interview questions focused on each country's overall COVID-19 vaccination campaign and policies, participants' perspectives regarding the involvement of nurses in the planning, design, and implementation of the mass vaccination program observed outcomes, and the impact of nursing on the outcomes. Interviews were recorded, transcribed, translated where necessary, coded, and thematically analyzed., Results: Main areas of involvement identified by participants were membership in advising and decision-making committees, operationalization planning, implementation and coordination processes, education efforts, and nurses' interactions with the media. Seven themes emerged among perceived facilitators of nursing involvement: existing systems and infrastructure, nursing profession-related skills and competencies, communication and messaging, multidisciplinary and interagency work, recognition and visibility of nurses and nursing, trust in nurses, and nursing pride. Meanwhile, perceived barriers included lack of a voice, recognition and appreciation for nursing, workforce-related challenges, decentralization of responsibility and authority, supply and access issues, downstream effects of the pandemic, and non-COVID-related nursing barriers. Three main themes emerged among suggestions made by participants for improved involvement of nursing: culture change within nursing and healthcare, opportunities and momentum to build upon, and perceptions of responsible parties., Conclusions: Nurses play a central role in providing health services but are inconsistently included in the policy, planning, and decision-making processes. Findings highlighted the critical importance of nursing leadership roles and expanded roles for nurses. Nursing should be represented by high-level leaders as part of multidisciplinary decision-making groups, educational initiatives for involvement in health policy should be implemented in nursing schools and continuing education, and advocacy and inclusion efforts should utilize bottom-up and top-down approaches concurrently., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Middle East respiratory syndrome coronavirus (MERS-CoV), a highly pathogenic coronavirus in the same Betacoronavirus genus and Coronaviridae family as SARS-CoV-2, continues to post a threat to human health. Mortality remains high; therefore, there is a need to develop effective vaccines to prevent MERS-CoV infection. The receptor-binding domain (RBD) within the MERS-CoV spike (S) protein is a critical vaccine target. The latest mRNA technology has enabled rapid development of much-needed vaccines with high efficiency and scalable manufacturing capacity. Here, we designed a mRNA vaccine encoding the RBD of MERS-CoV S protein (RBD-mRNA) and evaluated its immunogenicity and protective efficacy in a mouse model. The data showed that nucleoside-modified RBD-mRNA, but not RBD-mRNA lacking the nucleoside modification, was stable and elicited broadly and durable neutralizing antibody and cellular immune responses, which neutralized the original strain and multiple MERS-CoV variants. Among all immunization routes tested, the intradermal route was appropriate for this RBD-mRNA to induce strong B-cell responses and the highest neutralizing antibody titers. Importantly, injection of nucleoside-modified RBD-mRNA through the intradermal route protected immunized mice against challenge with MERS-CoV. This protection correlated with serum neutralizing antibody titers. Overall, we have developed an effective MERS-CoV RBD-based mRNA vaccine (with potential for further development) that prevents infection by divergent strains of MERS-CoV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

SARS-CoV-2, the causative agent of COVID-19 encodes at least 16 nonstructural proteins of variably understood function. Nsp3, the largest nonstructural protein contains several domains, including a SARS-unique domain (SUD), which occurs only in Sarbecovirus . The SUD has a role in preferentially enhancing viral translation. During isolation of mouse-adapted SARS-CoV-2, we isolated an attenuated virus that contained a single mutation in a linker region of nsp3 (nsp3-S676T). The S676T mutation decreased virus replication in cultured cells and primary human cells and in mice. Nsp3-S676T alleviated the SUD translational enhancing ability by decreasing the interaction between two translation factors, Paip1 and PABP1. We also identified a compensatory mutation in the nucleocapsid (N) protein (N-S194L) that restored the virulent phenotype, without directly binding to SUD. Together, these results reveal an aspect of nsp3-N interactions, which impact both SARS-CoV-2 replication and, consequently, pathogenesis.

Objectives: The predictors of SARS-CoV-2 reinfection are unclear. We examined predictors of reinfection with pre-Omicron and Omicron variants among COVID-19-recovered individuals., Methods: Randomly selected COVID-19-recovered patients (N = 1004) who donated convalescent plasma during 2020 were interviewed between August 2021 and March 2022 regarding COVID-19 vaccination and laboratory-proven reinfection. The sera from 224 (22.3%) participants were tested for antispike (anti-S) immunoglobulin G and neutralizing antibodies., Results: The participants' median age was 31.1 years (78.6% males). The overall reinfection incidence rate was 12.8%; 2.7% versus 21.6% for the pre-Omicron (mostly Delta) versus Omicron variants. Negative associations were found between fever during the first illness and pre-Omicron reinfection: relative risk 0.29 (95% confidence interval 0.09-0.94), high anti-N level at first illness and Omicron reinfection: 0.53 (0.33-0.85), and overall reinfection: 0.56 (0.37-0.84), as well as between subsequent COVID-19 vaccination with the BNT162b2 vaccine and pre-Omicron 0.15 (0.07-0.32), Omicron 0.48 (0.25-0.45), and overall reinfections 0.38 (0.25-0.58). These variables significantly correlated with immunoglobulin G anti-S follow-up levels. High pre-existing anti-S binding and neutralizing antibody levels against the SARS-CoV-2 Wuhan and Alpha strains predicted protection against Omicron reinfections., Conclusion: Strong immune responses after the first COVID-19 infection and subsequent vaccination with the BNT162b2 vaccine provided cross-protection against reinfections with the Delta and Omicron variants., Competing Interests: Declarations of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The SARS-CoV-2 Omicron subvariants BA.1 and BA.2 exhibit reduced lung cell infection relative to previously circulating SARS-CoV-2 variants, which may account for their reduced pathogenicity. However, it is unclear whether lung cell infection by BA.5, which displaced these variants, remains attenuated. Here, we show that the spike (S) protein of BA.5 exhibits increased cleavage at the S1/S2 site and drives cell-cell fusion and lung cell entry with higher efficiency than its counterparts from BA.1 and BA.2. Increased lung cell entry depends on mutation H69Δ/V70Δ and is associated with efficient replication of BA.5 in cultured lung cells. Further, BA.5 replicates in the lungs of female Balb/c mice and the nasal cavity of female ferrets with much higher efficiency than BA.1. These results suggest that BA.5 has acquired the ability to efficiently infect lung cells, a prerequisite for causing severe disease, suggesting that evolution of Omicron subvariants can result in partial loss of attenuation., (© 2023. The Author(s).)

Broadly protective coronavirus vaccines are an important tool for protecting against future SARS-CoV-2 variants and could play a critical role in mitigating the impact of future outbreaks or pandemics caused by novel coronaviruses. The Coronavirus Vaccines Research and Development (R&D) Roadmap (CVR) is aimed at promoting the development of such vaccines. The CVR, funded by the Bill & Melinda Gates Foundation and The Rockefeller Foundation, was generated through a collaborative and iterative process, which was led by the Center for Infectious Disease Research and Policy (CIDRAP) at the University of Minnesota and involved 50 international subject matter experts and recognized leaders in the field. This report summarizes the major issues and areas of research outlined in the CVR and identifies high-priority milestones. The CVR covers a 6-year timeframe and is organized into five topic areas: virology, immunology, vaccinology, animal and human infection models, and policy and finance. Included in each topic area are key barriers, gaps, strategic goals, milestones, and additional R&D priorities. The roadmap includes 20 goals and 86 R&D milestones, 26 of which are ranked as high priority. By identifying key issues, and milestones for addressing them, the CVR provides a framework to guide funding and research campaigns that promote the development of broadly protective coronavirus vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Galit Alter: Employed by Moderna. Dan Barouch: Co-inventor on vaccine patents that have been licensed to Janssen. Ralph Baric: Holds IP on sarbecovirus universal vaccine design, on the SAB for VacArt, and has collaborated with Adiago and NIH on Moderna mRNA vaccines. Norman Baylor: Provides regulatory advice and strategy to the regulated biopharma industry. Luciana Borio: In addition to holding a position at the Council on Foreign Relations, is a venture partner at Arch Venture Partners, an early-stage life sciences and biotechnology venture firm. Rachel Chikwamba: Has a grant with the Bill & Melinda Gates Foundation on localizing biologics manufacturing in South Africa. Cheryl Cohen: Has received grant support from Sanofi Pasteur, the US Centers for Disease Control and Prevention (CDC), Wellcome Trust, the Programme for Applied Technologies in Health (PATH), the Bill & Melinda Gates Foundation, and the South African Medical Research Council (SA-MRC). Bruce Gellin: Works for The Rockefeller Foundation, which is one of the funders of this work. Jonathan Heeney: Funded by CEPI and the Bill & Melinda Gates Foundation/Flu Lab to develop pre-pandemic vaccines for coronaviruses and influenza, respectively. Florian Krammer: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays (US Provisional Application Numbers: 62/994,252, 63/018,457, 63/020,503 and 63/024,436) and NDV-based SARS-CoV-2 vaccines (US Provisional Application Number: 63/251,020) which list FK as co-inventor. Patent applications were submitted by the Icahn School of Medicine at Mount Sinai. Mount Sinai is seeking to commercialize a mucosal NDV-based SARS-CoV-2 vaccine; therefore, the institution and its faculty inventors could benefit financially. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. FK has consulted for Merck, Seqirus, Curevac, and Pfizer, and is currently consulting for Pfizer, Third Rock Ventures, Merck, and Avimex. The FK laboratory is also collaborating with Pfizer on animal models for SARS-CoV-2. Teresa Lambe: Vaccine Taskforce via NIHR Support: Grant to support the running of the trial paid to University of Oxford. AstraZeneca Support: Support for medical writing. Vaccitech Consultant Fees for an unrelated project. Seqirus honoraria: Meeting relating to influenza meeting—unrelated work. Named as an inventor on a patent application for a vaccine against SARS CoV-2. Jason McLellan: Is an inventor on patents and patent applications regarding coronavirus vaccines and antibodies. Angela Mehr: Holds small amount of shares in AstraZeneca and Moderna. Kayvon Modjarrad: Current affiliation is as an employee of Pfizer, Inc. Peter Openshaw: Has participated in scientific advisory boards for GSK, Moderna, Janssen, Seqirus, and Pfizer. Peter Paradiso: Consultant to Pfizer, Member of Board of Directors at Dynavax. Stanley Plotkin: Consultant to Moderna, Sanofi, Merck, Janssen, Inovio, NTx Bio, Codagenix, Vaxinnity, Valneva, Meissa, and Rational. Gregory Poland: Offers consultative advice on COVID-19 vaccine development to AstraZeneca, Pfizer, Medicago, Johnson&Johnson/Janssen, Novavax, and Moderna. GAP has received grant funding from ICW Ventures for preclinical studies on a peptide-based COVID-19 vaccine for which he holds a patent. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with Mayo Clinic Conflict of Interest policies. Andrew Pollard: Oxford University has an agreement with AstraZeneca for development of a COVID-19 vaccine. AJP led the clinical development of the Oxford-AstraZeneca vaccine. He is chair of the UK Government's Joint Committee on Vaccination and Immunisation, but does not participate in the COVID-19 committee. Melanie Saville: Has shares with Sanofi, a vaccine company; is an employee of CEPI, an organization that funds SARS CoV 2 and broadly protective coronavirus vaccine development. Lin-fa Wang: Co-inventor of patents on test, vaccine, and monoclonal antibodies for SARS-related coronaviruses. Daniela Weiskopf: The La Jolla Institute for Immunology has filed for patent protection for various aspects of T-cell epitope and vaccine design work. E. John Wherry: Is a member of the Parker Institute for Cancer Immunotherapy. EJW is an advisor for Danger Bio, Janssen, Merck, Marengo, New Limit, Pluto Immunotherapeutics, Related Sciences, Santa Ana Bio, Synthekine, and Surface Oncology. EJW is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. Michael Worobey: Has received consulting fees on SARS-CoV-2 and the COVID-19 pandemic.], (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium., (© 2023 New York Academy of Sciences.)

Coronaviruses (CoVs) of genera α, β, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication., (© 2022 Wiley Periodicals LLC.)

Loss of olfactory function has been commonly reported in SARS-CoV-2 infections. Recovery from anosmia is not well understood. Previous studies showed that sustentacular cells, and occasionally olfactory sensory neurons (OSNs) in the olfactory epithelium (OE), are infected in SARS-CoV-2-infected patients and experimental animals. Here, we show that SARS-CoV-2 infection of sustentacular cells induces inflammation characterized by infiltration of myeloid cells to the olfactory epithelium and variably increased expression of proinflammatory cytokines. We observed widespread damage to, and loss of cilia on, OSNs, accompanied by downregulation of olfactory receptors and signal transduction molecules involved in olfaction. A consequence of OSN dysfunction was a reduction in the number of neurons in the olfactory bulb expressing tyrosine hydroxylase, consistent with reduced synaptic input. Resolution of the infection, inflammation, and olfactory dysfunction occurred over 3-4 weeks following infection in most but not all animals. We also observed similar patterns of OE infection and anosmia/hyposmia in mice infected with other human coronaviruses such as SARS-CoV and MERS-CoV. Together, these results define the downstream effects of sustentacular cell infection and provide insight into olfactory dysfunction in COVID-19-associated anosmia.

We propose the term "common cold coronaviruses," or ccCoV, to describe the four human coronaviruses commonly associated with upper respiratory tract disease - coronaviruses 229E, OC43, NL63, and HKU1. This will differentiate these previously described coronaviruses from those causing more severe disease in humans - including the viruses severe acute respiraotry syndrome coronavirus (SARS-CoV), the Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2., Competing Interests: Potential conflicts of interest. S. P. has received research support from the National Institutes of Health (grant numbers PO1 AI060699 and R01AI AI129269) and from BioAge and ATI. J. A. E. has been a consultant for Sanofi Pasteur, Meissa Vaccines, Moderna, and AstraZeneca, and receives research support from AstraZeneca, GlaxoSmithKline, Merck, and Pfizer, unrelated to this topic. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

COVID-19 is the latest zoonotic RNA virus epidemic of concern. Learning how it began and spread will help to determine how to reduce the risk of future events. We review major RNA virus outbreaks since 1967 to identify common features and opportunities to prevent emergence, including ancestral viral origins in birds, bats, and other mammals; animal reservoirs and intermediate hosts; and pathways for zoonotic spillover and community spread, leading to local, regional, or international outbreaks. The increasing scientific evidence concerning the origins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is most consistent with a zoonotic origin and a spillover pathway from wildlife to people via wildlife farming and the wildlife trade. We apply what we know about these outbreaks to identify relevant, feasible, and implementable interventions. We identify three primary targets for pandemic prevention and preparedness: first, smart surveillance coupled with epidemiological risk assessment across wildlife-livestock-human (One Health) spillover interfaces; second, research to enhance pandemic preparedness and expedite development of vaccines and therapeutics; and third, strategies to reduce underlying drivers of spillover risk and spread and reduce the influence of misinformation. For all three, continued efforts to improve and integrate biosafety and biosecurity with the implementation of a One Health approach are essential. We discuss new models to address the challenges of creating an inclusive and effective governance structure, with the necessary stable funding for cross-disciplinary collaborative research. Finally, we offer recommendations for feasible actions to close the knowledge gaps across the One Health continuum and improve preparedness and response in the future.

Multiple SARS-CoV-2 variants are identified with higher rates of transmissibility or greater disease severity. Particularly, recent emergence of Omicron variant with rapid human-to-human transmission posts new challenges to the current prevention strategies. In this study, following vaccination with an mRNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD-mRNA), we detected serum antibodies that neutralized pseudoviruses expressing spike (S) protein harboring single or multiple mutations, as well as authentic SARS-CoV-2 variants, and evaluated its protection against SARS-CoV-2 infection. The vaccine induced durable antibodies that potently neutralized prototypic strain and B.1.1.7 lineage variant pseudoviruses containing N501Y or D614G mutations alone or in combination with a N439K mutation (B.1.258 lineage), with a L452R mutation (B.1.427 or B.1.429 lineage), or a L452R-E484Q double mutation (B.1.617.1 variant), although neutralizing activity against B.1.1.7 lineage variant containing 10 amino acid changes in the S protein was slightly reduced. The RBD-mRNA-induced antibodies exerted moderate neutralization against authentic B.1.617.2 and B.1.1.529 variants, and pseudotyped B.1.351 and P.1 lineage variants containing K417N/T, E484K, and N501Y mutations, the B.1.617.2 lineage variant harboring L452R, T478K, and P681R mutations, and the B.1.1.529 lineage variant containing 38 mutations in the S protein. Particularly, RBD-mRNA vaccine completely protected mice from challenge with a virulent mouse-adapted SARS-CoV-2 variant. Among these lineages, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 belong to Alpha, Beta, Gamma, Delta, and Omicron variants, respectively. Our observations reveal that RBD-mRNA vaccine is promising and highlights the need to design novel vaccines with improved neutralization against current and future pandemic SARS-CoV-2 variants., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Safe, passive immunization methods are required against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants. Immunization of chickens with antigen is known to induce specific IgY antibodies concentrated in the egg yolk and has a good safety profile, high yield of IgY per egg, can be topically applied, not requiring parenteral delivery. Our data provide the first evidence of the prophylactic efficacy of Immunoglobulin Y antibodies against SARS-CoV-2 in mice. Lohmann hens were injected with recombinant SARS-CoV-2 RBD protein; IgY-Abs were extracted from the eggs and characterized using SDS-PAGE. Antiviral activity was evaluated using plaque reduction neutralization tests. In additional experiments, IgY-RBD efficacy was examined in mice sensitized to SARS-CoV-2 infection by transduction with Ad5-hACE2 (mild disease) or by using mouse-adapted virus (severe disease). In both cases, prophylactic intranasal administration of IgY-Abs reduced SARS-CoV-2 replication, and reduced morbidity, inflammatory cell infiltration, hemorrhage, and edema in the lungs and increased survival compared to control groups that received non-specific IgY-Abs. These results indicate that further evaluation of IgY-RBD antibodies in humans is warranted., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2 has mutated frequently since its first emergence in 2019. Numerous variants, including the currently emerging Omicron variant, have demonstrated high transmissibility or increased disease severity, posing serious threats to global public health. This study describes the identification of an immunodominant non-neutralizing epitope on SARS-CoV-2 receptor-binding domain (RBD). A subunit vaccine against this mutant RBD, constructed by masking this epitope with a glycan probe, did not significantly affect RBD's receptor-binding affinity or antibody-binding affinity, or its ability to induce antibody production. However, this vaccine enhanced the neutralizing activity of this RBD and its protective efficacy in immunized mice. Specifically, this vaccine elicited significantly higher-titer neutralizing antibodies than the prototypic RBD protein against Alpha (B.1.1.7 lineage), Beta (B.1.351 lineage), Gamma (P.1 lineage), and Epsilon (B.1.427 or B.1.429 lineage) variant pseudoviruses containing single or combined mutations in the spike (S) protein, albeit the neutralizing antibody titers against some variants were slightly lower than against original SARS-CoV-2. This vaccine also significantly improved the neutralizing activity of the prototypic RBD against pseudotyped and authentic Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants, although the neutralizing antibody titers were lower than against original SARS-CoV-2. In contrast to the prototypic RBD, the mutant RBD completely protected human ACE2 (hACE2)-transgenic mice from lethal challenge with a prototype SARS-CoV-2 strain and a Delta variant without weight loss. Overall, these findings indicate that this RBD vaccine has broad-spectrum activity against multiple SARS-CoV-2 variants, as well as the potential to be effective and have improved efficacy against Omicron and other pandemic variants. IMPORTANCE Several SARS-CoV-2 variants have shown increased transmissibility, calling for a need to develop effective vaccines with broadly neutralizing activity against multiple variants. This study identified a non-neutralizing epitope on the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, and further shielded it with a glycan probe. A subunit vaccine based on this mutant RBD significantly enhanced the ability of prototypic RBD against multiple SARS-CoV-2 variants, including the Delta and Omicron strains, although the neutralizing antibody titers against some of these variants were lower than those against original SARS-CoV-2. This mutant vaccine also enhanced the protective efficacy of the prototypic RBD vaccine against SARS-CoV-2 infection in immunized animals. In conclusion, this study identified an engineered RBD vaccine against Omicron and other SARS-CoV-2 variants that induced stronger neutralizing antibodies and protection than the original RBD vaccine. It also highlights the need to improve the effectiveness of current COVID-19 vaccines to prevent pandemic SARS-CoV-2 variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuing to spread globally, contributing to the persistence of the COVID-19 pandemic. Increasing resources have been focused on developing vaccines and therapeutics that target the Spike glycoprotein of SARS-CoV-2. Recent advances in microfluidics have the potential to recapitulate viral infection in the organ-specific platforms, known as organ-on-a-chip (OoC), in which binding of SARS-CoV-2 Spike protein to the angiotensin-converting enzyme 2 (ACE2) of the host cells occurs. As the COVID-19 pandemic lingers, there remains an unmet need to screen emerging mutations, to predict viral transmissibility and pathogenicity, and to assess the strength of neutralizing antibodies following vaccination or reinfection. Conventional detection of SARS-CoV-2 variants relies on two-dimensional (2-D) cell culture methods, whereas simulating the micro-environment requires three-dimensional (3-D) systems. To this end, analyzing SARS-CoV-2-mediated pathogenicity via microfluidic platforms minimizes the experimental cost, duration, and optimization needed for animal studies, and obviates the ethical concerns associated with the use of primates. In this context, this review highlights the state-of-the-art strategy to engineer the nano-liposomes that can be conjugated with SARS-CoV-2 Spike mutations or genomic sequences in the microfluidic platforms; thereby, allowing for screening the rising SARS-CoV-2 variants and predicting COVID-19-associated coagulation. Furthermore, introducing viral genomics to the patient-specific blood accelerates the discovery of therapeutic targets in the face of evolving viral variants, including B1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), c.37 (Lambda), and B.1.1.529 (Omicron). Thus, engineering nano-liposomes to encapsulate SARS-CoV-2 viral genomic sequences enables rapid detection of SARS-CoV-2 variants in the long COVID-19 era., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Rheumatic diseases patients receiving Rituximab had severe COVID-19 disease. Although they had impaired humoral immune responses following COVID-19 vaccine, they had preserved cellular immune responses. Waning of COVID-19 antibody responses was observed within six months post vaccination among immunocompromised patients. Recent reports showed fatal outcome of breakthrough SARS-CoV-2 infections among vaccinated high-risk rheumatic diseases patients receiving Rituximab. SAR-CoV-2 serological tests were not performed., Objective: Evaluation of COVID-19 vaccine humoral responses and breakthrough infections among low risk fully vaccinated rheumatic patients during the Delta Variant Era., Methods: A case series of 19 fully vaccinated patients with rheumatic diseases were followed to determine post vaccine SARS-CoV-2 neutralizing antibody titers and to monitor the development of breakthrough infections up to eight months post vaccine at our tertiary care center in Jeddah, Saudi Arabia from 1st April until 30th November 2021., Results: The mean age of patients was 49 years old. 10% of patients were receiving Rituximab. 73% of patients had positive SARS-CoV-2 serological testing post second vaccine. Two mild breakthrough COVID-19 infections were diagnosed six months post second dose of vaccine. Patients were less than 65 years, did not receive Rituximab, did not have interstitial lung diseases and had positive post vaccine serological testing., Conclusions: We demonstrated high SARS-CoV-2 neutralizing antibodies seroprevalence and self-limiting breakthrough infections in low risk rheumatic diseases patients during the Delta Era. Future studies are needed to study the outcome of rheumatic diseases patients in the Era of Omicron in view of viral immune escape responses., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways—interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)—activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung–derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Repeated serosurveys are an important tool for understanding trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination. During 1 September 2020-20 March 2021, the NYC Health Department conducted a population-based SARS-CoV-2 antibody prevalence survey of 2096 NYC adults who either provided a blood specimen or self-reported the results of a previous antibody test. The serosurvey, the second in a series of surveys conducted by the NYC Health Department, aimed to estimate SARS-CoV-2 antibody prevalence across the city and for different groups at higher risk for adverse health outcomes. Weighted citywide prevalence was 23.5% overall (95% confidence interval (CI) 20.1-27.4) and increased from 19.2% (95% CI 14.7-24.6) before coronavirus disease 2019 vaccines were available to 31.3% (95% CI 24.5-39.0) during the early phases of vaccine roll-out. We found no differences in antibody prevalence by age, race/ethnicity, borough, education, marital status, sex, health insurance coverage, self-reported general health or neighbourhood poverty. These results show an overall increase in population-level seropositivity in NYC following the introduction of SARS-CoV-2 vaccines and highlight the importance of repeated serosurveys in understanding the pandemic's progression.

Importance: The rise of novel, more infectious SARS-CoV-2 variants has made clear the need to rapidly deploy large-scale testing for COVID-19 to protect public health. However, testing remains limited due to shortages of personal protective equipment (PPE), naso- and oropharyngeal swabs, and healthcare workers. Simple test methods are needed to enhance COVID-19 screening. Here, we describe a simple, and inexpensive spit-test for COVID-19 screening called Patient Self-Collection of Sample-CoV2 ( PSCS-CoV2 )., Objective: To evaluate an affordable and convenient test for COVID-19., Methods: The collection method relies on deep throat sputum (DTS) self-collected by the subject without the use of swabs, and was hence termed the Self-Collection of Sample for SARS-CoV-2 (abbreviated PSCS-CoV2 ). We used a phenol-chloroform extraction method for the viral RNA. We then tested for SARS-CoV-2 using real-time reverse transcription polymerase chain reaction with primers against at least two coding regions of the viral nucleocapsid protein (N1 and N2 or E) of SARS-CoV-2. We evaluted the sensitivity and specificity of our protocol. In addition we assess the limit of detection, and efficacy of our Viral Inactivating Solution. We also evaluated our protocol, and pooling strategy from volunteers on a local college campus., Results: We show that the PSCS-CoV2 method accurately identified 42 confirmed COVID-19 positives, which were confirmed through the nasopharyngeal swabbing method of an FDA approved testing facility. For samples negative for COVID-19, we show that the cycle threshold for N1, N2, and RP are similar between the PSCS-CoV2 and nasopharynx swab collection method ( n  = 30). We found a sensitivity of 100% (95% Confidence Interval [CI], 92-100) and specifity of 100% (95% CI, 89-100) for our PSCS-CoV2 method. We determined our protocol has a limit of detection of 1/10,000 for DTS from a COVID-19 patient. In addition, we show field data of the PSCS-CoV2 method on a college campus. Ten of the twelve volunteers (N1 < 30) that we tested as positive were subsequently tested positive by an independent laboratory. Finally, we show proof of concept of a pooling strategy to test for COVID-19, and recommend pool sizes of four if the positivity rate is less than 15%., Conclusion and Relevance: We developed a DTS-based protocol for COVID-19 testing with high sensitivity and specificity. This protocol can be used by non-debilitated adults without the assistance of another adult, or by non-debilitated children with the assistance of a parent or guardian. We also discuss pooling strategies based on estimated positivity rates to help conserve resources, time, and increase throughput. The PSCS-CoV2 method can be a key component of community-wide efforts to slow the spread of COVID-19., Competing Interests: The authors declare there are no competing interests., (©2022 Huang et al.)

SARS-CoV-2 continues to evolve into variants of concern (VOC), with greatest variability in the multidomain, entry-facilitating spike proteins. To recognize the significance of adaptive spike protein changes, we compare variant SARS-CoV-2 virus particles in several assays reflecting authentic virus-cell entry. Virus particles with adaptive changes in spike amino-terminal domains (NTDs) are hypersensitive to proteolytic activation of membrane fusion, an essential step in virus-cell entry. Proteolysis is within fusion domains (FDs), at sites over 10 nm from the VOC-specific NTD changes, indicating allosteric inter-domain control of fusion activation. In addition, NTD-specific antibodies block FD cleavage, membrane fusion, and virus-cell entry, suggesting restriction of inter-domain communication as a neutralization mechanism. Finally, using structure-guided mutagenesis, we identify an inter-monomer β sheet structure that facilitates NTD-to-FD transmissions and subsequent fusion activation. This NTD-to-FD axis that sensitizes viruses to infection and to NTD-specific antibody neutralization provides new context for understanding selective forces driving SARS-CoV-2 evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures., (© 2022. Springer Nature Limited.)

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations 1 . Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility 2 . The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern 2 . Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D 2 (PGD 2 )) and a phospholipase (phospholipase A2 group 2D (PLA 2 G2D)) contributed to poor outcomes in aged mice 3,4 . mRNA expression of PLA 2 G2D and prostaglandin D 2 receptor (PTGDR), and production of PGD 2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA 2 G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA 2 G2D-PGD 2 /PTGDR pathway is a useful target for therapeutic interventions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The recent emergence of B.1.1.529, the Omicron variant 1,2 , has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs.  3,4 ), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data., (© 2022. The Author(s).)

The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2'-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5' cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5' cap in vitro . Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHV nsp14-Y414A ) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHV nsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHV nsp14-Y414A mutant was restored in Ifnar -/- mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2 nsp14-Y420A ) also significantly decreased N7-methyltransferase activity in vitro , and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2 nsp14-Y420A Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development.

The Middle East Respiratory Syndrome-coronavirus (MERS-CoV) is the second of three zoonotic coronaviruses to infect humans since 2002, causing severe pneumonia. Unlike SARS-CoV-1 and SARS-CoV-2, the causes of the severe acute respiratory syndrome and Covid-19, respectively, MERS-CoV is enzootic in dromedary camels, a domestic/companion animal present across Africa, the Middle East and Central or South Asia and is sporadically transmitted to humans. However, it does not transmit readily from human to human except in hospital and household settings. Human MERS disease is reported only from the Arabian Peninsula (and only since 2012 even though the virus was detected in camels from at least the early 1990's) and in travelers from this region. Remarkably, no zoonotic MERS disease has been detected in Africa or Asia, even in areas of high density of MERS-CoV infected dromedaries. Here, we review aspects of MERS biology and epidemiology that might contribute to this lack of correlation between sites of camel infection and human zoonotic disease. Since MERS-CoV or MERS-like CoV have pandemic potential, further investigations into this disparity is critical, to forestall pandemics caused by this virus., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has resulted in a pandemic that has had widespread effects on human activities. The clinical presentation of severe COVID-19 includes a broad spectrum of clinical disease, most notably acute respiratory distress syndrome, cytokine release syndrome (CRS), multiorgan failure, and death. Direct viral damage and uncontrolled inflammation have been suggested as contributory factors in COVID-19 disease severity. The COVID-19 pandemic has emphasized the critical role of an effective host immune response in controlling a virus infection and demonstrated the devastating effect of immune dysregulation. Understanding the nature of the immune response to SARS-CoV-2 pathogenesis is key to developing effective treatments for COVID-19. Here, we describe the nature of the dysregulated host immune response in COVID-19, identify potential mechanisms involved in CRS, and discuss potential strategies that can be used to manage immune dysregulation in COVID-19.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age., Competing Interests: The authors have declared that no competing interests exist.

Human coronaviruses cause a wide spectrum of disease, ranging from mild common colds to acute respiratory distress syndrome and death. Three highly pathogenic human coronaviruses - severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and SARS-CoV-2 - have illustrated the epidemic and pandemic potential of human coronaviruses, and a better understanding of their disease-causing mechanisms is urgently needed for the rational design of therapeutics. Analyses of patients have revealed marked dysregulation of the immune system in severe cases of human coronavirus infection, and there is ample evidence that aberrant immune responses to human coronaviruses are typified by impaired induction of interferons, exuberant inflammatory responses and delayed adaptive immune responses. In addition, various viral proteins have been shown to impair interferon induction and signalling and to induce inflammasome activation. This suggests that severe disease associated with human coronaviruses is mediated by both dysregulated host immune responses and active viral interference. Here we discuss our current understanding of the mechanisms involved in each of these scenarios., (© 2021. Springer Nature Limited.)

Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a 10-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads . Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva., (© 2021 ABRF.)