In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age.

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.

Select Agent Regulatory Challenges in a Patient Care Setting: Review and Recommendations.

The Federal Select Agent Program ensures the safe and secure possession, use, and transfer of biological select agents and toxins through the select agent regulations (42 CFR §73, 7 CFR §331, and 9 CFR §121). These regulations are primarily written for interpretation by diagnostic and research laboratories, with limited text pertaining to the care of individuals infected with a select agent. The regulations applicable to patient care settings are ambiguous, resulting in challenges with regulatory compliance. The COVID-19 pandemic called attention to these shortcomings and the need to clarify and modify the select agent regulations. In this article, we discuss 3 select agent regulation phrases regarding patient care that need clarification-specifically, the window of time to transfer, patient care setting, and conclusion of patient care-and provide recommendations for improvement. These recommendations include implementing minimum security standards to safeguard patient specimens against theft, loss, or release prior to the appropriate transfer or destruction of the material and increasing the time allowed for the transfer or destruction of specimens before entities are subject to the select agent regulations. We encourage the Federal Select Agent Program to release a policy statement clarifying the select agent regulations regarding patient care discussed herein and to lengthen the designated time to destroy or transfer agents to a registered entity. Addressing these challenges will aid in compliance with the select agent regulations in patient care settings.

GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic disease in humans. There is a great need for effective vaccines and therapeutics against CCHFV for humans, as none are currently internationally approved. Recently, a monoclonal antibody against the GP38 glycoprotein protected mice against lethal CCHFV challenge. To show that GP38 is required and sufficient for protection against CCHFV, we used three inactivated rhabdoviral-based CCHFV-M vaccines, with or without GP38 in the presence or absence of the other CCHFV glycoproteins. All three vaccines elicited strong antibody responses against the respective CCHFV glycoproteins. However, only vaccines containing GP38 showed protection against CCHFV challenge in mice; vaccines without GP38 were not protective. The results of this study establish the need for GP38 in vaccines targeting CCHFV-M and demonstrate the efficacy of a CCHFV vaccine candidate based on an established vector platform.

In silico Design of a Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Multi-Epitope Antigen for Vaccine Development.

Objective: There is no licensed vaccine available to prevent the severe tick-borne disease Crimean-Congo hemorrhagic fever (CCHF), caused by the CCHF virus (CCHFV). This study sought to show that a combination of computational methods and data from published literature can inform the design of a multi-epitope antigen for CCHFV that has the potential to be immunogenic. Methods: Cytotoxic and helper T-cell epitopes were evaluated on the CCHFV GPC using bioinformatic servers, and this data was combined with work from previous studies to identify potentially immunodominant regions of the GPC. Regions of the GPC were selected for generation of a model multi-epitope antigen in silico, and the percent residue identity and similarity of each region was compared across sequences representing the widespread geographical and ecological distribution of CCHFV. Results: Eleven multi-epitope regions were joined together with flexible linkers in silico to generate a model multi-epitope antigen, termed EPIC, which included 812 (75.7%) of all predicted epitopes. EPIC was predicted to be antigenic by two independent bioinformatic servers, suggesting that multi-epitope antigens should be explored further for CCHFV vaccine development. Conclusion: The results presented within this manuscript provide information for potential targets within the CCHFV GPC for guiding future vaccine development.

Design and evaluation of neutralizing and fusion inhibitory peptides to Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.

Crimean-Congo hemorrhagic fever virus in tortoises and Hyalomma aegyptium ticks in East Thrace, Turkey: potential of a cryptic transmission cycle.

BACKGROUND: Recent reports have demonstrated the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) genomic material in Hyalomma aegyptium ticks feeding primarily on tortoises belonging to the genus Testudo. This raises the question if these ticks and their hosts play a role in the natural transmission dynamics of CCHFV. However, the studies are limited, and assessing the relevance of H. aegyptium in perpetuating the virus in nature, and a potential spillover to humans remains unknown. This study aimed to detect CCHFV in H. aegyptium ticks and their tortoise hosts in the East Thrace region of Turkey, where H. aegyptium is the most common human-biting tick and where a high density of tortoises of the genus Testudo can be found. METHODS: During the study period, 21 blood samples from different tortoises (2 T. hermanni and 19 T. graeca), 106 tick pools (containing 448 males, 152 females, 93 nymphs and 60 larvae) collected from 65 tortoises (5 T. hermanni and 60 T. graeca), 38 adult unfed questing ticks (25 males and 13 females, screened individually) and 14 pools (containing 8 nymphs and 266 larvae) of immature unfed questing ticks collected from the ground were screened for CCHFV genome by nested PCR and partial genomes sequenced. RESULTS: As a result of the screening of these 179 samples, 17 (9.5%) were detected as positive as follows: 2 of 21 blood samples (9.52%), 13 (containing 18 nymphs in 3 pools, and 52 males and 8 females in 10 pools) of 106 tick pools from tortoises (12.26%), and 2 of 38 adult questing ticks (5.26%). No positive result was determined in 14 pools of immature questing ticks. CONCLUSIONS: Previous studies have shown that reptiles can participate in the transmission of arthropod-borne viruses, but they may contribute to different aspects of the disease ecology and evolution of tick-borne viral pathogens. Our results indicate the presence of CCHFV in questing and feeding H. aegyptium ticks as well as tortoise hosts. This may indicate that CCHFV circulates in a cryptic transmission cycle in addition to the primary transmission cycle that could play a role in the natural dynamic of the virus and the transmission to humans.

Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain.

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

In vivo antiviral host response to SARS-CoV-2 by viral load, sex, and age.

Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3 , and Th1 chemokines CXCL9/10/11 , as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2 , increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3 , as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-κB signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.