The mRNA vaccine, a swift warhead against a moving infectious disease target.

INTRODUCTION: The rapid development of mRNA vaccines against SARS-CoV-2 has revolutionized vaccinology, offering hope for swift responses to emerging infectious diseases. Initially met with skepticism, mRNA vaccines have proven effective and safe, reducing vaccine hesitancy amid the evolving COVID-19 pandemic. The COVID-19 pandemic has demonstrated that the time required to modify mRNA vaccines to counter new mutant strains is significantly shorter than the time it takes for pathogens to mutate and generate new variants that can thrive in vaccinated populations. This highlights the notion that mRNA vaccine technology appears to be outpacing viruses in the ongoing evolutionary race. AREAS COVERED: This review article offers valuable insights into several crucial aspects of mRNA vaccine development and deployment, including the fundamentals of mRNA vaccine design and synthesis, the utilization of delivery systems, considerations regarding vaccine safety, the longevity of the immune response, strategies for modifying the original mRNA vaccine to address emerging mutant strains, as well as addressing vaccine hesitancy and potential approaches to mitigate reluctance. EXPERT OPINION: Challenges such as stability, storage, manufacturing complexities, production capacity, allergic reactions, long-term effects, accessibility, and misinformation must be addressed. Despite these hurdles, mRNA vaccine technology holds promise for revolutionizing future vaccination strategies.

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.

From Tick to Test: A Comprehensive Review of Tick-Borne Disease Diagnostics and Surveillance Methods in the United States.

Tick-borne diseases (TBDs) have become a significant public health concern in the United States over the past few decades. The increasing incidence and geographical spread of these diseases have prompted the implementation of robust surveillance systems to monitor their prevalence, distribution, and impact on human health. This comprehensive review describes key disease features with the geographical distribution of all known tick-borne pathogens in the United States, along with examining disease surveillance efforts, focusing on strategies, challenges, and advancements. Surveillance methods include passive and active surveillance, laboratory-based surveillance, sentinel surveillance, and a One Health approach. Key surveillance systems, such as the National Notifiable Diseases Surveillance System (NNDSS), TickNET, and the Tick-Borne Disease Laboratory Network (TBDLN), are discussed. Data collection and reporting challenges, such as underreporting and misdiagnosis, are highlighted. The review addresses challenges, including lack of standardization, surveillance in non-human hosts, and data integration. Innovations encompass molecular techniques, syndromic surveillance, and tick surveillance programs. Implications for public health cover prevention strategies, early detection, treatment, and public education. Future directions emphasize enhanced surveillance networks, integrated vector management, research priorities, and policy implications. This review enhances understanding of TBD surveillance, aiding in informed decision-making for effective disease prevention and control. By understanding the current surveillance landscape, public health officials, researchers, and policymakers can make informed decisions to mitigate the burden of (TBDs).

Development of a Syndromic Molecular Diagnostic Assay for Tick-Borne Pathogens Using Barcoded Magnetic Bead Technology.

Infectious disease diagnostics often depend on costly serological testing with poor sensitivity, low specificity, and long turnaround time. Here, we demonstrate proof of the principle for simultaneous detection of two tick-borne pathogens from a single test sample using barcoded magnetic bead technology on the BioCode 2500 system. Specific primer sets complementary to the conserved genes of Anaplasma phagocytophilum and Borrelia burgdorferi were used in PCR amplification of the target, followed by the hybridization of the resulting biotinylated PCR products with specific probes tethered to the barcoded magnetic beads for simultaneous detection, using a fluorophore with high quantum yield. The assay has an extremely high signal to background ratio, with a limit of detection (LOD) of 2.81 50% tissue culture infection dose (TCID50)/mL and 1 CFU/mL for A. phagocytophilum and B. burgdorferi, respectively. The observed LOD for gene blocks was 1.8 copies/reaction for both the pathogens. The assay demonstrated 100% positive and negative agreement on performance evaluation using patient specimens and blood samples spiked with 1 × LOD of pathogen stock. No cross-reactivity was observed with other related tick-borne pathogens and genomic DNA of human, cattle, and canine origin. The assay can be upgraded to a sensitive and cost-effective multiplex diagnostic approach that can simultaneously detect multiple clinically important tick-borne pathogens in a single sample with a short turnaround time. IMPORTANCE The low pathogen load in the tick-borne disease test samples and the lack of highly sensitive multiplex diagnostic approaches have impacted diagnosis during clinical testing and limited surveillance studies to gauge prior insight about the prevalence of tick-borne infections in a geographical area. This article demonstrates proof of the principle for simultaneous detection of two important tick-borne pathogens from a single test sample using digital barcoded magnetic bead technology. Using a fluorophore of high quantum yield, the diagnostic approach showed high sensitivity and specificity. The LOD was 1.8 genome copies per reaction for both A. phagocytophilum and B. burgdorferi. The assay can be upgraded for the detection of all clinically important tick-borne pathogens from a single patient sample with high sensitivity and specificity. The assay can provide a diagnostic answer to the clinician in a short turnaround time to facilitate speedy therapeutic intervention to infected patients and implement public health measures to prevent community spread.

SARS-CoV-2 Nucleocapsid Protein Is a Potential Therapeutic Target for Anticoronavirus Drug Discovery.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CLpro, and Mpro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.

A novel compound targets the feline infectious peritonitis virus nucleocapsid protein and inhibits viral replication in cell culture.

Feline infectious peritonitis (FIP) is a serious viral illness in cats, caused by feline coronavirus. Once a cat develops clinical FIP, the prognosis is poor. The effective treatment strategy for coronavirus infections with immunopathological complications such as SARS-CoV-2, MERS, and FIP is focused on antiviral and immunomodulatory agents to inhibit virus replication and enhance the protective immune response. In this article we report the binding and conformational alteration of feline alphacoronavirus (FCoV) nucleocapsid protein by a novel compound K31. K31 noncompetitively inhibited the interaction between the purified nucleocapsid protein and the synthetic 5' terminus of viral genomic RNA in vitro. K31 was well tolerated by cells and inhibited FCoV replication in cell culture with a selective index of 115. A single dose of K31inhibited FCoV replication to an undetectable level in 24 h post treatment. K31 did not affect the virus entry to the host cell but inhibited the postentry steps of virus replication. The nucleocapsid protein forms ribonucleocapsid in association with the viral genomic RNA that serves as a template for transcription and replication of the viral genome. Our results show that K31 treatment disrupted the structural integrity of ribonucleocapsid in virus-infected cells. After the COVID-19 pandemic, most of the antiviral drug development strategies have focused on RdRp and proteases encoded by the viral genome. Our results have shown that nucleocapsid protein is a druggable target for anticoronavirus drug discovery.

Purification of Crimean Congo Hemorrhagic Fever Virus (CCHFV) Nucleocapsid Protein Using Detergent Gradient and Free Thawing.

Protein aggregation remains a major challenge in the purification of recombinant proteins in both eukaryotic and prokaryotic expression systems. One such protein is the nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV), which has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the detergent gradient purification approach reported here, the freshly purified protein by NiNTA chromatography was mixed with the dilution buffer containing a high detergent concentration, followed by overnight freezing at -80 °C. Thawing the resulting mixture at room temperature triggered the formation of a detergent concentration gradient containing the active protein in the low detergent concentration zone towards the top of the gradient. The inactive aggregates migrated to the high detergent concentration zone towards the bottom of the gradient. The method prevented further aggregation and retained the activity of the native protein despite numerous freeze-thaw cycles. This simple approach creates an appropriate microenvironment towards the top of the gradient for correctly folded proteins, and it prevents aggregation by rapidly separating the preformed early aggregates from the correctly folded protein in the mixture. This unique approach will be of potential importance for the biotechnological industry, as well as other fields of protein biochemistry that routinely purify recombinant proteins and face the challenges of protein aggregation. Graphical abstract.

Current and Future Molecular Diagnostics of Tick-Borne Diseases in Cattle.

Ticks and tick-borne diseases such as babesiosis, anaplasmosis, ehrlichiosis, Lyme disease, Crimean Congo hemorrhagic fever, and Rocky Mountain spotted fever pose a significant threat to animal and human health. Tick-borne diseases cause billions of dollars of losses to livestock farmers annually. These losses are partially attributed to the lack of sensitive, robust, cost effective and efficient diagnostic approaches that could detect the infectious pathogen at the early stages of illness. The modern nucleic acid-based multiplex diagnostic approaches have been developed in human medicine but are still absent in veterinary medicine. These powerful assays can screen 384 patient samples at one time, simultaneously detect numerous infectious pathogens in each test sample and provide the diagnostic answer in a few hours. Development, commercialization, and wide use of such high throughput multiplex molecular assays in the cattle tick-borne disease surveillance will help in early detection and control of infectious pathogens in the animal reservoir before community spread and spillover to humans. Such approaches in veterinary medicine will save animal life, prevent billions of dollars of economic loss to cattle herders and reduce unwanted stress to both human and animal health care systems. This literature review provides recent updates on molecular diagnostics of tick-borne pathogens and discusses the importance of modern nucleic acid high throughput multiplex diagnostic approaches in the prevention of tick-borne infection to livestock.

A novel approach for the purification of aggregation prone proteins.

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

Hantaviruses use the endogenous host factor P58IPK to combat the PKR antiviral response.

Hantavirus nucleocapsid protein (NP) inhibits protein kinase R (PKR) dimerization by an unknown mechanism to counteract its antiviral responses during virus infection. Here we demonstrate that NP exploits an endogenous PKR inhibitor P58IPK to inhibit PKR. The activity of P58IPK is normally restricted in cells by the formation of an inactive complex with its negative regulator Hsp40. On the other hand, PKR remains associated with the 40S ribosomal subunit, a unique strategic location that facilitates its free access to the downstream target eIF2α. Although both NP and Hsp40 bind to P58IPK, the binding affinity of NP is much stronger compared to Hsp40. P58IPK harbors an NP binding site, spanning to N-terminal TPR subdomains I and II. The Hsp40 binding site on P58IPK was mapped to the TPR subdomain II. The high affinity binding of NP to P58IPK and the overlap between NP and Hsp40 binding sites releases the P58IPK from its negative regulator by competitive inhibition. The NP-P58IPK complex is selectively recruited to the 40S ribosomal subunit by direct interaction between NP and the ribosomal protein S19 (RPS19), a structural component of the 40S ribosomal subunit. NP has distinct binding sites for P58IPK and RPS19, enabling it to serve as bridge between P58IPK and the 40S ribosomal subunit. NP mutants deficient in binding to either P58IPK or RPS19 fail to inhibit PKR, demonstrating that selective engagement of P58IPK to the 40S ribosomal subunit is required for PKR inhibition. Cells deficient in P58IPK mount a rapid PKR antiviral response and establish an antiviral state, observed by global translational shutdown and rapid decline in viral load. These studies reveal a novel viral strategy in which NP releases P58IPK from its negative regulator and selectively engages it on the 40S ribosomal subunit to promptly combat the PKR antiviral responses.

Hantavirus Induced Kidney Disease.

Hantavirus induced hemorrhagic fever with renal syndrome (HFRS) is an emerging viral zoonosis affecting up to 200,000 humans annually worldwide. This review article is focused on recent advances in the mechanism, epidemiology, diagnosis, and treatment of hantavirus induced HFRS. The importance of interactions between viral and host factors in the design of therapeutic strategies is discussed. Hantavirus induced HFRS is characterized by thrombocytopenia and proteinuria of varying severities. The mechanism of kidney injury appears immunopathological with characteristic deterioration of endothelial cell function and compromised barrier functions of the vasculature. Although multidisciplinary research efforts have provided insights about the loss of cellular contact in the endothelium leading to increased permeability, the details of the molecular mechanisms remain poorly understood. The epidemiology of hantavirus induced renal failure is associated with viral species and the geographical location of the natural host of the virus. The development of vaccine and antiviral therapeutics is necessary to avoid potentially severe outbreaks of this zoonotic illness in the future. The recent groundbreaking approach to the SARS-CoV-2 mRNA vaccine has revolutionized the general field of vaccinology and has provided new directions for the use of this promising platform for widespread vaccine development, including the development of hantavirus mRNA vaccine. The combinational therapies specifically targeted to inhibit hantavirus replication and vascular permeability in infected patients will likely improve the disease outcome.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein harbors distinct RNA-binding sites in the stalk and head domains.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

Hantavirus induced cardiopulmonary syndrome: A public health concern.

Hantavirus cardiopulmonary syndrome is characterized by pulmonary capillary leakage and alveolar flooding, resulting in 50% mortality due to fulminant hypoxic respiratory failure. In addition, depression of cardiac function ensues, which complicates the picture with cardiogenic shock. Early diagnosis and appropriate use of extracorporeal membrane oxygenation (ECMO) are amongst the lifesaving interventions in this fatal illness. However, a recent case report demonstrates that implementation of high volume continuous hemofilteration along with protective ventilation reverses the cardiogenic shock within few hours in hantavirus infected patients. This review article is focused on the recent advances in clinical features, diagnosis, management, epidemiology, and pathogenesis of hantavirus induced cardiopulmonary syndrome. It provides information for clinicians to help in correct diagnosis during the early stages of viral infection that could improve the prognosis of this viral illness.