Replication models have been developed to describe the replication dynamics of a variety of viruses to better understand the kinetics and key contributing factors affecting infectivity and spread. However, accurate representations of the dynamics of virus replication observed in vitro and in vivo are often limited due to the failure of these models to account for both environmental influences, such as temperature, and the variety of possible mechanisms employed by viruses to spread. Several major families of viruses including paramyxoviruses, pneumoviruses and coronaviruses, induce and use the formation of syncytia, large multinucleated cell masses formed through fusion of cells, to aid in spread to neighbouring susceptible cells. In this study, we evaluate and compare both the dynamics and roles of temperature and syncytia formation on the replication of two different fusogenic viruses in vitro : human respiratory syncytial virus (hRSV) and a murine coronavirus, mouse hepatitis virus (MHV). Thermal stability, replication kinetics and both the rates and dynamics of syncytia formation were evaluated for hRSV and MHV. These data were then incorporated into a novel and improved replication model for each of the two viruses, which provides new insights into the contributions of both temperature and syncytia formation in the replication of fusogenic viruses.

Omsk hemorrhagic fever virus (OHFV) is a tick-borne flavivirus associated with acute febrile illness and hemorrhagic disease in humans. OHFV was first identified in 1947 and since its identification has largely remained endemic to the Omsk region of the Russian Federation, where it continues to result in illnesses each year. However, the 2 primary tick vectors of OHFV exhibit ranges that extend far beyond the Omsk region, and the recent positive confirmation of OHFV in samples outside of the historic endemic range, in both the Russian Federation and neighboring Kazakhstan, to the south raises new concerns about the future potential of the pathogen. In this review, we comprehensively discuss the emergence, virology, ecology, and disease of OHFV.

The increasing prevalence of antibiotic resistance and the limited availability of antiviral therapeutics for pathogens such as human respiratory syncytial virus (hRSV) underscore the need for novel, plant-derived antimicrobial substances. In this study, we evaluated the antiproliferative, antibacterial, and antiviral activities of aqueous leaf extracts from two plants commonly found in North America, Osage orange (M. pomifera) and spearmint (M. spicata). Both extracts exhibited no significant cytotoxic or morphologic impact on HEp-2 human cancer cells up to 25 mg/mL. However, both extracts demonstrated strong dose-dependent antibacterial activity, significantly inhibiting replication of E. coli and S. aureus at concentrations ≥ 1 mg/mL. Antiviral assays revealed that both extracts inhibited hRSV infectivity, with spearmint extract showing higher potency (EC50 = 1.01 mg/mL) compared to Osage orange (EC50 = 3.85 mg/mL). Gas chromatography–mass spectrometry (GC-MS) identified three major extract constituents: 3-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol (Osage orange), and R-(-)-carvone (spearmint). Among these, only carvone significantly inhibited hRSV in vitro, suggesting its key role in spearmint’s antiviral activity. These findings highlight the therapeutic potential of Osage orange and spearmint leaf extracts, particularly as sources of water-soluble compounds with antimicrobial properties, and support further investigation into their mechanisms of action and broader clinical relevance.

With new emerging diseases such as COVID-19 and an increasing incidence of cancer, there remains a significant need for investigating new therapeutic options to treat a wide range of ailments and disorders. Peppermint (Mentha × piperita) and white snakeroot (Ageratina altissima) have been used medicinally by native people in the Midwestern United States for centuries. However, the antiproliferative and antimicrobial properties of the aqueous extracts of these plants remain unclear. In this study, we evaluate the therapeutic potential of peppermint and white snakeroot aqueous leaf extracts by examining their activity against mammalian cancer cells, bacteria, and viruses. Both peppermint and snakeroot extracts showed no reductions in viability at concentrations lower than 25 mg/mL and 10 mg/mL, respectively, in two different cancer lines, HEp-2 and DBT-9 cells, in vitro. While treatment with the snakeroot extract resulted in significant disruption to cytoskeletal organization in HEp-2 cells at a concentration of 10 mg/mL, peppermint and snakeroot extracts did not have a major impact on the viability or proliferation of the cancer cell lines tested. Peppermint and snakeroot were then evaluated for antibacterial activity against four different bacterial pathogens. Significant inhibition of bacterial replication was observed for E. coli (at concentrations greater than 0.1 mg/mL) and S. aureus (at concentrations greater than 1 mg/mL) treated with either peppermint or snakeroot extracts. No significant activity was observed against the bacterial strains P. aeruginosa and S. pyogenes. Peppermint (EC50 = 2.36 mg/mL) and snakeroot (EC50 = 2.64 mg/mL) significantly reduce infectivity and replication (at concentrations above 0.2 mg/mL) of the major human pathogen, human respiratory syncytial virus (hRSV). However, testing for antiviral activity against a mouse coronavirus (murine hepatitis virus, MHV) showed no impact on replication at concentrations up to 2.5 mg/mL. Lastly, chemical analysis of the extracts identified several prominent compounds, which were subsequently evaluated for their biological contributions to the observed plant extract phenotypes. Two of the identified compounds, 1,8-cineole (Eucalyptol) and menthol, show significant antimicrobial activity. We report that aqueous extracts of peppermint and white snakeroot exhibit specific antibacterial and antiviral activities that support further investigation for therapeutic potential.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

The recent emergence of SARS-CoV-2 in 2019 has highlighted the necessity of antiviral therapeutics for current and future emerging coronaviruses. Recently, the traditional herbal medicines baicalein, baicalin, and andrographolide have shown inhibition against the main protease of SARS-CoV-2. This provides a promising new direction for COVID-19 therapeutics, but it remains unknown whether these three substances inhibit other human coronaviruses. In this study, we describe the development of novel chimeric mouse hepatitis virus (MHV) reporters that express firefly luciferase (FFL) and the 3CLpro proteases of human coronaviruses HKU1 and OC43. These chimeric viruses were used to determine if the phytochemicals baicalein, baicalin, and andrographolide are inhibitory against human coronavirus strains HKU1 and OC43. Our data show that both baicalein and baicalin exhibit inhibition towards the chimeric MHV strains. However, andrographolide induces cytotoxicity and failed to demonstrate selective toxicity towards the viruses. This study reports the development and use of a safe replicating reporter platform to investigate potential coronavirus 3CLpro inhibitors against common-cold human coronavirus strains HKU1 and OC43.

Traditional approaches employing natural plant products to treat a wide array of ailments have been documented and described for thousands of years. However, there remains limited scientific study of the therapeutic potential or effectiveness of ethnobotanical applications. Increases in the incidence of cancer and emerging infectious diseases demonstrate a growing need for advances in the development of therapeutic options. In this study, we evaluate the therapeutic potential of aqueous extracts prepared from four plants, purple aster (Symphyotrichum novae-angliae (L.) Nemsom), common sage (Salvia lyrata (L.)), northern spicebush (Lindera benzoin (L.) Blume), and lamb’s ear (Stachys byzantina (K.) Koch)) traditionally used in Native American medicine in Indiana, USA. Using a combination of cytotoxicity assays, immunofluorescence microscopy, and antiviral assays, we found that sage and spicebush extracts exhibit cytotoxic and antiproliferative effects on HeLa cell proliferation and that sage, spicebush, and aster extracts were capable of significantly inhibiting human respiratory syncytial virus (hRSV), a major respiratory pathogen of infants and the elderly. Chemical analysis of the four extracts identified four major compounds which were subsequently evaluated to identify the responsible constituents in the extracts. While none of the identified compounds were shown to induce significant impacts on HeLa cell proliferation, two of the compounds, (1S)-(-)-Borneol and 5-(hydroxymethyl)-furfural, identified in sage and spicebush, respectively, were shown to have antiviral activities. Our data suggest that several of the extracts tested exhibited either anti-proliferative or antiviral activity supporting future further analysis.

Sin Nombre virus (SNV) is an emerging virus that was first discovered in the Four Corners region of the United States in 1993. The virus causes a disease known as Hantavirus Pulmonary Syndrome (HPS), sometimes called Hantavirus Cardiopulmonary Syndrome (HCPS), a life-threatening illness named for the predominance of infection of pulmonary endothelial cells. SNV is one of several rodent-borne hantaviruses found in the western hemisphere with the capability of causing this disease. The primary reservoir of SNV is the deer mouse (Peromyscus maniculatus), and the virus is transmitted primarily through aerosolized rodent excreta and secreta. Here, we review the history of SNV emergence and its virus biology and relationship to other New World hantaviruses, disease, treatment, and prevention options.

Approximately 15–30% of all cases of the common cold are due to human coronavirus infections. More recently, the emergence of the more severe respiratory coronaviruses, SARS-CoV and MERS-CoV, have highlighted the increased pathogenic potential of emergent coronaviruses. Lastly, the current emergence of SARS-CoV-2 has demonstrated not only the potential for significant disease caused by emerging coronaviruses, but also the capacity of novel coronaviruses to promote pandemic spread. Largely driven by the global response to the COVID-19 pandemic, significant research in coronavirus biology has led to advances in our understanding of these viruses. In this review, we evaluate the virology, emergence, and evolution of the four endemic coronaviruses associated with the common cold, their relationship to pandemic SARS-CoV-2, and discuss the potential for future emergent human coronaviruses.

Australian bat lyssavirus (ABLV) is a negative-sense, single-stranded RNA rhabdovirus capable of causing fatal acute encephalitis in humans with similar pathogenesis to its closest serologic relative, rabies virus (RABV). In this review, we describe emergence and classification of ABLV, its known virology, reservoirs, and hosts, as well as both the pathogenesis and treatment approaches currently employed for presumed infections. ABLV was first identified in New South Wales, Australia in 1996 and emerged in humans months later in Queensland, Australia. Only five known bat reservoirs, all of which fall within the Pteropus and Saccolaimus genera, have been identified to date. Although ABLV antigens have been identified in bats located outside of Australia, the three known human ABLV infections to date have occurred within Australia. As such, there remains a potential for ABLV to expand its presence within and beyond Australia. ABLV infections are currently treated as if they were RABV infections by administering neutralizing antibodies against RABV at the site of the wound and employing the rabies vaccine upon possible exposures. Due to its recent emergence, there is still much left unknown about ABLV, posing concerns with how to safely and effectively address current and future ABLV infections.

The emergence of SARS-CoV-2 and the worldwide COVID-19 pandemic triggered considerable attention to the emergence and evolution of novel human pathogens. Bourbon virus (BRBV) was first discovered in 2014 in Bourbon County, Kansas, USA. Since its initial discovery, several cases of BRBV infection in humans have been identified in Kansas, Oklahoma, and Missouri. BRBV is classified within the Thogotovirus genus; these negative-strand RNA viruses appear to be transmitted by ticks, and much of their biology remains unknown. In this review, we describe the emergence, virology, geographic range and ecology, and human disease caused by BRBV and discuss potential treatments for active BRBV infections. This virus and other emerging viral pathogens remain key public health concerns and require continued surveillance and study to mitigate human exposure and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein spike for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live, attenuated, recombinant human respiratory syncytial virus expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200-fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunoglobulin A in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against virus variants of concern Alpha, Beta, and Delta. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in Phase 1 clinical trials as an intranasal COVID-19 vaccine.

Antimicrobial peptides are proteins that have been found to be an important factor in the natural immune response to a variety of pathogens. Respiratory syncytial virus (RSV) is a respiratory pathogen with the capability to cause serious upper and lower respiratory infections in infants and children and is a major viral cause of infant mortality. There is currently no functional vaccine for the virus, as recent efforts have been hindered by the virus's low immunogenicity, its ability to effectively mutate, and underlying instabilities of potential vaccines. Previous studies have shown that antimicrobial peptides may affect viral replication and spread of RSV. Our study evaluates the susceptibility of chimeric strains of RSV that express different fusion (F) and attachment (G) proteins to susceptibilities to inactivation by LL-37 and human beta-defensins (hBDs) hBD-1, hBD-3, and hBD-4. We show that LL-37 and hBD-3 result in dose-dependent, strain-independent inactivation of RSV, whereas treatment with either hBD-1 or hBD-4 appears more variable between strains. This suggests a potential role of the viral structural proteins in mitigating the inhibitory effects of the peptides. This study provides the first evidence of the sensitivity of RSV to several hBDs and indicates a role of LL-37 and beta-defensins in both limiting establishment of natural RSV infections and in the therapeutic treatment of severe RSV disease.

Abstract Background Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. Methods Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. Results Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. Conclusions Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Severe acute respiratory syndrome coronavirus-2 is the etiological agent of the ongoing pandemic of coronavirus disease-2019, a multi-organ disease that has triggered an unprecedented global health and economic crisis. The virally encoded 3C-like protease (3CLpro ), which is named after picornaviral 3C protease (3Cpro ) due to their similarities in substrate recognition and enzymatic activity, is essential for viral replication and has been considered as the primary drug target. However, information regarding the cellular substrates of 3CLpro and its interaction with the host remains scarce, though recent work has begun to shape our understanding more clearly. Here we summarized and compared the mechanisms by which picornaviruses and coronaviruses have evolved to evade innate immune surveillance, with a focus on the established role of 3Cpro in this process. Through this comparison, we hope to highlight the potential action and mechanisms that are conserved and shared between 3Cpro and 3CLpro . In this review, we also briefly discussed current advances in the development of broad-spectrum antivirals targeting both 3Cpro and 3CLpro .

Coronavirus protease nsp5 (Mpro, 3CLpro) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.

Respiratory syncytial virus (RSV) is a significant cause of respiratory illness in young children and adults worldwide. There is currently no vaccine or targeted antiviral for RSV. RSV is an enveloped, filamentous, negative-strand RNA virus. Individual virions vary in both diameter and length, with an average diameter of ∼130 nm and ranging from ∼500 nm to over 10 μm in length. The RSV matrix (M) protein is peripherally associated with the interior of the viral membrane. Though the general arrangement of structural proteins within the virion is known, the molecular organization of M and other structural proteins was previously unknown. Here, using whole-cell cryo-electron tomography and sub-tomogram averaging, we show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle of ∼47° to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. Finally, we report that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers. These results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets.

Respiratory syncytial virus (RSV) is a leading viral cause of pediatric respiratory infections and early infant mortality. Despite extensive development efforts currently underway, there remain no vaccines available for the prevention of RSV. RSV is an enveloped, negative-strand RNA virus that utilizes two different proteins (G and F) to mediate attachment and entry into host cells. These G and F proteins are the primary determinants of viral strain-specific differences and elicit protective neutralizing antibodies during natural infection in humans. Earlier studies have demonstrated that these proteins play an additional role in regulating the stability of RSV particles in response to temperature and pH. However, it remains unclear how much variability exists in the stability of RSV strains and what contribution changes in temperature and pH make to the clearance of virus during an active infection. In this study, we evaluated the impacts of changes in temperature and pH on the inactivation of four different chimeric recombinant RSV strains that differ exclusively in G and F protein expression. Using these data, we developed predictive mathematical models to examine the specific contributions and variations in susceptibility that exist between viral strains. Our data provide strain-specific clearance rates and temperature-pH landscapes that shed light on the optimal contributions of temperature and pH to viral clearance. These provide new insight into how much variation exists in the clearance of a major respiratory pathogen and may offer new guidance on optimization of viral strains for development of live-attenuated vaccine preparations.

Human coronaviruses are enveloped, positive-strand RNA viruses which cause respiratory diseases ranging in severity from the seasonal common cold to SARS and COVID-19. Of the 7 human coronaviruses discovered to date, 3 emergent and severe human coronavirus strains (SARS-CoV, MERS-CoV, and SARS-CoV-2) have recently jumped to humans in the last 20 years. The COVID-19 pandemic spawned by the emergence of SARS-CoV-2 in late 2019 has highlighted the importance for development of effective therapeutics to target emerging coronaviruses. Upon entry, the replicase genes of coronaviruses are translated and subsequently proteolytically processed by virus-encoded proteases. Of these proteases, nonstructural protein 5 (nsp5, Mpro, or 3CLpro), mediates the majority of these cleavages and remains a key drug target for therapeutic inhibitors. Efforts to develop nsp5 active-site inhibitors for human coronaviruses have thus far been unsuccessful, establishing the need for identification of other critical and conserved non-active-site regions of the protease. In this study, we describe the identification of an essential, conserved horseshoe-shaped region in the nsp5 interdomain loop (IDL) of mouse hepatitis virus (MHV), a common coronavirus replication model. Using site-directed mutagenesis and replication studies, we show that several residues comprising this horseshoe-shaped region either fail to tolerate mutagenesis or were associated with viral temperature-sensitivity. Structural modeling and sequence analysis of these sites in other coronaviruses, including all 7 human coronaviruses, suggests that the identified structure and sequence of this horseshoe regions is highly conserved and may represent a new, non-active-site regulatory region of the nsp5 (3CLpro) protease to target with coronavirus inhibitors.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.