The 37(th) International Conference on Antiviral Research (ICAR) was held in Gold Coast, Australia, May 20-24, 2024. ICAR 2024 featured over 75 presentations along with two poster sessions and special events, including those specifically tailored for trainees and early-career scientists. The meeting served as a platform for the exchange of cutting-edge research, with presentations and discussions covering novel antiviral compounds, vaccine development, clinical trials, and therapeutic advancements. A comprehensive array of topics in antiviral science was covered, from the latest breakthroughs in antiviral drug development to innovative strategies for combating emerging viral threats. The keynote presentations provided fascinating insight into two diverse areas fundamental to medical countermeasure development and use, including virus emergence at the human-animal interface and practical considerations for bringing antivirals to the clinic. Additional sessions addressed a variety of timely post-pandemic topics, such as the hunt for broad spectrum antivirals, combination therapy, pandemic preparedness, application of in silico tools and AI in drug discovery, the virosphere, and more. Here, we summarize all the presentations and special sessions of ICAR 2024 and introduce the 38(th) ICAR, which will be held in Las Vegas, USA, March 17-21, 2025.

Development of broad-spectrum antiviral therapies is critical for outbreak and pandemic preparedness against emerging and reemerging viruses. Viruses inducing hemorrhagic fevers cause high morbidity and mortality in humans and are associated with several recent international outbreaks, but approved therapies for treating most of these pathogens are lacking. Here, we show that 4'-fluorouridine (4'-FlU; EIDD-2749), an orally available ribonucleoside analog, has antiviral activity against multiple hemorrhagic fever viruses in cell culture, including Nipah virus, Crimean-Congo hemorrhagic fever virus, orthohantaviruses, and arenaviruses. We performed preclinical in vivo evaluation of oral 4'-FlU against two arenaviruses, Old World Lassa virus (LASV) and New World Junín virus (JUNV), in guinea pig models of lethal disease. 4'-FlU demonstrated both advantageous pharmacokinetic characteristics and high efficacy in both of these lethal disease guinea pig models. Additional experiments supported protection of the infected animals even when 4'-FlU delivery was reduced to a low dose of 0.5 milligram per kilogram. To demonstrate clinical utility, 4'-FlU treatment was evaluated when initiated late in the course of infection (12 or 9 days after infection for LASV and JUNV, respectively). Delayed treatment resulted in rapid resolution of clinical signs, demonstrating an extended window for therapeutic intervention. These data support the use of 4'-FlU as a potent and efficacious treatment against highly pathogenic arenaviruses of public health concern with a virus inhibition profile suggesting broad-spectrum utility as an orally available antiviral drug against a wide variety of viral pathogens.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes human disease ranging from subclinical to a fatal hemorrhagic syndrome. Determinants of CCHF pathogenesis are largely unknown and animal models that recapitulate human disease are limited. A recently described mouse model uses a monoclonal antibody (mAb 5A3) targeting the interferon (IFN) alpha/beta receptor to suppress type I IFN responses, making animals transiently susceptible to infection. To advance utility of this model, we investigated effects of challenge route, timing of 5A3 delivery, mouse sex and age, and virus strain on clinical course and outcome. C57BL/6J mice received mAb 5A3 -1, 0, or -1/+1 days post-infection (dpi). Subsets were challenged with CCHFV strain Turkey04 or IbAr10200 subcutaneously or intraperitoneally, and serially euthanized 3- and 7-dpi, when meeting euthanasia criteria or at study completion (14 dpi). CCHFV-IbAr10200-infected mice almost uniformly succumbed to infection, whereas CCHFV-Turkey04-infected mice transiently lost weight but survived. These results were consistent regardless of mAb timing or route of challenge. Viral replication and dissemination were comparable between the two strains at 3 dpi. However, in the plasma and livers of non-survivors, expression of proinflammatory cytokines/chemokines that correspond with macrophage activation and recruitment were significantly elevated. Lethal disease was also associated with elevated levels of macrophage activation marker CD163 in plasma. Further, mouse macrophages were more permissive to IbAr1200 infection in vitro, suggesting tropism for these cells may influence pathogenesis. Our data suggest that early inflammation may be a critical determinant of CCHF outcome and therapeutics to control inflammation may be worthwhile targets for future investigation.

Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo.

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination conferred 100% protection from lethal outcome and was associated with mild clinical disease, while the NP + GP38 combination conferred even more robust protection by reducing morbidity compared to mice receiving NP alone. Thus, recombinant CCHFV NP alone is a promising vaccine candidate conferring 100% survival against heterologous challenge. Moreover, incorporation of GP38 should be considered as it further enhances subunit vaccine efficacy by reducing morbidity in surviving animals.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar(-/-) mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar(-/-) mice with CCHF VRP 28, 14, 7, or 3 days prior to challenge, all known to confer complete protection, significantly reduced CCHFV viral load, mucosal shedding, and markers of clinical disease, with greater reductions associated with longer vaccination periods. Interestingly, there were no significant differences in innate immune responses, T cell activation, or antibody titers after challenge between groups of mice vaccinated a week or more before challenge, but higher anti-NP antibody avidity and effector function (ADCD) were positively associated with longer vaccination periods. These findings support the importance of antibody-mediated responses in VRP vaccine-mediated protection against CCHFV infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection.

Type of feed is an important consideration in herbivore colony management, yet limited studies report on the effects of diet on common conditions such as urolithiasis in guinea pigs. Urolithiasis is a well-documented cause of lower urinary tract disease in guinea pigs, with calcium carbonate uroliths reported as the predominant calculi formed in the guinea pig urinary tract. A calcium-rich diet has been suggested as a risk factor for of urolithiasis, with numerous commercially available guinea pig diets formulated for adults avoiding ingredients that are higher in calcium. Due to the high incidence of urolithiasis in our strain 13/N guinea pig colony, we conducted a prospective control study following the implementation of dietary changes aimed at improving overall urinary tract health and reducing risk factors for urolithiasis, thus improving colony welfare. A control group was kept on the original ad libitum alfalfa hay-based pellet diet with restricted loose timothy hay (control diet, 14 juveniles and 24 adults). An experimental group was placed on a portioned, 1 oz daily, timothy hay-based pellet diet with ad libitum loose timothy hay (experimental diet, 21 juveniles and 23 adults). Juveniles and adults were followed for a total of 14 and 26 wk, respectively. Longitudinal blood and urine samples were collected to evaluate blood chemistry and urinary parameters, along with weight and body condition scores to assess general health. Overall, dietary changes did not improve parameters associated with improved urinary tract health or reduced risk of urolithiasis; feeding strategy was not found to meaningfully affect calcium crystalluria, urine protein, urine specific gravity, or renal values. These data support alfalfa hay-based pellet or timothy hay-based pellet, when fed with loose timothy hay, as viable options and suggest that practices aimed at reducing dietary calcium by reducing pelleted diet portions are insufficient to mitigate risk factors for urolithiasis in guinea pigs.

The Third International Conference on Crimean-Congo Hemorrhagic Fever (CCHF) was held in Thessaloniki, Greece, September 19-21, 2023, bringing together a diverse group of international partners, including public health professionals, clinicians, ecologists, epidemiologists, immunologists, and virologists. The conference was attended by 118 participants representing 24 countries and the World Health Organization (WHO). Meeting sessions covered the epidemiology of CCHF in humans; Crimean-Congo hemorrhagic fever virus (CCHFV) in ticks; wild and domestic animal hosts; molecular virology; pathogenesis and animal models; immune response related to therapeutics; and CCHF prevention in humans. The concluding session focused on recent WHO recommendations regarding disease prevention, control strategies, and innovations against CCHFV outbreaks. This meeting report summarizes lectures by the invited speakers and highlights advances in the field.

Nipah virus (NiV) is a highly pathogenic paramyxovirus. The Syrian hamster model recapitulates key features of human NiV disease and is a critical tool for evaluating antivirals and vaccines. Here we describe longitudinal humoral immune responses in NiV-infected Syrian hamsters. Samples were obtained 1-28 days after infection and analyzed by ELISA, neutralization, and Fc-mediated effector function assays. NiV infection elicited robust antibody responses against the nucleoprotein and attachment glycoprotein. Levels of neutralizing antibodies were modest and only detectable in surviving animals. Fc-mediated effector functions were mostly observed in nucleoprotein-targeting antibodies. Antibody levels and activities positively correlated with challenge dose.

Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean-Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

Nipah virus (NiV) is a highly pathogenic paramyxovirus with a high case fatality rate. Due to its high pathogenicity, pandemic potential, and lack of therapeutics or approved vaccines, its study requires biosafety level 4 (BSL4) containment. In this report, we developed a novel neutralization assay for use in biosafety level 2 laboratories. The assay uses a recombinant vesicular stomatitis virus expressing NiV glycoprotein and a fluorescent protein. The recombinant virus propagates as a replication-competent virus in a cell line constitutively expressing NiV fusion protein, but it is restricted to a single round of replication in wild-type cells. We used this system to evaluate the neutralization activity of monoclonal and polyclonal antibodies, plasma from NiV-infected hamsters, and serum from human patients. Therefore, this recombinant virus could be used as a surrogate for using pathogenic NiV and may constitute a powerful tool to develop therapeutics in low containment laboratories.

Defective interfering (DI) genomes restrict viral replication and induce type-I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity and determine their long-term effects. To address this, we generated a recombinant (r) canine distemper virus (CDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues which are a major site of CDV replication. Our established pipeline for detection, generation and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome.Importance Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes could be harnessed therapeutically. Negative strand RNA virus research has provided a rich pool of natural DI genomes over many years and they are probably the best understood in vitro. Here, we demonstrate identification, synthesis, production and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.

The 36th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held March 13-17, 2023, in Lyon, France, and concurrently through an interactive remote meeting platform. Here we provide a report summarizing the presentations at the 36th ICAR, including the ISAR speaker awards. We also detail special events, sessions, and additional awards conferred at the meeting. ICAR returned to in-person meetings in 2022, convening in Seattle, WA, USA. The 36th ICAR is the first in-person meeting of the society in Europe since the beginning of the COVID-19 pandemic, which restricted most events to virtual attendance to help mitigate the spread and subsequent public health impact of SARS-CoV-2. An exceptionally high number of registrants and record attendance at this year's ICAR, along with a vast array of demonstrable expertise in a variety of antiviral research-related fields, reflected a strong and growing antiviral research community committed to improving health outcomes from viral diseases, including SARS-CoV-2, and to future pandemic preparedness. This report highlights the breadth of expertise, quality of research, and notable advancements that were contributed by members of ISAR and other participants at the meeting. ICAR aims to continue to provide a platform for sharing information, fostering collaborations, and supporting trainees in the field of antiviral research. The 37th ICAR will be held in Gold Coast, Australia, May 20-24, 2024.

Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA)-EBOVs, including one based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 80% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using hand-held blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

Guinea pigs are important animal models for human disease, and both outbred and inbred lines are utilized in biomedical research. The optimal maintenance of guinea pig colonies, commercially and in research settings, relies on robust informed breeding programs, however, breeding data on specialized inbred strains are limited. Here, we investigated the effects of parental age, parity, and pairing approaches on mean total fetus count, percentage of female pups in the litter, and pup survival rate after 10 days in strain 13/N guinea pigs. Our analysis of colony breeding data indicates that the average litter size is 3.3 pups, with a 25.2% stillbirth rate, a failure-to-thrive outcome in 5.1% of pups, and a 10 day survival rate of 69.7%. The only variable to significantly affect the reproductive outcomes examined was parental age (p < 0.05). In comparison to adults, both juvenile and geriatric sows had lower total fetus counts; juvenile boars had a higher percentage of females in litters, and geriatric boars had a lower 10 day survival rate of pups. These studies provide valuable information regarding the reproductive characteristics of strain 13/N guinea pigs, and support a variety of breeding approaches without significant effects on breeding success.

The 35th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held in Seattle, Washington, USA, on March 21-25, 2022 and concurrently through an interactive remote meeting platform. This report gives an overview of the conference on behalf of the society. It provides a general review of the meeting and awardees, summarizing the presentations and their main conclusions from the perspective of researchers active in many different areas of antiviral research and development. Through ICAR, leaders in the field of antiviral research were able to showcase their efforts, as participants learned about key advances in the field. The impact of these efforts was exemplified by many presentations on SARS-CoV-2 demonstrating the remarkable response to the ongoing pandemic, as well as future pandemic preparedness, by members of the antiviral research community. As we address ongoing outbreaks and seek to mitigate those in the future, this meeting continues to support outstanding opportunities for the exchange of knowledge and expertise while fostering cross-disciplinary collaborations in therapeutic and vaccine development. The 36th ICAR will be held in Lyon, France, March 13-17, 2023.

Development of lethal models of Ebola virus disease has been achieved by the serial passage of virus isolates from human cases in mice and guinea pigs. Use of mice infected with non-adapted virus has been limited due to the absence of overt clinical disease. In recent years, newly recognized sequelae identified in human cases has highlighted the importance of continued investigations of non-lethal infection both in humans and animal models. Here, we revisit the use of rodent-adapted and non-adapted Ebola virus (EBOV) variants in mice to investigate infection tolerance and future utility of these models in pathogenesis and therapeutic intervention studies. We found that like non-adapted wild-type EBOV, guinea pig-adapted EBOV results in widespread tissue infection, variably associated with tissue pathology, and alterations in clinical and immunological analytes in the absence of overt disease. Notably, infection with either non-lethal variant does not greatly differ from lethal mouse-adapted EBOV until near the time end-point criteria are reached in these mice, supporting use of these models of virus tolerance for continued investigations of non-lethal infection and sequelae.

Human infection with Sosuga virus (SOSV), a recently discovered pathogenic paramyxovirus, has been reported in one individual to date. No animal models of disease are currently available for SOSV. Here, we describe initial characterization of experimental infection in Syrian hamsters, including kinetics of virus dissemination and replication, and the corresponding clinical parameters, immunological responses, and histopathology. We demonstrate susceptibility of hamsters to infection in the absence of clinical signs or significant histopathologic findings in tissues.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.

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.

Syrian hamsters are a key animal model of SARS-CoV-2 and other respiratory viruses and are useful for the evaluation of associated medical countermeasures. Delivery of an infectious agent or intervention to the respiratory tract mirrors natural routes of exposure and allows for the evaluation of clinically relevant therapeutic administration. The data to support instillation or inoculation volumes are important both for optimal experimental design and to minimize or avoid effects of diluent alone, which may compromise both data interpretation and animal welfare. Here we investigate four intranasal (IN) instillation volumes in hamsters (50, 100, 200, or 400 µL). The animals were monitored daily, and a subset were serially euthanized at one of four pre-determined time-points (1, 3, 7, and 14 days post-instillation). Weight, temperature, oxygen saturation, CBC, radiographs, and respiratory tissue histopathology were assessed to determine changes associated with instillation volume alone. With all the delivery volumes, we found no notable differences between instilled and non-instilled controls in all of the parameters assessed, except for histopathology. In the animals instilled with 200 or 400 µL, inflammation associated with foreign material was detected in the lower respiratory tract indicating that higher volumes may result in aspiration of nasal and/or oropharyngeal material in a subset of animals, resulting in IN instillation-associated histopathology.

ABSTRACTLassa fever (LF) is endemic to broad regions of West Africa. Infection with Lassa virus (LASV), the etiologic agent of LF, results in a spectrum of clinical signs in humans, including severe and lethal hemorrhagic disease. Person-to-person transmission occurs through direct contact with body fluids or contaminated bedding and clothing. To investigate transmission risk in acute LASV infection, we evaluated viral RNA and infectious virus obtained from conjunctival, nasal, oral, genital, and rectal swab specimens from guinea pigs modeling lethal and non-lethal LF. Viral RNA and infectious virus were detected in all specimen types beginning 8 days post infection, prior to onset of fever. In the pre-clinical and clinical period, virus was isolated from a subset of nasal, oral, genital, and rectal swabs, and from all conjunctival swabs. Overall, conjunctival and nasal specimens most frequently yielded infectious virus. These findings indicate mucosal transmission risk based on virus isolation from various sites early in infection and support potential utility of minimally invasive specimen evaluation by RT-qPCR for LASV diagnostics.

Defective interfering particles (DIs) contain a considerably smaller genome than the parental virus but retain replication competency. As DIs can directly or indirectly alter propagation kinetics of the parental virus, they offer a novel approach to antiviral therapy, capitalizing on knowledge from natural infection. However, efforts to translate in vitro inhibition to in vivo screening models remain limited. We investigated the efficacy of virus-like particles containing DI genomes (therapeutic infectious particles [TIPs]) in the Syrian hamster model of lethal Nipah virus (NiV) disease. We found that coadministering a high dose of TIPs intraperitoneally with virus challenge improved clinical course and reduced lethality. To mimic natural exposure, we also evaluated lower-dose TIP delivery and virus challenge intranasally, finding equally efficacious reduction in disease severity and overall lethality. Eliminating TIP replicative capacity decreased efficacy, suggesting protection via direct inhibition. These data provide evidence that TIP-mediated treatment can confer protection against disease and lethal outcome in a robust animal NiV model, supporting further development of TIP treatment for NiV and other high-consequence pathogens. IMPORTANCE Here, we demonstrate that treatment with defective interfering particles (DIs), a natural by-product of viral infection, can significantly improve the clinical course and outcome of viral disease. When present with their parental virus, DIs can directly or indirectly alter viral propagation kinetics and exert potent inhibitory properties in cell culture. We evaluated the efficacy of a selection of virus-like particles containing DI genomes (TIPs) delivered intranasally in a lethal hamster model of Nipah virus disease. We demonstrate significantly improved clinical outcomes, including reduction in both lethality and the appearance of clinical signs. This work provides key efficacy data in a robust model of Nipah virus disease to support further development of TIP-mediated treatment against high-consequence viral pathogens.

Lassa virus (LASV) causes mild to severe hemorrhagic fever disease in humans. Strain 13/N guinea pigs are highly susceptible to infection with LASV strain Josiah (clade IV), providing a critical model system for therapeutics and vaccine development. To develop additional models of disease, we detail the clinical course in guinea pigs infected with 5 geographically and genetically diverse LASV strains. Two of the developed models (LASV clades II and III) were then used to evaluate efficacy of a virus replicon particle (VRP) vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.

Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses.