James A. Ellison

Bats are major reservoirs of viruses that can be transmitted to humans in zoonotic outbreaks. Antibody-mediated immunity plays an important role in shaping viral evolution and immune evasion but remains understudied in bats. All known mammals have a single immunoglobulin heavy chain (IgH) gene locus and up to two light chain loci. We have identified dual IgH loci on separate chromosomes in 26 bat species, highlighting extreme variation of immunogenetic architecture in order Chiroptera. In a model species, Eptesicus fuscus , we leveraged single-cell transcriptomes to confirm functional rearrangement and expression of both loci, but with different mechanisms for generating antibody diversity and function. These results provide a foundation for analysis of humoral immunity and pathogen response in bats.

The genetic locus encoding immunoglobulin heavy chains (IgH) is critical for vertebrate humoral immune responses and diverse antibody repertoires. Immunoglobulin and T cell receptor loci of most bat species have not been annotated, despite the recurrent role of bats as viral reservoirs and sources of zoonotic pathogens. We investigated the genetic structure and function of IgH loci across the largest bat family, Vespertilionidae, focusing on big brown bats (Eptesicus fuscus ). We discovered that E. fuscus and ten other species within Vespertilionidae have two complete, functional, and distinct immunoglobulin heavy chain loci on separate chromosomes. This locus organization is previously unknown in mammals, but is reminiscent of more limited duplicated loci in teleost fish. Single cell transcriptomic data validate functional rearrangement and expression of immunoglobulin heavy chains of both loci in the expressed repertoire of Eptesicus fuscus , with maintenance of allelic exclusion, bias of usage toward the smaller and more compact IgH locus, and evidence of differential selection of antigen-experienced B cells and plasma cells varying by IgH locus use. This represents a unique mechanism for mammalian humoral immunity and may contribute to bat resistance to viral pathogenesis.

Background: Rabies expert on demand (REOD) telehealth service is provided by the U.S. Centers for Disease Control and Prevention (CDC) to assist public health practitioners, health providers, and the public to interpret national and international rabies prevention guidelines. REOD is staffed by subject matter experts of the CDC Poxvirus and Rabies Branch to assess each unique situation and provide evidence‐based guidance to stakeholders. This study aims to describe the utilization of a rabies telehealth system and provide insight into common consultations.Methods: A cross‐sectional study of the nature of inquiries to REOD was done using the data collected from September 1, 2017 to September 30, 2021. An inquiry tracking form and Microsoft Access database were developed to document all inquiries received. Inquired ones were summarized to determine the frequency of inquiries by month, category, and location.Results: Over a 49‐month period, REOD received 5228 inquiries. Peak inquiries (n = 108) occurred during August 2019. The most frequent inquiries received pertained to risk assessment and management of rabies exposures (n = 1109), requests for testing assistance (n = 912), consultation for suspected human rabies (n = 746), rabies exposures and post‐bite treatment occurring internationally (n = 310), and consultation for deviations in the recommended pre‐ and postexposure prophylaxis regimen (n = 300).Conclusion: REOD is a global resource for consultation related to managing rabies exposures, diagnostic issues, and rabies control strategies. REOD is a regularly utilized CDC service, as the demand for up‐to‐date rabies guidance remains high. REOD fulfills a critical role for the interpretation and consultation on rabies prevention guidelines to stakeholder.

Mongooses, a nonnative species, are a known reservoir of rabies virus in the Caribbean region. A cross-sectional study of mongooses at 41 field sites on the US Virgin Islands of St. Croix, St. John, and St. Thomas captured 312 mongooses (32% capture rate). We determined the absence of rabies virus by antigen testing and rabies virus exposure by antibody testing in mongoose populations on all three islands. USVI is the first Caribbean state to determine freedom-from-rabies for its mongoose populations with a scientifically-led robust cross-sectional study. Ongoing surveillance activities will determine if other domestic and wildlife populations in USVI are rabies-free.

Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 10⁵ plaque-forming units (PFU; 90% lethal dose [LD₉₀]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID–1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID–1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [C_max]) and the time of the last quantifiable concentration (AUC_last) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival. IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today9s current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

Background: The District of Columbia (DC), a major metropolitan area, continues to see community transmission of SARS-CoV-2. While serologic testing does not indicate current SARS-CoV-2 infection, it can indicate prior infection and help inform local policy and health guidance. The DC Department of Health (DC Health) conducted a community-based survey to estimate DC’s SARS-CoV-2 seroprevalence and identify seropositivity-associated factors. Methods: A mixed-methods cross-sectional serology survey was conducted among a convenience sample of DC residents during July 27 – August 21, 2020. Free serology testing was offered at three public test sites. Participants completed an electronic questionnaire on household and demographic characteristics, COVID-like illness (CLI) since January 1, 2020, comorbidities, and SARS-CoV-2 exposures. Univariate and bivariate analyses were conducted to describe the sample population and assess factors associated with seropositivity. Results: Among a sample of 671 participants, 51 individuals were seropositive, yielding an estimated seroprevalence of 7.6%. More than half (56.9%) of the seropositive participants reported no prior CLI; nearly half (47.1%) had no prior SARS-CoV-2 testing. Race/ethnicity, prior SARS-CoV-2 testing, prior CLI, employment status, and contact with confirmed COVID-19 cases were associated with seropositivity (P– August 21, 2020 estimated that 7.6% of the convenience sample had antibodies to SARS-CoV-2, indicating prior infection. At the time of this survey, most of the participants reported that they had not been previously infected with or tested for SARS-CoV-2. These findings highlight both the value of serologic surveillance in complementing other surveillance methods, and the importance of continued prevention and mitigation measures, such as maintaining physical distances of at least 6 feet, avoiding crowds and poorly ventilated spaces, practicing frequent hand hygiene, and wearing face masks properly and consistently around people who do not live with you.

First responders are at increased risk of occupational exposure to SARS-CoV-2 while providing frontline support to communities during the COVID-19 pandemic. In the District of Columbia (DC), first responders were among the first people exposed to and infected with SARS-CoV-2, with over 200 first responders diagnosed with COVID-19 by May 15, 2020. From June – July 2020, DC Health conducted a serologic survey to estimate SARS-CoV-2 seroprevalence and assess risk factors and occupational exposures among a convenience sample of first responders in DC. Of the 310 first responders tested, 3.5% (n = 11) had anti-SARS-CoV-2 antibodies. Seropositivity varied by occupation, with 4.8% (3/62) of firefighters; 3.6% (8/220) of police officers; and no paramedics (0/10) or administration and support staff (0/18) testing positive. Type and consistency of personal protective equipment (PPE) use also varied: all paramedics (n=10) reported wearing a N95 respirator all or most of the time, compared to 83.3% of firefighters, 38.8% of police officers, and 23.5% of administration and support staff (p<0.001). All paramedics reported wearing gloves all or most of the time, compared to 80.0% of firefighters, 27.8% of administration and support staff, and 24.3% of police (p<0.001). The relatively low seroprevalence among first responders highlights the benefits of continuous training on and reinforcement of the proper use of PPE while performing job duties to mitigate potential transmission within and between first responders and the community. Summary: Understanding occupational exposure to and infection with SARS-CoV-2 among first responders is important for workforce planning and emergency preparedness and response. Seroprevalence among first responders (3.5%; 11/310) who participated in a survey conducted by the District of Columbia Department of Health (DC Health) from May 28 – July 15 was 48% lower than reported in the DC community (6.7%; 876/12990). The lower prevalence of SARS-CoV-2 among first responders highlights the importance of continuous training on and reinforcement of the proper use of personal protective equipment (PPE). Proper use of PPE is a critical mitigation strategy to reduce transmission among and between first responders and the community.

We report mortality events in a group of 123 common vampire bats (Desmodus rotundus) captured in México and housed for a rabies vaccine efficacy study in Madison, Wisconsin. Bat mortalities occurred in México and Wisconsin, but rabies cases reported herein are only those that occurred after arrival in Madison (n = 15). Bats were confirmed positive for rabies virus (RABV) by the direct fluorescent antibody test. In accordance with previous reports, we observed long incubation periods (more than 100 days), variability in clinical signs prior to death, excretion of virus in saliva, and changes in rabies neutralizing antibody (rVNA) titers post-infection. We observed that the furious form of rabies (aggression, hyper-salivation, and hyper-excitability) manifested in three bats, which has not been reported in vampire bat studies since 1936. RABV was detected in saliva of 5/9 bats, 2–5 days prior to death, but was not detected in four of those bats that had been vaccinated shortly after exposure. Bats from different capture sites were involved in two separate outbreaks, and phylogenetic analysis revealed differences in the glycoprotein gene sequences of RABV isolated from each event, indicating that two different lineages were circulating separately during capture at each site.

Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2–2 nM and 20–600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model (n = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.

Monkeypox virus (MPXV) is a member of the genus Orthopoxvirus, endemic in Central and West Africa. This viral zoonosis was introduced into the United States in 2003 via African rodents imported for the pet trade and caused 37 human cases, all linked to exposure to MPXV-infected black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs have since become a useful model of MPXV disease, utilized for testing of potential medical countermeasures. In this study, we used recombinant MPXV containing the firefly luciferase gene (luc) and in vivo imaging technology to characterize MPXV pathogenesis in the black-tailed prairie dog in real time. West African (WA) MPXV could be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver as early as day 6 post infection (p.i.). By day 9 p.i., lesions became visible on the skin and in some cases in the spleen. After day 9 p.i., luminescent signal representing MPXV replication either increased, indicating a progression to what would be a fatal infection, or decreased as infection was resolved. Use of recombinant luc+ MPXV allowed for a greater understanding of how MPXV disseminates throughout the body in prairie dogs during the course of infection. This technology will be used to reduce the number of animals required in future pathogenesis studies as well as aid in determining the effectiveness of potential medical countermeasures.