Charles E. Rupprecht

Background/Objectives: Lyssaviruses pose a persistent global public health threat, underscoring the need for broad-spectrum neutralizing antibodies to improve rabies post-exposure prophylaxis (PEP). The humanized monoclonal antibody cocktail SYN023 is approved for PEP in China and under active global regulatory review. This study aimed to validate pseudovirus-based neutralization assays, verify SYN023 epitope prediction accuracy, and systematically characterize its pan-lyssavirus neutralizing activity. Methods: Assay validation was performed by directly comparing neutralization titers obtained with authentic rabies virus (RABV) and pseudotyped virus. Epitope analysis was performed, followed by in vivo evaluation in a canine PEP challenge model. The pseudovirus-based neutralization assay was used to assess SYN023 broad-spectrum activity across diverse lyssaviruses. Results: Neutralization titers from authentic and pseudotyped viruses showed high concordance (R² = 0.8341, p < 0.0001), supporting the pseudovirus system as a reliable surrogate assay. Epitope analysis confirmed that SYN023’s epitope is fully conserved in the highly virulent New York City (NYC) RABV strain. In canine PEP challenge studies using the NYC strain, SYN023 conferred strong protection at 0.1–0.5 mg/kg. SYN023 exhibited potent neutralizing activity against phylogenetically diverse non-rabies lyssaviruses, including Mokola virus (MOKV; EC₅₀ = 14.74 µg/mL), whereas three clinically validated reference antibodies failed to neutralize MOKV even at concentrations up to 1000 µg/mL. Conclusions: This study demonstrates that the pseudovirus-based neutralization assay is a reliable surrogate assay. It also confirms the functional accuracy of the epitope mapping strategy and demonstrates the broad neutralization spectrum of SYN023 against a diverse panel of lyssaviruses.

Background: Lyssaviruses remain a global public health threat, requiring broad-spectrum, high-potency neutralizing antibodies as part of recommended human rabies post-exposure prophylaxis (PEP). A humanized monoclonal antibody cocktail, SYN023, is approved for PEP in China and is under global regulatory review. Its epitope mapping data are promising. However, the neutralization breadth across diverse lyssaviruses and the reliability of pseudovirus-based neutralization assays still require comprehensive validation to support worldwide regulatory approval and clinical use. Methods: First, the correlation between authentic lyssaviruses virus and pseudotyped viruses neutralization was established experimentally to verify the reliability of the pseudovirus-based neutralization assay. Second, epitope mapping was performed using a highly virulent rabies virus (RABV) variant (New York City strain: NYC) provided by the U.S. Department of Agriculture, followed by in vivo PEP efficacy studies in a standardized canine model to functionally validate the mapped epitopes. Third, the neutralizing activity of SYN023 was evaluated against a phylogenetically diverse panel of non-RABV lyssaviruses and directly benchmarked against clinically validated comparator monoclonal antibodies, thereby providing robust, quantitative empirical evidence supporting its broad-spectrum neutralization capability. Results: A strong positive correlation was observed between neutralization titers elicited by authentic lyssaviruses and pseudotyped viruses (Pearson R2 = 0.83, P < 0.0001), validating the pseudovirus-based assay as a robust surrogate for authentic virus neutralization. Specifically, SYN023 recognized and potently neutralized the epitope mapped on the prototypic NYC RABV strain. The in vivo PEP studies conducted in a standardized canine challenge model, using the NYC strain as the challenge virus, demonstrated high protective efficacy at SYN023 doses ranging from 0.1 to 0.5 mg/kg. Furthermore, SYN023 exhibited broad, potent, and reproducible neutralizing activity against a phylogenetically diverse panel of non-RABV lyssaviruses, including European bat lyssavirus 1 (EBLV1), European bat lyssavirus 2 (EBLV2), Australian bat lyssavirus (ABLV), Bokeloh bat lyssavirus (BBLV), Taiwan bat lyssavirus 1 (TWBLV1), Taiwan bat lyssavirus 2 (TWBLV2), Kotalahti bat lyssavirus (KBLV), Divača bat lyssavirus (DBLV), Gannoruwa bat lyssavirus (GBLV), Duvenhage virus (DUVV), Khujand virus (KHUV), Aravan virus (ARAV), Mokola virus (MOKV), Lagos bat virus (LBV) and Shimoni bat virus (SHIBV). Notably, SYN023 achieved robust neutralization of MOKV (EC50 = 14.74 µg/mL), whereas clinically referenced monoclonal antibodies—Rabishield, Ormutivimab and GR1801—exhibited no detectable neutralizing activity against MOKV even at concentrations up to 1,000 µg/mL. This pan-lyssavirus neutralizing capacity was attributable mechanistically to SYN023’s targeting of a highly conserved conformational epitope within the viral glycoprotein, which is structurally preserved across all major lyssavirus phylogroups. Conclusion: This study demonstrated that the pseudovirus-based neutralization assay is a reliable surrogate platform. In addition, it confirmed the functional accuracy of the epitope mapping strategy, showed the protective efficacy against a virulent RABV challenge in dogs, and demonstrated the broad neutralization spectrum of the SYN023 antibody cocktail against both RABV and a phylogenetically diverse panel of non-RABV lyssaviruses.

Bats are crucial reservoirs for coronaviruses, yet surveillance in the southeastern United States remains limited. Using carcasses submitted from public health surveillance for rabies, we screened 170 non-rabid bat carcasses from Alabama using a tiered real-time PCR framework targeting broad Alpha- and Betacoronavirus, alongside specific Sarbecovirus and Merbecovirus lineages. Overall, coronavirus prevalence was 3.5% (6/170), with all positives resolving as Alphacoronavirus. Detections spanned six counties and three bat taxa: Tadarida brasiliensis, Myotis spp., and Eptesicus fuscus, with the latter accounting for most cases (4/6; 66.7%). Sequence and phylogenetic analyses revealed two distinct evolutionary lineages. One isolate, ID-51 (E. fuscus), exhibited relative conservation, sharing 95.3% nucleotide and 93.0% amino acid identity with known references, forming a distinct sibling branch. Conversely, a highly divergent cluster of five isolates (ID-06, -145, -147, -169, -175) displayed remarkably low nucleotide similarities (83.7%–84.3%) to GenBank references despite maintaining high amino acid conservation (98.3%–100%). Topological shifts between nucleotide and amino acid phylogenies demonstrated genetic variation within this novel cluster. These findings underscore a previously uncharacterized genetic diversity of coronaviruses in regional US wildlife, highlighting the necessity of expanded local genomic surveillance.

Introduction: Mass dog rabies vaccination is the most effective strategy for preventing human rabies and interrupting transmission. However, achieving high vaccination coverage in dog populations remains challenging in many low‐ and middle‐income countries (LMICs), due to multiple barriers. In Nigeria, Africa's most populous country, rabies remains a major public health burden, with domestic dogs as the primary reservoir. Efforts to scale up canine rabies vaccination are limited by the lack of a coordinated national programme and challenges related to affordability, service availability, infrastructure, and owner awareness. Methods: We conducted a cross‐sectional study of dog‐owning households in three Nigerian states and the Federal Capital Territory (FCT) and developed a probabilistic model to uncover contextual factors associated with perceived barriers to canine rabies vaccination among owners of unvaccinated dogs. Results: Posterior distributions of our model estimates indicate that respondents reporting not knowing how to vaccinate their dogs against rabies were more likely to have tertiary education, prior awareness of rabies, use dogs for security purposes, urban residence, and residence in areas with higher household poverty. In contrast, respondents reporting knowledge of rabies transmission via bites from rabid dogs, access to local veterinary services, good road networks, and ownership of partially confined dogs were less likely to perceive this barrier. Respondents who were students, had attained tertiary education, resided in urban areas, and were in the FCT and Anambra State were more likely to perceive the rabies vaccine as too costly. Conversely, respondents from medium‐sized households, those who relied on traditional care in the event of a dog bite, and owners of partially confined dogs were also more likely to perceive the rabies vaccine as too costly. Finally, respondents who had heard about rabies and those with tertiary education were less likely to perceive the rabies vaccine as unimportant. Conclusions: Our study identified key barriers to canine rabies vaccination in Nigeria, including lack of knowledge, perceived high cost, and low perceived importance. These were influenced by socioeconomic factors, place of residence, and dog ownership practices, while rabies awareness, veterinary access, good road infrastructure, and partial confinement reduced these perceptions. These findings highlight the need for targeted, context‐specific strategies to improve affordability, access, and infrastructure, particularly among disadvantaged households.

Rabies is an acute, progressive, viral encephalitis. Warm-blooded vertebrates are susceptible. Major reservoirs reside in the Chiroptera and Carnivora. Among the latter, representatives include dogs, ferret badgers, foxes, jackals, mongooses, raccoons, and skunks. Within the Carnivora, pinnipeds represent a diverse group of >30 extant species. These marine mammals range from the Arctic to Antarctica, but there is no review about rabies in this group. Apparently, only a single 1980 case of rabies occurred from Svalbard in a ringed seal (Phoca hispida). However, in 2024, incidental cases appeared within South African Cape fur seals (Arctocephalus pusillus). Retrospective testing of archival material identified suspect cases dating back to 2022. Currently, more than 80 cases have been documented in seals. Moreover, a new 2025 focus appeared in Namibia and cases in Angola are predictable. Viral characterization supports spillover infection via rabid black-backed jackals (Lupulella mesomelas). A host shift appears likely, with ongoing seal intraspecific transmission. Given the unique nature of this epizootic, implications for the southern hemisphere abound. Unfortunately, comprehensive data are lacking on pinniped specimens examined outside of southern Africa. For example, although Antarctica is considered ‘rabies-free’, minimal international standards for support are unmet. No routine laboratory-based surveillance occurs. This enzootic rabies focus among seals in southern Africa presents unique challenges for the region and a rare opportunity for considering broader surveillance. Besides targeted parenteral vaccination of fur seals, local engagement involves vagrant species, including elephant (Mirounga leonina) and leopard seals (Hydrurga leptonyx). The void of regional pinniped surveillance, especially encompassing the Southern Ocean would require considerable proactive local resolution and much wider collaboration regarding future concerns to both public health and conservation biology.

Bats (Chiroptera) represent nearly one-fifth of all mammalian species and play vital ecological roles as pollinators, pest controllers, and reservoirs of zoonotic pathogens. Accurate identification of bat species is essential for biodiversity monitoring, conservation, and disease surveillance. Traditional methods based on morphology or acoustic calls are often limited by overlapping features, while DNA barcoding using the cytochrome oxidase I (COI) gene can be hindered by sequence variability. In this study, we developed a simple, single-step PCR assay targeting a short, variable region of the mitochondrial 12S rRNA gene. Alignment of sequences from 232 bat species allowed the design of a single primer pair producing a 203–224 bp amplicon that successfully distinguished all species analyzed. The assay achieved 100% amplification success across 241 bat samples, with 97.2% concordance between molecular and morphological identification. Two samples showed sequence divergence suggestive of an undescribed species. Overall, ten bat species from six genera were identified, with Eptesicus fuscus being the most frequent. This assay offers a practical and robust approach for bat identification, supporting biodiversity assessment and pathogen surveillance in ecological and public health research.

Lyssaviruses are RNA viruses in the Family Rhabdoviridae, Genus Lyssavirus. They represent the causative agents of acute, progressive encephalitis, known historically as rabies. Regardless of specific etiology, their collective viral morphology, biochemistry, pathobiology, associated clinical signs, diagnosis, epizootiology, and management are essentially the same. Despite centuries of clinical recognition, these quintessential neurotropic agents remain significant pathogens today, with substantive consequences to agriculture, public health, and conservation biology. Notably, the singular morbidity caused by lyssaviruses is incurable and constitutes the highest case fatality of any viral disease. All warm-blooded vertebrates are believed to be susceptible. The dog is the only domestic animal that serves as a reservoir, vector, and victim. In contrast, felids are effective vectors, but not reservoirs. All other rabid domestic species, such as livestock, constitute spillover infections, as a bellwether to local lyssavirus activity. Frequently, professional confusion abounds among the veterinary community, because although the viral species Lyssavirus rabies is inarguably the best-known representative in the Genus, at least 20 other recognized or putative members of this monophyletic group are known. Frequently, this is simply overlooked. Moreover, often the ‘taxonomic etiology’ (i.e., ‘Lyssavirus x’) is mistakenly referenced in a biopolitcal context, instead of the obvious clinical illness (i.e., ‘rabies’). Global consternation persists, if localities believe they are ‘disease-free’, when documented lyssaviruses circulate or laboratory-based surveillance is inadequate to support such claims. Understandably, professional chagrin develops when individuals mistake the epidemiological terminology of control, prevention, elimination, etc. Management is not simple, given that the only licensed veterinary and human vaccines are against rabies virus, sensu lato. There are no adequate antiviral drugs for any lyssaviruses or cross-reactive biologics developed against more distantly related viral members. While representative taxa among the mammalian Orders Chiroptera, Carnivora, and Primates exemplify the major global reservoirs, which mammalian species are responsible for the perpetuation of other lyssaviruses remains a seemingly academic curiosity. This zoonosis is neglected. Clearly, with such underlying characteristics as a fundamental ‘disease of nature’, rabies, unlike smallpox and rinderpest, is not a candidate for eradication. With the worldwide zeal to drive human fatalities from canine rabies viruses to zero by the rapidly approaching year 2030, enhanced surveillance and greater introspection of the poorly appreciated burden posed by rabies virus and diverse other lyssaviruses may manifest as an epidemiological luxury to the overall global program of the future.

A Pictorial Human Case of “Furious Rabies”

Human rabies cases today are predominantly associated with infection from rabid domestic dogs. Unlike dogs, a common global reservoir species that perpetuates rabies viruses (RABV) within their populations, domestic cats are much less frequently reported or vaccinated. Epidemiologically, cats are important vectors of lyssaviruses but are not viral reservoirs. Typically, cats are incidental hosts only, infected with the predominant lyssavirus in their geographic locale. Human cases associated with rabid cats have occurred in Africa, Asia, Europe and throughout the Americas. As adept, solitary hunters, wild and domestic felids are at risk of lyssavirus infection based upon interactions with infected prey, such as bats, or from transmission by other mesocarnivores, such as rabid dogs, foxes, jackals, raccoons, and skunks. Current veterinary vaccines provide safe and effective immunity in cats against phylogroup I lyssaviruses, such as RABV, but not against divergent lyssaviruses in phylogroups II-IV. With the focus upon the global elimination of canine rabies, the emergence of rabies in cats represents a concerning trend. Clearly, education about the occurrence of rabies in cats needs to be improved, as well as the routine vaccination of cats to reduce the associated risks to public health, agriculture, and conservation biology from a One Health perspective.

Journal of Wildlife Diseases publishes work on infectious, parasitic, toxic, nutritional, physiologic, and neoplastic diseases impacting wild animals.