Monkeypox Virus in Animals: Current Knowledge of Viral Transmission and Pathogenesis in Wild Animal Reservoirs and Captive Animal Models

Mpox, formerly called monkeypox, is now the most serious orthopoxvirus (OPXV) infection in humans. This zoonotic disease has been gradually re-emerging in humans with an increasing frequency of cases found in endemic areas, as well as an escalating frequency and size of epidemics outside of endemic areas in Africa. Currently, the largest known mpox epidemic is spreading throughout the world, with over 85,650 cases to date, mostly in Europe and North America. These increased endemic cases and epidemics are likely driven primarily by decreasing global immunity to OPXVs, along with other possible causes. The current unprecedented global outbreak of mpox has demonstrated higher numbers of human cases and greater human-to-human transmission than previously documented, necessitating an urgent need to better understand this disease in humans and animals. Monkeypox virus (MPXV) infections in animals, both naturally occurring and experimental, have provided critical information about the routes of transmission; the viral pathogenicity factors; the methods of control, such as vaccination and antivirals; the disease ecology in reservoir host species; and the conservation impacts on wildlife species. This review briefly described the epidemiology and transmission of MPXV between animals and humans and summarizes past studies on the ecology of MPXV in wild animals and experimental studies in captive animal models, with a focus on how animal infections have informed knowledge concerning various aspects of this pathogen. Knowledge gaps were highlighted in areas where future research, both in captive and free-ranging animals, could inform efforts to understand and control this disease in both humans and animals

Rabies Outbreak in Captive Big Brown Bats (Eptesicus fuscus) Used in a White-nose Syndrome Vaccine Trial

An outbreak of rabies occurred in a captive colony of wild-caught big brown bats (Eptesicus fuscus). Five of 27 bats exhibited signs of rabies virus infection 22–51 d after capture or 18–22 d after contact with the index case. Rabid bats showed weight loss, aggression, increased vocalization, hypersalivation, and refusal of food. Antigenic typing and virus sequencing confirmed that all five bats were infected with an identical rabies virus variant that circulates in E. fuscus in the US. Two bats with no signs of rabies virus infection were seropositive for rabies virus-neutralizing antibodies; the brains of these bats had no detectable viral proteins by the direct fluorescence antibody test. We suspect bat-to-bat transmission of rabies virus occurred among our bats because all rabies-infected bats were confined to the cage housing the index case and were infected with viruses having identical sequences of the entire rabies nucleoprotein gene. This outbreak illustrates the risk of rabies virus infection in captive bats and highlights the need for researchers using bats to assume that all wild bats could be infected with rabies virus

Immunogenicity, Safety, and Anti-Viral Efficacy of a Subunit SARS-CoV-2 Vaccine Candidate in Captive Black-Footed Ferrets (<i>Mustela nigripes</i>) and Their Susceptibility to Viral Challenge

A preliminary vaccination trial against the emergent pathogen, SARS-CoV-2, was completed in captive black-footed ferrets (Mustela nigripes; BFF) to assess safety, immunogenicity, and anti-viral efficacy. Vaccination and boosting of 15 BFF with purified SARS-CoV-2 S1 subunit protein produced a nearly 150-fold increase in mean antibody titers compared to pre-vaccination titers. Serum antibody responses were highest in young animals, but in all vaccinees, antibody response declined rapidly. Anti-viral activity from vaccinated and unvaccinated BFF was determined in vitro, as well as in vivo with a passive serum transfer study in mice. Transgenic mice that received BFF serum transfers and were subsequently challenged with SARS-CoV-2 had lung viral loads that negatively correlated (p < 0.05) with the BFF serum titer received. Lastly, an experimental challenge study in a small group of BFF was completed to test susceptibility to SARS-CoV-2. Despite viral replication and shedding in the upper respiratory tract for up to 7 days post-challenge, no clinical disease was observed in either vaccinated or naive animals. The lack of morbidity or mortality observed indicates SARS-CoV-2 is unlikely to affect wild BFF populations, but infected captive animals pose a potential risk, albeit low, for humans and other animals

Virally-vectored vaccine candidates against white-nose syndrome induce anti-fungal immune response in little brown bats (Myotis lucifugus)

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P \u3c 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS

Further Assessment of Monkeypox Virus Infection in Gambian Pouched Rats (<i>Cricetomys gambianus</i>) Using In Vivo Bioluminescent Imaging

<div><p>Monkeypox is a zoonosis clinically similar to smallpox in humans. Recent evidence has shown a potential risk of increased incidence in central Africa. Despite attempts to isolate the virus from wild rodents and other small mammals, no reservoir host has been identified. In 2003, <i>Monkeypox virus</i> (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (<i>Cricetomys gambianus</i>). Therefore, we investigated the potential reservoir competence of the Gambian pouched rat for MPXV by utilizing a combination of in vivo and in vitro methods. We inoculated three animals by the intradermal route and three animals by the intranasal route, with one mock-infected control for each route. Bioluminescent imaging (BLI) was used to track replicating virus in infected animals and virological assays (e.g. real time PCR, cell culture) were used to determine viral load in blood, urine, ocular, nasal, oral, and rectal swabs. Intradermal inoculation resulted in clinical signs of monkeypox infection in two of three animals. One severely ill animal was euthanized and the other affected animal recovered. In contrast, intranasal inoculation resulted in subclinical infection in all three animals. All animals, regardless of apparent or inapparent infection, shed virus in oral and nasal secretions. Additionally, BLI identified viral replication in the skin without grossly visible lesions. These results suggest that Gambian pouched rats may play an important role in transmission of the virus to humans, as they are hunted for consumption and it is possible for MPXV-infected pouched rats to shed infectious virus without displaying overt clinical signs.</p></div

Dorsal and ventral views of Gambian pouched rats (<i>Cricetomys gambianus</i>) infected intranasally with MPXV/Luc+.

<p>Gambian pouched rats infected intranasally with <i>Monkeypox virus</i> that expresses firefly luciferase (MPXV/Luc+) show luminescence (viral replication) at the initial site of inoculation, in the nasal area. In two animals (GPR 6 and GPR 14), there is luminescence in the region of the submandibular lymph nodes, a common site of secondary replication for Orthopoxviruses. In GPR 14, luminescence is evident in distant sites from day 8 to day 22 post-infection. Despite evidence of viral replication, none of these animals showed any grossly visible skin lesions.</p

Luminescence of Gambian Pouched Rats (<i>Cricetomys gambianus</i>) infected with Monkeypox/Luc+ by intradermal (A) and intranasal (B) routes.

<p>Total luminescence (p/s/cm³/str), an estimate of viral load, peaks in all animals between days 8 and 14 and returned to background levels by day 21 presumably in relation to viral load. The total luminescence of the negative control (GPR 51), taken at day 0, is shown as a dashed line (A), to estimate background luminescence of Gambian pouched rats. The total luminescence of the sentinel animal (GPR 7) is shown in B. It remained similar to that of the negative control.</p

Dorsal and ventral views of Gambian pouched rats (<i>Cricetomys gambianus</i>) infected intradermally with MPXV/Luc+.

<p>Gambian pouched rats that were infected intradermally with <i>Monkeypox virus</i> that expresses firefly luciferase (MPXV/Luc+) show luminescence, indicative of virus replication, at the initial site of inoculation, over the dorsal scapulae, and then at distant sites. The intensity of light produced is used to estimate the quantity of replicating MPXV/Luc+. By day 7, luminescence is visible in the oronasal area of all three infected rats. This is corroborated by tests for viral shedding; all three animals had detectable virus in oral and nasal swabs. Viral replication continues in GPR1, especially in the location of numerous abdominal skin lesions. GPR 5 developed systemic infection, with a large amount of luminescence detected in the abdominal and thoracic regions by day 14.</p

Anti-Orthopox virus antibody titers of animals infected intradermally (A) and intranasally (B) with <i>Monkeypox virus</i> expressing luciferase (MPXV/Luc+).

<p>Antibody titers indicate that animals develop a strong humoral immune response around 3 weeks, which coincides with the time at which luminescence and viral shedding begins to cease. Orthopox antibodies are strongly cross-reactive, so antibody responses to <i>Vaccinia virus</i>, as in this method, very closely approximate titers to <i>Monkeypox virus</i>.</p