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

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, Monkeypox virus (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (Cricetomys gambianus). 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.     

Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4×10⁴ PFU, 1×10⁵ PFU, or 1×10⁶ PFU resulted in lethality for 70% of the animals, whereas a dose of 4×10⁵ PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.     

A Novel Highly Reproducible and Lethal Nonhuman Primate Model for Orthopox Virus Infection

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1–3 days after onset of symptoms, even when very low infectious viral doses of 5×10² pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID₅₀ (minimal monkey infectious dose 50%) of 8.3×10² pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.     

Monkeypox disease transmission in an experimental setting: prairie dog animal model

Monkeypox virus (MPXV) is considered the most significant human public health threat in the genus Orthopoxvirus since the eradication of variola virus (the causative agent of smallpox). MPXV is a zoonotic agent endemic to forested areas of Central and Western Africa. In 2003, MPXV caused an outbreak in the United States due to the importation of infected African rodents, and subsequent sequential infection of North American prairie dogs (Cynomys ludovicianus) and humans. In previous studies, the prairie dog MPXV model has successfully shown to be very useful for understanding MPXV since the model emulates key characteristics of human monkeypox disease. In humans, percutaneous exposure to animals has been documented but the primary method of human-to-human MPXV transmission is postulated to be by respiratory route. Only a few animal model studies of MPXV transmission have been reported. Herein, we show that MPXV infected prairie dogs are able to transmit the virus to naive animals through multiple transmission routes. All secondarily exposed animals were infected with MPXV during the course of the study. Notably, animals secondarily exposed appeared to manifest more severe disease; however, the disease course was very similar to those of experimentally challenged animals including inappetence leading to weight loss, development of lesions, production of orthopoxvirus antibodies and shedding of similar levels or in some instances higher levels of MPXV from the oral cavity. Disease was transmitted via exposure to contaminated bedding, co-housing, or respiratory secretions/nasal mucous (we could not definitively say that transmission occurred via respiratory route exclusively). Future use of the model will allow us to evaluate infection control measures, vaccines and antiviral strategies to decrease disease transmission.     

Transmissibility of the Monkeypox Virus Clades via Respiratory Transmission: Investigation Using the Prairie Dog-Monkeypox Virus Challenge System

Monkeypox virus (MPXV) is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV) related to MPXV) and cessation of routine smallpox vaccination (with the live OPXV vaccinia), there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively). Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model.     

Comparison of West African and Congo Basin Monkeypox Viruses in BALB/c and C57BL/6 Mice

Although monkeypox virus (MPXV) studies in wild rodents and non-human primates have generated important knowledge regarding MPXV pathogenesis and inferences about disease transmission, it might be easier to dissect the importance of virulence factors and correlates of protection to MPXV in an inbred mouse model. Herein, we compared the two clades of MPXV via two routes of infection in the BALB/c and C57BL/6 inbred mice strains. Our studies show that similar to previous animal studies, the Congo Basin strain of MPXV was more virulent than West African MPXV in both mouse strains as evidenced by clinical signs. Although animals did not develop lesions as seen in human MPX infections, localized signs were apparent with the foot pad route of inoculation, primarily in the form of edema at the site of inoculation; while the Congo Basin intranasal route of infection led to generalized symptoms, primarily weight loss. We have determined that future studies with MPXV and laboratory mice would be very beneficial in understanding the pathogenesis of MPXV, in particular if used in in vivo imaging studies. Although this mouse model may not suffice as a model of human MPX disease, with an appropriate inbred mouse model, we can unravel many unknown aspects of MPX pathogenesis, including virulence factors, disease progression in rodent hosts, and viral shedding from infected animals. In addition, such a model can be utilized to test antivirals and the next generation of orthopoxvirus vaccines for their ability to alter the course of disease.     

猴痘的流行现状及防控

猴痘(monkeypox)是由猴痘病毒感染所致的人兽共患病,主要发生在非洲中部、西部地区。猴痘病毒可感染多种哺乳类动物,主要在动物中流行,人接触感染动物后可被传染。猴痘的临床表现与天花相似(发热、皮疹等),但症状较轻。天花疫苗接种可提供预防猴痘的免疫保护力。然而,因全球天花被消灭而停止接种天花疫苗后,猴痘成为最可能威胁人类的正痘病毒性疾病。近期,其散发病例在欧洲多地出现。2022年5月7日英国报道了猴痘疫情。随后,欧洲报道猴痘确诊和疑似病例超过100例。猴痘主要传播途径包括接触感染动物、与患者直接接触或间接接触。2022年5月20日,世界卫生组织就此次猴痘疫情召开了紧急会议,旨在提高对猴痘的认识,做好防范应对准备。世界卫生组织、美国疾病预防控制中心、英国卫生部门报告了相关疫情并制定了相应的防控措施。截至2022年5月28日我国尚无输入性猴痘报道,但因国际交往频繁等仍须提高警惕。本文介绍了猴痘流行现状及有关防控信息,以供借鉴。     

The evolving epidemiology of monkeypox virus

Monkeypox, caused by the monkeypox virus (MPXV), is a zoonotic disease endemic mainly in West and Central Africa. As of 27 September 2022, human monkeypox has occurred in more than 100 countries (mostly in non-endemic regions) and caused over 66,000 confirmed cases, which differs from previous epidemics that mainly affected African countries. Due to the increasing number of confirmed cases worldwide, the World Health Organization (WHO) has declared the monkeypox outbreak as a Public Health Emergency of International Concern on July 23, 2022. The international outbreak of human monkeypox represents a novel route of transmission for MPXV, with genital lesions as the primary infection, and the emergence of monkeypox in the current outbreak is also new, as novel variants emerge. Clinical physicians and scientists should be aware of this emerging situation, which presents a different scenario from previous outbreaks. In this review, we will discuss the molecular virology, evasion of antiviral immunity, epidemiology, evolution, and detection of MPXV, as well as prophylaxis and treatment strategies for monkeypox. This review also emphasizes the integration of relevant epidemiological data with genomic surveillance data to obtain real-time data, which could formulate prevention and control measures to curb this outbreak.     

Elucidating the role of the complement control protein in monkeypox pathogenicity

Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ～10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ～24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus.     

Effective antiviral treatment of systemic orthopoxvirus disease: ST-246 treatment of prairie dogs infected with monkeypox virus

Smallpox preparedness research has led to development of antiviral therapies for treatment of serious orthopoxvirus infections. Monkeypox virus is an emerging, zoonotic orthopoxvirus which can cause severe and transmissible disease in humans, generating concerns for public health. Monkeypox virus infection results in a systemic, febrile-rash illness closely resembling smallpox. Currently, there are no small-molecule antiviral therapeutics approved to treat orthopoxvirus infections of humans. The prairie dog, using monkeypox virus as a challenge virus, has provided a valuable nonhuman animal model in which monkeypox virus infection closely resembles human systemic orthopoxvirus illness. Here, we assess the efficacy of the antiorthopoxvirus compound ST-246 in prairie dogs against a monkeypox virus challenge of 65 times the 50% lethal dose (LD(50)). Animals were infected intranasally and administered ST-246 for 14 days, beginning on days 0, 3, or after rash onset. Swab and blood samples were collected every 2 days and analyzed for presence of viral DNA by real-time PCR and for viable virus by tissue culture. Seventy-five percent of infected animals that received vehicle alone succumbed to infection. One hundred percent of animals that received ST-246 survived challenge, and animals that received treatment before symptom onset remained largely asymptomatic. Viable virus and viral DNA were undetected or at greatly reduced levels in animals that began treatment on 0 or 3 days postinfection, compared to control animals or animals treated post-rash onset. Animals treated after rash onset manifested illness, but all recovered. Our results indicate that ST-246 can be used therapeutically, following onset of rash illness, to treat systemic orthopoxvirus infections.     

Record Chromosome Number in a Mammal?

CROSS has reported^{1, 2} chromosome complements in rodents ranging up to 2n=84 (in the North American geomyid Geomys breviceps) and 2n=86 (in the heteromyid Dipodomys merriami). Matthey has contested³ this claim; he maintained that in Geomys bursarius 2n =70 or 72 and he doubted whether a higher number had been confirmed. Numbers in the 2n=70 to 80 range have since been reported⁴ in several genera of Canidae and Ursidae. In general, rodent chromosomes fall into the lower group numbers but, among murines, Lophuromys aquillus is reported to have a chromosome complement of 70 (ref. 5) and Cricetomys gambianus one of 78 (ref, 6). In the gerbilline, Meriones shawii, the number can vary up to 78 (ref. 7).     

Identification of Wild-Derived Inbred Mouse Strains Highly Susceptible to Monkeypox Virus Infection for Use as Small Animal Models

Infection with monkeypox virus (MPXV) causes disease manifestations in humans that are similar, although usually less severe, than those of smallpox. Since routine vaccination for smallpox ceased more than 30 years ago, there is concern that MPXV could be used for bioterrorism. Thus, there is a need to develop animal models to study MPXV infection. Accordingly, we screened 38 inbred mouse strains for susceptibility to MPXV. Three highly susceptible wild-derived inbred strains were identified, of which CAST/EiJ was further developed as a model. Using an intranasal route of infection with an isolate of the Congo Basin clade of MPXV, CAST/EiJ mice exhibited weight loss, morbidity, and death in a dose-dependent manner with a calculated 50% lethal dose (LD₅₀) of 680 PFU, whereas there were no deaths of BALB/c mice at a 10,000-fold higher dose. CAST/EiJ mice exhibited greater MPXV sensitivity when infected via the intraperitoneal route, with an LD₅₀ of 14 PFU. Both routes resulted in MPXV replication in the lung, spleen, and liver. Intranasal infection with an isolate of the less-pathogenic West African clade yielded an LD₅₀ of 7,600 PFU. The immune competence of CAST/EiJ mice was established by immunization with vaccinia virus, which induced antigen-specific T- and B-lymphocyte responses and fully protected mice from lethal doses of MPXV. The new mouse model has the following advantages for studying pathogenesis of MPXV, as well as for evaluation of potential vaccines and therapeutics: relative sensitivity to MPXV through multiple routes, genetic homogeneity, available immunological reagents, and commercial production.Monkeypox virus (MPXV), a member of the orthopoxvirus genus of the Chordopoxvirinae subfamily of the Poxviridae, was isolated in 1958 from lesions in a cynomolgous monkey that had been imported from Africa (27). The first human infections with MPXV were reported in 1972, and since then more than two thousand cases have been recorded, most in the Democratic Republic of the Congo and lesser numbers in West African countries (reviewed by Parker et al. [18]). The mortality from human monkeypox in the Congo is estimated to be 10% of infected individuals with clinical symptoms that mimic smallpox, which is caused by another member of the orthopoxvirus genus: variola virus. However, whereas the host range of variola virus is restricted to humans, serological studies indicate that MPXV naturally infects a large number of animal species, particularly squirrels and nonhuman primates. The sporadic occurrence of human monkeypox is thought to arise from close proximity and handling of infected animals. In this respect, a self-limited outbreak in the United States was traced to a shipment of West African rodents (19). Although monkeypox is a minor public health problem when compared historically to smallpox, the potential for expansion of the MPXV host range and adaptations to enhance human transmission make it prudent to continue careful surveillance. Moreover, the potential use of MPXV for bioterrorism has led to its inclusion as a select agent in the United States (http://www.selectagents.gov).Animal models are crucial for studying virus pathogenesis, and MXPV is no exception. Ground squirrels (22, 26), black-tailed prairie dogs (9, 11, 13, 30), and African dormice (23) are highly susceptible to MPXV. However, as experimental systems, each has limitations with regard to unavailability of commercial breeding, genetic heterogeneity and absence of immunological and other reagents. Laboratory mice, including BALB/c, C57BL/6, and several other mouse strains tested, were found to be resistant to MPXV disease unless impaired in innate or acquired immunity (10, 17, 24). In the present study, we tested a large group of distinct inbred strains of mice chosen for genetic diversity, inclusion of classical and wild-derived strains, and commercial availability. Of 38 inbred mouse strains tested, three wild-derived strains were highly susceptible to MPXV. One of these, CAST/EiJ, was further characterized with regard to MPXV strain sensitivity, route of inoculation, virus dissemination, immune response, and protection by vaccination and drug treatment.     

Establishment of the black-tailed prairie dog (Cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high- and low-dose monkeypox virus challenges

The 2003 monkeypox virus (MPXV) outbreak and subsequent laboratory studies demonstrated that the black-tailed prairie dog is susceptible to MPXV infection and that the ensuing rash illness is similar to human systemic orthopoxvirus (OPXV) infection, including a 7- to 9-day incubation period and, likely, in some cases a respiratory route of infection; these features distinguish this model from others. The need for safe and efficacious vaccines for OPVX in areas where it is endemic or epidemic is important to protect an increasingly OPXV-naïve population. In this study, we tested current and investigational smallpox vaccines for safety, induction of anti-OPXV antibodies, and protection against mortality and morbidity in two MPXV challenges. None of the smallpox vaccines caused illness in this model, and all vaccinated animals showed anti-OPXV antibody responses and neutralizing antibody. We tested vaccine efficacy by challenging the animals with 10⁵ or 10⁶ PFU Congo Basin MPXV 30 days postvaccination and evaluating morbidity and mortality. Our results demonstrated that vaccination with either Dryvax or Acambis2000 protected the animals from death with no rash illness. Vaccination with IMVAMUNE also protected the animals from death, albeit with (modified) rash illness. Based on the results of this study, we believe prairie dogs offer a novel and potentially useful small animal model for the safety and efficacy testing of smallpox vaccines in pre- and postexposure vaccine testing, which is important for public health planning.     

Deletion of the monkeypox virus inhibitor of complement enzymes locus impacts the adaptive immune response to monkeypox virus in a nonhuman primate model of infection

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.     

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening

With the rising number of patients with human immunodeficiency virus (HIV)/AIDS in developing countries, the control of mycobacteria is of growing importance. Previous studies have shown that rodents and insectivores are carriers of mycobacteria. However, it is not clear how widespread mycobacteria are in these animals and what their role is in spreading them. Therefore, the prevalence of mycobacteria in rodents and insectivores was studied in and around Morogoro, Tanzania. Live rodents were trapped, with three types of live traps, in three habitats. Pieces of organs were pooled per habitat, species, and organ type (stratified pooling); these sample pools were examined for the presence of mycobacteria by PCR, microscopy, and culture methods. The mycobacterial isolates were identified using phenotypic techniques and sequencing. In total, 708 small mammals were collected, 31 of which were shrews. By pool prevalence estimation, 2.65% of the animals were carriers of mycobacteria, with a higher prevalence in the urban areas and in Cricetomys gambianus and the insectivore Crocidura hirta. Nontuberculous mycobacteria (Mycobacterium chimaera, M. intracellulare, M. arupense, M. parascrofulaceum, and Mycobacterium spp.) were isolated from C. gambianus, Mastomys natalensis, and C. hirta. This study is the first to report findings of mycobacteria in African rodents and insectivores and the first in mycobacterial ecology to estimate the prevalence of mycobacteria after stratified pool screening. The fact that small mammals in urban areas carry more mycobacteria than those in the fields and that potentially pathogenic mycobacteria were isolated identifies a risk for other animals and humans, especially HIV/AIDS patients, that have a weakened immune system.     

A Mouse Model of Lethal Infection for Evaluating Prophylactics and Therapeutics against Monkeypox Virus

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola, the etiological agent of smallpox. In humans, MPXV causes a disease similar to smallpox and is considered to be an emerging infectious disease. Moreover, the use of MPXV for bioterroristic/biowarfare activities is of significant concern. Available small animal models of human monkeypox have been restricted to mammals with poorly defined biologies that also have limited reagent availability. We have established a murine MPXV model utilizing the STAT1-deficient C57BL/6 mouse. Here we report that a relatively low-dose intranasal (IN) infection induces 100% mortality in the stat1^−/− model by day 10 postinfection with high infectious titers in the livers, spleens, and lungs of moribund animals. Vaccination with modified vaccinia virus Ankara (MVA) followed by a booster vaccination is sufficient to protect against an intranasal MPXV challenge and induces an immune response more robust than that of a single vaccination. Furthermore, antiviral treatment with CMX001 (HDP-cidofovir) and ST-246 protects when administered as a regimen initiated on the day of infection. Thus, the stat1^−/− model provides a lethal murine platform for evaluating therapeutics and for investigating the immunological and pathological responses to MPXV infection.During the early smallpox-free epoch, the orthopoxviruses were of minor bioterroristic concern due to the largely vaccinated population; however, this has changed with the increased risk of bioterrorism, and variola virus (VARV) and monkeypox virus (MPXV) are considered to have significant potential to become bioterror agents (36, 37). VARV, the etiological agent of smallpox, is officially stored at two WHO secure laboratories in the United States and Russia; however, there is concern that covert stocks exist. Furthermore, we are currently faced with the possibility of intentional release of wild-type or genetically modified VARV. Of most concern would be viruses encoding human interleukin-4 (IL-4), which could significantly increase virulence, as demonstrated with the mousepox/ectromelia virus (ECTV) model (18). As a result of the cessation of routine vaccination and the high number of individuals that are contraindicated for vaccination, the human population lacks solid “herd immunity” to naturally circulating orthopoxviruses. One such virus that is of particular concern is MPXV, due to its ability to infect humans, its mortality rate of approximately 10% (depending on the strain), its propensity to infect a large number of species, its apparently increasing transmissibility in the human population, and its reportedly expanding host range (36). One such example of increasing host range was observed during 2003 in the United States, where imported African rodents transmitted MPXV to native prairie dogs, which acted as an “amplification reservoir” that allowed for the transmission of MPXV to humans (14).To date, MPXV animal models for efficacy testing of prophylactics and therapeutics have been restricted to nonhuman primates and nonmurine small animal models, such as the 13-lined ground squirrel (Spermophilus tridecemlineatus) (49, 55), the black-tailed prairie dog (Cynomys ludovicianus) (14, 17, 22, 60), and the African dormouse (Graphiurus kelleni) (50). Because the ground squirrel and the prairie dog are difficult to propagate, have low fecundity rates, and have complex husbandry requirements, they must be obtained from their natural habitat and therefore have unknown health statuses (16, 58). Conversely, the African dormouse has many characteristics similar to those of laboratory mice and can be easily propagated in a research vivarium. The disadvantage to this model is that there are few commercially available reagents for characterizing the animals'' response to infection, and their biology is poorly understood (50).Suckling white mice have been shown to be highly susceptible to MPXV inoculations by various routes. Eight-day-old white mice developed disease and died following intraperitoneal or intranasal (IN) inoculations with 1.2 × 10⁶ PFU. Injection into the footpad also induced severe disease and death following 6 × 10² PFU inoculations. Disease symptoms included flabbiness; loss of appetite; and following footpad infections, edema of the foot. Similar symptoms were observed following inoculation by the oral route, which induced 40% lethality. Intradermal inoculations with MPXV resulted in 50% death. The intranasal route of infection was determined to induce the highest level of lethality, causing 100% death in mice as old as 15 days, compared to only 14% and 60% lethality in 12-day-old mice infected orally or via the footpad, respectively (24, 51). Recently, Osorio et al. showed that 4-week-old SCID-BALB/c mice are susceptible to 10⁵ PFU intraperitoneal (IP) MPXV inoculations, resulting in a mean day of death of 9 days postinfection (p.i.) (33). Unfortunately the IP route of infection does not model the natural transmission route of MPXV.Several factors make young (<15 days old) white mice a poor choice for studying MPXV. First, mice do not become fully immunocompetent until approximately 4 or 5 weeks old; therefore, the opportunity to study the immune response to infection is hampered. Moreover, immunoimmature animals cannot be used as models to study MPXV infections in immunocompetent humans. Second, a functioning immune system works in synergy with antiviral therapies to provide protection against viral challenge; thus, antiviral efficacy cannot be properly evaluated. Third, the relatively short susceptible time window of birth to 15 days old makes large-scale experiments impractical. Fourthly, young immunoimmature mice cannot be used to study vaccination efficacy. To this end, we sought to identify adult mice that are susceptible to lethal MPXV challenges and can be used for antiviral and vaccination efficacy studies.In the present study we found that most common strains of adult immunocompetent laboratory mice are resistant to MPXV. We also found that type 1 and type 2 interferon (IFN) receptor-null mice were resistant. Because strains lacking STAT1, a key protein involved in type 1 and 2 IFN signaling networks, have been shown to be sensitive to a wide number of viral and bacterial infections (13, 15, 30, 46, 52-54), we evaluated their sensitivities to MPXV challenges. We found that C57BL/6 mice lacking stat1 (C57BL/6 stat1^−/−) were highly sensitive to MPXV and that 129 mice lacking stat1 were sensitive but to a lesser degree than the C57BL/6 stat1^−/− animals. In this report, we show that the disease course in MPXV-infected C57BL/6 stat1^−/− mice, that is, weight loss and death by day 10 postinfection, is similar to that observed in wild-type mice infected with ECTV, the etiological agent of mousepox (11). Further, we reveal that antiviral therapy with CMX001 or ST-246 protects mice to a degree similar to that of vaccination with Dryvax or modified vaccinia virus Ankara (MVA), supporting the use of the C57BL/6 stat1^−/− as a model to evaluate orthopoxvirus prophylactics and therapeutics.     

Monkeypox virus: a re-emergent threat to humans

Human monkeypox (MPX) is a rare zoonotic infection characterized by smallpox-like signs and symptoms. It is caused by monkeypox virus (MPXV), a double stranded DNA virus belonging to the genus Orthopoxvirus. MPX was first identified in 1970 and mostly prevailed in the rural rainforests of Central and West Africa in the past. Outside Africa, MPX was reported in the United Kingdom, the USA, Israel, and Singapore. In 2022, the resurgence of MPX in Europe and elsewhere posed a potential threat to humans. MPXV was transmitted by the animals-human or human-human pathway, and the symptoms of MPXV infection are similar to that of smallpox, but in a milder form and with lower mortality (1%–10%). Although the smallpox vaccination has been shown to provide 85% protection against MPXV infection, and two anti-smallpox virus drugs have been approved to treat MPXV, there are still no specific vaccines and drugs against MPXV infection. Therefore it is urgent to take active measures including the adoption of novel anti-MPXV strategies to control the spread of MPXV and prevent MPX epidemic. In this review, we summarize the biological features, epidemiology, pathogenicity, laboratory diagnosis, and prevention and treatment strategies on MPXV. This review provides the basic knowledge for prevention and control of future outbreaks of this emerging infection.     

Molecular evolution of poxviruses

Previous restriction fragment length polymorphism analysis divided variola virus (VARV) strains into two subtypes, one of which included West African and South American isolates. This allowed a dating to be introduced for the first time in estimation of the VARV evolution rate. The results were used to analyze the molecular evolution of the total family Poxviridae. Comparisons of the known nucleotide sequences were performed for the extended conserved central genome region in 42 orthopoxvirus strains and for the eight genes of multisubunit RNA polymerase in 65 viruses belonging to various genera of the family Poxviridae. Using the Bayesian dating method, the mutation accumulation rate of poxviruses was estimated at (1.7–8.8) × 10^?6 nucleotide substitutions per site per year. Computations showed that the modern poxvirus genera started diverging from an ancestral virus more than 200 thousand years ago and that an ancestor of the genus Orthopoxvirus emerged 131 ± 45 thousand years ago. The other genera of mammalian poxviruses with a low GC content diverged approximately 110–90 thousand years ago. The independent evolution of VARV started 3.4 ± 0.8 thousand years ago. It was shown with the example of VARV and the monkeypox virus (MPXV) that divergent evolution of these orthopoxviruses started and the West African subtypes of VARV and MPXV were formed as geographical conditions changed to allow isolation of West African animals from other African regions.     

Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Live and carcass weights of giant rat Cricetomys gambianus Waterhouse and domestic rabbit Oryctolagus cuniculus L

This paper presents a record of the live and carcass weights of the African giant rat Cricetomys gambianus Waterhouse and the domestic rabbit Oryctolagus cuniculus L. Male and female giant rats have an average killing out percentage of 51.4% (n= 10, S.D. ± 3.8) and 516% (n = 7, S.D. ± 3.47) respectively. The difference between the killing-out percentages of both sexes was not statistically significant at 5% probability level. There was also no significant difference at 5% probability level between the killing-out percentages of giant rats and domestic rabbit. Very low fat content was observed in the carcasses of wild giant rats.