Comparative Pathology of Zoonotic Orthopoxviruses

This review provides a brief history of the impacts that a human-specific Orthopoxvirus (OPXV), Variola virus, had on mankind, recalls how critical vaccination was for the eradication of this disease, and discusses the consequences of discontinuing vaccination against OPXV. One of these consequences is the emergence of zoonotic OPXV diseases, including Monkeypox virus (MPXV). The focus of this manuscript is to compare pathology associated with zoonotic OPXV infection in veterinary species and in humans. Efficient recognition of poxvirus lesions and other, more subtle signs of disease in multiple species is critical to prevent further spread of poxvirus infections. Additionally included are a synopsis of the pathology observed in animal models of MPXV infection, the recent spread of MPXV among humans, and a discussion of the potential for this virus to persist in Europe and the Americas.


Significance of Zoonotic Viruses
The ongoing Coronavirus pandemic has highlighted the serious impacts that contagion can have on human health, behavior, and quality of life. The stressors that pandemics put on the economic and political stability are also profound. Preemptive community awareness campaigns and adequately prepared responses to emerging zoonotic viruses are critical to minimize the negative effects of emerging diseases. Zoonotic viruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have the ability to spread from one or more non-human animal species to humans (Homo sapiens). There is significant variability between viruses in mode of transmission, rate of spread, morbidity and mortality caused in different species, and ability to adapt and persist in a population. There are several known zoonotic poxviruses that are being diagnosed in humans more frequently and have emerged in areas of the globe where they are not yet endemic. This review provides descriptions of zoonotic OPXV disease in veterinary species and in humans. This knowledge is critical to minimize the spread of poxviral diseases and understand their potential to emerge in areas of the world where they are not considered endemic.

Smallpox
There is evidence that the causative agent of smallpox, Variola virus (VARV), caused mortality in humans for centuries. Egyptian mummies dating from 1186-1070 B.C. appear to have smallpox lesions [1]. Four clinical presentations of smallpox are recognized: ordinary, modified-type, flat-type, and hemorrhagic smallpox. Flat-type smallpox is often fatal in unvaccinated individuals. Hemorrhagic smallpox can be fatal in unvaccinated and vaccinated people alike [2]. There are two variants of VARV; the more deadly strain, VARV major, causes severe disease with up to 30% fatality in unvaccinated people. The VARV minor strain causes morbidity, but the mortality rate following infection is <1% [3,4].
Discontinuation of mandatory vaccination programs avoided the risk of complications caused by vaccination. Although smallpox was no longer a disease of concern, the vaccine provided protection against other pathogenic Orthopoxviruses (OPXV) that cause disease in people and are endemic in various regions of the world: Vaccinia virus (VACV) in Asia and South America, Monkeypox virus (MPXV) in Africa, Cowpox virus (CPVX) in Europe, and Camelpox virus (CPXV) in the Middle East. Outbreaks, evolution of OPXV genomes, and emergence of zoonotic OPXV could increase transmission and worsen the severity of OPXV diseases [9][10][11]. Early, rapid, and specific diagnosis of both known and emerging OPXV is critical to prevent large outbreaks of OPXV [12].

Seroprevalence in the Middle East
The seroprevalence of anti-OPXV antibodies in animal populations in the Middle East is not well described. Camelpox virus seems to have a restricted host range as compared to other zoonotic OPXVs.

Potential for Orthopoxvirus Bioterrorism
Historically, smallpox has been used as a bioweapon. In 1763, the English purposefully donated blankets from a smallpox ward to North American Indians who were fighting against them with the French [3]. Given the severe consequences of the spread of smallpox through the human population, the zoonotic potential of many OPXV, the ability of OPXV to spread via respiratory droplets, and the lack of antibody protection in the vast majority of humans, the potential for the development of an OPXV as a bioweapon is considered high [25]. As such, animal models of smallpox have been developed to understand the disease in more detail, guard against alterations of OPXV that could advance their potential as bioweapons, and develop safe and effective vaccines and treatments for both weaponized poxviruses and naturally emerging OPXVs. An excellent discussion of animal models of OPXV was published in 2010 [26].

Vaccinia virus
Vaccinia virus (VACV) is the OPXV that was used to vaccinate people throughout the world against smallpox. Extensive research has been conducted using VACV infection in animal models to study OPXV biology and understand VARV pathogenesis in humans. There are several variants of VACV that have been named according to the species or location they were first identified in. These include Rabbitpox virus (RPXV, New York 1932 and Utrecht 1941), Buffalopox virus (BPXV, northern India 1967), Horsepox virus (HSPV, Mongolia 1976), and Brazilian VACV (Brazil 1999) [27,28]. Different variants of VACV have variable pathogenicity in different species. This is likely due to alterations in gene expression by the variants. For example, HSPV is known to encode genes that are not expressed by other VACV variants [29]. Phylogenetic analysis of poxviruses suggests that HSPV and RPXV share a viral ancestor that separates them from other VACV variants [30].
VACV is considered endemic in areas of South America and Asia. The virus causes disease in several animals (Table 1), including rodents, cattle, horses, monkeys, and humans [31]. Experimental transmission of VACV from cow feces to mice has been documented. No signs of poxviral disease were observed in the mice; however, anti-OPXV antibodies were detected after mice were exposed to cow feces containing VACV DNA [32]. The number of human cases of VACV infection continues to increase as the number of people vaccinated against OPXV decreases.

Vaccinia virus in South America
VACV infections in humans were first reported in Brazil in 1999. There are two genetically different groups of VACV circulating in Brazil. The two groups of VACV grow differently in BSC-40 cell cultures; Group 1 Brazilian VACVs form large viral plaques, while Group 2 Brazilian VACVs form small plaques. Group 1 Brazilian VACVs lack the viral A56R gene and are typically less virulent [89,90]. B-type viral inclusions are often detected within skin lesions of affected veterinary species and humans [91].
Rodents are a likely reservoir for VACV. High titers of virus were isolated from the peritoneum and testicles of a non-symptomatic house mouse (Mus musculus) during a 2005 outbreak in Mariana County [77]. VACV DNA was found in 6.4% of rodents tested, and anti-OPXV antibodies were found in 10.5% of animal sera collected. Overall, 25.7% of the rodents and 7.6% of the marsupials tested were considered positive for exposure to VACV [92].
The incubation period for VACV in cows is 1-7 days. VACV-infected cows present with exanthematous lesions on their teats and udders. These can be extensive, bloody, and painful. Nursing calves can develop lesions on their lips and nose [31]. Milk production is decreased, particularly in cows that have secondary mastitis [89]. Milk can contain VACV particles [93,94].
Horses with VACV infection often present with painful lesions on the lips and nose. Udders can also be affected. Nursing foals can have lesions in their mouths. The lesions are proliferative or papular and may ulcerate. Lesions typically last 6-12 days before healing [31].
The incubation period for VACV in humans is approximately 3-5 days. Active disease symptoms usually last 4 weeks; however, secondary bacterial infections can prolong the disease course. Humans infected with VACV can experience fever, headache, and malaise for 2-5 days. Lesions are usually first observed on the hands and arms [31,89,95]. The lesions may spread to the mouth, scrotum, and/or vulva and are often itchy. Initially, lesions appear as papules that develop into pustules, then become necrotic and ulcerated over approximately 12 days. Eventually, the lesions scab over. Lymphadenopathy is common. Less common signs of disease include anorexia, dehydration, nausea, and sweating [31]. Decreases in T-cell activation have been documented in people infected with Brazilian VACV [96,97]. Downregulation of co-stimulatory molecules (CD80/86) on B-cells and CD14 + cells was reported [97]. Stimulation of peripheral blood mononuclear cells by VACV appears variable and may depend on the technique or timing of sample collection following infection [96,97]. There is strong evidence that immunosuppressed people can experience a more severe disease course after zoonotic transmission of VACV, just as they can following vaccination [91].

Vaccinia virus in Asia
Buffalopox virus (BPXV) is the most common strain of VACV infection reported in humans in India. Outbreaks have occurred in Bangladesh, Egypt, India, Indonesia, Iran, Nepal, and Pakistan [98]. In buffalo (Bubalus spp.) and cows (Bos taurus), it causes lesions on teats and udders, inguinal area, ears, and eyelids [98][99][100][101]. Decreased milk production is observed with or without secondary mastitis [99,100]. Between 1992 and 1996, buffalo and humans from Maharashtra State had evidence of BPXV infection. Manifestations of the disease included cases in children with disseminated pox lesions on their face, arms, and rump [101]. Another outbreak occurred in Maharashtra State in 2008 and 2009 that involved 351 humans and several buffalo. Attack rates of 6.6% in humans and 11.7% in buffalo were reported. Most cases in humans occurred in males 20-59 years of age. Disease progression in humans is similar to that seen with other subtypes of VACV, including fever; malaise; painful skin lesions on the hands, feet, face, and mouth; and lymphadenopathy [99,100]. In a 2017 outbreak in Moinabad, skin lesions in humans took up to 6 weeks to scab over [102].
In the Punjab province of Pakistan, buffalo cows with wart-like nodules and inflammatory skin lesions on their teats and udders were tested for BPXV. PCR detected VACV in 49 of the 63 scabs collected. Positive animals had peripheral blood neutrophilia and lymphocytosis [103].

Monkeypox virus
Monkeypox virus (MPXV) was first described as pox-like outbreaks in two groups of cynomolgus monkeys (Macaca fascicularis) at the State Serum Institute in Copenhagen, Denmark. A total of 20 to 30% of monkeys housed together were affected by a rash that progressed to papules, which covered the trunk, tail, face, limbs, hands, and feet. Lesions ulcerated, then formed crusts, which fell off, leaving scars after a period of two to four months. The animals were healthy otherwise, and no lesions were observed in internal organs at necropsy [104]. The first human MPXV infection was reported in 1970 [72]. There are two clades of MPXV that are genetically distinct: one isolated in West Africa and one in the Congo Basin. The Congo Basin MPXV is more virulent than the west African MPXV [105]. The fatality rate associated with the Congo Basin MPXV is estimated to be 10.6%, while the West African MPXV is less than 3.6%. MPXV genomes detected in humans during the 2022 outbreak belong to the West African clade [106]. Transmission of MPXV occurs via contact with skin lesions and respiratory droplets. The disease can also be spread through scratches and bite wounds from infected animals [107,108]. Human-to-human transmission has been well documented [60,[109][110][111][112][113][114]. The incubation period (between infection and onset of clinical disease) is estimated to be 7-18 days in humans [115][116][117].

Clinical Signs
There is evidence of asymptomatic OPXV infection (presumed to be caused by MPXV) in both vaccinated and unvaccinated people in Ghana [118]. If asymptomatic individuals shed MPXV in oropharyngeal (or other) secretions, they could contribute to inadvertent human-to-human spread of the disease. Classic symptoms of MPXV include fever, pruritis, skin and mucocutaneous lesions, malaise, headache, backache, and lymphadenopathy. Often, disease symptoms are more severe in unvaccinated individuals [119,120]. Disease sequalae observed in unvaccinated people during an outbreak in Zaire (1980)(1981)(1982)(1983)(1984)(1985) included secondary bacterial infections in 19% unvaccinated people, bronchopneumonia (12%), vomiting and diarrhea leading to dehydration (7%), corneal ulceration (4%), septicemia (<1%), encephalitis (<1%), and death (11%) [120]. In a study of a human outbreak of Congo Basin MPXV in the Kivu provinces (2011-2015), the fever lasted 1-4 days, and skin lesions began as macules that progressed to papules, vesicles, pustules, then crusts, and scarred 14-24 days after macules were detected [115]. Lesions can occur all over the body, including the mouth and genitalia [18]. The timing of disease was slightly different in an outbreak of West African MPXV in the United States (2003). The fever lasted 2-13 days, and the rash lasted 7-24 days [117]. The timing of the onset of fever and development of a rash differed between individuals who were scratched or bitten by an infected animal and individuals who simply handled infected animals [108]. Oropharyngeal lesions occurred in 76% of unvaccinated people as compared to 47% of vaccinated people [119]. Virus replication within lesions in the upper respiratory tract likely contributes to spread of the virus in respiratory droplets. Lymphadenopathy is an important feature of MPXV infection that is not observed during VARV infection (smallpox). Lymphadenopathy begins 1-2 days before the onset of the rash and occurs in approximately 85-90% of unvaccinated people infected with MPXV [18,119,121,122]. Coughing and/or conjunctivitis occur in ≥30% of unvaccinated people [119].
The median age of humans infected with MPXV in the Democratic Republic of the Congo between 1996 and 1997 was 10 years old, and disease was slightly more common in men (56.8%) [60]. A review of 1057 confirmed MPXV cases in the Democratic Republic of the Congo between 2011 and 2015 indicated that approximately 60% of cases were men and that most were between 10 and 19 years old [122]. A study of an outbreak of 122 cases in Nigeria in 2017 found that the median age of affected individuals was 29 years old, and 69% of cases occurred in males [123]. In a past outbreak, the incidence of cases in unvaccinated people was over 2.5× greater than in vaccinated people [122]. More recently, it has been estimated that 80-90% of cases of human MPXV are diagnosed in unvaccinated people [124]. In people who have not been vaccinated for smallpox, case fatality rates between 9.8% and 13% have been reported [18,[119][120][121]. Deaths occur more often in the very young and in immunosuppressed people [111,119,120] Natural infection with MPXV has been detected in many species (Table 1) [67]. The first non-captive species that MPXV was diagnosed in was a Thomas's rope squirrel (Funisciurus anerthrus) [63]. Fatal MPXV infection was described in an infant sooty mangabey (Cercocebus atys) from the Ivory Coast. The monkey was found dead with disseminated red crusts on its extremities and abdomen. Eosinophilic cytoplasmic viral inclusion bodies and severe secondary bacterial infection were observed in histologic sections of skin. MPXV DNA was isolated from several organs [68]. Disease in wild chimpanzees (Pan troglodytes) at the Taï National Park in the Ivory Coast was described in three infant chimpanzees, one of whom died. The chimpanzees had a cutaneous rash, coughing, and respiratory distress. MPXV DNA was detected in 62/492 (12.6%) fecal, 11/55 (20%) urine, and 5/26 (19.2%) fruit wedge samples tested. MPXV DNA was also detected in maggots and materials around the individual that died from disease [125].

Pathology
There are very few reports of alterations in complete blood counts and serum biochemistry profiles of people infected with MPXV, but leukocytosis (45% of patients), thrombocytopenia (35%), hypoalbuminemia (50%), decreased urea nitrogen concentration (61%), and increased alanine aminotransferase and aspartate aminotransferase have been documented [117]. The combination of increased aminotransferases and decreased albumin and urea nitrogen is highly suggestive of liver damage and decreased liver function. Experimental infection of cynomolgus monkeys with very high doses of aerosolized MPXV-Zaire caused anemia, leukocytosis, hypoalbuminemia, increased liver enzymes, and azotemia [126,127].
Histopathologic findings in MPVX-infected skin lesions are remarkably similar in humans and cynomolgus monkeys. Intraepidermal vesicles and pustules can be observed. As lesions progress, they become ulcerated and form crusts with marked suppurative inflammation within and below the crusts. Around the crust, there is significant epidermal hyperplasia and ballooning degeneration of keratinocytes associated with a ring of edema and mononuclear inflammation (macrophages and lymphocytes) that extends into the dermis. Keratinocytes often contain intracytoplasmic inclusion bodies [117,126].

Experimental Infection
Guinea pigs (Cavia porcellus), golden hamsters (Mesocricetus auratus), European rabbits (Oryctolagus cuniculus), and most laboratory mice are relatively resistant to MPXV infection [67,128]. Experimental infection of 38 strains of mice found CAST/EiJ, PERA/EiJ, and MOLF/EiJ mice susceptible, likely because they are immunodeficient [129]. Neither Balb/c nor C57BL/6 mice showed signs of disease after intradermal (i.d.) or intranasal (i.n.) inoculation with 10 5 pfu MPXV-west African. Mild disease was observed in Balb/c mice but not C57BL/6 mice, similarly inoculated with MPXV-Zaire [130]. This is one of the reasons that other experimental models of MPXV have been investigated. More critically, it is important to know what the disease looks like in: (1) species that are naturally susceptible to MPXV infection, (2) species that cohabitate with humans living in areas where MPXV is endemic, and (3) species that may become reservoirs of MYXV if the disease spreads to other regions of the world.
Kellen's dormouse (Graphiurus kelleni) was tested for susceptibility to MPXV infection. Intradermal and i.n. inoculation caused loss of body weight, decreased activity, hunched posture, unkempt haircoats, dehydration, conjunctivitis, and death in most animals tested. Symptom severity was dose dependent with 100% mortality at 200 pfu, 63% at 20 pfu, and 0% at 2 pfu following i.n. inoculation. The virus was detected in nasal lavage fluid as early as 2 days post-inoculation (dpi) and in several organs (including lung) at 4 dpi. Necropsy findings were somewhat different from those in cynomolgus monkeys that were given far higher doses of MPXV. Similarities included necrotizing lymphadenopathy and necrotizing hepatitis. In the dormice, necrosis was also detected in the spleen, thymus, and bone marrow. The lungs, intestinal tract, gall bladder, and brain had hemorrhagic lesions. Additionally, rhinitis with syncytial cell formation and B-type viral inclusions were reported [58].
Black-tailed prairie dogs (Cynomys ludovicianus) have been experimentally infected i.n. with 10,000 pfu MPXV-US. The animals (n = 3) had to be euthanized within 16 dpi. All had skin and/or oral lesions. Virus replication was detected in the lymph nodes at 5 dpi. Histologically, they had necrosis in skin lesions and lymphoid tissues. One prairie dog was co-housed with an experimentally infected animal and contracted the disease. Lymphoplasmacytic interstitial pneumonia was documented, although two of the prairie dogs also had lung mites [131]. At 6 × 10 3 pfu MPXV i.n., the signs of disease included weight loss and development of skin lesions. Serum biochemical abnormalities (including increased alkaline phosphatase, alanine transferase, glucose, and globulins, as well as decreased albumin) were reported and correlated with evidence of hepatic necrosis. Transmission of the virus was observed in animals that shared bedding or were co-housed with infected prairie dogs [132].
Inoculation of Thomas's rope squirrel (Funisciurus anerythrus) with 10 6 pfu MYXV-Zaire i.n. caused slightly less fatality than that observed in prairie dogs infected with MPXV-US (75% as compared to 100%). Clinical signs included ocular and oral lesions seen 6 and 8 dpi, respectively, tachypnea 9 dpi, and skin lesions 11 dpi. A sentinel animal became infected and was tachypneic. Histopathology of skin lesions of animals infected i.n. were similar to those reported in other species with epidermal necrosis and ulceration, suppurative inflammation within and under ulcers, and surrounding epidermal spongiosis and edema associated with mononuclear inflammation. Reactive endothelium and fibrin deposition, which is noted in several other poxvirus lesions, were also described. Half of the animals inoculated by scarification or i.d. injection had to be euthanized, but these animals developed skin lesions much sooner (3 dpi). However, histopathology results suggested that their lesions were less severe with epithelial hyperplasia and lymphoplasmacytic vasculitis. Lymphoplasmacytic interstitial pneumonia and lymphoid hyperplasia were also noted. Low numbers of animals had necrosis in lymph nodes, a finding that seems to be common in other species. The virus was detected in oral secretions from all animals from 3 dpi up to 25 dpi [133].
Intranasal or intraperitoneal infection of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) with 10 5.1 pfu MPXV-west African US caused anorexia and lethargy but no other signs of disease. However, all animals died between 6 and 9 dpi. Hepatic and splenic necrosis and consolidation of lung tissue were observed in i.p.-inoculated animals. Intranasal inoculation caused multifocal steatosis of the liver, interstitial pneumonia, and necrosis in some lymph nodes. Basophilic viral inclusions were observed in hepatocytes. Immunohistochemistry indicated viral antigen was also present in mesothelial cells and adipocytes of some animals. The virus was isolated from blood, oral swabs, and all organs tested [134].
Gambian pouched rats (Cricetomys gambianus) were inoculated with 4 × 10 4 pfu MPXV by scarification [135] or with very high doses (10 6 pfu) of MPXV i.d. or i.n. [136]. Fatality occurred in one rat infected i.d., but no signs of disease were observed in a sentinel rat exposed to the infected animals. However, significant amounts of virus were detected in saliva of the i.d.-and i.n.-infected animals [136].

Epidemiology
In 2014 and 2016, outbreaks of MPXV were observed in captive chimpanzees in Cameroon, but no symptoms were reported in humans. In 2018, 63 people who worked with the chimpanzees or lived in the nearby village were tested for exposure to OPXV. These people were born after 1980 (after mandatory smallpox vaccinations were discontinued). Serum anti-OPXV IgG was detected in 6.3% and IgM in 1.6% of these individuals, indicating they had been exposed to an OPXV [118]. A study that surveyed 939 people in the Democratic Republic of the Congo identified age (≥18 years-old), sex (male), and occupation (hunter) as potential risk factors for contracting MPXV [137]. A review of 71 documents in-dicated that MPXV outbreaks occur more commonly in men, people <30 years old, people with animal contact, and in rural areas [138].
Between 1970 and 1983, 80% of human MPXV cases (n = 155) occurred in Zaire [121]. In 2003, 37 people were diagnosed with and 10 people were suspected of having MPXV infection in the United States [108]. Several species of animals imported from Ghana had been housed with prairie dogs. Disease symptoms in prairie dogs (including watery eyes, congestion, and skin lesions) were noted after distribution of several animals to private households. Humans became ill within 4-21 days of direct contact with the prairie dogs. Histopathology of skin lesions was typical of poxvirus infection [140]. In the Democratic Republic of the Congo, the incidence of MPXV infection was 0.64 cases/100,000 people in 2001. In 2013, case incidence rose to 2.82/100,000 people [124]. It is clear that the disease is becoming more common in humans, likely due to the lack of protection from OPXV that previously had been provided by widespread smallpox vaccination.
Over 120 cases of human MPXV were reported in countries outside of Africa in the week before 20 May 2022 [141]. As of 27 June 2022, 3413 human MPXV infections have been reported across >50 countries in Europe, Latin America, the Middle East, North America, and Asia [142]. Many of the cases are associated with men traveling by plane from Nigeria to other countries [143][144][145]. At least two people coming from Nigeria infected with MPXV stopped in Canada before traveling to the United Kingdom and the United States [146]. Many of these cases are occurring in men who likely have had direct contact with lesions during sex or indirect contact via fomites [147].
CPXV lesions appear similar to those caused by other OPXVs; however, the infected cells contain eosinophilic A-type (Cowdry) viral inclusions as opposed to the more basophilic B-type inclusions usually observed with other OPXVs. The virus also forms red, hemorrhagic pocks on chicken chorioallantoic membranes, rather than inflammatory, white pocks that are observed with other OPXVs.
Short-tailed field voles (Microtus agrestis) are likely a reservoir for CPXV. A negative correlation between CPXV infection and survival has been reported in this species in England [157]. Likewise, common voles (Microtus arvalis) can be infected with CPXV. Following experimental oronasal inoculation with 10 4 pfu of CPXV isolated from a wild common vole, skin lesions in experimentally infected voles were not observed, but virus was shed in oropharyngeal secretions 4-8 days after inoculation [158]. Bank voles (Myodes glareolus) inoculated by the oronasal route with CPXV show no clinical signs of disease and do not shed detectable amounts of virus, but they do seroconvert. Seroconversion was also observed in low numbers of voles that were in contact with the inoculated voles [159]. In a study of bank voles collected from 2017-2018 in Germany, CPXV DNA was detected in 5/509 (0.98%) of nasal septum samples, and virus was isolated from one animal [160]. In France, montane water voles (Arvicola scherman) are reported to have a CPXV seroprevalence of 41.8% (n = 158) [161].
Rats are known to acquire natural infections with CPVX. Experimental infection of Wistar rats and fancy rats (Rattus norvegicus domestica) via i.n. or i.n. and scarification with 10 6 pfu of CPXV causes disease within 11 days [162]. Disease signs in rats include ulcerative skin lesions, unkempt fur, sneezing, ocular discharge, dyspnea, anorexia, and lethargy [162][163][164][165]. Pneumonia and abdominal distention were observed in some studies [164,165]. In one study, histologic sections showed necrotizing lesions in the skin and upper respiratory tract with eosinophilic cytoplasmic inclusion bodies. Some rats had bronchopneumonia, pulmonary edema, emphysema, and/or meningoencephalitis. Rats that were in contact with the infected animals developed cutaneous lesions on day 10 but had a milder disease course and recovered from CPXV infection within 36 days [163]. Rats have been shown to shed virus as early as 5 days and up to 21 days after exposure [162].
Cats likely acquire CPXV from infected rats [166]. Cats with CPXV infection often present with skin lesions on the head, neck, and forelimbs [149]. Conjunctiva and mucous membranes can also be affected [153]. Lesions begin as erythematous macules or papules that develop into nodules, which ulcerate and eventually crust over within 4-5 weeks. Histologically, a necrotizing dermatitis with ballooning degeneration of epithelial cells is observed. Lesions can become secondarily infected with bacteria. Lymphadenopathy has been reported [153]. Occasionally, severe systemic infection or necrotizing pneumonia occurs, which can be fatal. Viral inclusions can be observed in bronchoalveolar lavage samples from some cats with CPXV pneumonia [167]. Fatalities are more common in young kittens and immunosuppressed adults [166]. The OPXV seropositivity in 226 cats tested in Italy was 33.3% [154]. Evidence for the spread of CPXV via fomites has been documented [149].
CPXV can cause severe disease in many species housed in zoos. In Germany, MVA vaccination is used to protect zoo animals from CPXV infection [48]. Fatal CPXV infections can occur in banded mongooses (Mungos mungo) [44,48] and jaguarundi (Herpailurus yagouaroundi) [48]. Infected mongooses were anorexic, lethargic, and dyspneic. Imbalance, petechia, pox-like skin lesions, mucosal ulcers, and lymphadenopathy were observed. Histopathology revealed necrotizing and inflammatory lesions in lymph nodes, skin, liver, and spleen. A brown rat in the enclosure also had skin lesions consistent with OPXV infection [44]. As expected, CPXV infection severity varies between species. Four cotton-top tamarins (Saguinus oedipus) housed in an animal park in Germany died from CPXV infection in 2010. The animals had alopecia and some ulcerations of skin and mucocutaneous junctions. CPXV was isolated from the skin of these animals. CPXV DNA was detected by PCR in oral swabs from common marmosets that were housed next to them. The marmosets showed no clinical signs of disease [168]. There are many other examples of CPXV infection in zoo animals. CPXV DNA was isolated from a giant anteater (Myrmecophaga tridactyla) that was anorexic, lethargic, and had vesicular and ulcerated skin lesions on its nose, mouth, vulva, flanks, and feet. Clinical pathology abnormalities included neutrophilia with a left shift, hypoglobulinemia, and increased alkaline phosphatase. The anteater survived infection with supportive care [169]. CPXV infection of Patagonian maras (Dolichotis patagonum) was detected in captive animals in the Netherlands. Animals had conjunctivitis and were anorexic. At necropsy, ulcers were observed on the tongue and buccal mucosa, and petechial hemorrhages were noted on the heart. Histopathology indicated there was lymph node and splenic lymphoid hyperplasia, renal tubular necrosis, and pulmonary edema. CPXV DNA was isolated from these animals [42].
Hoofstock are also susceptible to CPXV infection. In cattle, CPXV causes ulcerated lesions on the udder and teats. Lesions can become generalized. The disease course typically lasts 4 weeks [170]. CPXV can cause abortion and premature birth in horses. The mares are typically asymptomatic, but papules on the skin and ulcers in the mouth that contain A-type inclusion bodies can be detected in the foals [171,172]. CPXV disease in alpaca ranged in severity from localized lesions to fatalities. Seroprevalence varied from 16.1% to 81.2% in four herds that were affected by the outbreak. CPXV DNA and anti-OPXV antibodies were found in common voles, striped field mice, and bank voles captured at the alpaca farms [173].
A localized skin lesion with fatigue, fever, and lymphadenopathy are often observed in humans infected with CPXV, but generalized CPXV infections have been reported. The generalized disease causes ulcerated nodules in several areas of the body, sometimes including the groin. Skin lesions typically heal within 2-4 weeks, unless secondary bacterial infection occurs [174]. The disease can be fatal in immunosuppressed people [175].

Camelpox virus
In dromedary camels (Camelus dromedarius) and Bactrian camels (C. bactrianus), Camelpox virus (CMLV) can cause fever; nasal discharge; pox on the head, neck, throat, lips, extremities, inguinal and perianal areas, and scrotum; respiratory stress; and gastrointestinal upset [37,176]. Morbidity is often low (1.1%), and most affected camels are >2 years old [176]. However, fatality rates of 12-25% have been reported [37,176]. Histologically, skin lesions have dermatitis with ballooning of keratinocytes. Less commonly, lymphoid hyperplasia is observed. Bronchitis or pneumonia can occur in animals that succumb to disease [176]. A human outbreak of CMLV in Rajasthan, India, occurred in three people in 2009. People reported fever, itching, erythema, and edema that developed into classic poxvirus lesions on the hands, including vesicles that ulcerated approximately 7 days and formed crusts approximately 15 days after signs of disease began. Virus DNA was isolated from the lesions on one person. Virus was isolated from 14 dromedary camels associated with the outbreak [37]. Camel-to-human transmission of CMLV was also reported in a 2014 outbreak of poxvirus infection in eastern Sudan. Humans experienced fever, malaise, itching, erythema that progressed to nodules 7-10 days after contact with affected camels, and clearance of skin lesions 30-40 days after the onset of disease. Viral DNA was detected in two dromedary camels and three out of four humans tested. Virus was isolated from one of the affected people [177].

Alaskapox virus
In 2015, an Alaskan woman presented with a pox lesion on her shoulder and lymphadenopathy. She reported 5 days of fever, malaise, and fatigue. The lesion took 6 months to heal. It was unclear if the disease was transmitted through contact with a human or indigenous small mammals [178]. Genetic testing indicated the virus is a unique OPXV [35].

Akhmeta virus
In 2013, genetically distinct OPXV DNA was isolated from the skin lesions of two Georgian milkers with symptoms of fever, eschars, hand swelling, and lymphadenopathy. Cows, rodents, and shrews in the area tested positive for anti-OPXV antibodies [34].

Discussion
Zoonotic OPXV are endemic in specific regions of the world. In the past, they were rarely diagnosed in humans, and outbreaks were relatively easy to contain. This seems to be changing due to the lack of protective immunity in the human population following cessation of smallpox vaccination. It is possible that zoonotic OPXV will become endemic in new areas of the globe due to human spread of the diseases into native populations of animals that are susceptible to the viruses. This necessitates that OPXV diseases are recognized rapidly in non-human species that are in contact with people and can contribute to spread of the disease. Table 2 provides a summary of pathology observed in key species affected by zoonotic OPXV. Disease signs are quite similar in most species, but minimal disease severity in some species can make diagnosis difficult. Additionally, it is nearly certain that additional species are infected by zoonotic OPXVs, including bats [179,180]. Surveillance and reporting of OPXV pathology in new species will be important to help monitor the spread of OPXV diseases. Given the large number of species that MPXV is known to infect, it is likely to become endemic outside of Africa if the disease is not contained. -Isolation of the affected individuals and ring vaccination of people in contact with contagious animals and people are currently the best ways to prevent the spread of zoonotic OPXV diseases. The ability of countries to respond to OPXV infections rapidly and effectively has been tested by the MPXV outbreaks occurring throughout the globe. The cooperation of countries with the World Health Organization and strong leadership to coordinate public health agencies will be critical to manage OPXV, as it was during the campaign to eradicate smallpox. Continued advancement of our understanding of OPXV biology and pathology in different species is needed to protect people and animals from expansion of zoonotic OPXV disease.

Conflicts of Interest:
The author declares no conflict of interest.