Scott Parker, Anthony Nuara, R Mark Buller, Denise A Schultz. Future Microbiology. Volume 2, Issue 1, February 2007.
Cases of human monkeypox are increasing in Africa, and for the first time, human monkeypox was observed in the western hemisphere during an outbreak in 2003. This may be due to a combination of factors, including a broader interface between humans and monkeypox virus (MPXV)-infected animals through ecosystem degradation, the cessation of vaccination against smallpox, an increasing susceptibility of humans to severe monkeypox disease due to poor nutrition and coinfections with other pathogens, and an increase in the human-to-human transmissibility of the virus. Other factors may include an adaptation of MPXV to additional animal species in extended geographical regions or the expansion of the geographical range of reservoir species. Regardless of the reason(s), the increasing number of cases provides an opportunity to enhance virus transmissibility within human populations, which will be discussed in the context of the natural lifecycle of MPXV in humans and the genetic potential of this virus.
Discovery
In 1958, MPXV was isolated from vesiculo-pustular lesions of infected cynomolgus macaques imported to the State Serum Institute in Copenhagen, Denmark, where it was identified as a member of the orthopoxvirus genus (family Poxviridae and subfamily Chordopoxvirinae ). Orthopoxviruses have host specificities ranging from narrow (e.g., ectromelia and variola) to broad (e.g., cowpox and vaccinia). MPXV belongs to the latter group and has the capability to infect humans and animals, such as rodents and nonhuman primates. The MPXV virion morphology is consistent with other orthopoxviruses; that is, a 200-250-nm brick-shaped enveloped virus with characteristic surface tubules and a dumbell-shaped core component.
Recognition of human monkeypox as an epizootic disease
Highly susceptible nonhuman primates infected with MPXV have near identical clinical manifestations as humans infected with variola (the causative agent of smallpox). Despite this similarity, MPXV remained primarily of academic interest throughout the 1960s. The attitude of the scientific community changed upon the realization that MPXV could lethally infect humans in known smallpox-free locales. Between 1970 and 1971, six cases of human MPXV infection were reported in Liberia, Sierra Leone and Nigeria, which had been free of smallpox for at least 1 year. In addition, the first human monkeypox case discovered during this same period was in a 9-month-old child in Zaire (now the Democratic Republic of the Congo [DRC]). From 1970 to 1979, 47 human cases of monkeypox were identified, 38 were from Zaire, and nearly all were conterminous with the tropical rain forest. A total of 79 cases of human monkeypox were subsequently reported over the next 12 years. Of the 59 cases recorded between 1970 and 1980, 47 (80%) occurred in the tropical rainforest of the DRC. Of these, 23 patients (49%) had severe disease and eight died (17%). Although MPXV infection of humans is not as virulent as that of variola virus, MPXV is still capable of human-to-human transmission. For example, four out of 47 (9%) cases between 1970 and 1980 were suspected to be from human-to-human transmission, with the remaining 43 (91%) human cases acquired from contact with animals.
Clinical features of human monkeypox
The most severe human MPXV infections have been reported in the Congo Basin, whereas attenuated human infections have generally occurred in West-African countries. In both regions, human infections generally resulted from handling MPXV-infected animal tissues. In the 2003 US outbreak, infections were initiated by a number of routes, which appeared to affect the clinical manifestations of the disease. The smallpox-like rash produced in the skin of MPXV-infected humans is preceded by two phases: an incubation period of 10-14 days, which is followed by a 1-3 day interval characterized by a prodromal fever, malaise and severe swollen lymph nodes. Most patients report chills, sweats, and, less frequently, headaches, backaches, sore throats, coughing and shortness of breath. Patients often present with severe lymphadenopathy of the neck, inguinal and axillary regions. This distinguishing lymphadenopathy can facilitate differentiation from variola and varicella-zoster virus (VZV; chickenpox) infections; however, diagnosis cannot be made solely on clinical observations. Of patients clinically diagnosed in Zaire (between 1981 and 1986) with chickenpox, atypical chickenpox and undetermined skin rashes, 3, 7 and 6% were actually infected with MPXV, as determined by laboratory tests, respectively. Similarly, up to 23% of patients clinically diagnosed as infected with monkeypox were instead infected with VZV. The development of the monkeypox rash is similar to that of smallpox. With each infection, there is a 2-4 week period where macules develop and sequentially transform into papules, vesicles and pustules. Finally, the pustules umbilicate, scab and desquamate. The monkeypox rash usually occurs in a centrifugal distribution, but can have a centripetal distribution in a smaller number of cases. Monkeypox skin lesions generally range from 0.5 to 1 cm in diameter, and appear on the trunk and spread over the body to the soles of the feet, hands, oral mucosa and genitalia. Secondary infections are unsurprisingly common and may contribute to mortality, which is approximately 10%. In some cases, patients with smallpox develop a hemorrhagic form of the disease; there is no evidence to suggest this occurs in MPXV-infected individuals, but it does occur, to a certain degree, in African dormice after experimental infection with MPXV (Schultz, Pers. Comm.)
MPXV from West Africa is attenuated and less transmissible than the MPXV from the Congo Basin. The US MPXV outbreak of 2003 was due to MPXV-infected West-African rodents imported from Ghana. During this outbreak, 26% of 69 patients clinically diagnosed with human monkeypox were hospitalized but, in the majority of cases, this action was more of a precautionary measure. Similar to human monkeypox cases in West Africa, no case fatalities or person-to-person transmission was observed in the 37 laboratory-confirmed US cases. All patients acquired the infection from direct or indirect contact with black-tailed prairie dogs that had prior contact with MPXV-infected African rodents. Both groups of animals were destined for sale in the US pet industry. Skin lesions on these patients were generally found at, or close to, the site of a bite or scratch caused by a MPXV-infected prairie dog. Although this outbreak was likely contained, a different contrivance may have emerged if the more aggressive Congo Basin strain had accompanied the African rodent shipment into the USA. This episode demonstrates the ease with which MPXV can penetrate the species—species interface, and it is fortunate that the virus appears to have failed in establishing itself in a US animal reservoir(s).
Epidemiology of human monkeypox in Africa
One could speculate that human MPXV infections have been occurring in Africa for centuries and were masked under the guise of smallpox. This possibility lead the WHO to initiate a MPXV surveillance program in the DRC between 1981 and 1986 in order to determine whether MPXV could fill the niche vacated by variola. This program was hampered by the clinical similarity of human monkeypox and VZV, and the lack of a robust immunoassay to specifically detect antibodies to MPXV. Frequently, cases were reported as suspected MPXV infections, but could have been VZV, since these cases were not confirmed by diagnostic tests. To confound matters, MPXV and VZV can cause simultaneous infections. For example, a 1997 WHO bulletin documented a large MPXV outbreak in the DRC, which disclosed a rate of human-to-human transmission as high as 73%, a surprising 2.5-fold higher than previously recorded. A follow-up report suggested that the increased transmissibility and lower case-fatality rate observed in this outbreak could have been due to the inclusion of VZV infections. In 1981, a serological survey of 10,300 unvaccinated central and West-African children was conducted to detect hemagglutinin-inhibiting (HI; orthopoxvirus) antibodies. This test revealed that 13% of West-African children (Ivory Coast and Sierra Leone) and 19% of central African children (DRC) were positive for orthopoxvirus antibodies. As a follow-up test, a less sensitive but more specific radioimmunoassay adsorption test for MPXV antibodies was utilized. This test indicated that 17% of the HI-positive sera contained MPXV-specific antibodies.
Between the 1981 and 1986 period, 404 human monkeypox cases were reported in Africa, 386 of the 404 (96%) cases were from the DRC. Of these 386 cases, 338 (88%) fell into the intensive surveillance area within the DRC, where 203 (60%) cases were primary and 42 (12%) cases were considered co-primary as the initiation of illness fell within 7 days of the onset of the rash in the primary case. The remaining 93 (28%) cases were secondary cases where 69 were first generation, 19 were second generation and five were third or fourth generations. Of these cases, 72% were determined to be from an animal source. This 1981-1986 study found a case-fatality rate of 10%. The secondary attack rate (among close susceptible contacts), which is a measure of the risk of person-to-person transmission, was 9%. Furthermore, mathematical modeling experiments, based on outbreaks in the DRC, concluded that MPXV could not transmit indefinitely in the unvaccinated human population without zoonotic amplification.
Reported cases dwindled after the 1981-1986 surveillance program with no reports between 1992 and 1995, possibly as a result of less surveillance. Since this respite, an upsurge was noted with 71 cases between February and August of 1996, where six (8%) deaths occurred in a human population of 15,698. In addition, in July of 1996, 42 cases, including three (7%) deaths, occurred in a village populated with 346 inhabitants known to hunt squirrels. In the latter episode, a male, identified epidemiologically as the primary case, is believed to have directly or indirectly passed the infection through eight members of his clan and to the village inhabitants. These findings were interpreted as enhanced human-to-human transmission in the Congo Basin compared with previous studies. A further 511 cases were reported in 1997. Reported cases continued to rise between 1998 and 2002, with 1265 reported cases in the DRC, but only 215 specimens were collected and 88 (41%) specimens tested positive for MPXV. This suggested that human monkeypox cases may be lower (∼518 rather than 1265). Moreover, in 2003 a hospital in the Republic of the Congo reported six generations of human-to-human MPXV transmission, suggesting that the transmission efficiency may also be increasing.
The reason for the upsurge in cases is not known, but many Africans may be more susceptible to severe disease due to poor nutrition or coinfections with other pathogens. Two reports have documented co-infections of MPXV and VZV, with one fatal outcome. It is known that AIDS increases the morbidity and mortality of humans when they are concurrently infected with Mycobacterium tuberculosis, and a similar effect would be expected following co-infection with MPXV. Another likely contributing factor could be the cessation of smallpox vaccination by the WHO circa 1980, as vaccination for smallpox is 85% effective against severe monkeypox disease 3-19 years following immunization. Alternatively, there may be more frequent contact between humans and infected animals as the ecosystem is degraded. Lastly, the broad host range of MPXV may permit additional species to become reservoirs or incidental hosts, which increase the exposure risks for humans. This latter possibility will be considered in greater detail later.
Genetic basis of monkeypox virus virulence & transmissibility
Despite clinical disease similarities, MPXV is not considered to be the direct ancestor to variola virus, rather both viruses evolved from progenitor poxvirus(es) most similar to the cowpox virus lineage. MPXV isolates originating from West Africa appear to be less virulent and/or transmissible to humans and nonhuman primates than those from the Congo Basin. Genomic differences amongst MPXV isolates have been mapped using restriction fragment-length polymorphisms (RFLP) and DNA sequencing techniques. Reminiscent of other orthopoxviruses, MPXV strains demonstrate gene conservation towards the center of the genome, while variation frequency increases towards the terminal regions. Sequence analysis of the genomes of several Congo Basin and West-African MPXV isolates revealed approximately 95% identity amongst all MPXV isolates; however, this value approaches 99% when comparing between isolates from West or Congo Basin African regions, thereby enabling separation into two clades based on both sequence homology and disease severity. Consistent with the aforementioned RFLP analysis, the greatest diversity between the two clades is localized to the terminal regions that encode for predicted host-response modifier (HRM) proteins.
A candidate gene responsible for the difference in virulence and transmissibility between the Congo Basin and West-African clades is the monkeypox ortholog of the smallpox inhibitor of complement enzymes (SPICE). The monkeypox ortholog of SPICE (monkeypox inhibitor of complement enzymes [MOPICE]) is present in Congo Basin isolates, but absent in sequenced West-African viruses. Lack of MOPICE in the West-African MPXV isolates could enhance virion or virus-infected cell susceptibility to host-derived complement-mediated lysis, contributing to a lower viremia and to less severe disease, less seeding of the respiratory mucosa and less transmissibility (see later). MOPICE from Congo Basin isolates has a C-terminal truncation resulting in loss of one of the four complement control domains present in SPICE and other orthopoxvirus orthologs. Despite this truncation, studies have demonstrated complement-inhibiting activity of the MOPICE protein, although the activity is far lower than that reported for SPICE. Presence of MOPICE in the more virulent and transmissible Congo Basin MPXV isolates and SPICE in variola, combined with the absence of MOPICE in the less virulent West-African monkeypox isolates, makes members of the PICE family candidates for virulence and transmissibility factors associated with human orthopoxvirus disease. Other candidate open-reading frames that are possibly important for virulence and transmissibility of Congo Basin MPXV isolates are: D10L (host range), B10R (virulence factor for myxoma virus), B14R (interleukin [IL]-1 binding protein) and B19R (serine protease inhibitor-1).
An important question is whether virulence and transmissibility vary independently or are linked, as suggested by preliminary comparisons of the Congo Basin and West-African strains. The answer is not apparent when other poxviruses are examined. For example, following release of the leporipoxvirus myxoma into Australian wild-rabbit populations in 1950, the myxoma virus population became dominated by strains of reduced virulence that were more efficiently transmitted by mosquito vectors. Similarly, Fenner observed that the Moscow and mouse-passaged Hampstead strains of the orthopoxvirus ectromelia were similarly virulent for the mouse, but the mouse-passage Hampstead strain was less transmissible. And finally, in the late 19th Century, mild forms of smallpox, alastrim and amaas, with case-fatality rates of approximately 1% or less, were observed in unvaccinated individuals in North America and South Africa, respectively. These milder clinical forms of smallpox became known as variola minor, leading to the usage of variola major for classical smallpox. Compared with variola major, variola minor is characterized with less severe constitutional symptoms, and smaller and fewer lesions, which evolve more rapidly. Since less virus was found in the oropharyngeal secretions of persons with variola minor, inherent transmissibility was probably lower than that of variola major; however, due to the less severe disease, patients infected with variola minor were more mobile during the infectious period, and thus the secondary attack rate for variola major and minor were similar at 58 and 61%, respectively. Taken together, these studies suggest that MPXV transmissibility could evolve independently from changes in virulence.
Ecology of monkeypox virus
Numerous field studies completed in the lowland tropical forests of central and West Africa in the latter part of the 20th Century have confirmed that MPXV infects many animal species. Table 1 lists species (species groups where n > 20) in rank order of seroprevalence of antimonkeypox antibodies, using species-specific serologic assays, such as radioimmunoassay adsorption (RIAA) or immunofluoresence assay adsorption (IFAA) tests (bold face entries). These species have some similar and dissimilar traits based on diet and habitat preferences; approximately 40% are arboreal, 40% are semiterrestrial and 20% are terrestrial. The lowland tropical forest where they dwell is comprised of primary and secondary forest with varying densities of undergrowth. In general, the arboreal species inhabit the middle and upper canopy strata of the forest. Semiterrestrial species inhabit the ground-level undergrowth and lower forest strata. Terrestrial species inhabit the ground and undergrowth strata of the forest. Considering this habitat spectrum, MPXV infects animal species that inhabit all strata of the lowland tropical forest within central and West Africa.
Seroprevalence studies suggest that there may be no one reservoir of MPXV; rather, several animal species may support MPXV in nature. For example, two genera and six species of squirrels and five genera and nine species of nonhuman primates had MPXV-specific antibodies (Table 1). Virus has been isolated from an animal in nature only once, and the source was a diseased squirrel of the species Funisciurus anerythrus. However, in the 2003 shipment of African rodents that introduced MPXV into the USA, cell culture and/or PCR assays demonstrated MPXV in Funiscuirus spp. (rope squirrel), Cricetomys spp. (giant pouched rat) and Graphiurus spp. (african dormouse). The lack of MPXV antibodies in sera from certain animal species may also be informative; 579 sera from a sampling of terrestrial rodents (genera: Lemniscomys, Lophuromys, Thamnomys, Oenomys, and Praomys ) were negative for MPXV antibodies. Only one study has evaluated the potential of MPXV transmission from an insect (ants) to a rodent (squirrel). This study exposed ants to MPXV-infected squirrel tissue before placing the ants with an uninfected squirrel; the conclusion was that MPXV did not transmit from the ants to the squirrel. The paucity of studies to evaluate any role of insect species in the transmission of MPXV is apparent. Consequently, the role of insects in the natural lifecycle of MPXV may be worth evaluating. The presence of MPXV antibodies in so many distinct species and virus detection in specimens from Funisciurus spp. , Cricetomys spp. and Graphiurus spp. suggest the natural lifecycle to be a complex interaction of reservoir hosts and incidental species. This broad host range is a cause for concern as it may facilitate the adaptation of MPXV to new hosts in new regions.
Increased number of animal hosts for monkeypox virus
We still do not understand the ecology of MPXV with respect to reservoir and incidental hosts; nor do we understand the potential for virus transmission to humans from animal species within and outside their geographical range. Until 2003, human MPXV infections had remained localized to a handful of countries in central and West Africa, with the majority of cases in the DRC. Given that sustained human-to-human transmission was considered unlikely and that the expected reservoir(s) was most likely a native African animal species, the West gave little consideration to MPXV as an important zoonotic agent (although monkeypox was placed on the US government’s select agent list of potential bioterrorism agents prior to 2003). This opinion changed in the summer of 2003 when human monkeypox was reported in Midwestern states of the USA. The 2003 US MPXV outbreak was instructive as it added to the breadth of host species capable of supporting MPXV replication, and underscored the potential for MPXV to expand its geographic range as a result of commerce or environmental degradation. It highlights how inadequate quarantine and import regulations or noncompliance with regulations can result in the introduction of exotic pathogens in shipments of live animals into Western countries. Importation of African rodents into the US is now banned, as is the interstate movement of all prairie dogs and some species of African rodents. However, US cities with large populations of African immigrants often play host to vibrant black-market trading in native African bush meats, some of which are not cooked, skinned or disemboweled before entering the USA. Although bush meat could be a vehicle for the importation of exotic agents into the USA, bush meat was not implicated in the 2003 monkeypox outbreaks in the USA.
The CDC tested animals from the 2003 shipment of African rodents that entered the USA and confirmed the presence of MPXV by PCR and virus isolation. Surprisingly, no human infections were attributed to contact with these animals; rather, most patients had direct contact with native prairie dogs, which had originally been housed in close contact with the imported African rodents. However, it was reported that some individuals became infected with MPXV via indirect exposure. In one such case, a human female became infected with MPXV after a prairie dog was taken into her home for a brief period while she was absent; fomites or aerosols were the suspected exposure route(s). Ulcerative lesions were observed on lips, tongues and buccal mucosae of surviving experimentally infected prairie dogs and MPXV could be cultured from nasal discharge and the oropharynx for up to 22 days, indicating that transmission is possible via respiratory secretions and mucocutaneous contact. Experimental intranasal and subcutaneous inoculations of prairie dogs resulted in 60 and 100% mortality respectively, and transmission-relevant tissues and secretions were shown to have high levels of infectivity. PCR testing of tissues from animals that cohabited with animals infected with MPXV during the 2003 US outbreak indicated that Cricetus spp. (hamster), Gerbillus spp. (gerbil) and Chinchilla spp. (chinchilla) are also potential hosts for MPXV. The broad host range of MPXV underscores the real risk that an introduced pathogen can enter a new animal reservoir in a new region from which it can cause epizootic infections, and be resistant to eradication.
Expansion of epizootic MPXV on the African continent may be indicated by a recent outbreak in Bentiu, Sudan. In October 2005, an 8-month-old infant presented with a generalized vesiculopapular rash in Bentiu, which is approximately 300 miles northeast of the DRC border and at least 100 miles from the edge of the tropical rainforest. The WHO Reference Center for Orthopoxviruses at the CDC, Atlanta, USA, confirmed that specimens from this index case contained virus related to the more virulent Congo Basin clade. Subsequently, 18 suspected cases of MPXV were clinically diagnosed in Bentiu, with no associated deaths, and there have been no reports of new cases since November, 2005. If this outbreak arose from an animal source, it could represent MPXV adaptation to new hosts or the expanding range of traditional hosts due to changes in habitat. Animal habitat changes, some caused by agricultural expansion, climate change and urbanization, can increase human exposure to a greater number and diversity of small animal populations, which may support pathogens. The maintenance of Lassa fever virus in Mastomys spp. is an example of this principle. As the Bentiu region is a grassy plain with few trees in a subtropical climate zone (savannah), maintenance of MPXV may not be inexorably linked to species of the lowland tropical forests of central and West Africa. Interestingly, the range of putative reservoir species Heliosciurus rufobrachium and H. gambianus includes parts of Sudan. Other animal species listed in Table 1 and found in Sudan include Atherus africanus, Sus scrofa, and Cercopithecus aethiops.
Conclusions
The emergence of human MPXV likely resulted from a number of risk factors, including cessation of smallpox vaccination and an increase in geographical range, and numbers, of reservoir or incidental animal host(s). The US outbreak underpinned how a stable virus with a broad host range has global reach to cause human disease. It is fortuitous that this outbreak was not due to the more aggressive Congo Basin MPXV strains. Time will tell whether MPXV has established itself into a native US animal reservoir and if future outbreaks will occur. The adaptation of poxviruses to new animal hosts has occurred previously. Variola virus likely entered the human population from an animal reservoir and vaccinia has adapted to new hosts on several occasions. When vaccination with vaccinia was practiced, it was not unusual for domestic animals to become infected, and in certain instances the virus persisted. In 1985, vaccinia virus was isolated from scabs taken from pox lesions on buffaloes in five different districts of Maharashtra State, India. Buffalopox (a subspecies of vaccinia) outbreaks continued in this region until 1996 with human infections and possible subclinical disease in endemic areas. In addition, since 1999, vaccinia virus has been making increasing infectious outbreaks in cattle and dairy workers based in Minas Gerais state, Brazil. In both the Brazilian and Indian outbreaks, the source of vaccinia virus was thought to be the live vaccine used during the smallpox eradication period. Similarly, in 1932, vaccinia virus appeared to adapt to a rabbit host, thereby causing a new disease that was highly lethal and transmissible to contact rabbits by airborne infection. The isolated agent was named rabbitpox virus, and recent sequencing of its genome confirmed it as a strain of vaccinia virus. From our experience with vaccinia virus, we should not be surprised if MPXV continues to adapt to new animal species, this has already happened in prairie dogs (in the USA), and MPXV may have adapted to new species in Sudan, thereby facilitating the 2005 outbreak in humans.
Currently, human monkeypox is a minor public health consideration compared with that of smallpox in the pre-1980s era; however, MPXV is becoming a more common infection in central Africa, and there is a rise in the number of transmission generations observed during at least one outbreak. Currently MPXV outbreaks could be contained by quarantine and the traditional ring vaccination strategy that was effectively used in the smallpox eradication program. In the future, there may also be antiviral drug options. Cidofovir, an antiviral drug with activity against virtually all DNA viruses, including monkeypox, can be considered in severe cases. A prodrug, such as hexadecyloxy-propyl-cidofovir (HDP-cidofovir), and ST-246, have also provided encouraging results when used to therapeutically treat poxvirus infections in small mammals and nonhuman primates. In addition, highly specific assays are now available that facilitate quick and accurate diagnosis of MPXV infections using real-time PCR approaches.
Future perspective: can monkeypox virus evolve from a zoonotic to an endemic infection?
Based on the 1981-1986 surveillance program, most human monkeypox cases occurred as single sporadic infections after contact with animals or animal tissues. The overall first-generation secondary attack rate of monkeypox in nonvaccinated household contacts was approximately 9% compared with 58% for smallpox. The monkeypox attack rate decreased precipitously over the second and third generations, and fourth-generation infections were extremely rare. The evolution of MPXV from a zoonotic disease in which humans are incidental hosts to a human disease of endemic character will require genetic changes that enhance MPXV transmissibility.
Perpetual transmission of MPXV in human populations will require a secondary attack rate that approaches the rate observed for variola virus. The secondary attack rate of a population is based on the frequency of infected persons having transmitted sufficient virus to cause recognizable clinical disease or seroconversion in a human contact. The transmission cycle begins with infection of the respiratory epithelium, followed by movement of the virus through the lymphatic system to differentially infect all major internal organs (primary viremia). Released virus from internal organs and lymphoid tissue are transported in the blood (secondary viremia) to the cornified and mucosal epithelium to form the exanthem and enanthem, respectively. Virus-containing secretions from the oropharynx are responsible for initiation of the next transmission cycle, which explains the long delay in a patient becoming infectious. The culmination of the natural lifecycle, and the test of virus fitness for this viral species, is transmission to a new host.
This transmission cycle can be broken into at least six critical steps, beginning with the release of virions from lesions in the oropharyngeal mucosa and their aerosolization into the next host’s breathing space. For variola major, this period of infectiousness occurs optimally between 12 and 19 days post infection correlating with titers of over 10 2 pock-forming units/ml in the oropharyngeal secretions. The highest viral titers were noted on days 16 and 17 post infection. Any viral mutations culminating in higher titers or the presence of virus in the oropharyngeal secretions for longer periods will likely increase virus transmissibility.
Transmission requires stability of the virus outside the body. Epidemiologic studies suggest that the variola virion, and by analogy the monkeypox virion, is exhaled as a large droplet aerosol that can dry to a droplet nuclei in flight, considering variables as residence time in the air, ambient temperature and humidity. The droplet nuclei in its simplest form is an approximate 0.5 µµm diameter particle containing a single protein and salt-encased virion. However, the size may be larger as studies measuring the recovery of variola virus from the vicinity of patients detected most virions on solid surfaces near the patient, and little if any virus in the air. There is no evidence implicating either the extracellular enveloped virus or intracellular mature virus as the infectious form of the virus, although our current model of virus release from infected cells would favor the extracellular enveloped virus as the virion form entering the respiratory gases. The infectiousness of the poxvirus virion is highly stable, as indicated by the 1970 Meschede Hospital and 1971 Aralsk smallpox outbreaks that were initiated by airborne variola virus over remarkable distances. There is no reason to think that MPXV virions are any less stable than those of variola.
The cell types first infected during a respiratory infection of humans with either variola or monkeypox viruses, and the location of these cells in the respiratory tract, are not known, as a primary lesion has not been described. However, there is a great body of evidence to suggest that a very small number of poxvirions are capable of initiating infection, while the dose required to induce clinical disease varies considerably. For example, only low levels of infectivity were detected in oropharyngeal secretions of infectious smallpox patients, and virus was virtually undetectable in the air of smallpox wards unless measured within a short distance of a patient’s mouth. This suggests the infectious dose is low. This notion is also supported by the Meschede Hospital and Aralsk smallpox outbreaks mentioned earlier. Similarly, low infectious doses were observed with vaccinia virus in rabbits (four plaque forming units [PFU]/100% seroconversion), rabbitpox virus in rabbits (15 PFU/86% mortality) and ectromelia virus in mice (0.6 PFU/50% mortality). There are no data to suggest that MPXV is less infectious than other orthopoxviruses.
Although the immune system is well equipped to detect infections early in the disease process, a wide-range of pathogens, including poxviruses, encode a group of host-specific proteins that neutralize the innate/immune response. In this regard, molluscum contagiosum virus has achieved the highest level of survivability for a human poxvirus pathogen, persisting in the superficial layers of the skin in an infectious form for months and sometimes years. MPXV also has a unique group of immune evasion molecules. The tuning of any number of these immune evasion molecules could lengthen the survival time of MPXV-infected cells, resulting in incremental increases of progeny virus during the progression of the infection, culminating in a greater secondary viremia, leading to additional respiratory mucosal lesions, and increasing numbers of aerosolized virions expelled from the respiratory system. In particular, the E3L ortholog deserves mention. It has been conserved in all sequenced poxviruses, and a vaccinia virus mutant lacking it replicates less efficiently in primary cells and is highly attenuated following intranasal inoculation of BALB/c mice compared with wild-type virus. The E3L gene codes for an inhibitor of the activation of interferon-induced antiviral pathways PKR (double stranded [ds]RNA-dependent protein kinase) and OAS (oligoadenylate synthetase). The N-terminal domain of E3L shows sequence similarity with a group of host Z-DNA binding proteins, ADAR-1 and Dlm, and has been shown to be important in the spread of vaccinia virus in the mouse. Interestingly, this domain is disrupted in the MPXV ortholog, thereby suggesting it is not fully functional in MPXV. Repair of this gene in nature resulting in enhanced transmission is not out of the question, as such an ADAR-1/E3L chimera was able to partially complement a deletion of the N-terminal domain of E3L in the vaccinia/mouse model.
Since smallpox is thought to lack a primary lesion at the sites of entry, spread of virus within the host is key to clinical disease and transmission potential. The primary viremia denotes virus replication and spread that occurs between the initial site(s) of infection and lymphoid tissues, and other organ systems. During primary viremia, little or no virus can be found in the blood, as it is efficiently removed by the reticuloendothelial system. There are no data describing the primary viremia of variola or monkeypox viruses in humans during the first 10-12 days following infection; rather, the proposed movement of virus in monocytes from the respiratory epithelium to draining lymph nodes is based on the mousepox model, and, cell-to-cell spread of MPXV in tissue is thought to be mediated by the extracellular enveloped form of the virus, as in vaccinia infections. The magnitude of the primary viremia is dependant on the immune evasion factors mentioned earlier, factors that protect virions and/or virus-infected cells, and factors that govern viral tropism. An example of a virus-encoded molecule that could protect monkeypox virions or MPXV-infected cells from host factors or cells is MOPICE (see earlier). Viral tropism genes ensure that sufficient cell types and numbers are fully permissive for replication to result in efficient transmission of virus to contacts. Although it is likely that poxviruses bind to most cells, enter them and transcribe early genes to some degree, completion of the replication cycle is more dependant on host cell metabolism than has been thought previously. For example, intermediate and late virus transcription require host-derived transcription factors, quiescent cells must be activated by virus-encoded growth factors or other molecules, and apoptotic pathways triggered as a consequence of infection must be blocked. Mutations of MPXV genes that govern viral tropism have great potential to enhance transmissibility.
The secondary viremia is described as the movement of virus from the infected lymphoid tissues and internal organs to the mucosal and cornified epithelium. All of the viral functions that are important for maximizing the magnitude of the primary viremia are likely important in the secondary viremia. Virus is readily detected in blood during the secondary viremia, possibly as a result of virus-mediated toxicity on the reticuloendothelial system, which facilitates clearance of blood-borne pathogens. The severity of the exanthem and enanthem is probably proportional to the concentration of the virions in the bloodstream during the secondary viremia, with each epithelial lesion initiated by one virion. Transmissibility of this virus will depend upon the number of virus lesions in the host oropharyngeal mucosa, viral survivability in the face of the host immune response, and the ability of this virus to produce numerous, viable virions within these lesions, thereby allowing a high number of virions to be exhaled via the respiratory tract of the lung.
It is clear from our recent history with vaccinia virus that orthopoxviruses are capable of infecting and maintaining themselves in new hosts in the absence of human intervention. There is no reason to believe that MPXV cannot do the same, given our understanding of the genetic potential of the MPXV genome. Although sufficient therapeutic and prophylactic options are available to prevent such a variant from taking over the niche once held by variola virus, it could become a nuisance pathogen if its ability to transmit continually in human populations was combined with the use of reservoir hosts that were both abundant and found in close proximity to humans.
The discussion above concerns the natural evolution of MPXV into a pathogen with enhanced transmissibility in human populations. An unfortunate circumstance is the possibility of terrorists or rogue nations bioengineering MPXV to enhance its virulence and/or transmissibility. Although variola would be the first choice for a would-be terrorist, the option of MPXV might also be tempting, considering it is readily available in Africa, the molecular biology of poxviruses is fairly well understood and genetic tinkering is possible in standard equipped laboratories. Although data in the public domain suggest that the virulence of MPXV could be enhanced by the expression of human type 2 cytokines, such as IL-4, the engineered genetic manipulations that would be required to enhance transmissibility are less clear.
African animals known to have been naturally infected with monkeypox virus: Funisciurus lemniscatus, Funisciurus anerythrus, Heliosciurus gambianus, Heliosciurus rufobrachium, Funisciurus iIsabella, Cercopithecus ascanius, Cercopithecus mona, Atherurus africanus, Colobus badius, Allenopithecus nigroviridis, Cercocebus galeritus, Cercopithecus aethiops, Cercopithecus nictitans, Cercopithecus petaurista, Funisciurus congicus, Pan troglodytes, Sus scrofa, Cricetomys emini, Petrodromus tetradactylus.